A class of selective mtorc1 degraders based on autophagy-lysosome pathway and preparation method and application thereof

Abstract

The application discloses a kind of selective mTORC1 degradation agent based on autophagy-lysosome pathway and its preparation method and application, belong to the field of biological medicine technology.The application based on autophagy degradation proposes the concept of selective mTORC1 degradation, designs and synthesizes three kinds of rapamycin derivatives, and the in-vitro anti-MCF7 tumor cell activity of representative compound can be comparable with positive drug rapamycin;The rapamycin derivative obtained by the application can realize the selective degradation of mTORC1, and the degradation process is related to autophagy, while the protein level in mTORC2 is not affected.The selective mTORC1 degradation agent obtained by the application can bring new ideas and potential drug research and development direction for the treatment of related diseases, and also provides a powerful tool for in-depth study of the function and regulation mechanism of mTORC1.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a selective mTORC1 degradation agent based on the autophagy-lysosome pathway and a preparation method and application thereof. BACKGROUND

[0002] Mammalian target of rapamycin (mTOR) is a serine / threonine protein kinase with a molecular weight of 289 kDa, belonging to the phosphatidylinositol 3-kinase-related kinase (PIKK) family. mTOR plays an important role in the regulation of cell life activities and metabolism, and its signaling pathway is closely related to many human diseases, such as tumors, neurodegenerative diseases, diabetes, immunity, etc. In cells, mTOR mainly exerts its function by participating in the formation of two different complexes, mTORC1 and mTORC2. mTORC1 can regulate the translation of messenger RNA and cell size through various mechanisms, mainly including the phosphorylation of ribosomal S6 kinase (S6K) and eukaryotic initiation factor 4E-binding protein (4EBP). mTORC1 is usually activated in the presence of sufficient amino acids, which promotes the production of proteins, nucleotides and lipids, and promotes cell growth by promoting anabolism and inhibiting catabolism, and may trigger the occurrence and development of tumors. mTORC2 regulates cell metabolism, proliferation and survival by phosphorylating AGC family kinases such as protein kinase B (AKT). Since mTOR signaling pathway controls cell metabolism, growth, proliferation and survival, sustained overactivation of mTOR signaling will lead to increased cell metabolic level, sustained growth and proliferation, prolonged cell life and even cell immortalization, which can directly or indirectly induce various types of cancer, neurodegenerative diseases, obesity, diabetes, chronic inflammatory diseases and dry eye, etc. And inhibiting this state can effectively delay or treat related diseases caused by overactivation of mTOR. Therefore, the research of mTOR inhibitors is a popular field at present, and many mTOR inhibitors have been developed. Current mTOR inhibitors are mainly divided into the following three categories: allosteric mTOR inhibitors represented by rapamycin and its derivatives (first generation), ATP-competitive mTOR inhibitors (second generation), and mTOR inhibitors covalently linked by a linker between rapamycin and an ATP-competitive mTOR inhibitor (third generation). Among them, ATP-competitive mTOR inhibitors can simultaneously inhibit the activity of mTORC1 and mTORC2, and the inhibition of mTORC2 activity will reduce the activation of AKT, which has two potential drawbacks, one is to induce hyperglycemia, and the other is to relieve the feedback inhibition of AKT on receptor tyrosine kinase (RTK) expression, which can cause greater toxic side effects and drug resistance after clinical application. Therefore, compared with pan-mTOR inhibitors, i.e. inhibitors that simultaneously inhibit the activity of mTORC1 and mTORC2, mTORC1 selective inhibitors have lower toxicity and are less likely to cause adaptive resistance.

[0003] In recent years, targeted protein degradation has been widely applied to various proteins as a major new drug development model in the past two decades, providing a highly potential treatment strategy for the treatment of diseases such as cancer and inflammation. Among them, proteolysis targeting chimeras (PROTAC) as a potential targeted protein degradation technology is a heterobifunctional small molecule connected by a part that can recruit E3 ubiquitin ligase and a ligand of target protein (POI). It can realize the degradation of target protein by promoting the close proximity of E3 ubiquitin ligase and POI. PROTAC molecules exhibit high efficiency, low drug resistance, and other characteristics, and can also target "undruggable" proteins, so its application prospect is extremely broad. PROTAC based on ATP-competitive mTOR inhibitor MLN0128 as POI can achieve mTOR protein degradation, but due to its poor activity and selectivity, there is no further related research [1] . And PROTAC using rapamycin as POI can only achieve FKBP12 protein degradation and cannot achieve mTOR degradation [2] . The core of PROTAC is that it recruits E3 ligase to the vicinity of the target protein to form a ternary complex, induces ubiquitination of the target protein, and degrades it through the proteasome. Rapamycin and its analogs first form a binary complex with FKBP12 protein, and the activity of mTOR is inhibited by the complex. Therefore, it is difficult for PROTAC using rapamycin as POI to form a ternary complex with mTOR and induce its ubiquitination and proteasome degradation. However, autophagy-lysosome is another important protein degradation system in eukaryotic cells in addition to the ubiquitin-proteasome system, and has also been gradually applied to the field of targeted protein degradation. Targeted degradation technology based on autophagy-lysosome pathway mainly includes: autophagosome-tethering compound (ATTEC) [3-5] , autophagy-targeting chimera (AUTAC) [6] and AUTOTAC [7] , etc.

[0004] ATTEC initially refers to a class of molecular glue that can tightly bind mutant huntingtin (mHTT) protein and LC3 protein together, and promote the degradation of mHTT based on the autophagy-lysosome pathway. Later, researchers designed these ATTEC small molecules as LC3 binding ligands, and connected them with different target protein ligands through a linker, thus obtaining a class of heterobifunctional autophagy degraders, further expanding the concept of ATTEC, and successfully degrading BRD4, NAMPT and other proteins [3,5] . AUTAC also belongs to a class of heterobifunctional molecules, which consists of a target protein ligand, a degradation tag (cGMP analogue), and a linker. In cells, AUTAC first binds to the target protein, simulates "modification" through the degradation tag, and induces K63 polyubiquitination of the target protein. The ubiquitinated target protein is then selectively recognized and bound by the autophagy receptor p62, which further interacts with LC3, thereby mediating the recruitment of the target protein to the autophagosome and ultimately degrading it in the lysosome. However, the mechanism of how S-adenosylation modification induces K63 ubiquitination is still unclear. Researchers have successfully degraded MetAP2, FKBP12 and other proteins using this technology. In addition, AUTAC is also suitable for the targeted degradation of organelles such as mitochondrial fragments [6] . AUTOTAC can directly bind to the target protein and the ZZ binding domain of p62, forming a ternary complex and mediating the degradation of the target protein through the autophagy-lysosome pathway. In this process, the target protein does not need to undergo ubiquitination, but is directly initiated by the p62-dependent macroautophagy induction cascade. Studies have shown that AUTOTAC not only mediates the targeted degradation of monomeric proteins, but also mediates the degradation of protein aggregates, such as tau protein (P301L mutation) pathological aggregates in neurodegenerative diseases, providing a new technical means and treatment strategy for clearing pathological aggregates in neurodegenerative diseases [7] . The above three protein degradation technologies can achieve the degradation of protein complexes and even organelles, providing a theoretical basis for the development of selective mTORC1 degraders based on the autophagy-lysosome pathway.

[0005] Existing research on mTOR-related degraders mainly includes two parts: 1. PROTAC based on ATP-competitive mTOR inhibitor MLN0128 as POI to achieve mTOR protein degradation; 2. PROTAC based on rapamycin as POI. Among them, 1 has poor activity and cannot achieve selective mTORC1 degradation; 2 can only achieve FKBP12 protein degradation and cannot achieve mTOR protein degradation.

[0006] The following references to prior art documents or patent information are made:

[0007] [1] Zhang Q, Yan PZ, Zhao P, et al. Design, Synthesis, and Biological Evaluation of mTOR-Targeting PROTACs Based on MLN0128 and Pomalidomide [J]. Chem Pharm Bull (Tokyo), 2023, 71(2): 120-128.

[0008] [2] Sun XY, Wang J, Yao X, et al. A chemical approach for global protein knockdown from mice to non-human primates [J]. Cell Discovery, 2019, 5(1): 10.

[0009] [3] Pei JP, Pan XL, Wang AX, et al. Developing potent LC3-targeting AUTAC tools for protein degradation with selective autophagy [J]. Chemical Communications, 2021, 57(97): 13194-13197.

[0010] [4] Li ZY, Wang C, Wang ZY, et al. Allele-selective lowering of mutant HTT protein by HTT-LC3 linker compounds [J]. Nature, 2019, 575(7781): 203-+.

[0011] [5]Dong GQ, Wu Y, Cheng JF, et al. Ispinesib as an Effective Warhead for the Design of Autophagosome-Tethering Chimeras: Discovery of Potent Degraders of Nicotinamide Phosphoribosyltransferase (NAMPT) [J]. Journal of Medicinal Chemistry, 2022, 65(11): 7619-7628.

[0012] [6]Takahashi D, Arimoto H. Targeting selective autophagy by AUTAC degraders [J]. Autophagy, 2020, 16(4): 765-766.

[0013] [7]Ji CH, Kim HY, Lee MJ, et al. The AUTOTAC chemical biology platform for targeted protein degradation via the autophagy-lysosome system [J]. Nature Communications, 2022, 13(1): 904.

[0014] [8]CN202310124471.2. SUMMARY

[0015] In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide a kind of selective mTORC1 degrader based on autophagy-lysosome pathway and its preparation method and application,

[0016] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0017] The present application discloses a kind of selective mTORC1 degrader based on autophagy-lysosome pathway, its structural formula is as shown below:

[0018]

[0019] In the formula, R represents terminal autophagy-lysosome target protein modification label;

[0020] Linker represents intermediate linker.

[0021] The "autophagy-lysosome pathway" (ALP) described in the present application is an important pathway in the process of cellular autophagy, which involves the process of cells engulfing and degrading part or all of their organelles and proteins to maintain the balance of the intracellular environment and cope with environmental stress. Further, the target protein modification tags in the autophagy-lysosome pathway play a crucial role in the process of autophagy. These tags are usually used to mark proteins that need to be degraded, thereby guiding them into the autophagy-lysosome pathway for degradation.

[0022] Preferably, the terminal autophagy-lysosome target protein modification tag comprises an LC3 ligand, an S-adenosylation tag and a p62 binding ligand.

[0023] Preferably, R comprises a compound as shown in the following structure:

[0024] 1) LC3 ligand: and derivatives thereof;

[0025] 2) S-adenosylation tag: and derivatives thereof;

[0026] 3) p62 binding ligand:

[0027]

[0028] and derivatives thereof.

[0029] Preferably, Linker is selected from one of the following structures:

[0030]

[0031] The number of oxygen atoms in the polyethylene glycol chain is n, 0≤n≤5, n is an integer;

[0032]

[0033] The number of carbon atoms in the alkyl chain is m, where 1≤m≤20, m is an integer.

[0034] Preferably, the selective mTORC1 degradation agent based on the autophagy-lysosome pathway synthesized by the present application, in particular six structures, the structural formula is as follows:

[0035]

[0036]

[0037] The application also discloses a synthesis method of the selective mTORC1 degradation agent based on the autophagy-lysosome pathway.

[0038] Preferably, the compound A is composed of a terminal autophagy-lysosome target protein modification tag and a linker.

[0039] The terminal autophagy-lysosome target protein modification tag comprises an LC3 ligand, an S-adenosylation tag or a p62 binding ligand.

[0040] The linker is selected from a polyethylene glycol chain structure or an alkyl chain structure.

[0041] Preferably, the rapamycin donor has the following structural formula.

[0042]

[0043] Preferably, the copper sulfate solution has a concentration of 1 mol / L, the sodium ascorbate solution has a concentration of 1 mol / L, and the volume ratio of the copper sulfate solution to the sodium ascorbate solution is 1-2:1.

[0044] Preferably, the molar ratio of the compound A to the rapamycin donor is 1-1.2:1.

[0045] The application also discloses application of the selective mTORC1 degradation agent based on the autophagy-lysosome pathway in preparation of a medicine for treating cancer, a neurodegenerative disease, a metabolic disease or a chronic inflammatory disease.

[0046] Preferably, the cancer comprises breast cancer, liver cancer, prostate cancer, cervical cancer, skin cancer or colon cancer.

[0047] Preferably, the neurodegenerative disease comprises Alzheimer's disease.

[0048] Preferably, the metabolic disease comprises diabetes, hyperlipidemia or obesity.

[0049] Preferably, the chronic inflammatory disease comprises dry eye, chronic hepatitis or chronic pancreatitis.

[0050] Compared with the prior art, the application has the following beneficial effects:

[0051] The selective mTORC1 degradation agent based on the autophagy-lysosome pathway disclosed in the application mainly comprises three parts: a rapamycin donor, an intermediate linker and a terminal autophagy-lysosome target protein modification tag. In existing mTOR degradation agents, the ATP competitive mTOR inhibitor MLN0128 as the POI has poor PROTAC activity and poor selectivity for mTORC1, and the PROTAC with rapamycin as the POI can only achieve the degradation of FKBP12 protein and cannot achieve the degradation of mTOR, so the application first proposes a selective degradation agent for mTORC1, which can achieve selective degradation of mTORC1. At the same time, due to the terminal connection modification tag, the degradation process is related to autophagy, and the protein level in mTORC2 is not affected, which can effectively solve the problems that the existing mTOR inhibitors have limitations in therapeutic effect, drug resistance occurs after long-term use, and obvious toxic side effects caused by poor selectivity for mTORC1.

[0052] The rapamycin derivative obtained by the application brings a new idea and potential drug research and development direction for the treatment of related diseases (such as cancer, neurodegenerative diseases, metabolic diseases, chronic inflammatory diseases, etc.), and also provides a powerful tool for in-depth research on the function and regulation mechanism of mTORC1, and provides beneficial reference and inspiration for other similar researches.

[0053] Further, the application designs and synthesizes three types of rapamycin derivatives, and the in vitro anti-MCF7 tumor cell activity of representative compounds can be comparable to that of positive drug rapamycin. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 The concentration-dependent cell growth inhibition curve of MCF-7 cell lines treated with rapamycin and representative compounds for 72 hours;

[0055] Figure 2 The representative protein expression amount change graph in mTORC1 and mTORC2 in MCF-7 cell lines after rapamycin and 1-8-2 act for 48 hours;

[0056] Figure 3 The compound 1-8-1 induced mTORC1 protein degradation mechanism graph in MCF-7 cell lines.

[0057] Figure 4 The concentration (n=3) of A) mTORC1 and B) mTORC2 in MCF-7 cells after rapamycin and 1-8-2 act for 48h was determined by ELISA method. DETAILED DESCRIPTION

[0058] In the following, the technical solutions in the embodiments of the present application will be described clearly and completely with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative work should belong to the protection scope of the present application.

[0059] It should be noted that the terms "first", "second" and the like in the description and claims of the present application and the above-mentioned drawings are used to distinguish similar objects, and do not necessarily indicate a specific order or sequence. It should be understood that the data thus used can be interchanged under appropriate circumstances, so that the embodiments of the present application described herein can be implemented in an order other than that illustrated or described herein. In addition, the terms "comprise" and "have" and any variations thereof are intended to cover non-exclusive inclusion, for example, a process, method, system, product or device comprising a series of steps or units does not necessarily have to be limited to those steps or units clearly listed, but can include other steps or units not clearly listed or inherent to these processes, methods, products or devices.

[0060] The present application will be described in further detail below with reference to the drawings:

[0061] The present application aims to provide an mTORC1 selective degradation agent and a preparation method and application thereof.

[0062] Disclosed is a compound and a pharmaceutically acceptable salt thereof, the structural formula of which is shown in the following formula:

[0063]

[0064] R represents an end autophagy-lysosome target protein modification tag;

[0065] Linker represents an intermediate linker.

[0066] Further, R in the formula includes an LC3 ligand, an S-adenosylated tag or a p62 binding ligand; further, the structural formula of the LC3 ligand is shown in the following formula:

[0067]

[0068] Further, the structural formula of the S-adenosylated tag is shown in the following formula:

[0069]

[0070] Further, the structural formula of the p62 binding ligand is shown in the following formula:

[0071]

[0072]

[0073] Further, the Linker is selected from a polyethylene glycol chain structure or an alkyl chain structure.

[0074] Still further, the polyethylene glycol chain structure has n oxygen atoms, where 0≤n≤5, n is an integer; and the structure is as follows:

[0075]

[0076] The alkyl chain has m carbon atoms, where 1≤m≤20, m is an integer; and the structure is as follows:

[0077]

[0078] Particularly preferably, the present application produces six selective mTORC1 degradation agents, and the structures are as follows:

[0079]

[0080]

[0081] I. The following is the preparation of representative compounds. 1. Preparation of ATTEC

[0082]

[0083] Ethanol is used as a solvent, and compound a and compound 1-1 are used as raw materials to prepare compound 1-2 under the catalysis of piperidine; and compound 1-3 is prepared by removing the BOC protecting group of the compound 1-2 in a dichloromethane solution of trifluoroacetic acid;

[0084] The compound 1-3 and compound b are used as raw materials, DMF is used as a solvent, HATU is used as a condensing agent, and condensation reaction occurs under the basic conditions of N,N-diisopropylethylamine to prepare compound 1-4;

[0085]

[0086] Dichloromethane is used as a solvent, compound 1-5 is used as a raw material, and trifluoromethanesulfonic anhydride is used as an acylating agent to prepare compound 1-6 under the basic conditions of 2,6-dimethylpyridine;

[0087] Toluene is used as a solvent, and the compound 1-6 and rapamycin are used as raw materials to prepare compound 1-7 under the basic conditions of N,N-diisopropylethylamine under microwave conditions;

[0088]

[0089] Water and methanol as mixed solvent, with the compound 1-4 and compound 1-7 as raw materials, under the catalytic conditions of copper sulfate and sodium ascorbate, to prepare compound 1-8.

[0090] Take the synthesis of 1-8-2 as an example:

[0091]

[0092] The specific preparation process is as follows:

[0093] Into 15 mL of ethanol, add compound 1-1 (0.43 g, 1.1 mmol) and compound a (0.26 g, 1.0 mmol), and add piperidine (20 uL) as a catalyst, heat the system to reflux for 18 h, and cool to room temperature. A large amount of solid is precipitated in the system, which is directly filtered to obtain 0.60 g of yellow solid 1-2 with a yield of 95%. Dissolve the obtained 1-2 in 10 mL of dichloromethane, and add 3 mL of trifluoroacetic acid. Stir at room temperature overnight, and detect the reaction system by TLC until the reaction is completed. Evaporate the solvent, and directly perform the next step reaction without purification. Dissolve the residue obtained by rotary evaporation in 10 mL of DMF, and sequentially add compound b2 (0.22 g, 1.0 mmol), HATU (0.45 g, 1.2 mmol) and DIPEA (0.63 mL, 3.6 mmol). Stir the system at room temperature under nitrogen atmosphere for 12 h, and evaporate the solvent to obtain a crude product. Purify by silica gel column chromatography with petroleum ether / ethyl acetate = 2 / 1 as the mobile phase to obtain 709 mg of orange-yellow solid with a yield of 91%.

[0094] Synthesis of rapamycin moiety linker:

[0095]

[0096] Into a 100 mL flask, add 20 mL of dichloromethane, and add compound 1-5 (3.0 g, 30 mmol) and pyridine (2.9 mL, 36 mmol). Into a 50 mL constant pressure dropping funnel, add trifluoromethanesulfonic anhydride (6.1 mL, 36 mmol) and 20 mL of dichloromethane. Stir under ice-water bath to cool the reaction solution to 0°C, and slowly drop the solution of trifluoromethanesulfonic anhydride into the reaction system. After dropping, sequentially wash the system with water (10 mL x 3), saturated sodium chloride (10 mL), and anhydrous sodium sulfate. Evaporate the solvent to obtain a residue, which is separated by flash silica gel column chromatography with petroleum ether / ethyl acetate = 20 / 1 as the mobile phase to obtain 6.0 g of light brown transparent liquid with a yield of 93%.

[0097] In a glove box, rapamycin (91.4 mg, 0.1 mmol), compound 1-6 (218.0 mg, 1 mmol), DIPEA (0.23 mL, 1.3 mmol), triphenylphosphine oxide (56.0 mg, 0.2 mmol), molecular sieves (3A, 40.0 mg) and 0.17 mL of toluene were added into a 5 mL microwave tube. After sealing, the glove box was taken out and placed in a microwave reactor, and the temperature was set to 65 °C and the time was set to 5 minutes. After the reaction was completed, it was taken out and directly separated by low-temperature silica gel column chromatography with n-hexane / acetone = 5 / 1 as the mobile phase to obtain 61.0 mg of white foamy solid with a yield of 61%. 1H NMR (600 MHz, DMSO-d6) δ 6.49 - 6.44 (m, 1H), 6.42 - 6.38 (m, 1H), 6.25 - 6.20 (m, 1H), 6.17 - 6.10 (m, 2H), 5.46 (dd, J = 14.9, 9.5 Hz, 1H), 5.31 - 5.26 (m, 1H), 5.09 (d, J = 10.3 Hz, 1H), 5.01 - 4.95 (m, 1H), 4.94 (dd, J = 6.1, 2.2 Hz, 1H), 4.15 - 4.14 (m, 2H), 4.03 - 3.99 (m, 2H), 3.95 (d, J = 4.6 Hz, 1H), 3.65 - 3.59 (m, 4H), 3.55 - 3.51 (m, 3H), 3.46 - 3.42 (m, 1H), 3.41 (t, J = 2.4 Hz, 1H), 3.33 (s, 3H), 3.31 - 3.29 (m, 1H), 3.28 - 3.25 (m, 1H), 3.20 - 3.17 (m, 1H), 3.16 (s, 3H), 3.09 - 3.06 (m, 1H), 3.05 (s, 3H), 2.99 - 2.96 (m, 1H), 2.73 (dd, J = 17.8, 2.9 Hz, 1H), 2.42 - 2.35 (m, 2H), 2.25 - 2.20 (m, 1H), 2.10 (d, J = 11.9 Hz, 1H), 2.06 - 2.01 (m, 1H), 1.96 - 1.88 (m, 3H), 1.86 - 1.80 (m, 2H), 1.75 (s, 3H), 1.70 - 1.67 (m, 2H), 1.63 (s, 3H), 1.57 - 1.52 (m, 4H), 1.43 - 1.38 (m, 2H), 1.26 - 1.23 (m, 2H), 1.13 - 1.11 (m, 1H), 1.07 - 1.04 (m, 2H), 0.98 (d, J = 6.6 Hz, 3H), 0.96 - 0.93 (m, 2H), 0.87 (d, J = 6.6 Hz, 3H), 0.82 (d, J = 6.6 Hz, 3H), 0.77 (d, J = 6.8 Hz, 2H), 0.73 (d, J = 6.8 Hz, 3H), 0.67 - 0.61 (m, 1H). 13C NMR (150 MHz, DMSO-d6) δ 211.0, 208.1, 199.4, 169.7, 167.5, 139.8, 138.4, 137.7, 132.8, 130.9, 127.5, 125.4, 99.5, 86.0, 82.9, 82.7, 80.9, 77.5, 76.2, 74.1, 69.5, 69.4, 68.9, 66.7, 60.2, 57.9, 57.5, 57.5, 57.4, 56.0, 51.2, 45.7, 44.0, 38.7, 36.5, 35.7, 35.3, 33.8, 32.7, 31.4, 30.2, 26.9, 26.7, 25.0, 22.1, 21.6, 21.2, 20.9, 16.1, 16.0, 16.0, 15.2, 14.6, 14.0, 13.8, 10.9. HRMS (ESI): m / z calcd for C 56 H 85 NO 14 Na + ([M+Na] + ) = 1018.58623, found = 1018.59257.

[0098] Synthesis of 1-8-2:

[0099]

[0100] Into a 5 mL flask was added compound 1-4-2 (155.8 mg, 0.2 mmol) and compound 1-7 (199.3 mg, 0.2 mmol), then 2 mL of the previously prepared mixed solvent (methanol / water = 5 / 1) was added, followed by the addition of 0.15 mL of the previously prepared copper sulfate solution (1 mol / L) and 0.3 mL of sodium ascorbate solution (1 mol / L) in sequence, and stirred at room temperature overnight. The system was diluted with 30 mL of ethyl acetate, and the obtained organic phase was washed with water (10 mL x 3), saturated sodium chloride (10 mL), and anhydrous sodium sulfate in sequence. The solvent was removed by evaporation, and the obtained residue was separated by low-temperature silica gel column chromatography with n-hexane / acetone = 5 / 1 as the mobile phase to obtain 0.30 g of yellow solid with a yield of 84%, which was 1-8-2. 1H NMR (600 MHz, DMSO-d6) δ 10.85 (s, 1H), 8.80 (s, 2H), 8.12 - 8.07 (m, 2H), 8.04 (d, J = 9.4 Hz, 2H), 7.87 (s, 1H), 7.58 - 7.55 (m, 1H), 6.70 (d, J = 8.3 Hz, 1H), 6.47 (s, 1H), 6.42 - 6.37 (m, 1H), 6.25 - 6.19 (m, 1H), 6.16 - 6.04 (m, 2H), 5.49 - 5.41 (m, 1H), 5.29 (d, J = 4.8 Hz, 1H), 5.08 (d, J = 10.3 Hz, 1H), 5.00 - 4.95 (m, 1H), 4.95 - 4.91 (m, 1H), 4.58 - 4.49 (m, 6H), 4.46 (s, 2H), 4.02 (q, J = 5.6, 3.9 Hz, 2H), 3.95 (d, J = 4.6 Hz, 1H), 3.81 (t, J = 5.3 Hz, 3H), 3.67 - 3.59 (m, 4H), 3.54 - 3.52 (m, 6H), 3.51 - 3.49 (m, 6H), 3.43 (d, J = 11.7 Hz, 1H), 3.31 (s, 3H), 3.15 (s, 3H), 3.12 (s, 1H), 3.05 (s, 3H), 3.01 (s, 1H), 2.99 - 2.93 (m, 2H), 2.87 - 2.76 (m, 1H), 2.75 - 2.68 (m, 1H), 2.41 - 2.33 (m, 2H), 2.25 - 2.18 (m, 1H), 2.12 - 2.08 (m, 1H), 2.05 - 2.01 (m, 1H), 1.93 - 1.83 (m, 4H), 1.74 (s, 3H), 1.69 - 1.65 (m, 2H), 1.55 - 1.52 (m, 2H), 1.43 - 1.37 (m, 2H), 1.32 - 1.21 (m, 5H), 1.12 - 1.09 (m, 1H), 1.07 - 1.02 (m, 3H), 0.97 (d, J = 6.4 Hz, 3H), 0.94 (d, J = 6.4 Hz, 1H), 0.91 (d, J = 7.2 Hz, 1H), 0.86 (d, J = 6.6 Hz, 3H), 0.81 (d, J = 6.4 Hz, 3H), 0.76 (d, J = 6.8 Hz, 2H), 0.73 (d, J = 6.6 Hz, 3H), 0.69 (s, 1H), 0.66 - 0.59 (m, 1H). 13C NMR (150 MHz, DMSO-d6) δ 210.9, 208.0, 199.4, 197.0, 169.7, 168.0, 167.5, 167.0, 166.9, 166.8, 153.3, 152.8, 143.3, 141.0, 139.8, 139.1, 138.4, 138.1, 137.6, 136.7, 134.8, 134.2, 134.0, 133.8, 132.8, 131.1, 130.9, 128.9, 128.7, 127.6, 127.5, 125.3, 124.9, 123.5, 118.2, 117.5, 113.2, 112.5, 99.5, 85.9, 84.7, 82.9, 82.7, 76.2, 74.0, 71.5, 70.0, 69.2, 66.7, 64.0, 60.2, 58.0, 57.5, 57.4, 56.0, 55.4, 51.3, 49.9, 45.7, 44.0, 38.8, 36.5, 35.7, 35.3, 33.8, 32.7, 31.3, 30.1, 26.9, 26.7, 24.9, 22.1, 21.2, 20.9, 16.1, 16.0, 15.2, 14.6, 14.0, 13.8, 10.9. HRMS (ESI): m / z calcd for C 81 H 112 Br2IN6O 20 + ([M+H] + ) = 1773.53374, found = 1773.53725.

[0101] 2, Preparation of AUTAC is as follows:

[0102]

[0103] Compound 2-2 is prepared from compound 2-1 and di-tert-butyl dicarbonate as raw materials, with 4-dimethylaminopyridine as catalyst and ethanol as solvent;

[0104] Compound 2-3 is prepared by stirring compound 2-2 in sodium hydride solution in tetrahydrofuran;

[0105] Compound 2-4 is prepared from compound 2-3 and compound c under the catalytic conditions of DEAD and triphenylphosphine;

[0106] Compound 2-5 is prepared by removing the BOC group from compound 2-4 in formic acid solution;

[0107]

[0108] The compound 2-5 is brominated in bromine water to prepare the compound 2-6;

[0109] DMSO is used as a solvent, the compound 2-5 and the compound d are used as raw materials, and a nucleophilic substitution reaction occurs under the alkaline condition of potassium carbonate to prepare the compound 2-7;

[0110] The compound 2-7 and the compound e are used as raw materials, DMF is used as a solvent, EDC and HOBt are used as condensing agents, and a condensation reaction occurs under the alkaline condition of triethylamine to prepare the compound 2-8;

[0111]

[0112] Water and methanol are used as mixed solvents, the compound 2-8 and the compound 1-7 are used as raw materials, and a compound 2-9 is prepared under the catalytic condition of copper sulfate and sodium ascorbate.

[0113] The synthesis of 2-9-2 is taken as an example:

[0114]

[0115]

[0116] The specific preparation process is as follows:

[0117] Compound 2-1 (5.09 g, 30 mmol) is dissolved in 30 mL of DMSO solution, the temperature of the system is reduced to 0°C under ice-water bath condition, di-t-butyl dicarbonate (6.55 g, 30 mmol) and 4-dimethylaminopyridine (0.18 g, 1.5 mmol) are added. After stirring at room temperature overnight, 100 mL of ice water is directly added to the system, and 7.70 g of white solid is obtained by filtration, with a yield of 87%. The obtained white solid is completely dissolved in 50 mL of tetrahydrofuran, cooled to 0°C under ice-water bath condition, and sodium hydride (60%, 3.0 g, 75 mmol) is added in batches. After stirring at room temperature for 2 h, the system is washed with saturated sodium chloride (10 mL x 3) in sequence and dried with anhydrous sodium sulfate. The solvent is evaporated, and the obtained residue is separated by silica gel column chromatography, with dichloromethane / methanol = 20 / 1 as the mobile phase, to obtain 6.70 g of white solid, with a yield of 87%.

[0118] Compound 2-3 (2.70 g, 10 mmol) was dissolved in 50 mL of tetrahydrofuran, and then compound c (1.26 g, 10 mmol) and triphenylphosphine (6.56 g, 25 mmol) were added successively, and finally DEAD (4.40 g, 25 mmol) was added dropwise. After stirring at room temperature for 6 h, the solvent was evaporated. The residue was separated by silica gel column chromatography with dichloromethane / methanol = 30 / 1 as the mobile phase to obtain a crude product containing triphenylphosphine oxide. The crude product was dissolved in 20 mL of 80% formic acid solution, and heated to 75 °C in an oil bath, and stirred overnight. The solvent was evaporated, and the residue was separated by silica gel column chromatography with dichloromethane / methanol = 10 / 1 as the mobile phase to obtain 4.0 g of white solid with a yield of 60%.

[0119] Compound 2-5 (0.50 g, 1.9 mmol) was dissolved in 5 mL of toluene, and 1 mL of bromine water was added. After stirring at room temperature for 8 h, the system was diluted with 20 mL of ethyl acetate, and the obtained organic phase was washed successively with water (10 mL x 3), saturated sodium chloride (10 mL), and anhydrous sodium sulfate. The solvent was evaporated, and the obtained residue was separated by flash silica gel column chromatography with dichloromethane / methanol = 10 / 1 as the mobile phase to obtain 0.55 g of yellowish solid with a yield of 85%.

[0120] Compound 2-6 (0.50 g, 1.5 mmol) was dissolved in 10 mL of DMF, and then compound d (0.27 g, 1.6 mmol) and potassium carbonate (0.62 g, 4.5 mmol) were added successively. The system was heated to 75 °C in an oil bath, and stirred for 5 h, and then cooled to room temperature. 30 mL of hydrochloric acid solution (4N) was added slowly to the system to precipitate a solid, which was filtered to obtain 0.40 g of white powder with a yield of 63%.

[0121] Compound 2-7 (105.1 mg, 0.25 mmol) was dissolved in 2 mL of DMF, and then EDC (77.6 mg, 0.5 mmol), HOBt (67.5 mg, 0.5 mmol), and TEA (50.6 mg, 0.5 mmol) were added successively. After stirring at room temperature for 4 h, the solvent was evaporated. The residue was separated by silica gel column chromatography with dichloromethane / methanol = 30 / 1 as the mobile phase to obtain 75.0 g of white powder with a yield of 33%.

[0122]

[0123] Into a 5 mL flask was added compound 2-8-2 (155.5 mg, 0.25 mmol) and compound 1-7 (250.0 mg, 0.25 mmol), followed by 2 mL of a previously prepared mixed solvent (methanol / water = 5 / 1), then 0.375 mL of a previously prepared copper sulfate solution (1 mol / L) and 0.75 mL of a sodium ascorbate solution (1 mol / L) were added in sequence, and the system was stirred at room temperature overnight. The resulting organic phase was diluted with 30 mL of ethyl acetate, washed with water (10 mL x 3), saturated sodium chloride (10 mL), and dried over anhydrous sodium sulfate. The solvent was evaporated, and the resulting residue was separated by low-temperature silica gel column chromatography with a mobile phase of n-hexane / acetone = 4 / 1 to obtain 0.25 g of a yellowish solid with a yield of 62%, which was 2-9-2. 1H NMR (600 MHz, DMSO-d6) δ 10.64 (s, 1H), 8.41 (dd, J = 8.2, 2.5 Hz, 1H), 8.07 (t, J = 5.7 Hz, 1H), 8.03 (s, 1H), 7.21 (dd, J = 8.8, 5.5 Hz, 2H), 7.16 (t, J = 8.8 Hz, 2H), 6.53 (s, 2H), 6.47 (s, 1H), 6.44 - 6.37 (m, 1H), 6.25 - 6.20 (m, 1H), 6.17 - 6.05 (m, 2H), 5.46 (dd, J = 14.9, 9.6 Hz, 1H), 5.29 (d, J = 4.6 Hz, 1H), 5.11 - 5.05 (m, 3H), 5.00 - 4.95 (m, 1H), 4.94 (d, J = 7.9 Hz, 1H), 4.51 (s, 3H), 4.50 - 4.46 (m, 3H), 4.04 - 3.99 (m, 2H), 3.96 (d, J = 4.4 Hz, 1H), 3.79 (t, J = 5.3 Hz, 2H), 3.65 - 3.60 (m, 3H), 3.55 - 3.52 (m, 2H), 3.51 - 3.49 (m, 2H), 3.48 - 3.43 (m, 8H), 3.42 (d, J = 5.1 Hz, 1H), 3.37 (t, J = 6.1 Hz, 2H), 3.31 (s, 3H), 3.28 - 3.25 (m, 2H), 3.19 - 3.17 (m, 2H), 3.15 (s, 3H), 3.12 (s, 1H), 3.05 (s, 3H), 3.01 (s, 1H), 2.98 - 2.95 (m, 1H), 2.76 - 2.69 (m, 1H), 2.42 - 2.34 (m, 2H), 2.25 - 2.18 (m, 1H), 2.12 - 2.08 (m, 1H), 2.05 - 2.00 (m, 1H), 1.94 - 1.89 (m, 2H), 1.84 (s, 5H), 1.74 (s, 3H), 1.69 - 1.65 (m, 2H), 1.63 (s, 3H), 1.60 - 1.51 (m, 5H), 1.43 - 1.37 (m, 2H), 1.31 - 1.22 (m, 4H), 1.13 - 1.09 (m, 1H), 1.07 - 1.03 (m, 2H), 0.98 (d, J = 6.4 Hz, 3H), 0.94 (s, 1H), 0.86 (d, J = 6.4 Hz, 3H), 0.82 (d, J = 6.6 Hz, 3H), 0.77 (d, J = 6.8 Hz, 2H), 0.73 (d, J = 6.6 Hz, 3H), 0.66 - 0.60 (m, 1H). 13C NMR (150 MHz, DMSO-d6) δ 211.0, 208.1, 199.4, 170.8, 170.2, 169.9, 169.7, 167.5, 166.6, 162.8, 161.2, 156.1, 154.2, 153.2, 144.4, 142.7, 139.8, 138.4, 137.6, 133.1, 132.8, 130.9, 129.5, 129.5, 127.5, 127.5, 125.3, 124.7, 117.1, 116.0, 115.9, 99.5, 86.0, 82.9, 82.7, 76.2, 74.1, 70.1, 70.1, 70.0, 70.0, 69.9, 69.2, 69.0, 66.7, 64.0, 60.2, 57.5, 57.4, 56.0, 55.4, 52.7, 51.3, 49.8, 45.7, 45.1, 44.0, 39.1, 38.7, 36.5, 35.7, 35.3, 33.8, 32.7, 31.4, 30.2, 26.9, 26.7, 25.0, 23.0, 22.1, 21.2, 20.9, 16.1, 16.0, 15.2, 14.6, 14.0, 13.8, 10.9, 0.6. HRMS (ESI): m / z calcd for C 81 H 119 FN 11 O 20 S + ([M+H] + ) = 1616.83321, found = 1616.83698.

[0124] 3, Preparation of AUTOTAC is as follows:

[0125]

[0126] Compound 3-2 was prepared from compound 3-1 and bromobenzyl in acetonitrile as solvent under the alkaline action of sodium bicarbonate; compound 3-3 was prepared from compound 3-2 and compound f in DMF solution under the alkaline action of potassium carbonate with stirring;

[0127] Compound 3-4 was prepared from compound 3-3 in a dichloromethane solution of meta-chloroperoxybenzoic acid with stirring;

[0128]

[0129] Compound 3-5 was prepared from compound 3-4 and compound g in DMF;

[0130] Ethanol as solvent, with compound 3-5 and compound b as raw materials, nucleophilic substitution reaction to prepare compound 3-6;

[0131]

[0132] Water and methanol as mixed solvents, with compound 3-6 and compound 1-7 as raw materials, under the catalytic conditions of copper sulfate and sodium ascorbate to prepare compound 3-7.

[0133] Take the synthesis of 3-7-2 as an example:

[0134]

[0135] The specific preparation process is as follows:

[0136] Compound 3-1 (7.37g, 53.4mmol) was dissolved in 100mL acetonitrile solution, and benzyl bromide (6.76g, 53.4mmol) and sodium bicarbonate (5.83g, 69.4mmol) were added, and the system was heated to 80℃ and stirred overnight. 100mL hydrochloric acid (4N) was added, and the aqueous phase was extracted with ethyl acetate (80mL x 3), and the combined organic phase was washed with water (10mL x 3), saturated sodium chloride (10mL), and anhydrous sodium sulfate. The solvent was evaporated to obtain 0.80g of white powder solid, with a yield of 13%.

[0137] The obtained compound 3-2 (0.80g, 3.5mmol) was dissolved in DMF (5mL), and compound f (0.84g, 4.2mmol) and potassium carbonate (0.97g, 7.00mmol) were added, and the system was heated to 80℃ and stirred overnight. The solvent was removed, and the residue was separated by silica gel column chromatography, with petroleum ether / ethyl acetate=10 / 1 as the mobile phase, to obtain 1.20g of yellow solid, with a yield of 98%.

[0138] The obtained compound 3-3 (1.20 g, 3.5 mmol) was dissolved in dichloromethane, and m-chloroperbenzoic acid (0.90 g, 5.2 mmol) was further added. After stirring at room temperature for 4 h, the solvent was evaporated, and the residue was diluted with 30 mL of ethyl acetate, and then washed with saturated sodium carbonate solution (10 mL x 3), saturated sodium chloride solution (10 mL), and anhydrous sodium sulfate in sequence. After the solvent was evaporated, the residue was dissolved in methanol, and 20 mL of 6N sodium hydroxide solution was further added. After stirring at room temperature for 0.5 h, 4N hydrochloric acid solution was added to adjust the pH to 7, and then stirred for 0.5 h. The aqueous phase was extracted with ethyl acetate (40 mL x 3), and the combined organic phase was washed with water (10 mL x 3), saturated sodium chloride solution (10 mL), and anhydrous sodium sulfate in sequence. After the solvent was evaporated, the residue was separated by flash silica gel column chromatography with n-hexane / ethyl acetate = 7 / 3 as the mobile phase, and then dissolved in ethanol. Compound g (1.60 g, 17.3 mmol) and potassium hydroxide (0.23 g, 4.2 mmol) were further added. After stirring at room temperature overnight, the solvent was evaporated, and the residue was diluted with 30 mL of ethyl acetate, and then washed with saturated sodium chloride solution (10 mL x 3), saturated sodium chloride solution (10 mL), and anhydrous sodium sulfate in sequence. After the solvent was evaporated, the residue was separated by flash silica gel column chromatography with n-hexane / ethyl acetate = 10 / 1 as the mobile phase, and then 0.90 g of white powder was obtained as a solid with a yield of 67%.

[0139] The obtained compound 3-5 (0.10 g, 0.26 mmol) was dissolved in 4 mL of ethanol, and compound b2 (0.28 g, 1.28 mmol) was further added. After heating to 50°C and stirring for 5 h, the solvent was evaporated, and the residue was separated by silica gel column chromatography with dichloromethane / methanol = 3 / 1 as the mobile phase, and then 70.0 mg of colorless oil was obtained with a yield of 44%.

[0140]

[0141] In a 5 mL flask, compound 3-6-2 (70.0 mg, 0.11 mmol) and compound 1-7 (114.5 mg, 0.11 mmol) were added, and 2 mL of a previously prepared mixed solvent (methanol / water = 5 / 1) was further added. Then, 0.33 mL of a previously prepared copper sulfate solution (1 mol / L) and 0.17 mL of a sodium ascorbate solution (1 mol / L) were added in sequence. After stirring at room temperature overnight, the system was diluted with 30 mL of ethyl acetate, and then the obtained organic phase was washed with water (10 mL x 3), saturated sodium chloride solution (10 mL), and anhydrous sodium sulfate in sequence. After the solvent was evaporated, the residue was separated by low-temperature silica gel column chromatography with n-hexane / acetone = 2 / 1 as the mobile phase, and then 0.12 g of light yellow solid was obtained with a yield of 76%, which was 3-7-2. 1H NMR (600 MHz, DMSO-d6) δ 8.03 (s, 1H), 7.45 (d, J = 7.5 Hz, 2H), 7.37 (t, J = 7.6 Hz, 2H), 7.32 - 7.29 (m, 1H), 7.27 (d, J = 7.5 Hz, 2H), 7.22 - 7.15 (m, 4H), 6.92 (d, J = 8.8 Hz, 1H), 6.56 (d, J = 2.9 Hz, 1H), 6.47 (s, 1H), 6.42 - 6.38 (m, 2H), 6.24 - 6.19 (m, 1H), 6.17 - 6.08 (m, 2H), 5.46 (dd, J = 14.9, 9.6 Hz, 1H), 5.29 (d, J = 4.6 Hz, 1H), 5.09 (d, J = 10.1 Hz, 1H), 5.02 (s, 2H), 4.99 - 4.95 (m, 1H), 4.94 (d, J = 7.9 Hz, 1H), 4.52 (s, 2H), 4.50 (t, J = 5.2 Hz, 2H), 4.05 - 3.97 (m, 2H), 3.97 - 3.94 (m, 3H), 3.92 - 3.87 (m, 2H), 3.84 (d, J = 5.0 Hz, 1H), 3.80 (t, J = 5.2 Hz, 3H), 3.64 - 3.60 (m, 3H), 3.53 - 3.46 (m, 14H), 3.31 (s, 3H), 3.28 - 3.24 (m, 2H), 3.21 - 3.16 (m, 1H), 3.15 (s, 3H), 3.05 (s, 3H), 2.99 - 2.96 (m, 1H), 2.81 - 2.71 (m, 7H), 2.69 - 2.65 (m, 1H), 2.43 - 2.32 (m, 2H), 2.25 - 2.18 (m, 1H), 2.13 - 2.07 (m, 1H), 2.03 - 1.99 (m, 3H), 1.93 - 1.87 (m, 2H), 1.87 - 1.78 (m, 2H), 1.74 (s, 2H), 1.70 - 1.66 (m, 2H), 1.64 - 1.62 (m, 3H), 1.58 - 1.51 (m, 5H), 1.43 - 1.37 (m, 2H), 1.29 - 1.21 (m, 4H), 1.13 - 1.09 (m, 1H), 1.06 - 1.01 (m, 3H), 0.98 (d, J = 6.6 Hz, 3H), 0.95 (d, J = 6.4 Hz, 1H), 0.86 (d, J = 6.4 Hz, 3H), 0.82 (d, J = 6.2 Hz, 3H), 0.77 (d, J = 6.6 Hz, 2H), 0.73 (d, J = 6.8 Hz, 3H), 0.67 - 0.61 (m, 1H). 13C NMR (150 MHz, DMSO-d6) δ 209.8, 206.9, 198.3, 168.6, 166.4, 153.1, 149.2, 143.2, 141.5, 140.9, 140.8, 138.7, 137.2, 137.1, 136.5, 131.7, 129.8, 127.7, 127.7, 127.6, 127.0, 126.8, 126.4, 126.4, 125.2, 125.1, 124.2, 123.5, 123.5, 115.7, 104.1, 101.6, 98.4, 84.9, 81.8, 81.6, 75.1, 73.0, 70.6, 70.2, 69.0, 69.0, 68.9, 68.8, 68.7, 68.1, 67.9, 66.8, 65.6, 62.9, 56.4, 56.4, 56.3, 54.8, 54.3, 51.2, 50.1, 48.6, 47.8, 44.6, 42.9, 39.4, 37.6, 35.4, 34.6, 34.2, 32.7, 31.6, 30.9, 30.8, 30.3, 29.9, 29.0, 29.0, 25.8, 25.6, 23.8, 21.0, 19.7, 15.0, 14.9, 14.1, 12.9, 12.7, 9.8. HRMS (ESI): m / z calcd for C 89 H 130 N5O 21 + ([M+H] + ) = 1604.92528, found = 1604.92793.

[0142] II. Pharmacological Experiment Part

[0143] 1. In vitro anti-MCF7 tumor cell proliferation test

[0144] Human breast cancer cells MCF-7 were cultured in DMEM medium containing 10% fetal bovine serum (FBS) in a cell incubator at 37°C, 5% CO2. Cells were seeded into a black 96-well plate at 3000 cells per well (100 μL), and placed in a CO2 cell incubator. After the cells adhered and grew overnight, different concentrations of compounds (1 μL) were added, six replicate wells were set for each concentration, and DMSO control wells were set at the corresponding concentration. After 72 h of compound treatment, 100 μL of ATP detection reagent (CellTiter-Glo Luminescent Cell Viability Assay kit) was added, and the plate was shaken for 1 h. The luminescence was detected by analyst AD. According to the fluorescence value compared with the blank control, Prism was plotted and statistically analyzed. The results are shown inFigure 1 Compounds 1-8-2, 2-9-2 and 3-7-3 all showed good in vitro anti-MCF-7 tumor cell activity.

[0145] 2. Western Blot experiment

[0146] Human breast cancer cells MCF-7 were inoculated into six-well plates, and after adhering, they were treated with different concentrations of target compounds or positive drugs for 48 h. Then, 100 μL of RIPA lysis solution containing protease inhibitors was added to each well, and the cells were scraped with a clean gun head. All the scraped liquid was transferred to a 1.5 mL centrifuge tube. The centrifuge was pre-cooled to 4°C, and centrifuged at 12000 rpm for 20 min. 75 μL of supernatant was gently aspirated (be careful not to aspirate the white flocculent material). Protein quantification was performed using the BCA method according to the BCA kit instructions. 5x loading buffer was added to the protein solution (1:4 ratio of protein solution volume), vortexed, and then placed in boiling water for 5 min to obtain denatured protein samples. According to the instructions, prepare 8% separating gel and 5% concentrated gel (insert comb immediately after pouring gel and stand for 30 min). Add electrophoresis buffer to the electrophoresis instrument, slowly pull out the comb, then add denatured protein samples or protein marker to the gel well, and load 10 μg protein per well. Connect the power supply, electrophorese at 90V for 2h. After electrophoresis, cut the corresponding molecular weight bands according to the protein marker, and stack them in the transfer membrane clamp with the methanol-activated PVDF membrane. Transfer at 400mA for 30min. Place the protein-transferred PVDF membrane in the pre-configured 5% skim milk (configured with PBST, 5mL) and shake at room temperature for 1h. Absorb the blocking solution, wash the band with PBST twice, 2min each time. Absorb the PBST, add the diluted primary antibody BSA (5%, PBST configuration) solution, and incubate at 4°C for 13-16h. Absorb and recover the primary antibody, wash the band with TBST 3 times, 5min each time. Absorb the TBST, add the diluted secondary antibody with TBST, and shake at room temperature for 1h. Discard the secondary antibody and wash the band with PBST 3 times, 5min each time. Take out the band, evenly apply the prepared ECL luminescent liquid, and expose and save the image in the gel imager. From Figure 2The results can be seen that for the compounds with better activity, Western blotting experiments show that the compounds can degrade mTOR, Raptor and FKBP12 proteins in mTORC1, and the protein level in mTORC2 (such as Rictor protein) is not affected. At a concentration of 3.3 umol / L, the degradation degree of the related proteins is the most obvious; however, the positive drug rapamycin does not significantly affect the protein level of all related proteins at the same drug concentration, which confirms the rationality of introducing the degradation tag strategy in the structure of rapamycin in the present application. From Figure 3 The results can be seen that the mTORC1 protein degradation mechanism is only related to the autophagy-lysosome pathway, and is not related to the ubiquitin-proteasome pathway, which is consistent with the view proposed in the present application.

[0147] 3. Enzyme-linked immunosorbent assay (ELISA)

[0148] Human breast cancer cells MCF-7 were inoculated into a six-well plate, and after adhering, they were treated with different concentrations of target compounds or positive drugs for 48 h, then trypsinized and collected, and washed twice with phosphate buffer (PBS, pH = 7.4). The collected cells were diluted with PBS containing protease inhibitors, then ultrasonicated (2 kHz, 2 s x 3), centrifuged (1500 x g, 10 min) to remove the insoluble material, and then the protein concentration was determined by BCA reagent. A fixed amount of total protein (30 ug) was taken, and the protein concentration of mTORC1 and mTORC2 in different samples was detected by mTORC1 and mTORC2 enzyme-linked immunosorbent assay kits, and Prism was plotted and statistically analyzed. From Figure 4 It can be seen that the ELISA experiment can more directly detect that compared with the control group (i.e. the solvent group, 0.1% DMSO), the synthesized compound (1-8-2 as an example) can achieve selective degradation of mTORC1 protein ( Figure 4 A), while the protein level of mTORC2 is not affected ( Figure 4 B).

[0149] In summary, the present application proposes the concept of selective mTORC1 degradation based on autophagy degradation, designs and synthesizes a class of selective mTORC1 degradation agents based on the autophagy-lysosome pathway, and particularly synthesizes three types of representative compounds, i.e. rapamycin derivatives. The in vitro anti-MCF7 tumor cell activity of the representative compounds can be comparable to that of the positive drug rapamycin. The selective mTORC1 degradation agent obtained by the present application can achieve selective degradation of mTORC1, and the degradation process is related to autophagy, while the protein level in mTORC2 is not affected. Therefore, the selective mTORC1 degradation agent obtained by the present application can bring a new idea and a potential drug research and development direction for the treatment of related diseases (such as cancer, neurodegenerative diseases, obesity, diabetes, chronic inflammatory diseases, dry eye, etc.), and also provides a powerful tool for in-depth study of the function and regulation mechanism of mTORC1, and provides a beneficial reference and inspiration for other similar researches.

[0150] The above is only to illustrate the technical idea of the present application, and cannot limit the protection scope of the present application. Any modification made according to the technical idea of the present application on the basis of the technical scheme falls within the protection scope of the claims of the present application.

Claims

1. A class of selective mTORCI degraders based on the autophagy-lysosomal pathway, characterized in that, The structural formula is as follows: ； In the formula, R represents a terminal autophagy-lysosome target protein modification tag, which is an LC3 ligand, an S-adenosylation tag or a p62 binding ligand; The structural formula of the LC3 ligand is as follows: ； The structural formula of the S-adenosylation tag is as follows: The structural formula of the p62 binding ligand is as follows: , , or ; The Linker represents an intermediate linker, which is a polyethylene glycol chain structure or an alkyl chain structure; In the polyethylene glycol chain structure, the number of oxygen atoms is n, 0≤n≤5, and n is an integer; the structural formula is as follows: ； In the alkyl chain, the number of carbon atoms is m, 1≤m≤20, and m is an integer; the structural formula is as follows: 。 2. The class of selective mTORCI degraders based on autophagy-lysosome pathway according to claim 1, characterized in that, Specifically, the six selective mTORC1 degradation agents include the following structural formulas: 、 、 、 、 、 。 3. The method of synthesis of a selective mTORCI degrader based on the autophagy-lysosome pathway according to claim 1 or 2, characterized in that, The selective mTORC1 degradation agent based on the autophagy-lysosome pathway is prepared by using water and methanol as reaction solvents, using compound A and a rapamycin donor as reaction raw materials, and under the catalysis of copper sulfate and sodium ascorbate; Compound A is composed of a terminal autophagy-lysosome target protein modification tag and a Linker; The terminal autophagy-lysosome target protein modification tag is an LC3 ligand, an S-adenosylation tag or a p62 binding ligand; The Linker represents an intermediate linker, which is a polyethylene glycol chain structure or an alkyl chain structure. The structural formula of the rapamycin donor is as follows:

4. The method of synthesis of selective mTORCI degraders based on autophagy-lysosomal pathway according to claim 3, wherein, The concentration of the copper sulfate solution is 1 mol / L, the concentration of the sodium ascorbate solution is 1 mol / L, and the volume ratio of the copper sulfate solution to the sodium ascorbate solution is 1-2:

1. 。 5. The method of synthesis of selective mTORCI degraders based on autophagy-lysosomal pathway according to claim 3, wherein, The molar ratio of compound A to the rapamycin donor is 1-1.2:

1.

6. The method of synthesis of selective mTORCI degraders based on autophagy-lysosomal pathway according to claim 3, wherein, The cancer is breast cancer.

7. Use of the selective mTORCI degrader based on the autophagy-lysosome pathway according to claim 1 or 2 for the manufacture of a medicament for the treatment of cancer, characterized in that, 8. Use of the selective mTORC1 degradation agent based on the autophagy-lysosome pathway in the preparation of a medicament for treating a neurodegenerative disease, a metabolic disease or a chronic inflammatory disease mediated by mTORC1. The neurodegenerative disease is Alzheimer's disease; the metabolic disease is diabetes, hyperlipidemia or obesity; and the chronic inflammatory disease is dry eye, chronic hepatitis or chronic pancreatitis.

9. Use according to claim 8, characterized in that, ​

Images