Selective mTORC1 degradation agent based on autophagy-lysosome pathway as well as preparation method and application of selective mTORC1 degradation agent

Abstract

The invention discloses a selective mTORC1 degradation agent based on an autophagy-lysosome pathway as well as a preparation method and application of the selective mTORC1 degradation agent, and belongs to the technical field of biological medicines. The concept of selective mTORC1 degradation is provided based on autophagy degradation, three types of rapamycin derivatives are designed and synthesized, and the in-vitro anti-MCF7 tumor cell activity of a representative compound can be equivalent to that of a positive drug rapamycin; the obtained rapamycin derivative can achieve selective degradation of mTORC1, meanwhile, the degradation process is related to autophagy, and the protein level in mTORC2 is not affected. The selective mTORC1 degradation agent obtained by the invention can bring a brand new thought and a potential drug research and development direction for treatment of related diseases, and also provides a powerful tool for in-depth study of functions and regulation mechanisms of mTORC1.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a class of selective mTORC1 degraders based on the autophagy-lysosome pathway, and a preparation method and application thereof. Background Art

[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 cell life activities and metabolic regulation, 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 functions by participating in the formation of two different complexes, mTORC1 and mTORC2. mTORC1 can regulate the translation of messenger RNA and cell size through a variety of mechanisms, mainly including ribosomal S6 kinase (S6K) phosphorylation and eukaryotic initiation factor 4E binding protein (4EBP). mTORC1 is usually activated when amino acids are sufficient. This process promotes the production of proteins, nucleotides and lipids. It promotes cell growth by promoting anabolism and inhibiting catabolism, and may induce 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 the mTOR signaling pathway controls the metabolism, growth, proliferation and survival of cells, the continuous overactivation of the mTOR signal will lead to an increase in the level of cell metabolism, continuous growth and proliferation, extended cell lifespan and even cell immortalization, which can directly or indirectly induce various types of cancer, neurodegenerative diseases, obesity, diabetes, chronic inflammatory diseases and dry eye disease, etc. Inhibiting this state can effectively delay or treat related diseases caused by excessive activation of mTOR. Therefore, the study of mTOR inhibitors is a hot field at present, and many mTOR inhibitors have been developed. The 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 in which rapamycin and an ATP competitive mTOR inhibitor are covalently linked through a linker (third generation). Among them, ATP-competitive mTOR inhibitors can inhibit the activity of mTORC1 and mTORC2 at the same time, and their inhibition of mTORC2 activity will reduce the activation of AKT, which has two potential disadvantages: one is to induce hyperglycemia, and the other is to relieve the feedback inhibition of AKT on the expression of receptor tyrosine kinase (RTK), which is easy to cause greater toxic side effects and drug resistance after clinical application. Therefore, compared with pan-mTOR inhibitors, that is, inhibiting the activity of mTORC1 and mTORC2 at the same time, mTORC1 selective inhibitors are less toxic and induce less adaptive resistance.

[0003] In recent years, targeted protein degradation has been widely used in a variety of proteins as a major new drug development model in the past two decades, providing a highly promising therapeutic strategy for the treatment of diseases such as cancer and inflammation. Among them, protein degradation targeting chimeras (PROTAC) are a promising targeted protein degradation technology. They are heterobifunctional small molecules that are connected to the target protein (POI) ligand by a part that can recruit E3 ubiquitin ligase. It achieves the degradation of the target protein by bringing the E3 ubiquitin ligase and POI close to each other. PROTAC molecules exhibit characteristics such as high efficiency and low drug resistance. At the same time, they can also target "undruggable" proteins, and their application prospects are extremely broad. PROTACs based on the ATP-competitive mTOR inhibitor MLN0128 as POI can achieve the degradation of mTOR protein, but due to its poor activity and selectivity, there has been no further related research. [1] However, PROTAC using rapamycin as POI can only degrade FKBP12 protein, but cannot degrade mTOR. [2] . The core of PROTAC is that it recruits E3 ligases to the vicinity of the target protein to form a ternary complex, inducing ubiquitination of the target protein and degradation through the proteasome. Rapamycin and its analogs first form a binary complex with the FKBP12 protein, and the activity of mTOR is inhibited by the complex. Therefore, it is difficult for PROTAC obtained using rapamycin as the 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 besides the ubiquitin-proteasome system, and it is gradually being applied to the field of targeted protein degradation. Targeted degradation technologies based on the autophagy-lysosome pathway mainly include: autophagosome-tethering compound (ATTEC) [3-5] , autophagy-targeting chimera AUTAC [6] and AUTOTAC [7] wait.

[0004] ATTEC originally refers to a type of molecular glue that can tightly bind mutant huntingtin (mHTT) protein to LC3 protein, and they can promote the degradation of mHTT based on the autophagy-lysosome pathway. Later, according to the design concept of PROTAC, the researchers used these ATTEC small molecules as LC3 binding ligands and connected them to different target protein ligands through linkers, thus obtaining a type of autophagy degrader with heterobifunctionality, further expanding the concept of ATTEC and successfully achieving the degradation of proteins such as BRD4 and NAMPT. [3,5] . AUTAC also belongs to a class of heterobifunctional molecules, which consists of three parts: target protein ligand, degradation tag (cGMP analogue) and linker. In the cell, AUTAC will first bind to the target protein, simulate the "modification" through the degradation tag, and induce K63 polyubiquitination of the target protein. The ubiquitinated target protein will then be selectively recognized and bound by the autophagy receptor p62. p62 further interacts with LC3, thereby mediating the recruitment of the target protein into the autophagosome, and finally fusion with the lysosome for degradation. However, the mechanism of how S-guanylation modification induces K63 ubiquitination is still unclear. Researchers have successfully achieved the degradation of proteins such as MetAP2 and FKBP12 with the help of 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 to form a ternary complex, thereby mediating the degradation of the target protein through the autophagy-lysosomal pathway. In this process, the target protein does not need to undergo ubiquitination, but directly initiates the macroautophagy-induced cascade reaction in a p62-dependent manner. Studies have shown that AUTOTAC can not only mediate the targeted degradation of monomeric proteins, but also mediate the degradation of protein aggregates, such as the pathological aggregates in neurodegenerative diseases - tau protein (P301L mutation), which provides new technical means and therapeutic strategies for clearing pathological aggregates of 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] The 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, but not mTOR protein degradation.

[0006] The references to existing technical literature or patent information are as follows:

[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 AUTACdegraders[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].NatureCommunications,2022,13(1):904.

[0014] [8]CN202310124471.2. Summary of the invention

[0015] In order to overcome the shortcomings of the prior art, the present invention aims to provide a class of selective mTORC1 degraders based on the autophagy-lysosome pathway and a preparation method and application thereof.

[0016] In order to achieve the above object, the present invention adopts the following technical solutions:

[0017] The present invention discloses a class of selective mTORC1 degraders based on the autophagy-lysosome pathway, the structural formula of which is shown below:

[0018]

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

[0020] Linker stands for intermediate connector.

[0021] The "autophagy-lysosome pathway" (ALP) described in the present invention is an important pathway in the process of cellular autophagy, which involves the process in which cells engulf and degrade some or all of their own organelles and proteins to maintain the balance of the intracellular environment and cope with environmental pressure. Furthermore, the target protein modification tags in the autophagy-lysosome pathway play a vital role in the autophagy process. These tags are generally 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 includes an LC3 ligand, an S-guanylation tag, and a p62 binding ligand;

[0023] Preferably, R comprises a compound represented by the following structure:

[0024] 1) LC3 ligand: and its derivative structures;

[0025] 2) S-guanylation tag: and its derivative structures;

[0026] 3) p62 binding ligand:

[0027]

[0028] and its derivative structures.

[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, and n is an integer;

[0032]

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

[0034] Preferably, the selective mTORC1 degraders based on the autophagy-lysosome pathway synthesized by the present invention have six structures, and their structural formulas are as follows:

[0035]

[0036]

[0037] The present invention also discloses a method for synthesizing the above-mentioned selective mTORC1 degrader based on the autophagy-lysosome pathway, comprising: using water and methanol as reaction solvents, using compound A and rapamycin donor as reaction raw materials, and preparing the selective mTORC1 degrader based on the autophagy-lysosome pathway under the catalysis of copper sulfate and sodium ascorbate;

[0038] Preferably, compound A is composed of a terminal autophagy-lysosome target protein modification tag connected to a Linker;

[0039] Terminal autophagy-lysosome target protein modification tags include LC3 ligands, S-guanylation tags, or p62 binding ligands;

[0040] Linker selects polyethylene glycol chain structure or alkyl chain structure

[0041] Preferably, the rapamycin donor structure of the present invention is as follows:

[0042]

[0043] Preferably, copper sulfate adopts a copper sulfate solution with a concentration of 1 mol / L, sodium ascorbate adopts a sodium ascorbate solution with 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 compound A to rapamycin donor is 1-1.2:1.

[0045] The present invention also discloses the use of the selective mTORC1 degrader based on the autophagy-lysosome pathway in the preparation of drugs for treating cancer, neurodegenerative diseases, metabolic diseases or chronic inflammatory diseases.

[0046] Preferably, the cancer includes breast cancer, liver cancer, prostate cancer, cervical cancer, skin cancer or colon cancer, etc.;

[0047] Preferably, the neurodegenerative diseases include Alzheimer's disease, etc.

[0048] Preferably, the metabolic disease includes diabetes, hyperlipidemia or obesity;

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

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

[0051] The selective mTORC1 degrader based on the autophagy-lysosome pathway disclosed in the present invention mainly includes three parts: a rapamycin donor, an intermediate linker, and a terminal autophagy-lysosome target protein modification tag. Among the existing mTOR degraders, the PROTAC with the ATP competitive mTOR inhibitor MLN0128 as the POI has poor activity and poor selectivity for mTORC1. The PROTAC with rapamycin as the POI can only achieve the degradation of FKBP12 protein, but cannot achieve the degradation of mTOR. Therefore, the present invention proposes a selective degrader of mTORC1 for the first time, which can achieve the selective degradation of mTORC1. At the same time, due to the terminal connection modification tag, the degradation process is related to autophagy, while the protein level in mTORC2 is not affected, which can effectively solve the existing mTOR inhibitors. The common problems of limited therapeutic effects, drug resistance after long-term use, and poor selectivity for mTORC1 lead to obvious toxic side effects.

[0052] The rapamycin derivatives obtained by the present invention can bring new ideas and potential drug development directions for the treatment of related diseases (such as cancer, neurodegenerative diseases, metabolic diseases, chronic inflammatory diseases, etc.), and also provide a powerful tool for in-depth research on the function and regulatory mechanism of mTORC1, and provide useful reference and inspiration for other similar studies;

[0053] Furthermore, the present invention designs and synthesizes three types of rapamycin derivatives, and the in vitro anti-MCF7 tumor cell activity of the representative compounds is comparable to that of the positive drug rapamycin. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0055] Figure 2 The following is a graph showing changes in the expression of representative proteins in mTORC1 and mTORC2 in the MCF-7 cell line after 48 hours of treatment with rapamycin and 1-8-2;

[0056] Figure 3 Diagram of the mechanism of compound 1-8-1 inducing mTORC1 protein degradation in MCF-7 cell line.

[0057] Figure 4 After 48 h of treatment with rapamycin and 1-8-2, the concentrations of A) mTORC1 and B) mTORC2 in MCF-7 cells were measured by ELISA (n=3). DETAILED DESCRIPTION

[0058] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0059] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0060] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0061] The purpose of the present invention is to provide an mTORC1 selective degrader and a preparation method and application thereof.

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

[0063]

[0064] R represents the terminal autophagy-lysosomal target protein modification tag;

[0065] Linker stands for intermediate connector.

[0066] Further, in the formula, R includes LC3 ligand, S-guanylated tag or p62 binding ligand; further, the structural formula of LC3 ligand is as follows:

[0067]

[0068] Furthermore, the structural formula of the S-guanylated tag is shown below:

[0069]

[0070] Furthermore, the structural formula of the p62 binding ligand is shown below:

[0071]

[0072]

[0073] Furthermore, the linker selects a polyethylene glycol chain structure or an alkyl chain structure.

[0074] Furthermore, the number of oxygen atoms in the polyethylene glycol chain structure is n, 0≤n≤5, n is an integer; the structural formula is as follows:

[0075]

[0076] The number of carbon atoms in the alkyl chain is m, where 1≤m≤20, m is an integer, and the structural formula is as follows:

[0077]

[0078] Particularly preferably, the present invention prepares six selective mTORC1 degraders, and the structural formulas are as follows:

[0079]

[0080]

[0081] 1. The preparation steps of representative compounds are as follows:

[0082]

[0083] Using ethanol as solvent, compound a and compound 1-1 as raw materials, and under the catalytic action of piperidine, compound 1-2 is prepared; the BOC protecting group of compound 1-2 is removed in a dichloromethane solution of trifluoroacetic acid to prepare compound 1-3;

[0084] Compound 1-3 and compound b are used as raw materials, DMF is used as solvent, HATU is used as condensation agent, and a condensation reaction is carried out under the alkaline condition of N,N-diisopropylethylamine to prepare compound 1-4;

[0085]

[0086] Compound 1-6 was prepared using dichloromethane as solvent, compound 1-5 as raw material, trifluoromethanesulfonic anhydride as acylating agent, and 2,6-lutidine as alkaline condition;

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

[0088]

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

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

[0091]

[0092] The specific preparation process is as follows:

[0093] Compound 1-1 (0.43g, 1.1mmol) and compound a (0.26g, 1.0mmol) were added to 15mL ethanol, piperidine (20uL) was added as catalyst, the system was heated to reflux for 18h, cooled to room temperature, a large amount of solid was precipitated in the system, and 0.60g yellow solid 1-2 was obtained by direct suction filtration, and the yield was 95%. The obtained 1-2 was dissolved in 10mL dichloromethane, 3mL trifluoroacetic acid was added, and stirred overnight at room temperature, and the reaction system was detected by TLC until the reaction was completed, and the solvent was evaporated, and the next step reaction could be directly carried out without purification. The spin-dried residue was dissolved in 10mL DMF, and compound b2 (0.22g, 1.0mmol), HATU (0.45g, 1.2mmol) and DIPEA (0.63mL, 3.6mmol) were added successively. After the system was stirred at room temperature for 12h under a nitrogen atmosphere, the solvent was evaporated to obtain a crude product. The product was purified by silica gel column chromatography with the mobile phase being petroleum ether / ethyl acetate = 2 / 1 to obtain 709 mg of an orange-yellow solid with a yield of 91%.

[0094] Synthesis of rapamycin partial linker:

[0095]

[0096] 20 mL of dichloromethane was added to a 100 mL flask, and compound 1-5 (3.0 g, 30 mmol) and pyridine (2.9 mL, 36 mmol) were added. Trifluoromethanesulfonic anhydride (6.1 mL, 36 mmol) and 20 mL of dichloromethane were added to a 50 mL constant pressure dropping funnel. The reaction solution was stirred under an ice-water bath to cool to 0 ° C. The constant pressure dropping funnel was opened to slowly drip the solution of trifluoromethanesulfonic anhydride into the reaction system. After the dripping was completed, the system was washed with water (10 mL × 3), saturated sodium chloride (10 mL), and dried over anhydrous sodium sulfate. The solvent was evaporated and the obtained residue was separated by rapid silica gel column chromatography. The mobile phase was petroleum ether / ethyl acetate = 20 / 1 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 to a 5 mL microwave tube in sequence. After sealing, the glove box was taken out and placed in a microwave reactor, and the temperature was set to 65 ° C for 5 minutes. After the reaction was completed, the system was taken out and directly separated by low-temperature silica gel column chromatography, with the mobile phase being n-hexane / acetone = 5 / 1, to obtain 61.0 mg of a white foamy solid with a yield of 61%. 1H NMR(600MHz,DMSO-d6)δ6.49–6.44(m,1H),6.42–6.38(m,1H),6.25–6.20(m,1H),6.17–6.10(m,2H),5.46(dd,J=14.9,9.5Hz,1H),5.31–5.26(m,1H),5.09(d,J=10.3Hz,1H),5.01–4.95(m,1H),4.94(dd,J=6.1,2.2Hz,1H),4.15–4.14(m,2H),4.03–3.99(m,2H),3.95(d,J=4.6Hz,1H),3.65–3.59(m,4H),3.55–3.51(m,3H),3.46–3.42(m,1H),3.41(t,J=2.4Hz,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=17.8,2.9Hz,1H),2.42–2.35(m,2H),2.25–2.20(m,1H),2.10(d,J=11.9Hz,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=6.6Hz,3H),0.96–0.93(m,2H),0.87(d,J=6.6Hz,3H),0.82(d,J=6.6Hz,3H),0.77(d,J=6.8Hz,2H),0.73(d,J=6.8Hz,3H),0.67–0.61(m,1H). 13C NMR(150MHz,DMSO-d6)δ211.0,208.1,199.4,169.7,167.5,139.8,138.4,137.7,132.8,130.9,127 .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 / zcalcd.for C 56 H 85 NO 14 Na + ([M+Na] + )=1018.58623,found=1018.59257.

[0098] Synthesis of 1-8-2:

[0099]

[0100] Compound 1-4-2 (155.8 mg, 0.2 mmol) and compound 1-7 (199.3 mg, 0.2 mmol) were added to a 5 mL flask, and 2 mL of a pre-prepared mixed solvent (methanol / water = 5 / 1) was added, followed by 0.15 mL of a pre-prepared copper sulfate solution (1 mol / L) and 0.3 mL of a sodium ascorbate solution (1 mol / L), stirred at room temperature overnight, and the system was diluted with 30 mL of ethyl acetate, and the obtained organic phase was washed with water (10 mL × 3), saturated sodium chloride (10 mL), and dried over anhydrous sodium sulfate. The solvent was evaporated, and the obtained residue was separated by low-temperature silica gel column chromatography, with the mobile phase being n-hexane / acetone = 5 / 1, to obtain 0.30 g of a yellow solid with a yield of 84%, i.e., 1-8-2. 1H NMR(600MHz,DMSO-d6)δ10.85(s,1H),8.80(s,2H),8.12–8.07(m,2H),8.04(d,J=9.4Hz,2H),7.87(s,1H),7.58–7.55(m,1H),6.70(d,J=8.3Hz,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=4.8Hz,1H),5.08(d,J=10.3Hz,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=5.6,3.9Hz,2H),3.95(d,J=4.6Hz,1H),3.81(t,J=5.3Hz,3H),3.67–3.59(m,4H),3.54–3.52(m,6H),3.51–3.49(m,6H),3.43(d,J=11.7Hz,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=6.4Hz,3H),0.94(d,J=6.4Hz,1H),0.91(d,J=7.2Hz,1H),0.86(d,J=6.6Hz,3H),0.81(d,J=6.4Hz,3H),0.76(d,J=6.8Hz,2H),0.73(d,J=6.6Hz,3H),0.69(s,1H),0.66–0.59(m,1H). 13C NMR(150MHz,DMSO-d6)δ210.9,208.0,199.4,197.0,169.7,168.0,167.5,167.0,166.9,166.8,153.3,152.8,143.3,141.0,139.8,139.1,138 .4,138.1,137.6,136.7,134.8,134.2,134.0,133.8,132.8,131.1,13 0.9,128.9,128.7,127.6,127.5,125.3,124.9,123.5,118.2,117.5,11 3.2,112.5,99.5,85.9,84.7,82.9,82.7,76.2,74.0,71.5,70.0,69.2 ,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.forC 81 H 112 Br2IN6O 20 + ([M+H] + )=1773.53374,found=1773.53725.

[0101] 2. The preparation steps of AUTAC are as follows:

[0102]

[0103] Compound 2-2 was prepared using ethanol as solvent, compound 2-1 and di-tert-butyl dicarbonate as raw materials under the catalysis of 4-dimethylaminopyridine;

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

[0105] Compound 2-3 and compound c are used as raw materials, and compound 2-4 is prepared under the catalytic conditions of DEAD and triphenylphosphine;

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

[0107]

[0108] Compound 2-6 is prepared by brominating compound 2-5 in bromine water;

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

[0110] Compound 2-7 and compound e are used as raw materials, DMF is used as solvent, EDC and HOBt are used as condensation agents, and triethylamine is used as alkaline conditions to undergo a condensation reaction to prepare compound 2-8;

[0111]

[0112] Water and methanol were used as a mixed solvent, and the compound 2-8 and the compound 1-7 were used as raw materials, and the compound 2-9 was prepared under the catalytic conditions of copper sulfate and sodium ascorbate.

[0113] Take the synthesis of 2-9-2 as an example:

[0114]

[0115]

[0116] The specific preparation process is as follows:

[0117] Compound 2-1 (5.09 g, 30 mmol) was dissolved in 30 mL DMSO solution, the system temperature was lowered to 0°C under ice-water bath conditions, di-tert-butyl dicarbonate (6.55 g, 30 mmol) and 4-dimethylaminopyridine (0.18 g, 1.5 mmol) were added. After stirring overnight at room temperature, 100 mL of ice water was directly added to the system, and 7.70 g of white solid was obtained by suction filtration, with a yield of 87%. The obtained white solid was completely dissolved in 50 mL of tetrahydrofuran, cooled to 0°C under ice-water bath conditions, and sodium hydride (60%, 3.0 g, 75 mmol) was added in batches. After stirring at room temperature for 2 h, the system was washed with saturated sodium chloride (10 mL×3) in turn and dried over anhydrous sodium sulfate. The solvent was evaporated, and the obtained residue was separated by silica gel column chromatography, with the mobile phase being dichloromethane / methanol = 20 / 1, 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 compound c (1.26 g, 10 mmol) and triphenylphosphine (6.56 g, 25 mmol) were added in sequence, and finally DEAD (4.40 g, 25 mmol) was slowly added dropwise. After stirring for 6 h at room temperature, the solvent was evaporated. The residue was separated by silica gel column chromatography, and the mobile phase was dichloromethane / methanol = 30 / 1, and the crude product contained triphenylphosphine oxide. The crude product was dissolved in 20 mL of 80% formic acid solution, 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, and the mobile phase was dichloromethane / methanol = 10 / 1, and 4.0 g of white solid was obtained, and the yield was 60%.

[0119] Compound 2-5 (0.50 g, 1.9 mmol) was dissolved in 5 mL of toluene, 1 mL of bromine water was added, and 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 with water (10 mL × 3), saturated sodium chloride (10 mL), and dried over anhydrous sodium sulfate. The solvent was evaporated, and the obtained residue was separated by rapid silica gel column chromatography, with the mobile phase being dichloromethane / methanol = 10 / 1, to obtain 0.55 g of a light yellow solid, with a yield of 85%.

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

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

[0122]

[0123] Compound 2-8-2 (155.5 mg, 0.25 mmol) and compound 1-7 (250.0 mg, 0.25 mmol) were added to a 5 mL flask, and 2 mL of a pre-prepared mixed solvent (methanol / water = 5 / 1) was added, followed by 0.375 mL of a pre-prepared copper sulfate solution (1 mol / L) and 0.75 mL of a sodium ascorbate solution (1 mol / L), stirred at room temperature overnight, and the system was diluted with 30 mL of ethyl acetate, and the obtained organic phase was washed with water (10 mL × 3), saturated sodium chloride (10 mL), and dried over anhydrous sodium sulfate. The solvent was evaporated, and the obtained residue was separated by low-temperature silica gel column chromatography, with the mobile phase being n-hexane / acetone = 4 / 1, to obtain 0.25 g of a light yellow solid with a yield of 62%, i.e., 2-9-2. 1H NMR(600MHz,DMSO-d6)δ10.64(s,1H),8.41(dd,J=8.2,2.5Hz,1H),8.07(t,J=5.7Hz,1H),8.03(s,1H),7.21(dd,J=8.8,5.5Hz,2H),7.16(t,J=8.8Hz,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=14.9,9.6Hz,1H),5.29(d,J=4.6Hz,1H),5.11–5.05(m,3H),5.00–4.95(m,1H),4.94(d,J=7.9Hz,1H),4.51(s,3H),4.50–4.46(m,3H),4.04–3.99(m,2H),3.96(d,J=4.4Hz,1H),3.79(t,J=5.3Hz,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=5.1Hz,1H),3.37(t,J=6.1Hz,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=6.4Hz,3H),0.94(s,1H),0.86(d,J=6.4Hz,3H),0.82(d,J=6.6Hz,3H),0.77(d,J=6.8Hz,2H),0.73(d,J=6.6Hz,3H),0.66–0.60(m,1H). 13C NMR(150MHz,DMSO-d6)δ211.0,208.1,199.4,170.8,170.2,169.9,169.7,167.5,166.6,162.8,161.2,156.1,154.2,153.2,144.4,142.7,1 39.8,138.4,137.6,133.1,132.8,130.9,129.5,129.5,127.5,127.5 ,125.3,124.7,117.1,116.0,115.9,99.5,86.0,82.9,82.7,76.2,74. 1,70.1,70.1,70.0,70.0,69.9,69.2,69.0,66.7,64.0,60.2,57.5,5 7.4,56.0,55.4,52.7,51.3,49.8,45.7,45.1,44.0,39.1,38.7,36.5, 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. The preparation steps of AUTOTAC are as follows:

[0125]

[0126] Acetonitrile is used as a solvent, compound 3-1 and benzyl bromide are used as raw materials, and compound 3-2 is prepared under the alkaline action of sodium bicarbonate; compound 3-2 and compound f are used as raw materials, and compound 3-3 is prepared by stirring in a DMF solution under the alkaline action of potassium carbonate;

[0127] Compound 3-4 is prepared by stirring the compound 3-3 in a dichloromethane solution of m-chloroperbenzoic acid;

[0128]

[0129] Using the compound 3-4 and compound g as raw materials, compound 3-5 was prepared in DMF;

[0130] Ethanol is used as a solvent, and the compound 3-5 and compound b are used as raw materials to undergo a nucleophilic substitution reaction to prepare compound 3-6;

[0131]

[0132] Water and methanol were used as a mixed solvent, and the compound 3-6 and the compound 1-7 were used as raw materials, and the compound 3-7 was prepared under the catalytic conditions of copper sulfate and sodium ascorbate.

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

[0134]

[0135] The specific preparation process is as follows:

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

[0137] The obtained compound 3-2 (0.80 g, 3.5 mmol) was dissolved in DMF (5 mL), and compound f (0.84 g, 4.2 mmol) and potassium carbonate (0.97 g, 7.00 mmol) were added, and the system was heated to 80°C and stirred overnight. The solvent was removed, and the residue was separated by silica gel column chromatography with a mobile phase of petroleum ether / ethyl acetate = 10 / 1 to obtain 1.20 g of a light 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 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×3) and saturated sodium chloride (10 mL) in sequence, and dried over anhydrous sodium sulfate. After evaporating the solvent, the residue was dissolved in methanol, and 20 mL of 6N sodium hydroxide solution was added, and after stirring at room temperature for 0.5 h, 4N hydrochloric acid solution was added to adjust the pH to 7, and after stirring for 0.5 h, the aqueous phase was extracted with ethyl acetate (40 mL×3), and the combined organic phase was washed with water (10 mL×3) and saturated sodium chloride (10 mL) in sequence, and dried over anhydrous sodium sulfate. The solvent was evaporated, and the obtained residue was separated by rapid silica gel column chromatography, and the mobile phase was n-hexane / ethyl acetate = 7 / 3. The obtained oil was directly dissolved in ethanol, and compound g (1.60 g, 17.3 mmol) and potassium hydroxide (0.23 g, 4.2 mmol) were 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 × 3), saturated sodium chloride washing (10 mL), and dried over anhydrous sodium sulfate. After evaporating the solvent, the obtained residue was separated by rapid silica gel column chromatography, and the mobile phase was n-hexane / ethyl acetate = 10 / 1, and 0.90 g of white powdery solid was obtained, and the yield was 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 added. The system was heated to 50°C and stirred for 5 h, then the solvent was evaporated and the residue was separated by silica gel column chromatography with the mobile phase being dichloromethane / methanol = 3 / 1 to give 70.0 mg of a colorless oil with a yield of 44%.

[0140]

[0141] Compound 3-6-2 (70.0 mg, 0.11 mmol) and compound 1-7 (114.5 mg, 0.11 mmol) were added to a 5 mL flask, and 2 mL of a pre-prepared mixed solvent (methanol / water = 5 / 1) was added, followed by 0.33 mL of a pre-prepared copper sulfate solution (1 mol / L) and 0.17 mL of a sodium ascorbate solution (1 mol / L), stirred at room temperature overnight, and the system was diluted with 30 mL of ethyl acetate, and the obtained organic phase was washed with water (10 mL × 3), saturated sodium chloride (10 mL), and dried over anhydrous sodium sulfate. The solvent was evaporated, and the obtained residue was separated by low-temperature silica gel column chromatography, with the mobile phase being n-hexane / acetone = 2 / 1, to obtain 0.12 g of a light yellow solid with a yield of 76%, i.e., 3-7-2. 1H NMR(600MHz,DMSO-d6)δ8.03(s,1H),7.45(d,J=7.5Hz,2H),7.37(t,J=7.6Hz,2H),7.32–7.29(m,1H),7.27(d,J=7.5Hz,2H),7.22–7.15(m,4H),6.92(d,J=8.8Hz,1H),6.56(d,J=2.9Hz,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=14.9,9.6Hz,1H),5.29(d,J=4.6Hz,1H),5.09(d,J=10.1Hz,1H),5.02(s,2H),4.99–4.95(m,1H),4.94(d,J=7.9Hz,1H),4.52(s,2H),4.50(t,J=5.2Hz,2H),4.05–3.97(m,2H),3.97–3.94(m,3H),3.92–3.87(m,2H),3.84(d,J=5.0Hz,1H),3.80(t,J=5.2Hz,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=6.6Hz,3H),0.95(d,J=6.4Hz,1H),0.86(d,J=6.4Hz,3H),0.82(d,J=6.2Hz,3H),0.77(d,J=6.6Hz,2H),0.73(d,J=6.8Hz,3H),0.67–0.61(m,1H). 13C NMR(150MHz,DMSO-d6)δ209.8,206.9,198.3,168.6,166.4,153.1,149.2, 143.2,141.5,140.9,140.8,138.7,137.2,137.1,136.5,131.7,129.8,12 7.7,127.7,127.6,127.0,126.8,126.4,126.4,125.2,125.1,124.2,123. 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 ,56.3,54.8,54.3,51.2,50.1,48.6,47.8,44.6,42.9,39.4,37.6,35.4,3 4.6,34.2,32.7,31.6,30.9,30.8,30.3,29.9,29.0,29.0,25.8,25.6,23. 8,21.0,19.7,15.0,14.9,14.1,12.9,12.7,9.8.HRMS(ESI):m / zcalcd.for C 89 H 130 N5O 21 + ([M+H] + )=1604.92528,found=1604.92793.

[0142] 2. Pharmacological Experiments

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

[0144] Human breast cancer cells MCF-7 were cultured in a cell culture incubator at 37°C and 5% CO2 using DMEM medium containing 10% fetal bovine serum (FBS). The cells were inoculated into a black 96-well plate, with 3,000 cells (100 μL) per well, and placed in a CO2 cell culture incubator. After the cells adhered to the wall and grew overnight, different concentrations of compounds (1 μL) were added, and six replicate wells were set for each concentration, and DMSO control wells of the corresponding concentration were set. After the compound was exposed for 72 hours, 100 μL of ATP detection reagent (CellTiter-GloLuminescent Cell Viability Assay kit) was added. After shaking on a shaker for 1 hour, luminescence was detected using analyze AD. According to the fluorescence value compared with the blank control, Prism was used to draw a graph and statistically analyze. Results can be found in Figure 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. After the cells adhered to the plate, they were treated with different concentrations of target compounds or positive drugs for 48 hours. 100 μL of RIPA lysis buffer containing protease inhibitors was added to each well. Then the cells were scraped off with a clean gun tip and all the scraped liquid was transferred to a 1.5 mL centrifuge tube. Centrifuge at 12000 rpm for 20 minutes in a centrifuge precooled to 4°C, and 75 μL of supernatant was gently aspirated (be careful not to aspirate white flocs). Protein quantification was performed using the BCA method according to the instructions of the BCA kit. 5× loading buffer (1:4 volume ratio to protein solution) was added to the protein solution, vortexed and mixed, and then placed in boiling water for 5 minutes to obtain a denatured protein sample. Prepare 8% separation gel and 5% concentration gel according to the ratio in the instructions (insert the comb immediately after filling the gel and let it stand for 30 minutes). Add electrophoresis buffer to the electrophoresis instrument, slowly pull out the comb, and then add the denatured protein sample or protein marker to the gel well. The sample is loaded with 10μg protein per well. Turn on the power supply and run the electrophoresis at 90V for 2h. After the electrophoresis is completed, cut the band of the corresponding molecular weight according to the protein marker indication and stack it in the transfer folder together with the PVDF membrane excited by methanol. Transfer at a current of 400mA for 30min. Place the PVDF membrane with the transferred protein in the pre-prepared 5% skim milk powder (prepared with PBST, 5mL) and shake at room temperature for 1h. Aspirate the blocking solution and wash the bands with PBST shaking twice, 2min each time. Aspirate the PBST, add the BSA (5%, PBST) solution diluted with the primary antibody, and incubate at 4℃ for 13-16h. Aspirate and recover the primary antibody, and wash the bands with TBST shaking 3 times, 5min each time. Aspirate the TBST, add the secondary antibody diluted with TBST, and shake at room temperature for 1h. Discard the secondary antibody and wash the strips with PBST 3 times, 5 min each time. Take out the strips, evenly apply the prepared ECL luminescent solution, expose in a gel imager and save the image. Figure 2The results show that for the compounds with better activity, protein blotting experiments were performed, indicating that the compounds can simultaneously degrade mTOR, Raptor and FKBP12 proteins in mTORC1, while the protein levels in mTORC2 (such as Rictor protein) are not affected. When the dosage concentration is 3.3umol / L, the degree of degradation of related proteins is most obvious; however, at the same dosage concentration of the positive drug rapamycin, all related protein levels are not significantly affected, which confirms the rationality of the present invention's introduction of a degradation tag strategy into the rapamycin structure. From Figure 3 It can be seen from the results that the mTORC1 protein degradation mechanism is only related to the autophagy-lysosome pathway, but not to the ubiquitin-proteasome pathway, which is consistent with the viewpoint proposed in the present invention.

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

[0148] Human breast cancer cells MCF-7 were inoculated into six-well plates. After adherence, they were treated with different concentrations of target compounds or positive drugs for 48 hours, then digested with trypsin and collected, and washed twice with phosphate buffer (PBS, pH = 7.4). The collected cells were diluted with PBS containing protease inhibitors, then cleared by ultrasound (2kHz, 2s×3) and centrifugation (1500×g, 10min), and then the insoluble substances were removed. The protein concentration was determined using the BCA reagent. A fixed amount of total protein (30ug) was taken, and the protein concentrations of mTORC1 and mTORC2 in different samples were detected using mTORC1 and mTORC2 enzyme-linked immunosorbent assay kits. Prism was used to draw graphs and perform statistical analysis. From Figure 4 It can be seen that the ELISA experiment can more intuitively detect that compared with the control group (i.e., 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 was not affected ( Figure 4 (middle B).

[0149] In summary, the present invention proposes the concept of selective mTORC1 degradation based on autophagy degradation, designs and synthesizes a class of selective mTORC1 degraders based on the autophagy-lysosome pathway, and in particular, synthesizes three types of representative compounds, namely rapamycin derivatives, and the in vitro anti-MCF7 tumor cell activity of the representative compounds is comparable to that of the positive drug rapamycin. The selective mTORC1 degrader obtained by the present invention 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 degrader obtained by the present invention can bring new ideas and potential drug development directions 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 regulatory mechanism of mTORC1, and provides useful reference and inspiration for other similar studies.

[0150] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A selective mTORC1 degrader based on the autophagy-lysosome pathway, characterized in that: Its structural formula is as follows: In the formula, R represents the terminal autophagy-lysosomal target protein modification tag; Linker stands for intermediate connector.

2. A selective mTORC1 degrader based on the autophagy-lysosome pathway according to claim 1, characterized in that: R includes LC3 ligand, S-guanylation tag, or p62 binding ligand; The structural formula of LC3 ligand is shown below: The structural formula of the S-guanylated tag is shown below: The structural formula of the p62 binding ligand is shown below:

3. The selective mTORC1 degrader based on the autophagy-lysosome pathway according to claim 1, characterized in that: Linker can choose polyethylene glycol chain structure or alkyl chain structure.

4. The selective mTORC1 degrader based on the autophagy-lysosome pathway according to claim 3, characterized in that: The number of oxygen atoms in the polyethylene glycol chain structure is n, 0≤n≤5, n is an integer; the structural formula is as follows: The number of carbon atoms in the alkyl chain is m, where 1≤m≤20, m is an integer, and the structural formula is as follows:

5. The selective mTORC1 degrader based on the autophagy-lysosome pathway according to claim 1, characterized in that: Specifically, there are six selective mTORC1 degraders, and their structural formulas are as follows:

6. The method for synthesizing a selective mTORC1 degrader based on the autophagy-lysosome pathway according to any one of claims 1 to 5, characterized in that: include: Using water and methanol as reaction solvents, compound A and rapamycin donor as reaction raw materials, and under the catalysis of copper sulfate and sodium ascorbate, a selective mTORC1 degrader based on the autophagy-lysosome pathway is prepared; Among them, compound A is composed of a terminal autophagy-lysosome target protein modification tag connected to a linker; Terminal autophagy-lysosome target protein modification tags include LC3 ligands, S-guanylation tags, or p62 binding ligands; Linker can choose polyethylene glycol chain structure or alkyl chain structure.

7. The method for synthesizing a selective mTORC1 degrader based on the autophagy-lysosome pathway according to claim 6, characterized in that: The structural formula of rapamycin donor is as follows:

8. The method for synthesizing a selective mTORC1 degrader based on the autophagy-lysosome pathway according to claim 6, characterized in that: The copper sulfate adopts a copper sulfate solution with a concentration of 1 mol / L, and the sodium ascorbate adopts a sodium ascorbate solution with a concentration of 1 mol / L, and the volume ratio of the copper sulfate solution to the sodium ascorbate solution is 1-2:

1.

9. The method for synthesizing a selective mTORC1 degrader based on the autophagy-lysosome pathway according to claim 6, characterized in that: The molar ratio of compound A to rapamycin donor is 1-1.2:

1.

10. Use of a selective mTORC1 degrader based on the autophagy-lysosome pathway according to any one of claims 1 to 4 in a drug for treating cancer, neurodegenerative diseases, metabolic diseases or chronic inflammatory diseases; Preferably, the cancer includes breast cancer, liver cancer, prostate cancer, cervical cancer, skin cancer or colon cancer; Preferably, the neurodegenerative disease includes Alzheimer's disease; Preferably, the metabolic disease includes diabetes, hyperlipidemia or obesity; Preferably, the chronic inflammatory disease includes dry eye, chronic hepatitis or chronic pancreatitis.

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