Class of selective mtorc1 degraders based on autophagy-lysosome pathway, preparation method therefor, and use thereof

Abstract

Disclosed in the present invention are a class of selective mTORC1 degraders based on an autophagy-lysosome pathway, a preparation method therefor, and the use thereof. The prepared rapamycin derivative exhibits an in vitro anti-MCF7 tumor cell activity comparable to that of rapamycin, and can achieve the selective degradation of mTORC1, while the protein level in mTORC2 is not affected.

Description

A class of selective mTORC1 degraders based on the autophagy-lysosome pathway, their preparation methods and applications Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a class of selective mTORC1 degrading agents based on the autophagy-lysosome pathway, their preparation methods, and applications. Background Technology

[0002] Mammalian target of rapamycin (mTOR) is a 289 kDa serine / threonine protein kinase belonging to the phosphatidylinositol 3-kinase-associated kinase (PIKK) family. mTOR plays a crucial role in cellular life activities and metabolic regulation, and its signaling pathways are closely related to many human diseases, such as tumors, neurodegenerative diseases, diabetes, and immune disorders. Intracellularly, mTOR primarily functions by participating in the formation of two distinct complexes: mTORC1 and mTORC2. mTORC1 regulates messenger RNA translation and cell size through various mechanisms, mainly including ribosomal S6 kinase (S6K) phosphorylation and eukaryotic initiation factor 4E binding protein (4EBP). mTORC1 is typically activated under amino acid-sufficient conditions, promoting the production of proteins, nucleotides, and lipids. It promotes cell growth by stimulating anabolism and inhibiting catabolism, and may contribute to tumor development and progression. mTORC2, on the other hand, regulates cellular metabolism, proliferation, and survival by phosphorylating AGC family kinases, such as protein kinase B (AKT). Since the mTOR signaling pathway controls cellular metabolism, growth, proliferation, and survival, persistent overactivation of mTOR signaling leads to increased cellular metabolic levels, sustained growth and proliferation, prolonged cell lifespan, and even cell immortalization. This can directly or indirectly induce various types of cancer, neurodegenerative diseases, obesity, diabetes, chronic inflammatory diseases, and dry eye syndrome. Inhibiting this state can effectively delay or treat related diseases caused by mTOR overactivation. Therefore, research on mTOR inhibitors is currently a hot topic, and many mTOR inhibitors have been developed. Current mTOR inhibitors are mainly divided into three categories: allosteric mTOR inhibitors represented by rapamycin and its derivatives (first generation), ATP-competitive mTOR inhibitors (second generation), and mTOR inhibitors where rapamycin is covalently linked to an ATP-competitive mTOR inhibitor via a linker (third generation). Among them, ATP-competitive mTOR inhibitors can simultaneously inhibit the activity of mTORC1 and mTORC2. However, the inhibition of mTORC2 activity reduces AKT activation, which presents two potential drawbacks: firstly, it can induce hyperglycemia; secondly, it can remove the feedback inhibition of AKT on receptor tyrosine kinase (RTK) expression, easily leading to significant toxic side effects and drug resistance after clinical application. Therefore, compared to pan-mTOR inhibitors, which simultaneously inhibit the activity of mTORC1 and mTORC2, selective mTORC1 inhibitors are less toxic and induce less adaptive resistance.

[0003] In recent years, targeted protein degradation has emerged as a major novel drug development paradigm over the past two decades and has been widely applied to various proteins, providing a highly promising therapeutic strategy for diseases such as cancer and inflammation. Among these, protein degradation targeting chimeras (PROTACs) represent a promising targeted protein degradation technology. These are heterobifunctional small molecules that recruit E3 ubiquitin ligases to link with a target protein (POI) ligand. PROTACs achieve target protein degradation by bringing the E3 ubiquitin ligase and POI closer together. PROTAC molecules exhibit high efficiency and low drug resistance, and can also target "undruggable" proteins, making their application prospects extremely broad. PROTACs derived using the ATP-competitive mTOR inhibitor MLN0128 as a POI can degrade mTOR proteins; however, due to their poor activity and selectivity, further research has been limited. [1] However, PROTAC obtained using rapamycin as a POI can only degrade FKBP12 protein and cannot degrade mTOR. [2] The core of PROTAC lies in its ability to recruit E3 ligases to the vicinity of target proteins to form ternary complexes, inducing ubiquitination of the target proteins and their degradation via the proteasome. Rapamycin and its analogues, however, first form a binary complex with the FKBP12 protein, inhibiting mTOR activity. Therefore, PROTACs obtained using rapamycin as a point of interest (POI) are unlikely to form ternary complexes with mTOR and induce ubiquitination and proteasome degradation. However, autophagy-lysosomes are another important protein degradation system in eukaryotic cells besides the ubiquitin-proteasome system, and are increasingly being applied to targeted protein degradation. Targeted degradation technologies based on the autophagy-lysosome pathway mainly include: autophagosome-tethering compounds (ATTEC). [3-5] Autophagy-targeting chimera (AUTAC) [6] and AUTOTAC [7] wait.

[0004] ATTEC initially referred to a class of molecular gels that could tightly bind mutant huntingtin (mHTT) proteins to LC3 proteins, thereby promoting the degradation of mHTT via the autophagy-lysosomal pathway. Subsequently, following the design concept of PROTAC, researchers used these small ATTEC molecules as LC3-binding ligands, linking them to different target protein ligands via linkers, thus obtaining a class of heterobifunctional autophagic degradative agents. This further expanded the concept of ATTEC and successfully achieved the degradation of proteins such as BRD4 and NAMPT. [3,5] AUTAC, also a class of heterobifunctional molecules, consists of three parts: a target protein ligand, a degradation tag (a cGMP analog), and a linker. In the cell, AUTAC first binds to the target protein, mimicking "modification" through the degradation tag and inducing 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, mediating the recruitment of the target protein to the autophagosome, where it ultimately fuses with lysosomes for degradation. However, the mechanism by which S-guanylate modification induces K63 ubiquitination remains unclear. Researchers have successfully used this technology to degrade proteins such as MetAP2 and FKBP12. Furthermore, AUTAC is also suitable for the targeted degradation of organelles such as mitochondrial debris. [6] AUTOTAC can directly bind to the target protein and the ZZ-binding domain of p62, forming a ternary complex, which then mediates the degradation of the target protein via the autophagy-lysosomal pathway. In this process, the target protein does not require ubiquitination; instead, it directly initiates a macroautophagy-induced cascade reaction in a p62-dependent manner. Studies show that AUTOTAC can mediate not only the targeted degradation of monomeric proteins but also the degradation of protein aggregates, such as the pathological aggregate in neurodegenerative diseases—tau protein (P301L mutation). This provides a new technical means and therapeutic 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] Current research on mTOR-related degraders mainly includes two parts: 1. Degrading mTOR protein by using the ATP-competitive mTOR inhibitor MLN0128 as a POI (point of interest); 2. Degrading mTOR protein by using rapamycin as a POI. Of these, option 1 has poor activity and cannot selectively degrade mTORC1; option 2 can only degrade FKBP12 protein and cannot degrade mTOR protein.

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

[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 of the Invention

[0015] To overcome the shortcomings of the prior art, the present invention aims to provide a class of selective mTORC1 degrading agents based on the autophagy-lysosome pathway, their preparation method, and applications.

[0016] To achieve the above objectives, the present invention employs the following technical solution:

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

[0018] In the formula, R represents the terminal autophagy-lysosomal target protein modification tag;

[0019] Linker stands for intermediate connector.

[0020] The "autophagy-lysosome pathway" (ALP) described in this invention is a crucial pathway in the process of autophagy, involving the cell's phagocytosis and degradation of some or all of its own organelles and proteins to maintain cellular homeostasis and cope with environmental stresses. Furthermore, target protein modification tags in the autophagy-lysosome pathway play a vital role in autophagy. These tags are typically used to label proteins that need to be degraded, thereby guiding them into the autophagy-lysosome pathway for degradation.

[0021] Preferably, the terminal autophagy-lysosomal target protein modification tag includes an LC3 ligand, an S-guanylate tag, and a p62 binding ligand;

[0022] Preferably, R comprises a compound with the structure shown below:

[0023] 1) LC3 ligand: and its derived structures;

[0024] 2) S-guanylate tag: and its derived structures;

[0025] 3) p62 binding ligand: and its derived structures.

[0026] Preferably, the Linker is selected from one of the following structures:

[0027] The number of oxygen atoms in the polyethylene glycol chain is n, where 0 ≤ n ≤ 5, and n is an integer;

[0028] The number of carbon atoms in the alkyl chain is m, where 1 ≤ m ≤ 20, and m is an integer.

[0029] More preferably, the number of oxygen atoms n in the polyethylene glycol chain is an integer from 2 to 4, and the number of carbon atoms m in the alkyl chain is an integer from 6 to 12; this parameter range ensures that the degrading agent has both good solubility and mTORC1 targeting binding activity in tumor cells.

[0030] Preferably, the selective mTORC1 degrader based on the autophagy-lysosome pathway synthesized in this invention has six preferred structures, the structural formulas of which are as follows:

[0031] Preferably, the first compound among the above six specific compounds exhibits in vitro anti-MCF-7 tumor cell activity comparable to that of the positive control drug rapamycin. After 72 hours of treatment, a high concentration (3.3 μmol / L) can significantly degrade mTOR, Raptor, and FKBP12 proteins in the mTORC1 protein complex.

[0032] The present invention also discloses a method for synthesizing the above-mentioned selective mTORC1 degrader based on the autophagy-lysosome pathway. Water and methanol in a volume ratio of 5:1 are used as reaction solvents, and rapamycin donor is used as reaction raw material. The mixture is stirred overnight at room temperature under the catalysis of copper sulfate and sodium ascorbate to prepare the selective mTORC1 degrader based on the autophagy-lysosome pathway.

[0033] The structural formula of the rapamycin donor is as follows:

[0034] Furthermore, after the reaction was completed, the sample was diluted with ethyl acetate, washed with water, washed with saturated sodium chloride, dried with anhydrous sodium sulfate, and purified by low-temperature silica gel column chromatography, wherein the mobile phase for silica gel column chromatography was a mixture of n-hexane and acetone.

[0035] Preferably, the copper sulfate solution is 1 mol / L, 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.

[0036] The present invention also discloses the application of the above-mentioned selective mTORC1 degrader based on the autophagy-lysosome pathway in the preparation of drugs for regulating mTORC1 activity via the autophagy-lysosome pathway.

[0037] The regulation of mTORC1 activity specifically involves reducing the levels of mTOR, Raptor, and FKBP12 proteins in MCF-7 cells without affecting the Rictor protein level of mTORC2. The drug can be used to improve pathological conditions related to abnormal cell proliferation caused by excessive activation of mTORC1.

[0038] Applications in the preparation of drugs for treating cancer, neurodegenerative diseases, metabolic diseases, or chronic inflammatory diseases.

[0039] Preferably, the cancer includes breast cancer, liver cancer, prostate cancer, cervical cancer, skin cancer, or colon cancer, etc.

[0040] Preferably, the neurodegenerative disease includes Alzheimer's disease, etc.

[0041] Preferably, the metabolic diseases include conditions such as diabetes, hyperlipidemia, or obesity.

[0042] Preferably, the chronic inflammatory disease includes dry eye syndrome, chronic hepatitis, or chronic pancreatitis. Beneficial effects

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] This invention discloses a selective mTORC1 degrader based on the autophagy-lysosome pathway, mainly comprising three parts: a rapamycin donor, an intermediate linker, and a terminal autophagy-lysosome target protein modification tag. Existing mTOR degraders using the ATP-competitive mTOR inhibitor MLN0128 as a point-of-care (POI) have poor PROTAC activity and similarly poor selectivity for mTORC1. PROTACs using rapamycin as a POI can only degrade FKBP12 protein and cannot degrade mTOR. Therefore, this invention proposes for the first time a selective mTORC1 degrader that can achieve selective degradation of mTORC1. Furthermore, due to the terminal linker modification tag, this degradation process is related to autophagy, while the protein level in mTORC2 remains unaffected. This effectively addresses the limitations of existing mTOR inhibitors, such as limited therapeutic efficacy, drug resistance with long-term use, and significant toxic side effects due to poor selectivity for mTORC1.

[0045] The rapamycin derivative obtained by this 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.

[0046] Furthermore, this invention designed and synthesized three types of rapamycin derivatives, with representative compounds exhibiting in vitro anti-MCF7 tumor cell activity comparable to that of the positive control drug rapamycin. Attached Figure Description

[0047] Figure 1 shows the concentration-dependent cell growth inhibition curves of MCF-7 cell lines treated with rapamycin and representative compounds for 72 hours;

[0048] Figure 2 shows the changes in the expression levels of representative proteins mTORC1 and mTORC2 in the MCF-7 cell line after 48 hours of treatment with rapamycin and 1-8-2.

[0049] Figure 3 illustrates the mechanism by which compound 1-8-1 induces the degradation of mTORC1 protein in the MCF-7 cell line.

[0050] Figure 4 shows the concentrations of A) mTORC1 and B) mTORC2 in MCF-7 cells (n=3) after 48 h of treatment with rapamycin and 1-8-2. Detailed Implementation

[0051] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0052] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0053] The present invention will now be described in further detail with reference to the accompanying drawings:

[0054] The purpose of this invention is to provide a selective mTORC1 degrader, its preparation method, and its application.

[0055] A compound and its medicinal salt are disclosed, with the structural formula shown below:

[0056] R represents the terminal autophagy-lysosomal target protein modification tag;

[0057] Linker stands for intermediate connector.

[0058] Furthermore, R in the formula includes an LC3 ligand, an S-guanylate tag, or a p62-binding ligand; even further, the structural formula of the LC3 ligand is shown below:

[0059] Furthermore, the structural formula of the S-guanylate tag is shown below:

[0060] Furthermore, the structural formula for the p62-binding ligand is shown below:

[0061] Furthermore, Linker selects either a polyethylene glycol chain structure or an alkyl chain structure.

[0062] Furthermore, the number of oxygen atoms in the polyethylene glycol chain structure is n, where 0 ≤ n ≤ 5, and n is an integer; the structural formula is as follows:

[0063] The alkyl chain contains m carbon atoms, where 1 ≤ m ≤ 20, and m is an integer. The structural formula is as follows:

[0064] Particularly preferred, the present invention prepared six selective mTORC1 degrading agents, with the following structural formulas:

[0065] I. The following are the preparation methods of representative compounds.

[0066] 1. The preparation steps of ATTEC are as follows:

[0067] Using ethanol as a solvent, compound a and compound 1-1 were used as raw materials to prepare compound 1-2 under the catalysis of piperidine; compound 1-3 was prepared by removing the BOC protecting group from compound 1-2 in a dichloromethane solution of trifluoroacetic acid.

[0068] Using compounds 1-3 and compound b as raw materials, DMF as solvent, and HATU as condensing agent, a condensation reaction was carried out under alkaline conditions of N,N-diisopropylethylamine to prepare compounds 1-4.

[0069] Compounds 1-6 were prepared using dichloromethane as a solvent, compounds 1-5 as raw materials, and trifluoromethanesulfonic anhydride as an acylation reagent under alkaline conditions of 2,6-dimethylpyridine.

[0070] Compounds 1-7 were prepared using toluene as a solvent and compounds 1-6 and rapamycin as raw materials under alkaline conditions of N,N-diisopropylethylamine and microwave conditions.

[0071] Compounds 1-8 were prepared by using water and methanol as a mixed solvent, and compounds 1-4 and 1-7 as raw materials, under the catalytic conditions of copper sulfate and sodium ascorbate.

[0072] Taking the synthesis of 1-8-2 as an example:

[0073] The specific preparation process is as follows:

[0074] Compound 1-1 (0.43 g, 1.1 mmol) and compound a (0.26 g, 1.0 mmol) were added to 15 mL of ethanol, along with piperidine (20 μL) as a catalyst. The system was heated to reflux for 18 h, then cooled to room temperature. A large amount of solid precipitated in the system, which was directly filtered to obtain 0.60 g of yellow solid 1-2, with a yield of 95%. 1-2 was dissolved in 10 mL of dichloromethane, and 3 mL of trifluoroacetic acid was added. The mixture was stirred overnight at room temperature, and the reaction was monitored by TLC until completion. The solvent was evaporated to dryness, and the mixture could be directly proceeded to the next step without purification. The evaporated residue was dissolved in 10 mL of DMF, and compound b2 (0.22 g, 1.0 mmol), HATU (0.45 g, 1.2 mmol), and DIPEA (0.63 mL, 3.6 mmol) were added sequentially. The system was stirred at room temperature under a nitrogen atmosphere for 12 h, and the solvent was evaporated to dryness to obtain the crude product. Purification was performed by silica gel column chromatography with a mobile phase of petroleum ether / ethyl acetate = 2 / 1, yielding 709 mg of an orange-yellow solid, with a yield of 91%.

[0075] Synthesis of rapamycin partial linker:

[0076] Add 20 mL of dichloromethane to a 100 mL flask, along with compounds 1-5 (3.0 g, 30 mmol) and pyridine (2.9 mL, 36 mmol). Then, add trifluoromethanesulfonic anhydride (6.1 mL, 36 mmol) and 20 mL of dichloromethane to a 50 mL constant-pressure dropping funnel. Cool the reaction mixture to 0 °C with stirring in an ice-water bath. Open the constant-pressure dropping funnel and slowly add the trifluoromethanesulfonic anhydride solution dropwise into the reaction system. After the addition is complete, the system is washed sequentially with water (10 mL × 3), saturated sodium chloride (10 mL), and dried over anhydrous sodium sulfate. Remove the solvent by evaporation. Separate the residue by rapid silica gel column chromatography using petroleum ether / ethyl acetate = 20 / 1 as the mobile phase, yielding 6.0 g of a pale brown transparent liquid, with a yield of 93%.

[0077] In a glove box, rapamycin (91.4 mg, 0.1 mmol), compounds 1-6 (218.0 mg, 1 mmol), DIPEA (0.23 mL, 1.3 mmol), triphenylphosphine oxide (56.0 mg, 0.2 mmol), molecular sieve (3A, 40.0 mg), and 0.17 mL of toluene were added sequentially to a 5 mL microwave-safe tube. After sealing, the glove box was removed and placed in a microwave reactor, set to 65 °C for 5 minutes. After the reaction was complete, the tube was removed and the system was directly separated by low-temperature silica gel column chromatography with a mobile phase of n-hexane / acetone = 5 / 1, yielding 61.0 mg of a white bubbly 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)δ211.0,208.1,199.4,169.7,167.5,139.8,138.4,137.7,132.8,130.9,1 27.5,125.4,99.5,86.0,82.9,82.7,80.9,77.5,76.2,74.1,69.5,69.4,68.9,66.7,60.2,57.9, 57.5,57.5,57.4,56.0,51.2,45.7,44.0,38.7,36.5,35.7,35.3,33.8,32.7,31.4,30.2,26.9,2 6.7,25.0,22.1,21.6,21.2,20.9,16.1,16.0,16.0,15.2,14.6,14.0,13.8,10.9.HRMS(ESI):m / z calcd.for C 56 H 85 NO 14 Na + ([M+Na)) + )=1018.58623,found=1018.59257.

[0078] Synthesis of 1-8-2:

[0079] Compounds 1-4-2 (155.8 mg, 0.2 mmol) and 1-7 (199.3 mg, 0.2 mmol) were added to a 5 mL flask, along with 2 mL of a pre-prepared mixed solvent (methanol / water = 5 / 1). Then, 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) were added sequentially. The mixture was stirred overnight at room temperature. The system was diluted with 30 mL of ethyl acetate. The resulting organic phase was washed sequentially with water (10 mL × 3), saturated sodium chloride (10 mL), and dried over anhydrous sodium sulfate. The solvent was evaporated, and the residue was separated by low-temperature silica gel column chromatography using a mobile phase of n-hexane / acetone = 5 / 1, yielding 0.30 g of a yellow solid, with a yield of 84%, which was 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.for C 81 H 112 Br2IN6O 20 + ([M+H)) + )=1773.53374,found=1773.53725.

[0080] 2. The preparation steps of AUTAC are as follows:

[0081] Using ethanol as a solvent, compound 2-1 and di-tert-butyl dicarbonate were used as raw materials to prepare compound 2-2 under the catalysis of 4-dimethylaminopyridine.

[0082] Compound 2-3 was prepared by stirring the compound 2-2 in a tetrahydrofuran solution of sodium hydride;

[0083] Compound 2-4 was prepared from compounds 2-3 and compound c under the catalytic conditions of DEAD and triphenylphosphine.

[0084] Compound 2-5 was prepared by removing the BOC group from compound 2-4 in formic acid solution;

[0085] Compounds 2-6 were prepared by bromination of compounds 2-5 in bromine water;

[0086] Using DMSO as a solvent, compounds 2-5 and d were prepared by nucleophilic substitution reaction under potassium carbonate alkaline conditions;

[0087] Using compounds 2-7 and e as raw materials, DMF as solvent, EDC and HOBt as condensing agents, a condensation reaction was carried out under basic conditions of triethylamine to prepare compounds 2-8.

[0088] Compounds 2-9 were prepared by using water and methanol as a mixed solvent, and compounds 2-8 and 1-7 as raw materials, under the catalytic conditions of copper sulfate and sodium ascorbate.

[0089] Taking the synthesis of 2-9-2 as an example:

[0090] The specific preparation process is as follows:

[0091] Compound 2-1 (5.09 g, 30 mmol) was dissolved in 30 mL of DMSO solution. The system was cooled to 0 °C under an ice-water bath. 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 added directly to the system, and the mixture was filtered to obtain 7.70 g of white solid, with a yield of 87%. The obtained white solid was completely dissolved in 50 mL of tetrahydrofuran and cooled to 0 °C under an ice-water bath. Sodium hydride (60%, 3.0 g, 75 mmol) was added in batches. After stirring at room temperature for 2 h, the system was washed sequentially with saturated sodium chloride (10 mL × 3) and dried over anhydrous sodium sulfate. The solvent was evaporated, and the residue was separated by silica gel column chromatography with dichloromethane / methanol = 20 / 1 as the mobile phase, yielding 6.70 g of white solid, with a yield of 87%.

[0092] Compounds 2-3 (2.70 g, 10 mmol) were dissolved in 50 mL of tetrahydrofuran, followed by the sequential addition of compound c (1.26 g, 10 mmol) and triphenylphosphine (6.56 g, 25 mmol), and finally DEAD (4.40 g, 25 mmol) was slowly added dropwise. The mixture was stirred at room temperature for 6 h, and the solvent was removed by evaporation. The residue was separated by silica gel column chromatography with a mobile phase of dichloromethane / methanol = 30 / 1, yielding a crude product containing triphenylphosphine oxide. The crude product was dissolved in 20 mL of 80% formic acid solution, heated in an oil bath to 75 °C, and stirred overnight. The solvent was removed by evaporation, and the residue was separated by silica gel column chromatography with a mobile phase of dichloromethane / methanol = 10 / 1, yielding 4.0 g of a white solid, with a yield of 60%.

[0093] 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. The resulting organic phase was washed successively with water (10 mL × 3), saturated sodium chloride (10 mL), and dried over anhydrous sodium sulfate. The solvent was removed by evaporation, and the residue was separated by rapid silica gel column chromatography with dichloromethane / methanol = 10 / 1 as the mobile phase, yielding 0.55 g of a pale yellow solid, with a yield of 85%.

[0094] Compounds 2-6 (0.50 g, 1.5 mmol) were dissolved in 10 mL of DMF. Compound d (0.27 g, 1.6 mmol) and potassium carbonate (0.62 g, 4.5 mmol) were also added. The system was heated to 75 °C in an oil bath and stirred for 5 h, then cooled to room temperature. 30 mL of hydrochloric acid solution (4 N) was slowly added to the system, causing a solid to precipitate. The solid was filtered to obtain 0.40 g of a white powder, with a yield of 63%.

[0095] Compound 2-7 (105.1 mg, 0.25 mmol) was dissolved in 2 mL of 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 sequentially. The mixture was stirred at room temperature for 4 h, and the solvent was removed by evaporation. The residue was separated by silica gel column chromatography with dichloromethane / methanol = 30 / 1, yielding 75.0 g of a white powder solid, with a yield of 33%.

[0096] 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, along with 2 mL of a pre-prepared mixed solvent (methanol / water = 5 / 1). Then, 0.375 mL of a pre-prepared copper sulfate solution (1 mol / L) and 0.75 mL of sodium ascorbate solution (1 mol / L) were added sequentially. The mixture was stirred overnight at room temperature. The system was diluted with 30 mL of ethyl acetate. The resulting organic phase was washed sequentially with water (10 mL × 3), saturated sodium chloride (10 mL), and dried over anhydrous sodium sulfate. The solvent was evaporated, and the residue was separated by low-temperature silica gel column chromatography using a mobile phase of n-hexane / acetone = 4 / 1, yielding 0.25 g of a pale yellow solid, with a yield of 62%, which was 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.

[0097] 3. The preparation steps of AUTOTAC are as follows:

[0098] Compound 3-2 was prepared by using acetonitrile as a solvent and compound 3-1 and benzyl bromide as raw materials under the alkaline action of sodium bicarbonate; and compound 3-3 was prepared by stirring in DMF solution under the alkaline action of potassium carbonate using compound 3-2 and compound f as raw materials.

[0099] Compound 3-4 was prepared by stirring the compound 3-3 in a dichloromethane solution of m-chloroperoxybenzoic acid;

[0100] Compound 3-5 was prepared in DMF using compounds 3-4 and compound g as raw materials;

[0101] Compounds 3-6 were prepared by nucleophilic substitution reaction using ethanol as a solvent and compounds 3-5 and b as raw materials; compounds 3-7 were prepared by mixing water and methanol as a solvent and using compounds 3-6 and 1-7 as raw materials under the catalytic conditions of copper sulfate and sodium ascorbate.

[0102] Taking the synthesis of 3-7-2 as an example:

[0103] The specific preparation process is as follows:

[0104] 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. The system was heated to 80 °C and stirred overnight. 100 mL of hydrochloric acid (4 N) was added, and the aqueous phase was extracted with ethyl acetate (80 mL × 3). The combined organic phases were washed successively with water (10 mL × 3), saturated sodium chloride (10 mL), and dried over anhydrous sodium sulfate. The solvent was evaporated to give 0.80 g of a milky white powder, yield 13%.

[0105] 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. The system was heated to 80 °C and stirred overnight. After removing the solvent, the residue was separated by silica gel column chromatography with petroleum ether / ethyl acetate = 10 / 1 as the mobile phase, yielding 1.20 g of a pale yellow solid in 98% yield.

[0106] Compound 3-3 (1.20 g, 3.5 mmol) was dissolved in dichloromethane, and m-chloroperoxybenzoic 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, then washed successively with saturated sodium carbonate solution (10 mL × 3), saturated sodium chloride solution (10 mL), and dried over anhydrous sodium sulfate. After evaporation of the solvent, the residue was dissolved in methanol, and 20 mL of 6N sodium hydroxide solution was added. After stirring at room temperature for 0.5 h, the pH was adjusted to 7 with 4N hydrochloric acid solution, and after stirring for another 0.5 h, the aqueous phase was extracted with ethyl acetate (40 mL × 3). The combined organic phases were then washed successively with water (10 mL × 3), saturated sodium chloride solution (10 mL), and dried over anhydrous sodium sulfate. The solvent was removed by evaporation, and the residue was separated by rapid silica gel column chromatography with a mobile phase of hexane / ethyl acetate = 7 / 3. The resulting oily substance was directly soluble in ethanol, and compound g (1.60 g, 17.3 mmol) and potassium hydroxide (0.23 g, 4.2 mmol) were added. After stirring overnight at room temperature, the solvent was removed by evaporation, and the residue was diluted with 30 mL of ethyl acetate, then washed successively with saturated sodium chloride solution (10 mL × 3), followed by washing with saturated sodium chloride solution (10 mL), and dried over anhydrous sodium sulfate. After solvent removal, the residue was separated by rapid silica gel column chromatography with a mobile phase of hexane / ethyl acetate = 10 / 1, yielding 0.90 g of a white powdery solid, with a yield of 67%.

[0107] 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. The solvent was then removed by evaporation. The residue was separated by silica gel column chromatography with dichloromethane / methanol = 3 / 1 as the mobile phase, yielding 70.0 mg of a colorless oily substance, with a yield of 44%.

[0108] 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, along with 2 mL of a pre-prepared mixed solvent (methanol / water = 5 / 1). Then, 0.33 mL of a pre-prepared copper sulfate solution (1 mol / L) and 0.17 mL of sodium ascorbate solution (1 mol / L) were added sequentially. The mixture was stirred overnight at room temperature. The system was diluted with 30 mL of ethyl acetate. The resulting organic phase was washed sequentially with water (10 mL × 3), saturated sodium chloride (10 mL), and dried over anhydrous sodium sulfate. The solvent was evaporated, and the residue was separated by low-temperature silica gel column chromatography using a mobile phase of n-hexane / acetone = 2 / 1, yielding 0.12 g of a pale yellow solid, with a yield of 76%, which was 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, 127.7,127.7,127.6,127.0,126.8,126.4,126.4,125.2,125.1,124.2,1 23.5,123.5,115.7,104.1,101.6,98.4,84.9,81.8,81.6,75.1,73.0,70 .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.

[0109] II. Pharmacological Experiments

[0110] 1. In vitro anti-MCF7 tumor cell proliferation assay

[0111] Human breast cancer cells MCF-7 were cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 cell culture incubator. Cells were seeded into black 96-well plates at 3000 cells per well (100 μL) and placed in a CO2 cell culture incubator. After overnight cell adhesion, different concentrations of compounds (1 μL) were added, with six replicates for each concentration. A corresponding concentration of DMSO was also included as a control. After 72 h of compound treatment, 100 μL of ATP assay kit (CellTiter-Glo Luminescent Cell Viability Assay kit) was added, and the plates were shaken for 1 h. Luminescence was detected using Analyst AD, and Prism plots were performed based on the fluorescence values ​​compared to the blank control, followed by statistical analysis. The results are shown in Figure 1. Compounds 1-8-2, 2-9-2, and 3-7-3 all exhibited good in vitro anti-MCF-7 tumor cell activity.

[0112] 2. Western Blot Experiment

[0113] Human breast cancer cells (MCF-7) were seeded into six-well plates. After attachment, the cells were treated with different concentrations of the target compound or positive control for 48 hours. Then, 100 μL of RIPA lysis buffer containing a protease inhibitor was added to each well. Cells were scraped off using a clean pipette tip, and all the scraped liquid was transferred to a 1.5 mL centrifuge tube. The tube was centrifuged at 12,000 rpm for 20 min in a centrifuge pre-cooled to 4°C. 75 μL of the supernatant was gently aspirated (carefully avoiding aspirating any white flocculent material). Protein quantification was performed using the BCA method according to the BCA kit instructions. 5× loading buffer (1:4 volume ratio to protein solution) was added to the protein solution, vortexed, and then placed in boiling water for 5 min to obtain the denatured protein sample. An 8% separating gel and a 5% stacking gel were prepared according to the manufacturer's instructions (the gels were immediately combed after pouring and then allowed to stand for 30 min). Add electrophoresis buffer to the electrophoresis apparatus. Slowly remove the comb, then add the denatured protein sample or protein marker to each gel well, at a rate of 10 μg protein per well. Turn on the power and electrophoresis at 90V for 2 hours. After electrophoresis, cut the bands of the corresponding molecular weight according to the protein marker indications and stack them together with the methanol-excited PVDF membrane in the transfer clamp. Transfer at 400mA for 30 minutes. Place the PVDF membrane with transferred protein in a pre-prepared 5% skim milk powder solution (prepared with PBST, 5 mL) and incubate at room temperature with shaking for 1 hour. Aspirate the blocking solution and wash the bands twice with PBST, shaking for 2 minutes each time. Aspirate the PBST dry, add a diluted BSA (5%, prepared with PBST) solution, and incubate at 4°C for 13-16 hours. Aspirate and recover the primary antibody, wash the bands three times with TBST, shaking for 5 minutes each time. Aspirate the TBST dry, add a diluted secondary antibody solution, and incubate at room temperature with shaking for 1 hour. The secondary antibody was discarded, and the bands were washed three times with PBST for 5 min each time. The bands were then removed, evenly spread with the prepared ECL chemiluminescence solution, exposed in a gel imaging system, and the images were saved. As shown in Figure 2, Western blotting experiments on compounds with good activity indicate that the compounds can simultaneously degrade mTOR, Raptor, and FKBP12 proteins in mTORC1, while the protein levels in mTORC2 (e.g., Rictor protein) remain unaffected. The degradation of related proteins was most pronounced at a dosage concentration of 3.3 μmol / L; however, at the same dosage concentration, the levels of all related proteins were not significantly affected by the positive control drug rapamycin, confirming the rationality of the degradation tagging strategy introduced into the rapamycin structure in this invention. As shown in Figure 3, the mTORC1 protein degradation mechanism is only related to the autophagy-lysosome pathway and not to the ubiquitin-proteasome pathway, consistent with the viewpoint proposed in this invention.

[0114] 3. Enzyme-linked immunosorbent assay (ELISA)

[0115] Human breast cancer cells MCF-7 were seeded into six-well plates. After adhesion, the cells were treated with different concentrations of the target compound or positive control for 48 hours, followed by trypsin digestion and collection. The cells were then washed twice with phosphate-buffered saline (PBS, pH 7.4). The collected cells were diluted with PBS containing protease inhibitors, then sonicated (2 kHz, 2 s × 3), centrifuged (1500 × g, 10 min) to remove insoluble substances. Protein concentration was determined using BCA reagent. A fixed total protein amount (30 μg) was used, and the concentrations of mTORC1 and mTORC2 in different samples were detected using an mTORC1 and mTORC2 enzyme-linked immunosorbent assay (ELISA) kit. Prism plots were performed and statistical analysis was conducted. As shown in Figure 4, the ELISA experiment provides a more direct detection. Compared to the control group (i.e., the solvent group, 0.1% DMSO), the synthesized compound (1-8-2 as an example) selectively degraded mTORC1 protein in a dose-dependent manner (Figure 4A), while the mTORC2 protein level remained unaffected (Figure 4B).

[0116] In summary, this invention proposes the concept of selective mTORC1 degradation based on autophagy, and designs and synthesizes a class of selective mTORC1 degraders based on the autophagy-lysosomal pathway. Specifically, three representative compounds, namely rapamycin derivatives, were synthesized. The in vitro anti-MCF7 tumor cell activity of these representative compounds is comparable to that of the positive control drug rapamycin. The selective mTORC1 degraders obtained by this invention can achieve selective degradation of mTORC1, and this degradation process is related to autophagy, while the protein level in mTORC2 remains unaffected. Therefore, the selective mTORC1 degraders obtained by this invention can provide a new approach and potential drug development direction for the treatment of diseases related to the regulation of mTORC1 activity through the autophagy-lysosomal pathway (such as cancer, neurodegenerative diseases, obesity, diabetes, chronic inflammatory diseases, dry eye syndrome, etc.), and also provide a powerful tool for in-depth research on the function and regulatory mechanism of mTORC1, offering valuable reference and inspiration for other similar studies.

[0117] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A class of selective mTORC1 degrading agents based on the autophagy-lysosome pathway, characterized in that, Its structural formula is shown below: In the formula, R represents the terminal autophagy-lysosomal target protein modification tag; R is selected from LC3 ligand, S-guanylate tag or p62 binding ligand, wherein the LC3 ligand contains a fragment that specifically binds to the LIR domain of the LC3 protein, and the p62 binding ligand contains a substituent that binds to the ZZ domain of the p62 protein. Linker represents an intermediate connector; This selective mTORC1 degrader induces the degradation of the mTORC1 protein complex via the autophagy-lysosome pathway, without affecting the Rictor protein level in mTORC2.

2. The selective mTORC1 degrader based on the autophagy-lysosome pathway according to claim 1, characterized in that, The structural formula of the LC3 ligand is shown below: The structural formula of the S-guanylate tag is shown below: The structure of R is as follows: As shown; The structural formula for p62 binding ligands is shown below:

3. The selective mTORC1 degrading agent based on the autophagy-lysosome pathway according to claim 1, characterized in that, The Linker is selected from polyethylene glycol chain structure or alkyl chain structure; The linker in the polyethylene glycol chain structure has n oxygen atoms, where 0 ≤ n ≤ 5, and n is an integer; the structural formula is as follows: The linker in the alkyl chain structure has m carbon atoms, where 1 ≤ m ≤ 20, and m is an integer. The structural formula is as follows:

4. The selective mTORC1 degrader based on the autophagy-lysosome pathway according to claim 1, characterized in that, Specifically, it includes six selective mTORC1 degraders, with the following structural formulas:

5. The method for synthesizing the selective mTORC1 degrader based on the autophagy-lysosome pathway as described in any one of claims 1 to 4, characterized in that, include: Using water and methanol as reaction solvents and rapamycin donor as reaction raw material, a selective mTORC1 degrader based on the autophagy-lysosome pathway was prepared by stirring overnight at room temperature under the catalysis of copper sulfate and sodium ascorbate.

6. The method for synthesizing a selective mTORC1 degrader based on the autophagy-lysosome pathway according to claim 5, characterized in that, Copper sulfate and sodium ascorbate are both prepared using a 1 mol / L copper sulfate solution and a 1 mol / L sodium ascorbate solution, with a volume ratio of 1-2:

1.

7. The use of any one of the selective mTORC1 degraders based on the autophagy-lysosome pathway according to claims 1 to 4 in the preparation of a medicament for regulating mTORC1 activity via the autophagy-lysosome pathway.

8. The application according to claim 7, characterized in that, Regulating mTORC1 activity specifically involves reducing the levels of mTOR, Raptor, and FKBP12 proteins in mTORC1 in MCF-7 cells, without affecting the Rictor protein level of mTORC2.

9. The application according to claim 7, characterized in that, The drug is used to improve pathological conditions related to abnormal cell proliferation caused by excessive activation of mTORC1.

10. The application according to claim 7, characterized in that, Diseases that regulate mTORC1 activity through the autophagy-lysosomal pathway include 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 conditions such as diabetes, hyperlipidemia, or obesity.

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