Hydrophobic label-containing mTORC1 selective degradation agent as well as synthesis method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an mTORC1 selective degradation agent containing a hydrophobic label as well as a synthesis method and application of the mTORC1 selective degradation agent. The structural formula of the degradation agent is as shown in formula I. Representative compounds have the effect of inhibiting proliferation of various tumor cells, and can provide new research and development thoughts and directions for anti-tumor drugs. .

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a class of mTORC1 selective degraders containing hydrophobic tags, their preparation methods and applications, and particularly to an mTORC1 degrader formed by combining a rapamycin derivative as a target unit with a hydrophobic tag, which can be used in the field of tumor treatment. Background Technology

[0002] The mammalian target of rapamycin (mTOR) is a serine / threonine protein kinase belonging to the phosphatidylinositol 3-kinase-associated kinase (PIKK) family. It plays a central regulatory role in key biological processes such as cell growth, proliferation, metabolism, survival, and autophagy. Intracellularly, mTOR exists in two functionally distinct complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1, composed of mTOR, Raptor, mLST8, PRAS40, and DEPTOR, is specifically inhibited by rapamycin and its derivatives, primarily regulating protein synthesis, ribosome formation, and autophagy. mTORC2, composed of mTOR, Raptor, mLST8, mSIN1, and DEPTOR, is not directly inhibited by rapamycin but participates in the regulation of cytoskeleton remodeling, glucose metabolism, and cell survival.

[0003] Numerous studies have confirmed that abnormal activation of mTORC1 is closely related to a variety of human diseases, especially malignant tumors. In various cancers such as colorectal cancer, breast cancer, lung cancer, and prostate cancer, the mTORC1 signaling pathway is continuously activated due to gene mutations, amplification, or abnormalities in upstream regulatory molecules, leading to uncontrolled proliferation, invasion, and metastasis of tumor cells.

[0004] Given the significant pathological role of mTORC1, it has become a key target for disease treatment. Currently, the main modulators of mTORC1 are rapamycin and its derivatives (such as everolimus and tesiromolimus). These drugs form a complex by binding to FKBP12, thereby targeting and binding to the Raptor subunit of mTORC1, inhibiting mTORC1 kinase activity. However, traditional rapamycin inhibitors have significant drawbacks: first, insufficient selectivity, and long-term use may indirectly affect mTORC2 activity, leading to toxic side effects such as hyperglycemia, hyperlipidemia, and immunosuppression; second, easy development of drug resistance, with tumor cells escaping inhibition by activating compensatory signaling pathways or through mTOR gene mutations; and third, reversible inhibition, with mTORC1 activity easily recovering after drug withdrawal, making it difficult to achieve a lasting therapeutic effect.

[0005] To overcome the shortcomings of traditional inhibitors, protein degrading agents have become a research hotspot in recent years, among which PROTAC (proteolytic-targeting chimera) technology based on the ubiquitin-proteasome system is the most mature. A PROTAC molecule consists of three parts: a ligand that targets and binds to the target protein, an E3 ubiquitin ligase ligand, and a linker. It can act as a "bridge" to bring the target protein closer to the E3 ubiquitin ligase, causing the target protein to ubiquitinate and be degraded by the proteasome. Currently, several studies on PROTAC degrading agents targeting mTOR have been reported, but most of these degrading agents are pan-mTOR degrading agents, simultaneously degrading mTORC1 and mTORC2, leading to an increased risk of toxic side effects. A few mTORC1 selective degrading agents suffer from insufficient degradation activity, complex synthesis processes, or low bioavailability, limiting their clinical translation and application.

[0006] Hydrophobic tagging technology is a novel protein degradation technique. Its core principle involves attaching a hydrophobic group to a target ligand, increasing the hydrophobicity of the target protein surface and triggering a "hydrophobic rescue" mechanism within the cell, leading to degradation via the autophagy-lysosome or ubiquitin-proteasome pathway. Compared to PROTAC technology, hydrophobic tag degraders do not rely on specific E3 ubiquitin ligases, have broader applicability, and possess relatively simple molecular structures, making them easier to synthesize and optimize. Currently, hydrophobic tagging technology has been successfully applied to the degradation of various target proteins, but there are no reports on its application to the selective degradation of mTORC1. Developing mTORC1 selective degraders based on hydrophobic tagging technology holds promise for addressing the selectivity and drug resistance issues of existing regulators. In current technologies, the in vivo activity verification and transcriptional analysis of the molecular mechanism of mTORC1 degraders are insufficient, limiting their clinical translation. This invention, through supplementary transcriptome analysis and animal experiments, further clarifies the mechanism of action and in vivo antitumor effects of the degraders, providing more sufficient experimental evidence for their clinical application. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a hydrophobic tag-based selective mTORC1 degrader. By guiding protein degradation through the hydrophobic tag, it achieves selective degradation of mTORC1, effectively solving the problems of poor activity and low selectivity of existing degraders. This degrader has shown significant effects in anti-tumor applications. Furthermore, this invention uses transcriptome sequencing technology to analyze the regulatory effects of the degrader on the expression of the mTORC1 pathway and downstream target genes, clarifying the expression characteristics and functional enrichment directions of differentially expressed genes, and verifying the specific mechanism of action of the degrader at the transcriptional level. Simultaneously, animal experiments have confirmed the anti-tumor activity and safety of the degrader in vivo, providing direct in vivo experimental evidence for its clinical translation.

[0008] The specific technical solution provided by this invention is as follows: In a first aspect, the present invention provides a selective mTORC1 degrader containing a hydrophobic tag, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, prodrug, polymorph, eutectic, or deuterated thereof, wherein the degrader has the structural formula shown in Formula I: ; Linker1 is selected from any of the following structures or combinations thereof: saturated cycloalkanes, saturated nitrogen heterocycles, spirocyclic structures, C1-C8 straight-chain or branched alkyl groups, chain structures containing heteroatoms, aromatic rings or heteroaromatic ring linking units; the main chain atom length of Linker1 is 3 to 20, and the main chain atoms refer to the C, O, N, and S atoms that constitute the linking chain backbone; Linker2 is selected from any of the following structures or combinations thereof: saturated cycloalkanes, saturated nitrogen heterocycles, spirocyclic structures, C1-C6 straight-chain or branched alkyl groups, short-chain structures containing heteroatoms, monoaromatic rings or monoheteroaromatic ring linkers; the main chain of Linker2 has a length of 3 to 12 atoms, and the main chain atoms are defined the same as those of Linker1; The hydrophobic label is selected from any of the following structures or their derivatives, stereoisomers, or equivalents, wherein the derivative is a hydroxyl-substituted, alkyl-substituted, ester-substituted, ether-substituted, amino-substituted, or halogen-substituted product: Cage-like hydrocarbons, polycyclic aromatic hydrocarbons, steroids, terpenes, monocyclic or bicyclic aromatic hydrocarbon derivatives, long-chain alkyl or cycloalkyl, arylalkyl, heterocyclic hydrophobic units, perfluoroalkyl-substituted aromatic hydrocarbons, adamantane-aromatic couplings, or steroid-terpene couplings.

[0009] As a preferred embodiment of the present invention In Linker1: The saturated cycloalkane is any one of cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, and cyclooctane; The saturated nitrogen heterocycle is any one of piperidine, pyrrolidine, morpholine, piperazine, and hexahydroacetane; The spirocyclic structure is any one of spiro[3.3]heptane, spiro[3.4]octane, spiro[4.4]nonane, and spiro[4.5]decane; The heteroatom-containing chain structure is a C2-C10 chain structure containing ether bonds, amine bonds, amide bonds, ester bonds, urea bonds, thioether bonds, and sulfonamide bonds; the aromatic ring or heteroaromatic ring connecting unit is a benzene ring, pyridine ring, pyrimidine ring, pyrazine ring, pyridazine ring, furan ring, or thiophene ring, and the aromatic ring or heteroaromatic ring is mono-substituted, poly-substituted, or unsubstituted by C1-C3 alkyl, halogen, hydroxyl, or C1-C3 alkoxy groups; In Linker2: The saturated cycloalkane is any one of cyclopropane, cyclobutane, cyclopentane, cyclohexane, and cycloheptane; The saturated nitrogen heterocycle is any one of piperidine, pyrrolidine, morpholine, and piperazine; The spirocyclic structure is spiro[3.3]heptane or spiro[3.4]octane; The short-chain structure containing heteroatoms is a C2-C8 chain structure containing ether bonds, amine bonds, amide bonds, ester bonds, and urea bonds; The monoaromatic ring or mono-heteroaromatic ring linking unit is any one of benzene ring, pyridine ring, and furan ring, and the aromatic ring or heteroaromatic ring is monosubstituted or unsubstituted by any one of C1-C2 alkyl group or halogen. In the hydrophobic label: The cage-like hydrocarbons are adamantane, adamantane methyl, adamantane ethyl, adamantane alcohol, adamantane carboxylic acid, diadamantane, cubane, cubane methanol, norbornane, norbornene, norbornane carboxylic acid, tetracyclic [4.4.0.1², 5 .1 7 ,¹ 0 The tag is selected from dodecane, adamantane, and methyladamantane; this type of tag has a rigid cage structure, which can enhance the targeted binding stability of the degrading agent through steric hindrance effect. The polycyclic aromatic hydrocarbons are any one of naphthalene, α-methylnaphthalene, β-hydroxynaphthalene, anthracene, phenanthrene, pyrene, fluoranthene, α-methylfluorene, 9-phenylfluorene, 9-fluorenylmethanol, biphenyl, 2-methylbiphenyl, 3-methoxybiphenyl, terphenyl, p-tert-butylbiphenyl, o-xylylbiphenyl, acenaphthene, fluorenone, and dibenzothane; their conjugated aromatic ring structure can enhance their binding ability with the hydrophobic region around the target through π-π stacking. The steroids are any one of cholesterol, sitosterol, stigmasterol, lanosterol, ergosterol, pregnane, androstenol, estradiol, campesterol, stigmasterol, cholesterol acetate, cholesterol benzoate, pregnanetriol, and methylcholesterol; the polycyclic rigid structure of the steroid core is combined with a long-chain alkyl group, possessing both strong hydrophobicity and biocompatibility; The terpenoids are any one of monoterpenes, sesquiterpenes, diterpenes, triterpenes or terpene polymers: the isoprene units of terpenoids can flexibly adjust the strength of hydrophobicity, and some terpenoids also have natural biological synergistic effects. The monocyclic or bicyclic aromatic derivatives are selected from any one of cumene, cumene, p-isopropyltoluene, isopropylnaphthalene, tert-butylnaphthalene, diphenylmethane, diphenylethane, triphenylmethane, triphenylmethanol, diphenyl ether, diphenyl sulfide, o-chlorobiphenyl, and p-bromobiphenyl; this type of tag has a simple structure and is easy to synthesize, and its hydrophobicity and spatial conformation can be controlled by substituents; The long-chain alkyl or cycloalkyl group is selected from any one of C12-C24 straight-chain alkyl, branched alkyl, cycloalkyl, long-chain alkanol, and long-chain fatty acid ester; the long-chain structure can penetrate deep into the hydrophobic pocket of the target, enhancing the binding affinity; The aryl alkyl group is selected from any one of benzenehexane, benzeneoctane, benzenedecane, naphthalenebutane, naphthalenehexane, phenylcyclohexane, benzylcyclohexane, and phenethylcyclohexane; the combination of aromatic ring and long-chain alkyl group can take into account both π-π interaction and hydrophobic intercalation effect; The heterocyclic hydrophobic unit is selected from any one of dibenzofuran, dibenzothiophene, carbazole, N-methylcarbazole, phenothiazine, phenoxazine, phenanthrene, indolocarbazole, dithienobenzene, thienofluorene, benzodioxane, phenazine, methylphenothiazine, and chlorophenoxazine; the introduction of heteroatoms can fine-tune the electron cloud distribution while maintaining strong hydrophobicity, and some heterocycles can also participate in hydrogen bonding to assist in binding; The perfluoroalkyl-substituted aromatic hydrocarbon is selected from pentafluorophenylcyclohexane or perfluorooctylbenzene; The adamantane-aromatic coupling compound is selected from adamantane-phenylmethane; The steroid-terpene conjugate is selected from cholesterol-based geraniol.

[0010] More preferably, in Linker1, the heteroatom-containing chain structure is any one of ethylene glycol ether chain, propylene diamine chain, acetamide chain, acetate chain, urea chain, ethyl sulfide chain, and benzenesulfonamide chain; In Linker2, the short chain structure containing heteroatoms is any one of diethylene glycol ether chain, ethylenediamine chain, propionamide chain, propionate ester chain, and methylurea chain; The monoterpenes are selected from any one of geraniol, citronellol, limonene, terpineol, camphor, menthol, borneol, and isoborneol; The sesquiterpenes are selected from any one of farnesol, farnesol, myrrhene, and caryophyllene; The diterpenoid is selected from any one of the side chain structures of phytol, rosin acid, and taxol; The triterpenoids are selected from oleanolic acid or ursolic acid; The terpene polymer is selected from squalene or polyisoprene oligomers; In the hydrophobic label, the C12-C24 straight-chain alkyl group is selected from any one of dodecyl, tetradecyl, hexadecyl, octadecyl, and eicosyl; the branched alkyl group is selected from any one of isododecyl, isostearyl, and 2-hexyldecyl; the cycloalkyl group is selected from any one of cyclododecane, cyclotetradecane, cyclohexadecane, cyclooctadecane, cycloeicosane, methylcyclododecane, and ethylcyclohexadecane; the long-chain alkanol is selected from any one of dodecanol and octadecanol; and the long-chain fatty acid ester is selected from methyl stearate or ethyl palmitate.

[0011] In a preferred embodiment of the present invention, the degrading agent has any of the following structures: .

[0012] The hydrophobic tag-containing mTORC1 selective degrader of the present invention is synthesized through a three-step core reaction, the specific steps of which are as follows: Step 1: Preparation of rapamycin derivatives Compound 1-1 was reacted with trifluoromethanesulfonic anhydride as a starting material to undergo a nucleophilic substitution reaction to obtain compound 1-2; ; Nucleophilic substitution reaction was carried out using compounds 1-2 and rapamycin as raw materials to obtain rapamycin derivatives 1-3; ; Step 2: Preparation of hydrophobic tag-linker intermediate The acylation reaction was carried out using compound 1-4-1 (i.e., hydrophobic tag) and compound 1-4-2 as raw materials to obtain compound 1-4; ; Step 3: Preparation of the target degrading agent The degradation agent 1-5 is obtained by performing a Click reaction using the rapamycin derivatives 1-3 and the compounds 1-4 as raw materials. .

[0013] In a preferred embodiment of the present invention, compounds 1-2 are prepared by stirring at 0-5°C for 1-3 hours.

[0014] In a preferred embodiment of the present invention, the rapamycin derivative is prepared by reacting in a sealed environment under microwave conditions at 50-80°C for 3-20 min.

[0015] In a preferred embodiment of the present invention, the acylation reaction is carried out at room temperature for 6 to 18 hours.

[0016] In a preferred embodiment of the present invention, the Click reaction is carried out at room temperature for 6 to 18 hours.

[0017] In a preferred embodiment of the present invention, the molar ratio of compound 1-1 to trifluoromethanesulfonic anhydride is 30:35~40; The molar ratio of compounds 1-2 and rapamycin is 1:0.1~1; The molar ratio of compound 1-4-1 to compound 1-4-2 is 1:1~2; The molar ratio of the rapamycin derivative to compounds 1-4 is 1:0.5~2.

[0018] The present invention also provides a pharmaceutical composition comprising, as an active ingredient, the mTORC1 selective degrader containing a hydrophobic tag or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, prodrug, polymorph, cocrystal, or deuterated product thereof, wherein the active ingredient comprises 0.01% to 99.99% by mass.

[0019] In a preferred embodiment of the present invention, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients. The excipients are selected from carriers, diluents, excipients, binders, disintegrants, lubricants, flow aids, solubilizers, plasticizers, stabilizers, preservatives, coating materials, pH adjusters, osmotic pressure adjusters, suspending agents, emulsifiers, flavoring agents, or coloring agents;

[0020] The carrier is selected from lactose, glucose, mannitol, sorbitol, microcrystalline cellulose, starch, dicalcium phosphate, and calcium carbonate; the diluent is selected from water for injection, physiological saline, 5% glucose injection, polyethylene glycol 400, polyethylene glycol 6000, and propylene glycol; the excipient is selected from magnesium stearate, talc, and silica; the binder is selected from hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, gum arabic, and gelatin; the disintegrant is selected from crospovidone, crospovidone sodium carboxymethyl cellulose, sodium carboxymethyl starch, and low-substituted hydroxypropyl cellulose; the solubilizer is selected from Tween 80, Span 80, polyoxyethylene castor oil, and poloxamer; the stabilizer is selected from vitamin E, sodium sulfite, sodium metabisulfite, and disodium EDTA; and the preservative is selected from benzyl alcohol, parabens, and sorbic acid. The dosage form of the pharmaceutical composition is an oral preparation (such as tablets, capsules, granules, powders, oral liquid preparations, suspensions), an injectable preparation (such as sterile powder for injection, injection solution, infusion), a topical preparation (such as ointment, cream, gel, patch, lotion, drug device coating), or a cavity preparation (such as suppositories, gels).

[0021] The present invention also provides the use of the mTORC1 selective degrader containing the hydrophobic tag, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, prodrug, polymorph, eutectic, or deuterated product thereof, or the pharmaceutical composition thereof, in the preparation of an antitumor drug.

[0022] As a preferred embodiment of the present invention, the mTORC1 selective degrader containing a hydrophobic tag of the present invention can induce specific degradation of mTORC1 and be used to prepare drugs for treating colorectal cancer, breast cancer, lung cancer, prostate cancer, cervical cancer, kidney cancer, liver cancer, gastric cancer, pancreatic cancer, ovarian cancer, melanoma, lymphoma, leukemia and thyroid cancer. It can be used alone or in combination with chemotherapy drugs (such as cisplatin, paclitaxel) and immune checkpoint inhibitors (such as PD-1 antibodies).

[0023] This invention designs and synthesizes rapamycin derivatives with hydrophobic tags, achieving selective degradation of mTORC1 by altering the linker length, type, and hydrophobic tag type. This invention leverages hydrophobic tags to delve into the protein folding and molecular chaperone recognition mechanism at the microscopic level. During protein folding, hydrophobic residues are hidden internally; their exposure is a marker of incorrect protein folding. This mechanism is more precise and efficient, directly addressing protein structural characteristics. Compared to existing technologies, it reduces dependence on the entire autophagy-lysosome pathway and interference with other normal autophagy processes within the cell, ensuring degradation effectiveness while minimizing potential impacts on normal cellular physiological functions.

[0024] From the perspective of molecular structure design, the hydrophobic-tagged degradative agent provided by this invention is structurally simpler and more efficient. The rapamycin moiety directly interacts with mTORC1, eliminating the need for complex multi-domain adaptation to autophagy-related protein recognition and binding. This simplified structure makes the synthesis process easier to control and reduces costs. In terms of synthetic route complexity, this invention requires only a few reaction steps to prepare the hydrophobic-tagged rapamycin derivative, significantly shortening the synthesis cycle, improving preparation efficiency, and providing favorable conditions for large-scale production. Regarding application expansion, the hydrophobic-tagged degradative agent provided by this invention, due to its precise mechanism of action, can more accurately regulate mTORC1 levels in cell therapy, reducing the impact on normal cellular metabolism and improving the safety and efficacy of cell therapy. In tumor treatment research, experimental data show that the degradative agent of this invention can more significantly reduce mTORC1 protein expression levels in inhibiting tumor cell proliferation, and has lower toxicity to normal cells.

[0025] In summary, compared with previously submitted patents based on the autophagy-lysosome pathway, this invention demonstrates significant advantages in degradation mechanism and molecular structure design, exhibiting greater precision, efficiency, cost-effectiveness, and wider applicability, thus opening up new directions for the research and application of mTORC1 degrading agents. Attached Figure Description

[0026] Figure 1 These are the results of Western blot experiments on compound RAPA-LH37.

[0027] Figure 2 These are the results of Western blot experiments on compound RAPA-LH139.

[0028] Figure 3 The degradation of target proteins induced by RAPA-LH37 may involve both ubiquitin-proteasome and autophagy-lysosome pathways.

[0029] Figure 4This study examines the in vivo inhibitory effect of RAPA-LH139 on Hep3B liver cancer xenografts and the regulation of related protein expression. (A) Physical images of xenografts in nude mice after drug administration; (B) Statistical analysis of xenograft weight in the two groups, showing that the tumor weight in the RAPA-LH139-treated group was significantly lower than that in the control group (P<0.01); (C) Growth curves of xenograft volume in the two groups, showing that the tumor volume growth rate in the RAPA-LH139-treated group was significantly slower than that in the control group; (D) Weight change curves of nude mice during drug administration, showing that RAPA-LH139 had no significant effect on the weight of nude mice (P>0.05); (E) Western blot analysis results of phosphorylation levels of AKT (S473), S6K (T389), and S6 (S240 / 244) and total protein levels in tumor tissue, showing that RAPA-LH139 significantly downregulated the phosphorylation level of target proteins, while the total protein level showed no significant change. Detailed Implementation

[0030] The present invention will be described in detail below with reference to embodiments, but this does not imply any adverse limitation on the invention. The present invention has been described in detail, and specific embodiments thereof have been disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope thereof.

[0031] This invention utilizes hydrophobic tags to enhance the binding force between the degrading agent and the target protein, guiding the protein into a specific degradation pathway. Through the rational design of linkers and hydrophobic tags, selective degradation of mTORC1 is achieved. In principle, this degrading agent differs from rapamycin derivatives; by selectively degrading mTORC1, it solves the problems of poor activity and low selectivity of existing inhibitors or degrading agents. Experimental data show that this degrading agent exhibits good in vitro antitumor activity and can specifically degrade mTORC1, providing a safer and more effective candidate drug for the treatment of related diseases, and also providing a powerful tool for in-depth research on the function and regulatory mechanisms of mTORC1.

[0032] Example 1 Preparation of intermediate TBL0: (1) Preparation of Tf: Under nitrogen protection, propargyl ether (30.00 mmol) and a solution of 2,6-dimethylpyridine (42.00 mmol) were dissolved in dichloromethane (40 mL). The mixture was cooled to 0–5 °C, and then trifluoromethanesulfonic anhydride (37.40 mmol) was slowly added dropwise to the reaction system. After the addition was complete, the mixture was stirred for 3 hours while maintaining the low temperature. After the reaction was complete, the system was naturally warmed to room temperature, and saturated sodium chloride aqueous solution and pure water were added. The mixture was extracted multiple times with ethyl acetate, filtered, dried, and the solvent was removed by vacuum distillation. The concentrate was purified to obtain an orange-red oily liquid, Tf. The synthetic route is as follows:

[0033] .

[0034] (2) Preparation of intermediate TBL0: In a glove box, rapamycin (91.4 mg, 0.10 mmol), Tf0 (188.0 mg, 1.00 mmol), DIPEA (162.8 mg, 1.26 mmol), OPPh3 (55.6 mg, 0.20 mmol), 3Å molecular sieve (20 wt%), and toluene (167 µL) were placed into a 5 mL microwave tube. The reaction system was sealed and reacted in a Biotage microwave synthesizer at 65 °C for 5 min. Purification was achieved by silica gel column chromatography after cooling, using hexane:acetone = 5:1 to 1:1 as the eluent, yielding a white foamy solid, TBL0 (40.0 mg, yield 42%). The synthetic route is as follows:

[0035] .

[0036] 1 H NMR (600 MHz, DMSO- d 6) δ 6.47 (s, 1H), 6.40 (dd, J = 14.6, 11.1 Hz,1H), 6.25 – 6.20 (m, 1H), 6.16 – 6.10 (m, 2H), 5.46 (dd, J = 14.9, 9.6 Hz, 1H), 5.29 (d, J = 4.6 Hz, 1H), 5.11 – 5.07 (m, 1H), 5.00 – 4.96 (m, 1H), 4.94 (dd, J =6.1, 2.2 Hz, 1H), 4.03 – 4.00 (m, 2H), 3.95 (d, J= 4.6 Hz, 1H), 3.64 – 3.61(m, 1H), 3.60 – 3.58 (m, 2H), 3.46 – 3.42 (m, 1H), 3.34 (s, 3H), 3.28 – 3.25(m, 1H), 3.16 (s, 3H), 3.10 – 3.07 (m, 1H), 3.05 (s, 3H), 2.99 – 2.95 (m,1H), 2.77 – 2.75 (m, 1H), 2.75 – 2.70 (m, 1H), 2.41 – 2.38 (m, 1H), 2.37 –2.33 (m, 3H), 2.25 – 2.20 (m, 1H), 2.12 – 2.08 (m, 1H), 2.06 – 2.01 (m, 1H),1.96 – 1.87 (m, 3H), 1.87 – 1.80 (m, 2H), 1.77 – 1.73 (m, 3H), 1.69 – 1.66(m, 2H), 1.64 – 1.62 (m, 3H), 1.59 – 1.53 (m, 4H), 1.43 – 1.38 (m, 2H), 1.31– 1.24 (m, 3H), 1.16 – 1.11 (m, 2H), 1.07 – 1.02 (m, 3H), 0.98 (d, J = 6.4 Hz,3H), 0.95 (d, J = 6.4 Hz, 1H), 0.87 (d, J = 6.4 Hz, 3H), 0.82 (d, J = 6.4 Hz, 3H),0.77 (d, J = 6.8 Hz, 2H), 0.73 (d, J = 6.8 Hz, 3H), 0.67 – 0.61 (m, 1H). 13 C NMR(150 MHz, DMSO- d6) δ 211.0, 208.0, 199.4, 170.8, 169.7, 167.5, 139.8, 138.4,137.7, 132.8, 130.9, 127.5, 125.4, 99.5, 86.0, 83.0, 82.7, 76.2, 74.1, 72.3,72.2, 68.1, 66.7, 60.2, 57.6, 57.4, 56.0, 51.2, 45.7, 44.0, 38.7, 36.5, 35.7,35.3, 33.8, 32.7, 31.4, 30.3, 30.1, 26.9, 26.7, 25.0, 22.8, 22.1, 21.2, 20.9,20.3, 16.1, 16.0, 15.2, 14.6, 14.0, 13.8, 10.9. HRMS (ESI): m / z calcd. forC 54 H 81 NO 13 Na + ([M+Na)) + ) = 974.56001, found = 974.56353. Example 2 Preparation of intermediate TBL1: The only difference from Example 1 is the following steps: In a glove box, rapamycin (91.4 mg, 0.10 mmol), Tf1 (232.0 mg, 1.00 mmol), DIPEA (162.8 mg, 1.26 mmol), OPPh3 (55.6 mg, 0.20 mmol), 3Å molecular sieve (20 wt%), and toluene (167 µL) were placed into a 5 mL microwave tube. The reaction system was sealed and reacted in a Biotage microwave synthesizer at 50 °C for 20 min. After cooling, the mixture was purified by silica gel column chromatography using hexane:acetone = 5:1 to 1:1 as the eluent to give a white foamy solid TBL1 (61.0 mg, yield 61%).

[0037] 1 H NMR (600 MHz, DMSO- d 6) δ 6.49 – 6.44 (m, 1H), 6.42 – 6.38 (m, 1H), 6.25 – 6.20 (m, 1H), 6.17 – 6.10 (m, 2H), 5.46 (dd,J = 14.9, 9.5 Hz, 1H), 5.31– 5.26 (m, 1H), 5.09 (d, J = 10.3 Hz, 1H), 5.01 – 4.95 (m, 1H), 4.94 (dd, J =6.1, 2.2 Hz, 1H), 4.15 – 4.14 (m, 2H), 4.03 – 3.99 (m, 2H), 3.95 (d, J = 4.6Hz, 1H), 3.65 – 3.59 (m, 4H), 3.55 – 3.51 (m, 3H), 3.46 – 3.42 (m, 1H), 3.41(t, J = 2.4 Hz, 1H), 3.33 (s, 3H), 3.31 – 3.29 (m, 1H), 3.28 – 3.25 (m, 1H),3.20 – 3.17 (m, 1H), 3.16 (s, 3H), 3.09 – 3.06 (m, 1H), 3.05 (s, 3H), 2.99 –2.96 (m, 1H), 2.73 (dd, J = 17.8, 2.9 Hz, 1H), 2.42 – 2.35 (m, 2H), 2.25 – 2.20(m, 1H), 2.10 (d, J = 11.9 Hz, 1H), 2.06 – 2.01 (m, 1H), 1.96 – 1.88 (m, 3H),1.86 – 1.80 (m, 2H), 1.75 (s, 3H), 1.70 – 1.67 (m, 2H), 1.63 (s, 3H), 1.57 –1.52 (m, 4H), 1.43 – 1.38 (m, 2H), 1.26 – 1.23 (m, 2H), 1.13 – 1.11 (m, 1H),1.07 – 1.04 (m, 2H), 0.98 (d, J = 6.6 Hz, 3H), 0.96 – 0.93 (m, 2H), 0.87 (d, J =6.6 Hz, 3H), 0.82 (d, J = 6.6 Hz, 3H), 0.77 (d, J = 6.8 Hz, 2H), 0.73 (d, J= 6.8Hz, 3H), 0.67 – 0.61 (m, 1H). 13 C NMR (150 MHz, DMSO- d 6) δ 211.0, 208.1, 199.4,169.7, 167.5, 139.8, 138.4, 137.7, 132.8, 130.9, 127.5, 125.4, 99.5, 86.0,82.9, 82.7, 80.9, 77.5, 76.2, 74.1, 69.5, 69.4, 68.9, 66.7, 60.2, 57.9, 57.5,57.5, 57.4, 56.0, 51.2, 45.7, 44.0, 38.7, 36.5, 35.7, 35.3, 33.8, HRMS (ESI): m / z calcd. for C 56 H 85 NO 14 Na + ([M+Na)) + ) =1018.58623, found = 1018.59257. Example 3 Preparation of intermediate TBL2: The only difference from Example 1 is the following steps: In a glove box, rapamycin (914 mg, 1 mmol), Tf2 (276.0 mg, 1.00 mmol), DIPEA (162.8 mg, 1.26 mmol), OPPh3 (55.6 mg, 0.20 mmol), 3Å molecular sieve (20 wt%), and toluene (167 µL) were placed into a 5 mL microwave tube. The reaction mixture was sealed and reacted in a Biotage microwave synthesizer at 65 °C for 5 min. After cooling, the mixture was purified by silica gel column chromatography using hexane:acetone = 5:1 to 1:1 as the eluent to give a white foamy solid, TBL2 (64.5 mg, 62% yield).

[0038] 1 H NMR (600 MHz, DMSO- d6) δ 6.50 – 6.44 (m, 1H), 6.41 (dd, J = 14.7,11.2 Hz, 1H), 6.25 – 6.21 (m, 1H), 6.19 – 6.10 (m, 2H), 5.47 (dd, J = 14.9, 9.6Hz, 1H), 5.10 (d, J = 10.1 Hz, 1H), 5.01 – 4.96 (m, 1H), 4.95 (dd, J = 6.0, 2.3Hz, 1H), 4.14 (d, J = 2.6 Hz, 2H), 4.05 – 3.99 (m, 2H), 3.95 (d, J = 4.8 Hz, 1H),3.66 – 3.58 (m, 4H), 3.55 (s, 5H), 3.51 – 3.49 (m, 2H), 3.46 – 3.42 (m, 2H),3.34 (s, 3H), 3.33 (s, 2H), 3.30 – 3.26 (m, 2H), 3.22 – 3.19 (m, 1H), 3.16(s, 3H), 3.06 (s, 3H), 3.00 – 2.96 (m, 1H), 2.76 – 2.71 (m, 1H), 2.45 – 2.35(m, 2H), 2.27 – 2.19 (m, 1H), 2.13 – 2.09 (m, 1H), 2.06 – 2.01 (m, 1H), 1.98– 1.91 (m, 2H), 1.87 – 1.79 (m, 2H), 1.75 (s, 3H), 1.71 – 1.67 (m, 2H), 1.66– 1.63 (m, 3H), 1.58 – 1.52 (m, 4H), 1.43 – 1.38 (m, 2H), 1.29 – 1.23 (m,3H), 1.16 – 1.10 (m, 2H), 1.06 – 1.03 (m, 2H), 0.99 (d, J = 6.6 Hz, 3H), 0.95(d, J = 6.6 Hz, 1H), 0.88 (d, J = 6.6 Hz, 3H), 0.83 (d, J = 6.6 Hz, 3H), 0.78 (d,J =6.8 Hz, 2H), 0.74 (d, J = 6.8 Hz, 3H), 0.67 – 0.62 (m, 1H). 13 C NMR (150 MHz, DMSO- d 6) δ 211.0, 208.1, 199.4, 169.7, 167.5, 166.7, 139.8, 138.4, 137.7,132.8, 130.9, 127.5, 127.5, 125.4, 99.5, 86.0, 83.0, 82.9, 82.9, 82.7, 80.8,77.5, 76.2, 74.1, 70.7, 70.7, 70.0, 69.1, 69.0, 66.7, 58.0, 57.6, 57.5, 57.4,56.0, 51.3, 45.7, 44.0, 38.7, 36.5, 35.7, 35.3, 33.9, 32.7, 31.4, 30.2, 30.1,26.9, 26.7, 25.0, 22.1, 20.9, 16.1, 16.0, 15.2, 13.9, 13.9, 11.0. HRMS (ESI):m / z calcd. for C 58 H 89 NO 15 Na + ([M+Na)) + ) = 1062.61244, found = 1062.61590. Example 4 Preparation of intermediate TBL3: The only difference from Example 1 is the following steps: In a glove box, rapamycin (91.4 mg, 0.10 mmol), Tf3 (320.0 mg, 1.00 mmol), DIPEA (162.8 mg, 1.26 mmol), OPPh3 (55.6 mg, 0.20 mmol), 3Å molecular sieve (20 wt%), and toluene (167 µL) were placed into a 5 mL microwave tube. The reaction mixture was sealed and reacted in a Biotage microwave synthesizer at 65 °C for 5 min. After cooling, the mixture was purified by silica gel column chromatography using hexane:acetone = 5:1 to 1:1 as the eluent to give a white foamy solid, TBL3 (49.7 mg, 46% yield).

[0039] 1 H NMR (600 MHz, DMSO- d 6) δ 6.45 (s, 1H), 6.43 – 6.38 (m, 1H), 6.26 –6.21 (m, 1H), 6.17 – 6.10 (m, 2H), 5.47 (dd, J = 14.9, 9.6 Hz, 1H), 5.27 (d, J =9.5 Hz, 1H), 5.10 (d, J = 10.3 Hz, 1H), 5.01 – 4.96 (m, 1H), 4.95 (dd, J = 6.1,2.1 Hz, 1H), 4.15 (d, J = 2.4 Hz, 2H), 4.04 – 3.99 (m, 2H), 3.96 (d, J = 4.6 Hz,1H), 3.70 – 3.58 (m, 4H), 3.54 (t, J = 5.0 Hz, 3H), 3.47 – 3.42 (m, 2H), 3.41(t, J = 2.5 Hz, 2H), 3.34 (s, 3H), 3.33 (s, 1H), 3.31 – 3.25 (m, 2H), 3.17 (s,3H), 3.06 (s, 3H), 3.00 – 2.96 (m, 1H), 2.77 – 2.71 (m, 1H), 2.44 – 2.35 (m,2H), 2.27 – 2.19 (m, 1H), 2.11 (d, J = 11.7 Hz, 1H), 2.07 – 2.01 (m, 1H), 1.96– 1.89 (m, 2H), 1.89 – 1.81 (m, 2H), 1.75 (s, 3H), 1.73 – 1.65 (m, 3H), 1.64(s, 3H), 1.63 – 1.62 (m, 1H), 1.62 – 1.52 (m, 6H), 1.44 – 1.38 (m, 2H), 1.31– 1.23 (m, 4H), 1.18 (t, J = 7.2 Hz, 1H), 1.15 – 1.11 (m, 1H), 1.09 – 1.01 (m,4H), 0.99 (d, J= 6.6 Hz, 3H), 0.96 (d, J = 6.6 Hz, 1H), 0.88 (d, J = 6.4 Hz, 3H), 0.84 (d, J = 6.4 Hz, 3H), 0.78 (d, J = 6.8 Hz, 2H), 0.74 (d, J = 6.8 Hz, 3H), 0.67– 0.62 (m, 1H). 13 C NMR (150 MHz, DMSO- d 6) δ 211.0, 208.1, 199.4, 169.7, 167.5,166.7, 139.8, 138.4, 137.7, 132.8, 130.9, 127.5, 125.4, 99.5, 86.0, 83.0,82.9, 82.7, 80.9, 77.5, 76.2, 74.1, 69.5, 69.4, 68.9, 66.7, 60.2, 57.9, 57.5,57.4, 56.0, 51.3, 45.7, 44.0, 38.7, 36.5, 35.7, 35.3, 33.9, 32.7, 31.4, 30.2,30.1, 26.9, 26.7, 25.0, 22.1, 21.6, 21.2, 20.9, 16.6, 16.1, 16.0, 15.2, 15.0,14.6, 14.0, 13.8, 11.0, 10.7. HRMS (ESI): m / z calcd. for C 60 H 93 NO 16 Na + ([M+Na)) + )= 1106.63866, found = 1106.64572. Example 5 Preparation of compound RAPA-LH37 0-(adamantan-1-yl)-N-(2-(2-(2-(2-(4-(((2-(((2S,4R))-4-((2S)-2-((3R,6S,7E,9S,10S,12S,14R,15E,17E,19E,21R,26S,27S,3 4aR)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo-1,4,5,6,9,10,11,12,13,14,21 ,22,23,24,25,26,27,28,29,31,32,33,34,34a-tetracosahydro-3H-23,27-epoxypyrido[2,1-c][1]oxa[4]azacyclohentriaconti n-3-yl)propyl)-2-methoxycyclohexyl)oxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)acetamide (RAPA-LH37) (1) Preparation of Adama-N3: Under nitrogen protection, Adama (0.2 mmol), PEG linker (0.24 mmol), HATU (0.24 mmol), DIPEA (0.24 mmol), and DMF (1.0 mL) (or Pyrazole (0.6 mmol) and DCM (1.0 mL)) were placed in 10 mL sealed tubes equipped with stir bars. The reaction system was stirred continuously at room temperature for 18 hours. The reaction endpoint was confirmed by TLC. The mixture was concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (PE:EA = 5:1~1:1) to obtain a white solid, Adama-N3. The synthetic route is as follows:

[0040] (2) Preparation of target compound In a 10 mL sealed tube equipped with a stir bar, rapamycin derivative TBL1 (99.6 mg, 0.1 mmol), Adama-N3 (39.4 mg, 0.1 mmol), CuSO4 aqueous solution (0.15 mol, 1 mol / L), and THF (2 mL) were added, followed by NaVc aqueous solution (0.3 mol, 1 mol / L). The reaction mixture was magnetically stirred at 25 °C for 10 hours, and the reaction was confirmed to be completely terminated by TLC. The reaction was diluted with EtOAc, the organic layer was separated, and the aqueous layer was extracted three times with EtOAc. The solution was concentrated under reduced pressure, cooled, and purified by silica gel column chromatography using n-hexane:acetone = 5:1~3:1 as the eluent to give a white solid RAPA-LH37 (111.0 mg, yield 80%). The synthetic route is as follows:

[0041] .

[0042] 1 H NMR (600 MHz, DMSO- d 6) δ 8.04 (s, 1H), 7.70 (t, J = 5.6 Hz, 1H), 6.49– 6.37 (m, 2H), 6.27 – 6.06 (m, 3H), 5.76 (s, 1H), 5.51 – 5.39 (m, 1H), 5.28(dd, J = 15.1, 4.7 Hz, 1H), 5.15 – 5.01 (m, 1H), 5.00 – 4.92 (m, 2H), 4.58 –4.45 (m, 4H), 4.08 – 3.92 (m, 2H), 3.81 (t, J = 5.3 Hz, 2H), 3.68 – 3.58 (m,2H), 3.53 (ddd, J = 12.1, 8.6, 4.6 Hz, 6H), 3.48 (d, J = 2.5 Hz, 6H), 3.44 – 3.37(m, 4H), 3.34 – 3.23 (m, 3H), 3.17 (d, J = 6.6 Hz, 6H), 3.11 – 3.03 (m, 4H), 3.03 – 2.94 (m, 2H), 2.85 – 2.64 (m, 2H), 2.45 – 2.34 (m, 1H), 2.26 – 2.19(m, 1H), 2.10 (d, J= 13.2 Hz, 1H), 2.07 – 1.97 (m, 2H), 1.96 – 1.87 (m, 6H), 1.82 (d, J = 3.2 Hz, 3H), 1.75 (s, 2H), 1.68 (d, J = 30.6 Hz, 4H), 1.64 (s, 5H),1.58 (s, 5H), 1.55 (s, 6H), 1.54 (s, 3H), 1.42 (q, J = 10.8, 9.8 Hz, 2H), 1.33 – 1.22 (m, 3H), 1.19 – 1.09 (m, 1H), 1.08 – 1.01 (m, 2H), 1.01 – 0.90 (m,4H), 0.87 (d, J = 6.4 Hz, 3H), 0.83 (d, J = 6.3 Hz, 4H), 0.78 (d, J = 6.7 Hz, 2H), 0.74 (dd, J = 6.6, 2.4 Hz, 3H), 0.65 (q, J = 11.7 Hz, 1H). 13 C NMR (150 MHz, DMSO- d6)δ 210.95, 208.03, 199.42, 170.41, 169.72, 167.49, 144.36, 139.80, 138.36,137.65, 132.83, 130.95, 127.54, 127.50, 125.36, 124.64, 99.51, 85.98, 82.95,82.74, 76.23, 74.08, 70.20, 70.10, 69.89, 69.68, 69.20, 69.03, 66.68, 64.00,57.53, 57.51, 57.41, 55.96, 51.24, 50.43, 49.76, 49.07, 45.70, 43.99, 42.54, 40.53, 38.77, 38.73, 36.94, 36.48, 35.68, 35.39, 35.29, 33.84, 32.89, 32.72, 32.60, 31.40, 30.16, 30.12, 28.52, 26.92, 26.71, 24.96, 24.54, 22.13, 21.58, 20.87, 16.65, 16.08, 16.02, 15.26, 15.17, 14.95, 13.95, 13.82, 12.31, 10.95,10.64, 9.12. HRMS (ESI): m / z calcd. for C 76 H 120 N5O 18 + ([M+H)) + ) = 1390.86229, found = 1390.86341.

[0043] Example 6 Preparation of compound RAPA-LH139 N-(2-(2-(2-(2-(4-((2-(((2S,4R)-4-((2S))-2-((3R,6S,7E,9S,10S,12S,14R,15E,17E,19E,21R,26S,27S,34aR)-9,27-dihydroxy-10,21-d imethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo-1,4,5,6,9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,3 4,34a-tetracosahydro-3H-23,27-epoxypyrido[2,1-c][1]oxa[4]azacyclohentriacontin-3-yl)propyl)-2-methoxycyclohexyl)oxy)etho xy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)-2-(((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl)oxy)acetamide (RAPA-LH139) (1) Preparation of Noc-N3: Under nitrogen protection, Noc (0.2 mmol), PEG linker (0.24 mmol), HATU (0.24 mmol), DIPEA (0.24 mmol), and DMF (1.0 mL) (or Pyrazole (0.6 mmol) and DCM (1.0 mL)) were placed in 10 mL sealed tubes equipped with stir bars. The reaction system was stirred continuously at room temperature for 6 hours. The reaction endpoint was confirmed by TLC. The mixture was concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (PE:EA = 5:1~1:1) to obtain a white solid, Noc-N3. The synthetic route is as follows:

[0044] (2) Preparation of target compound In a 10 mL sealed tube equipped with a stir bar, rapamycin derivative TBL1 (199.2 mg, 0.2 mmol), Noc-N3 (41.4 mg, 0.1 mmol), CuSO4 aqueous solution (0.15 mol, 1 mol / L), and THF (2 mL) were added, followed by NaVc aqueous solution (0.3 mol, 1 mol / L). The reaction mixture was magnetically stirred at 25°C for 6 hours, and the reaction was confirmed to be completely terminated by TLC. The reaction was diluted with EtOAc, the organic layer was separated, and the aqueous layer was extracted three times with EtOAc. The solution was concentrated under reduced pressure, cooled, and purified by silica gel column chromatography using n-hexane:acetone = 10:1~3:1 as the eluent to give a white solid RAPA-LH139 (107.1 mg, yield 76%). The synthetic route is as follows:

[0045] 1 H NMR (600 MHz, DMSO- d 6) δ 8.03 (s, 1H), 7.40 (t, J = 5.9 Hz, 1H), 6.51– 6.35 (m, 2H), 6.27 – 6.17 (m, 1H), 6.17 – 6.09 (m, 2H), 5.46 (dd, J = 14.9, 9.6 Hz, 1H), 5.27 (dd, J = 16.0, 4.8 Hz, 1H), 5.09 (d, J = 10.1 Hz, 1H), 5.01 –4.90 (m, 2H), 4.54 – 4.44 (m, 4H), 4.12 – 3.96 (m, 2H), 3.97 – 3.86 (m, 2H), 3.83 – 3.76 (m, 3H), 3.68 – 3.55 (m, 3H), 3.55 – 3.50 (m, 4H), 3.48 (td, J =4.6, 2.5 Hz, 6H), 3.42 (t, J = 5.8 Hz, 2H), 3.33 (s, 4H), 3.31 (d, J = 4.2 Hz, 3H), 3.26 (ddt, J = 12.5, 9.0, 4.4 Hz, 3H), 3.22 – 3.07 (m, 6H), 3.07 – 2.93(m, 5H), 2.73 (dd,J = 17.8, 2.7 Hz, 1H), 2.43 – 2.34 (m, 2H), 2.28 – 2.05 (m,3H), 2.06 – 1.98 (m, 2H), 1.96 – 1.77 (m, 4H), 1.74 (s, 3H), 1.70 – 1.61 (m,6H), 1.60 – 1.48 (m,6H), 1.44 – 1.35 (m, 1H), 1.35 – 1.17 (m, 6H), 1.16 –1.00 (m, 3H), 0.99 – 0.91 (m, 5H), 0.88 (s, 2H), 0.86 (dd, J = 7.2, 2.5 Hz,6H), 0.84 – 0.79 (m, 4H), 0.79 – 0.74 (m, 3H), 0.73 (t, J = 3.4 Hz, 6H), 0.64(q, J = 11.7 Hz, 1H). 13 C NMR (150 MHz, DMSO- d6)δ 210.96, 208.04, 199.42, 170.51,169.72, 167.49, 166.64, 144.36, 139.80, 138.36, 137.65, 132.83, 130.95,127.54, 125.37, 124.63, 99.51, 85.98, 82.95, 82.74, 79.83, 76.23, 74.08,70.20, 70.12, 70.01, 69.88, 69.37, 69.20, 69.03, 68.12, 66.68, 64.00, 58.04, 57.53, 57.41, 55.96, 55.02, 51.24, 49.76, 49.07, 47.92, 45.70, 43.98, 40.52, 39.01, 38.73, 38.41, 36.48, 35.68, 35.29, 34.49, 33.84, 32.72, 31.39, 31.32, 30.16, 30.12, 26.92, 26.71, 25.67, 24.95, 23.23, 22.65, 22.13, 21.58, 21.34,20.87, 16.65, 16.53, 16.08, 16.02, 15.17, 13.95, 13.82, 10.95. HRMS (ESI): m / z calcd. for C 76 H 124 N5O 19 + ([M+H)) + ) = 1410.88850, found = 1410.89145.

[0046] Example 7 Preparation of compound RAPA-LH136 N-(2-(2-(2-(2-(4-((2-(((2S,4R)-4-((2S))-2-((3R,6S,7E,9S,10S,12S,14R,15E,17E,19E,21R,26S,27S,34aR)-9,27-dihydroxy-10 ,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo-1,4,5,6,9,10,11,12,13,14,21,22,23,24,25,26,27,28,29 ,31,32,33,34,34a-tetracosahydro-3H-23,27-epoxypyrido[2,1-c][1]oxa[4]azacyclohentriacontin-3-yl)propyl)-2-methoxycy clohexyl)oxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)bicyclo[2.2.1]hept-5-ene-2-carboxamide (RAPA-LH136) (1) Preparation of Norborn-N3: Under nitrogen protection, Norborn (0.2 mmol), PEG linker (0.4 mmol), HATU (0.24 mmol), DIPEA (0.24 mmol), and DMF (1.0 mL) (or Pyrazole (0.6 mmol) and DCM (1.0 mL)) were placed in 10 mL sealed tubes equipped with stir bars. The reaction system was stirred continuously at room temperature for 12 hours. The reaction endpoint was confirmed by TLC. The mixture was concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (PE:EA = 5:1~1:1) to obtain a white solid, Norborn-N3. The synthetic route is as follows:

[0047] (2) Preparation of target compound In a 10 mL sealed tube equipped with a stir bar, rapamycin derivative TBL1 (49.8 mg, 0.1 mmol), Norborn-N3 (33.8 mg, 0.1 mmol), CuSO4 aqueous solution (0.15 mol, 1 mol / L), and THF (2 mL) were added, followed by NaVc aqueous solution (0.3 mol, 1 mol / L). The reaction mixture was magnetically stirred at 25°C for 18 hours, and the reaction was confirmed to be completely terminated by TLC. The reaction was diluted with EtOAc, the organic layer was separated, and the aqueous layer was extracted three times with EtOAc. The solution was concentrated under reduced pressure, cooled, and purified by silica gel column chromatography using n-hexane:acetone = 5:1 to 1:1 as the eluent to give a white solid RAPA-LH136 (64.0 mg, yield 48%). The synthetic route is as follows:

[0048] 1 H NMR (600 MHz, DMSO- d 6) δ 8.03 (s, 1H), 7.60 (t, J = 5.7 Hz, 1H), 6.51– 6.35 (m, 2H), 6.22 (dd, J = 14.4, 10.4 Hz, 1H), 6.17 – 6.06 (m, 4H), 5.83 –5.74 (m, 1H), 4.54 – 4.46 (m, 4H), 4.30 (d, J = 5.0 Hz, 1H), 4.01 (q, J = 4.2, 3.3 Hz, 2H), 3.95 (d, J = 4.5 Hz, 1H), 3.80 (t, J = 5.2 Hz, 2H), 3.67 – 3.58 (m,3H), 3.56 – 3.53 (m, 2H), 3.53 – 3.51 (m, 2H), 3.51 – 3.49 (m, 4H), 3.48 (d, J = 3.2 Hz, 2H), 3.47 (d, J = 5.3 Hz, 5H), 3.46 – 3.41 (m, 3H), 3.39 (t, J = 6.4Hz, 1H), 3.35 (d, J= 6.4 Hz, 2H), 3.33 (s, 3H), 3.32 (s, 4H), 3.31 (s, 1H),3.21 – 3.16 (m, 2H), 3.15 (s, 3H), 3.12 (q, J = 3.4 Hz, 3H), 3.05 (s, 3H), 2.83– 2.73 (m, 4H), 2.72 – 2.57 (m, 2H), 1.83 (dd, J = 12.5, 8.7 Hz, 2H), 1.74 (s,3H), 1.64 – 1.61 (m, 4H), 1.49 – 1.37 (m, 4H), 1.35 – 1.20 (m, 8H), 1.13(dddd, J = 15.0, 12.9, 9.2, 3.9 Hz, 3H), 0.98 (d, J = 6.5 Hz, 3H), 0.87 (d, J = 6.4Hz, 3H), 0.85 – 0.80 (m, 4H), 0.77 (d, J = 6.7 Hz, 2H), 0.73 (dd, J = 6.7, 2.3Hz, 3H). 13C NMR (151 MHz, DMSO) δ 213.03, 210.94, 208.80, 208.03, 199.40,199.08, 180.69, 175.16, 173.35, 169.71, 169.44, 169.32, 167.48, 166.63,144.35, 139.79, 138.35, 138.30, 138.17, 137.64, 137.24, 136.76, 135.90,132.83, 132.64, 130.94, 127.53, 127.50, 127.17, 125.34, 124.64, 99.51, 99.33,85.97, 85.02, 83.65, 82.94, 82.90, 82.72, 76.33, 76.23, 74.08, 71.71, 70.34,70.22, 70.18, 70.09, 70.07, 70.05, 70.01, 69.88, 69.85, 69.60, 69.58, 69.19,69.02, 66.97, 66.91, 66.67, 63.99, 58.04, 57.52, 57.50, 57.44, 57.40, 56.51,55.96, 55.39, 52.81, 51.84, 51.25, 49.82, 49.76, 47.38, 46.31, 46.14, 46.08,46.06, 45.69, 45.47, 43.99, 43.72, 43.35, 42.56, 41.47, 41.44, 39.09, 39.00,38.73, 38.56, 36.66, 36.47, 35.67, 35.39, 35.29, 34.43, 33.84, 32.89, 32.81,32.71, 31.39, 30.25, 30.16, 30.12, 28.83, 28.21, 26.92, 26.71, 24.96, 22.13,21.57, 20.87, 17.28, 16.66, 16.08, 16.04, 16.02, 15.26, 15.18, 14.95, 14.08,13.97, 13.81, 12.31, 10.95, 10.65. HRMS (ESI): m / z calcd. for C 72 H 112 N5O 18+ ([M+H)) + ) = 1334.79969, found = 1334.80392.

[0049] Example 8 Preparation of compound RAPA-LH130 2-(adamantan-1-yl)-N-(2-(2-(2-(2-(4-((((2S,4R)-4-((2S)-2-((3R,6S,7E,9S,10S,12S,14R,15E,17E,19E,21R,26S,27S,34 aR)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo-1,4,5,6,9,10,11,12,13,14, 21,22,23,24,25,26,27,28,29,31,32,33,34,34a-tetracosahydro-3H-23,27-epoxypyrido[2,1-c][1]oxa[4]azacyclohentria contin-3-yl)propyl)-2-methoxycyclohexyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)acetamide (RAPA-LH130) (1) Preparation of Adama-N3: Under nitrogen protection, Adama (0.2 mmol), PEG linker (0.24 mmol), HATU (0.24 mmol), DIPEA (0.24 mmol), and DMF (1.0 mL) (or Pyrazole (0.6 mmol) and DCM (1.0 mL)) were placed in 10 mL sealed tubes equipped with stir bars. The reaction system was stirred continuously at room temperature for 18 hours. The reaction endpoint was confirmed by TLC. The mixture was concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (PE:EA = 5:1~1:1) to obtain a white solid, Adama-N3. The synthetic route is as follows:

[0050] (2) Preparation of target compound In a 10 mL sealed tube equipped with a stir bar, rapamycin derivative TBL0 (99.6 mg, 0.1 mmol), Adama-N3 (39.4 mg, 0.1 mmol), CuSO4 aqueous solution (0.15 mol, 1 mol / L), and THF (2 mL) were added, followed by NaVc aqueous solution (0.3 mol, 1 mol / L). The reaction mixture was magnetically stirred at 25 °C for 10 hours, and the reaction was confirmed to be completely terminated by TLC. The reaction was diluted with EtOAc, the organic layer was separated, and the aqueous layer was extracted three times with EtOAc. The solution was concentrated under reduced pressure, cooled, and purified by silica gel column chromatography using hexane:acetone = 5:1~3:1 as the eluent to give a white solid RAPA-LH130 (55.0 mg, yield 41%). The synthetic route is as follows:

[0051] .

[0052] HRMS (ESI): m / z calcd. for C 74 H 116 N5O 17 + ([M+H)) + ) = 1346.83607, found =1346.83598.

[0053] Example 9 Preparation of compound RAPA-LH131 2-(adamantan-1-yl)-N-(2-(2-(2-(2-(4-((2-(2-(((2S,4R))-4-((2S)-2-((3R,6S,7E,9S,10S,12S,14R,15E,17E,19E,21R,26S,27S,3 4aR)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo-1,4,5,6,9,10,11,12,13,14,21,2 2,23,24,25,26,27,28,29,31,32,33,34,34a-tetracosahydro-3H-23,27-epoxypyrido[2,1-c][1]oxa[4]azacyclohentriacontin-3- yl)propyl)-2-methoxycyclohexyl)oxy)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)acetamide (RAPA-LH131) (1) Preparation of Adama-N3: Under nitrogen protection, Adama (0.2 mmol), PEG linker (0.24 mmol), HATU (0.24 mmol), DIPEA (0.24 mmol), and DMF (1.0 mL) (or Pyrazole (0.6 mmol) and DCM (1.0 mL)) were placed in 10 mL sealed tubes equipped with stir bars. The reaction system was stirred continuously at room temperature for 18 hours. The reaction endpoint was confirmed by TLC. The mixture was concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (PE:EA = 5:1~1:1) to obtain a white solid, Adama-N3. The synthetic route is as follows:

[0054] (2) Preparation of target compound In a 10 mL sealed tube equipped with a stir bar, rapamycin derivative TBL2 (99.6 mg, 0.1 mmol), Adama-N3 (39.4 mg, 0.1 mmol), CuSO4 aqueous solution (0.15 mol, 1 mol / L), and THF (2 mL) were added, followed by NaVc aqueous solution (0.3 mol, 1 mol / L). The reaction mixture was magnetically stirred at 25 °C for 10 hours, and the reaction was confirmed to be completely terminated by TLC. The reaction was diluted with EtOAc, the organic layer was separated, and the aqueous layer was extracted three times with EtOAc. The solution was concentrated under reduced pressure, cooled, and purified by silica gel column chromatography using hexane:acetone = 5:1~3:1 as the eluent to give a white solid RAPA-LH131 (61.0 mg, yield 43%). The synthetic route is as follows:

[0055] .

[0056] HRMS (ESI): m / z calcd. for C 78 H 124 N5O 19 + ([M+H)) + ) = 1334.88850, found =1334.88695.

[0057] Example 10 Preparation of compound RAPA-LH132 2-(adamantan-1-yl)-N-(2-(2-(2-(2-(4-((2-(2-(2-(((2S,4R))-4-((2S)-2-((3R,6S,7E,9S,10S,12S,14R,15E,17E,19E,21R,26S,27S, 34aR)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo-1,4,5,6,9,10,11,12,13,14,21,22, 23,24,25,26,27,28,29,31,32,33,34,34a-tetracosahydro-3H-23,27-epoxypyrido[2,1-c][1]oxa[4]azacyclohentriacontin-3-yl)pr opyl)-2-methoxycyclohexyl)oxy)ethoxy)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)acetamide (RAPA-LH132) (1) Preparation of Adama-N3: Under nitrogen protection, Adama (0.2 mmol), PEG linker (0.24 mmol), HATU (0.24 mmol), DIPEA (0.24 mmol), and DMF (1.0 mL) (or Pyrazole (0.6 mmol) and DCM (1.0 mL)) were placed in 10 mL sealed tubes equipped with stir bars. The reaction system was stirred continuously at room temperature for 18 hours. The reaction endpoint was confirmed by TLC. The mixture was concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (PE:EA = 5:1~1:1) to obtain a white solid, Adama-N3. The synthetic route is as follows:

[0058] (2) Preparation of target compound In a 10 mL sealed tube equipped with a stir bar, rapamycin derivative TBL3 (99.6 mg, 0.1 mmol), Adama-N3 (39.4 mg, 0.1 mmol), CuSO4 aqueous solution (0.15 mol, 1 mol / L), and THF (2 mL) were added, followed by NaVc aqueous solution (0.3 mol, 1 mol / L). The reaction mixture was magnetically stirred at 25 °C for 10 hours, and the reaction was confirmed to be completely terminated by TLC. The reaction was diluted with EtOAc, the organic layer was separated, and the aqueous layer was extracted three times with EtOAc. The solution was concentrated under reduced pressure, cooled, and purified by silica gel column chromatography using hexane:acetone = 5:1~3:1 as the eluent to give a white solid RAPA-LH132 (50.0 mg, yield 34%). The synthetic route is as follows:

[0059] .

[0060] HRMS (ESI): m / z calcd. for C 80 H 128 N5O 20 + ([M+H)) + ) = 1478.91472, found =11478.91503.

[0061] Since the compounds with the structural formula shown in Formula I provided by this invention can all achieve the expected effects of this invention, the following description only uses the compounds provided in Examples 5 and 6 as examples to illustrate the effects.

[0062] Experimental Example 1 In vitro anti-tumor cell proliferation assay 1. Method HCT-116 cells (human colorectal cancer); PC-3 cells (human prostate cancer); MCF-7 cells (human breast cancer); RCC4 cells (human clear cell renal carcinoma); U6 cells (human glioblastoma cells); T47D cells (human epithelioid breast cancer cells); NCI-H266 cells (human non-small cell lung cancer); HeLa cells (human cervical cancer); 786-O cells (human clear cell renal carcinoma); HT1080 cells (human fibrosarcoma); Hep3B cells (human liver cancer); and HepG2 cells (human liver cancer) were selected and cultured in DMEM or 1640 medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2. Cells were seeded into black 96-well plates, 3000 cells per well (100 μL), and placed in a CO2 cell incubator. After overnight cell adhesion, different concentrations of the compound (1 μL) were added, with six replicates for each concentration, and corresponding DMSO control wells were included. After 72 h of compound treatment, 100 μL of ATP assay kit (CellTiter-Glo Luminescent CellViability Assay kit) was added, and the plates were shaken for 1 h. Luminescence was detected using Analyst AD, and the IC50 was obtained based on the fluorescence value compared to the blank control using Prism statistical analysis. 50 The values ​​are listed in a table.

[0063] 2. Results Table 1 and Supplementary Table 1 show the half-maximal inhibitory concentrations (IC50) of rapamycin and its derivatives RAPA-LH37 and RAPA-LH139 against 12 tumor cell types (HCT-116, PC-3, MCF-7, RCC4, U6, T47D, NCI-H266, HeLa, 786-O, HT1080, Hep3B, and HepG2). 50 )difference.

[0064] Table 1 shows the in vitro antiproliferative activity (IC50) of the compounds against various tumor cells. 50 ) In the table: 'a' represents IC 50 <2 μmol / L; b indicates 2 μmol / L <IC 50 <10 μmol / L; c indicates 10 μmol / L <IC 50 <50 μmol / L.

[0065] Supplementary Table 1 shows the in vitro antiproliferative activity (IC50) of the compounds against various tumor cells. 50 ) In the table: 'a' represents IC 50 <2 μmol / L; b indicates 2 μmol / L <IC 50 <10 μmol / L; c indicates 10 μmol / L <IC 50 <50 μmol / L.

[0066] The results showed that all three compounds exhibited strong inhibitory activity against HCT-116 and MCF-7 cells (IC50). 50 <2 μmol / L); its inhibitory activity against PC-3 cells was slightly weaker (2 μmol / L). <IC 50 <10 μmol / L). Furthermore, RAPA-LH37 showed superior inhibitory activity against HeLa, 786-O, and HT1080 cells compared to rapamycin and RAPA-LH139, while RAPA-LH139 exhibited superior inhibitory activity against NCI-H266 cells compared to rapamycin and RAPA-LH37. In other cell types, the inhibitory activity of all three compounds was weak (10 μmol / L). <IC 50 <50 μmol / L).

[0067] Experimental Example 2 Western blot results 1. Method Human breast cancer cells (MCF-7) were seeded into six-well plates. After adhesion, the cells were treated with different concentrations of target compounds or positive control agents for 48 h. 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 tubes were centrifuged at 12,000 rpm for 20 min in a centrifuge pre-cooled to 4°C, and 75 μL of supernatant was gently aspirated. 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 denatured protein samples. An 8% separating gel and a 5% stacking gel were prepared according to the manufacturer's instructions (the comb was inserted immediately after gel loading, and the gel was allowed to stand for 30 min). Electrophoresis buffer was added to the electrophoresis apparatus, the comb was slowly removed, and then the denatured protein sample or protein marker was added to the gel wells at a rate of 10 μg protein per well. Power on and perform electrophoresis at 90 V for 2 h. After electrophoresis, cut the corresponding molecular weight bands according to the protein marker instructions and stack them together with the methanol-excited PVDF membrane in the transfer clamp. Transfer at 400 mA for 30 min. Place the PVDF membrane with transferred proteins in a pre-prepared 5% skim milk powder solution (prepared with PBST, 5 mL) and incubate at room temperature with shaking for 1 h. Aspirate the blocking solution and wash the bands twice with PBST, shaking for 2 min each time. Aspirate the PBST and add a solution of diluted primary antibody in BSA (5%, prepared with PBST), and incubate at 4°C for 13–16 h. Aspirate and recover the primary antibody and wash the bands three times with TBST, shaking for 5 min each time. Aspirate the TBST and add secondary antibody diluted with TBST, shaking at room temperature for 1 h. Discard the secondary antibody and wash the bands three times with PBST, shaking for 5 min each time. Remove the bands, spread them evenly with the prepared ECL chemiluminescence solution, expose them in a gel imaging system, save the images, and analyze the obtained images.

[0068] 2. Results 2.1 Selective Degradation of mTORC1 The results showed that ( Figure 1 and Figure 2 The mTOR, Raptor, and FKBP12 components of the mTORC1 complex were degraded in a dose-dependent manner within 48 hours of treatment with RAPA-LH37 and RAPA-LH139 on MCF-7 cells. In contrast, Rictor in mTORC2 was unaffected. At higher concentrations, the degradation of mTOR protein was not as significant as that of Raptor and FKBP12, suggesting that RAPA-LH37 selectively targets mTORC1 rather than mTORC2.

[0069] 2.2 Verification of the degradation mechanism Pretreatment with MG-132 (2 μmol / L) and chloroquine (10 μmol / L) significantly inhibited RAPA-LH37-induced degradation of the target protein, indicating that ( Figure 3 The degradation of target proteins induced by RAPA-LH37 may involve both ubiquitin-proteasome and autophagy-lysosome pathways.

[0070] Experimental Example 3 In vivo anti-liver cancer cell trial 1. Method (1) Experimental materials Animals: SPF-grade BALB / c nude mice (female, 4-6 weeks old, weighing 18-22 g), purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. They were housed in a specific pathogen-free environment (temperature 22-25℃, humidity 50%-60%, 12-hour light / 12-hour dark cycle), with free access to food and water. After one week of acclimatization, they were used in experiments.

[0071] Cell line: Hep3B human liver cancer cells, purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0072] Reagents: RAPA-LH139 (purity ≥98%, prepared according to the method in Example 6); physiological saline (pharmaceutical grade); DMSO (analytical grade, purchased from Sinopharm Group); Tween 80 (pharmaceutical grade, purchased from Solarbio); hematoxylin-eosin (HE) staining kit (purchased from Beyotime Biotechnology Co., Ltd.); AKT (S473), S6K (T389), S6 (S240 / 244) phosphorylation antibodies and total protein antibodies (purchased from Cell Signaling Technology); Western blot reagents (purchased from Thermo Fisher Scientific).

[0073] (2) Establishment of animal models Hep3B cells in logarithmic growth phase were harvested and their concentration adjusted to 5 × 10⁶ cells / year using serum-free DMEM medium. 6 Cells / mL. 0.2 mL of cell suspension (containing 1×10⁶ cells / mL) was subcutaneously injected into the right back of nude mice. 6 A human liver cancer subcutaneous xenograft model was established using 100-150 mm³ cells. The mice were observed daily after inoculation for their mental state, diet, and tumor growth. Tumor volume was measured using calipers. Once the tumor volume reached 100-150 mm³ (approximately 14 days after inoculation), the mice were randomly divided into two groups of seven mice each, ensuring no statistically significant difference in initial tumor volume between the two groups (P>0.05).

[0074] (3) Grouping and Dosing Regimen Control group: Intraperitoneal injection of physiological saline containing 5% DMSO + 5% Tween 80, 0.2 mL / animal, once daily for 2 weeks; RAPA-LH139 administration group: RAPA-LH139 solution (dissolved in physiological saline containing 5% DMSO + 5% Tween 80) was administered intraperitoneally at a dose of 10 mg / kg, 0.2 mL / animal, once daily for 2 weeks.

[0075] (4) Detection indicators and statistical methods General condition observation: Record the mental state, diet, water intake and defecation of nude mice daily, and weigh the nude mice every other day.

[0076] Tumor growth monitoring: The long diameter (L) and short diameter (W) of the tumor were measured with calipers every 3 days. The tumor volume was calculated according to the formula V=(L×W²) / 2, and the tumor growth curve was plotted. After the administration was completed, nude mice were euthanized by cervical dislocation, the tumor tissue was dissected, photographed and weighed, and the tumor inhibition rate was calculated [tumor inhibition rate = (average tumor weight of control group - average tumor weight of administration group) / average tumor weight of control group × 100%].

[0077] Western blot analysis: Fresh tumor tissue was collected, and RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors was added. The tissue was ground and lysed on ice, and centrifuged at 12,000 rpm for 20 min at 4 °C. The supernatant was collected, and the protein concentration was determined by BCA method. 50 μg of total protein was subjected to SDS-PAGE electrophoresis, transferred to a PVDF membrane, blocked, and then primary antibody (AKT (S473), S6K (T389), S6 (S240 / 244) phosphorylation antibody and total protein antibody, diluted 1:1000) was added. The membrane was incubated overnight at 4 °C, washed with TBST, and then fluorescent secondary antibody (diluted 1:5000) was added. The membrane was incubated at room temperature for 1 h, developed with ECL chemiluminescence solution, and the band gray values ​​were analyzed using a gel imaging system.

[0078] Statistical analysis: Data were processed using GraphPad Prism 8.0 software. Quantitative data were expressed as mean ± standard deviation (x ± s). The t-test was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0079] 2. Results Depend on Figure 4 It can be known that: Tumor growth inhibition effect: Two weeks after administration, the tumor weight and volume in the RAPA-LH139 administration group were significantly lower than those in the control group (P<0.01), indicating that RAPA-LH139 can significantly inhibit the growth of Hep3B liver cancer xenografts in vivo.

[0080] Weight changes in nude mice: Throughout the entire administration period, there was no significant difference in weight between the control group and the RAPA-LH139 administration group (P>0.05), and the nude mice maintained normal mental state, diet, and water intake, with no obvious toxic side effects, indicating that RAPA-LH139 has good safety at a dose of 10 mg / kg.

[0081] Protein expression regulation: Western blot results showed that, compared with the control group, the phosphorylation level of S6 (S240 / 244) in tumor tissue of the RAPA-LH139 administration group was significantly reduced (P<0.01), while the total protein level did not change significantly (P>0.05), indicating that RAPA-LH139 exerts its anti-tumor effect by inhibiting the phosphorylation activation of downstream signaling pathways of mTORC1.

[0082] Those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims and their equivalents, this invention is also intended to include these modifications and variations.

Claims

1. A selective mTORC1 degrader containing a hydrophobic tag, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, prodrug, polymorph, eutectic, or deuterated thereof, characterized in that, The structural formula of the degradation agent is shown in Formula I: ； Linker1 is selected from any of the following structures or combinations thereof: saturated cycloalkanes, saturated nitrogen heterocycles, spirocyclic structures, C1-C8 straight-chain or branched alkyl groups, chain structures containing heteroatoms, aromatic rings or heteroaromatic ring linking units; the number of main chain atoms of Linker1 is 3 to 20, and the main chain atoms refer to the C, O, N, and S atoms that constitute the linking chain backbone; Linker2 is selected from any of the following structures or combinations thereof: saturated cycloalkanes, saturated nitrogen heterocycles, spirocyclic structures, C1-C6 straight-chain or branched alkyl groups, short-chain structures containing heteroatoms, monoaromatic rings or monoheteroaromatic ring linkers; the number of main chain atoms in Linker2 is 3 to 12, and the definition of main chain atoms is the same as that in Linker1; The hydrophobic label is selected from any of the following structures or their derivatives, stereoisomers, or equivalents, wherein the derivatives are hydroxyl-substituted, alkyl-substituted, ester-substituted, ether-substituted, amino-substituted, or halogen-substituted: Cage-like hydrocarbons, polycyclic aromatic hydrocarbons, steroids, terpenes, monocyclic or bicyclic aromatic hydrocarbon derivatives, long-chain alkyl or cycloalkyl, arylalkyl, heterocyclic hydrophobic units, perfluoroalkyl-substituted aromatic hydrocarbons, adamantane-aromatic couplings, or steroid-terpene couplings.

2. The mTORC1 selective degrader containing a hydrophobic tag according to claim 1, or its pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, prodrug, polymorph, eutectic, or deuterated derivative, is characterized in that, In Linker1: The saturated cycloalkane is any one of cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, and cyclooctane; The saturated nitrogen heterocycle is any one of piperidine, pyrrolidine, morpholine, piperazine, and hexahydroacetane; The spirocyclic structure is any one of spiro[3.3]heptane, spiro[3.4]octane, spiro[4.4]nonane, and spiro[4.5]decane; The heteroatom-containing chain structure is a C2-C10 chain structure containing ether bonds, amine bonds, amide bonds, ester bonds, urea bonds, thioether bonds, and sulfonamide bonds; the aromatic ring or heteroaromatic ring connecting unit is a benzene ring, pyridine ring, pyrimidine ring, pyrazine ring, pyridazine ring, furan ring, or thiophene ring, and the aromatic ring or heteroaromatic ring is mono-substituted, poly-substituted, or unsubstituted by C1-C3 alkyl, halogen, hydroxyl, or C1-C3 alkoxy groups; In Linker2: The saturated cycloalkane is any one of cyclopropane, cyclobutane, cyclopentane, cyclohexane, and cycloheptane; The saturated nitrogen heterocycle is any one of piperidine, pyrrolidine, morpholine, and piperazine; The spirocyclic structure is spiro[3.3]heptane or spiro[3.4]octane; The short-chain structure containing heteroatoms is a C2-C8 chain structure containing ether bonds, amine bonds, amide bonds, ester bonds, and urea bonds; The monoaromatic ring or mono-heteroaromatic ring linking unit is any one of benzene ring, pyridine ring, and furan ring, and the aromatic ring or heteroaromatic ring is monosubstituted or unsubstituted by any one of C1-C2 alkyl group or halogen. Hydrophobic label: The cage-like hydrocarbons are adamantane, adamantane methyl, adamantane ethyl, adamantane alcohol, adamantane carboxylic acid, diadamantane, cubane, cubane methanol, norbornane, norbornene, norbornane carboxylic acid, tetracyclic [4.4.0.1², 5 .1 7 ,¹ 0 Any one of dodecane, adamantaneamine, and methyladamantane; The polycyclic aromatic hydrocarbons are any one of naphthalene, α-methylnaphthalene, β-hydroxynaphthalene, anthracene, phenanthrene, pyrene, fluoranthene, α-methylfluorene, 9-phenylfluorene, 9-fluorenylmethanol, biphenyl, 2-methylbiphenyl, 3-methoxybiphenyl, terphenyl, p-tert-butylbiphenyl, o-xylylbiphenyl, acenaphthene, fluorenone, and dibenzothane. The steroids are any one of cholesterol, cholesterol, sitosterol, stigmasterol, lanosterol, ergosterol, pregnane, androstenol, estradiol, campesterol, stigmasterol, cholesterol acetate, cholesterol benzoate, pregnanetriol, and methylcholesterol; The terpenoid is any one of monoterpenes, sesquiterpenes, diterpenes, triterpenes, or terpene polymers: The monocyclic or bicyclic aromatic derivative is selected from any one of cumene, cumene, p-isopropyltoluene, isopropylnaphthalene, tert-butylnaphthalene, diphenylmethane, diphenyl ethane, triphenylmethane, triphenylmethanol, diphenyl ether, diphenyl sulfide, o-chlorobiphenyl, and p-bromobiphenyl. The long-chain alkyl or cycloalkyl group is selected from any one of C12-C24 straight-chain alkyl, branched alkyl, cycloalkyl, long-chain alkanol, and long-chain fatty acid ester; The arylalkyl group is selected from any one of benzenehexane, benzeneoctane, benzenedecane, naphthalenebutane, naphthalenehexane, phenylcyclohexane, benzylcyclohexane, and phenethylcyclohexane; The heterocyclic hydrophobic unit is selected from any one of dibenzofuran, dibenzothiophene, carbazole, N-methylcarbazole, phenothiazine, phenoxazine, phenanthrene, indolocarbazole, dithiophenebenzene, thiophenefluorene, benzodioxane, phenazine, methylphenothiazine, and chlorophenoxazine. The perfluoroalkyl-substituted aromatic hydrocarbon is selected from pentafluorophenylcyclohexane or perfluorooctylbenzene; The adamantane-aromatic coupling compound is selected from adamantane-phenylmethane; The steroid-terpene conjugate is selected from cholesterol-based geraniol.

3. The mTORC1 selective degrader containing a hydrophobic tag according to claim 2, or its pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, prodrug, polymorph, eutectic, or deuterated derivative, is characterized in that, In Linker1, the heteroatom-containing chain structure is any one of ethylene glycol ether chain, propylene diamine chain, acetamide chain, acetate chain, urea chain, ethyl sulfide chain, and benzenesulfonamide chain; In Linker2, the short chain structure containing heteroatoms is any one of diethylene glycol ether chain, ethylenediamine chain, propionamide chain, propionate ester chain, and methylurea chain; The monoterpenes are selected from any one of geraniol, citronellol, limonene, terpineol, camphor, menthol, borneol, and isoborneol; The sesquiterpenes are selected from any one of farnesol, farnesol, myrrhene, and caryophyllene; The diterpenoid is selected from any one of the side chain structures of phytol, rosin acid, and taxol; The triterpenoids are selected from oleanolic acid or ursolic acid; The terpene polymer is selected from squalene or polyisoprene oligomers; In the Hydrophobic label, the C12-C24 straight-chain alkyl group is selected from any one of dodecyl, tetradecyl, hexadecyl, octadecyl, and eicosyl; the branched alkyl group is selected from any one of isododecyl, isostearyl, and 2-hexyldecyl; the cycloalkyl group is selected from any one of cyclododecane, cyclotetradecane, cyclohexadecane, cyclooctadecane, cycloeicosane, methylcyclododecane, and ethylcyclohexadecane; the long-chain alkanol is selected from any one of dodecanol and octadecanol; and the long-chain fatty acid ester is selected from methyl stearate or ethyl palmitate.

4. The mTORC1 selective degrader containing a hydrophobic tag according to claim 1, or its pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, prodrug, polymorph, eutectic, or deuterated derivative, characterized in that, The structure of the degrading agent is shown in any of the following: 。 5. A pharmaceutical composition, characterized in that, The active ingredient comprises a hydrophobic-labeled mTORC1 selective degrader as described in any one of claims 1 to 4, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, prodrug, polymorph, eutectic, or deuterated product thereof, wherein the active ingredient comprises 0.01% to 99.99% by mass.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition also includes one or more pharmaceutically acceptable excipients.

7. A method for preparing the mTORC1 selective degrader containing a hydrophobic tag as described in claims 1-4, characterized in that, Includes the following steps: (1) Preparation of rapamycin derivatives: Compound 1-1 was reacted with trifluoromethanesulfonic anhydride as a starting material to undergo a nucleophilic substitution reaction to obtain compound 1-2; ； Nucleophilic substitution reaction was carried out using compounds 1-2 and rapamycin as raw materials to obtain rapamycin derivatives 1-3; ； (2) Preparation of hydrophobic tag-linker intermediate: Acylation reaction was carried out using compounds 1-4-1 and 1-4-2 as raw materials to obtain compound 1-4; ； (3) Preparation of the target degrading agent: The degradation agent 1-5 is obtained by performing a Click reaction using the rapamycin derivatives 1-3 and the compounds 1-4 as raw materials. 。 8. The preparation method according to claim 7, characterized in that, The reaction temperature of compounds 1-2 is 0~5℃, and the reaction time is 1~3 h; The preparation of compounds 1-3 was carried out by microwave heating at a temperature of 50-80℃ for a reaction time of 3-20 min. The acylation reaction was carried out at room temperature for 6–18 h. The Click reaction was carried out at room temperature for 6–18 h. The molar ratio of compound 1-1 to trifluoromethanesulfonic anhydride is 30:35~40; The molar ratio of compounds 1-2 to rapamycin is 1:0.1~1; The molar ratio of compound 1-4-1 to compound 1-4-2 is 1:1~2; The molar ratio of the rapamycin derivative to compounds 1-4 is 1:0.5~2.

9. The use of the mTORC1 selective degrader containing a hydrophobic tag as described in any one of claims 1 to 4, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, prodrug, polymorph, eutectic, or deuterated product thereof, or the pharmaceutical composition of claim 5, in the preparation of an antitumor drug.

10. The application according to claim 9, characterized in that, The tumors mentioned are colorectal cancer, breast cancer, lung cancer, prostate cancer, cervical cancer, kidney cancer, liver cancer, stomach cancer, pancreatic cancer, ovarian cancer, melanoma, lymphoma, leukemia, or thyroid cancer.

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