Mtor protein targeting degradation chimera and preparation method and application thereof

Abstract

This invention discloses an mTOR protein-targeting degradation chimera, its preparation method, and its applications. Based on the mTOR agonist MHY1485 and the E3 ubiquitin ligase CRBN protein ligand, this invention develops a novel mTOR protein-targeting degradation chimera. The chimera can bind to the mTOR protein and induce effective degradation, significantly downregulating mTOR protein levels. In the HeLa cell line, PD-M6 molecules exhibited the highest mTOR degradation rate, reaching 90%, providing a novel drug form for mTOR inhibition.

Description

(I) Technical Field

[0001] This invention belongs to the pharmaceutical field, and specifically relates to a mammalian target of rapamycin (mTOR) targeted degradation chimera, its preparation method, and its application in the degradation of mTOR in cancer cells. (II) Background Technology

[0002] Mammalian target of rapamycin (mTOR) is an atypical serine / threonine kinase that regulates cell proliferation, autophagy, and apoptosis through participation in multiple internal signaling pathways. As a signaling pathway closely related to cell growth and survival, mTOR plays a crucial role in various diseases, including cancer, arthritis, insulin resistance, and osteoporosis. In tumors, the mTOR signaling pathway is often overactivated, inducing tumor cell growth, migration, and invasion of healthy tissues, thus becoming an important target for anti-tumor therapy. In recent years, several novel mTOR inhibitors have entered clinical trials, and their combined use with other drugs has demonstrated high activity. The development of novel mTOR inhibitors continues to provide new options for cancer treatment, aiming to more effectively inhibit tumor growth and improve patient prognosis.

[0003] Protein-targeting chimeras (PROTACs) represent a novel strategy for drug development that induces the degradation of a target protein by simultaneously binding to both the target protein and an E3 ubiquitin ligase. Unlike traditional inhibitors, PROTACs do not require inhibition of the target protein's activity; instead, they provide binding activity to induce protein degradation via a ubiquitin-protease system. This event-driven pharmacological mode of action, controlling protein fate through degradation, offers a new avenue for drug development. This interaction-based technology can be used to target various protein types, including transcription factors, enzymes, and regulatory proteins, demonstrating significant potential for treating a wide range of diseases.

[0004] While current small-molecule inhibitors of mTOR can suppress its activity, they are less effective at reducing intracellular mTOR expression or preventing drug resistance. MHY1485 is an effective, cell-permeable mTOR agonist that also effectively inhibits autophagy; it can act as a ligand for mTOR and bind to it. However, whether a small-molecule inhibitor of mTOR can be prepared using the agonist MHY1485 through a protein-targeted degradation chimera remains unknown.

[0005] Therefore, this invention designs a class of PROTAC small molecule inhibitors based on the agonist MHY1485, which can utilize the intracellular ubiquitin-proteasome system to reduce intracellular mTOR expression, providing a new therapeutic strategy for solving the problem of mTOR in cancer treatment. (III) Summary of the Invention

[0006] The purpose of this invention is to provide an mTOR protein-targeting degradation chimera, its preparation method, and its applications. This invention utilizes a series of linking arms to connect the mTOR agonist MHY1485 and the E3 ubiquitin ligase CRBN ligand to obtain chimeras (PROTACs) capable of targeting and degrading the mTOR protein. These bifunctional small molecules possess a unique structure, with one end binding to mTOR and the other end recognizing and linking to the E3 ligase. Through the action of these chimeras, the intracellular ubiquitin-proteasome system can be utilized to guide mTOR into the intracellular proteasome degradation pathway, thereby reducing the intracellular mTOR expression level. This addresses the problem of incomplete mTOR inhibition by existing inhibitors, while also exhibiting low dosage and low toxicity, overcoming the potential toxicity and drug resistance issues of existing small molecule inhibitors. Therefore, the mTOR protein-targeting degradation chimera of this invention provides new possibilities and hope for the treatment of diseases related to mTOR activity and expression levels, demonstrating broad clinical application prospects.

[0007] The technical solution adopted in this invention is:

[0008] This invention provides an mTOR protein-targeted degradation chimera (i.e., a PD-M molecule) as shown in formula (Ⅰ).

[0009]

[0010] In formula (Ⅰ), R is any chemically feasible linking structure, preferably a C2-C11 straight-chain alkyl or alkoxy group, more preferably ethane, hexane, 3,6-dioxaoctane, or 3,6,9-trioxaundecanane.

[0011] Furthermore, the preferred mTOR protein-targeting degradation chimera shown in Formula (I) is one of the following:

[0012]

[0013]

[0014] This invention also provides a method for preparing the mTOR protein-targeted degradation chimera, the method comprising the following steps:

[0015] (1) Synthesis of the compound shown in formula (4): The compound shown in formula (3) was added to N,N-dimethylformamide (DMF), and then the compound shown in formula (9) was added dropwise. Then, N,N-diisopropylethylamine (DIEA) was slowly added dropwise. The mixture was stirred at 90°C for 6 hours and then cooled to 25°C. It was diluted with dichloromethane, washed once with water and once with saturated sodium chloride aqueous solution, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain a concentrate. It was dissolved in a small amount of ethyl acetate, and then methyl tert-butyl ether was added to precipitate a white solid. The solid was filtered, and the filter cake was dried under vacuum at 25°C. The white powder was collected to obtain the compound shown in formula (4). The compound shown in (4) is designated as intermediate 4; the molar ratio of the compound shown in formula (3) to the compound shown in formula (9) is 1:1-2 (preferably 1:1.5); the molar ratio of the compound shown in formula (3) to N,N-diisopropylethylamine is 1:1-3 (preferably 1:2); the volumetric amount of N,N-dimethylformamide used is 2-5 mL / mmol (preferably 3 mL / mmol) based on the amount of the compound shown in formula (3); the volumetric amount of dichloromethane used for dilution is 50-60 mL / mmol (preferably 57 mL / mmol) based on the amount of the compound shown in formula (3).

[0016] (2) Synthesis of the compound shown in formula (5): Intermediate 4 was added to a 1,4-dioxane solution with a hydrochloric acid concentration of 4.0 mol / L. The mixture was stirred at room temperature for 1.5 hours, and then methyl tert-butyl ether was added. A yellow solid was precipitated. The solid was filtered, and the filter cake was washed with diethyl ether and dried under vacuum at 25 °C to obtain the compound shown in formula (5), which is denoted as intermediate 5. The volume of the 1,4-dioxane solution used was 0.6-2.5 mL / mmol (preferably 0.8 mL / mmol) based on the amount of intermediate 4. The amount of diethyl ether used was 50-100 mL / mmol (preferably 80 mL / mmol) based on the amount of intermediate 4.

[0017] (3) Synthesis of the compound shown in formula (8): Sodium acetate (NaOAc) was added to acetic acid, followed by the compounds shown in formula (6) and (7). The mixture was stirred at 90°C for 8 hours and then cooled to room temperature. It was diluted with dichloromethane, washed once with water and once with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to dryness to obtain a white solid product, which is the compound shown in formula (8), denoted as intermediate 8. The compound shown in formula (6) and formula (7) The molar ratio of the compound shown is 1:1-2 (preferably 1:1.1); the molar ratio of the compound shown in formula (6) to sodium acetate is 1:1-5 (preferably 1:3); the volume of acetic acid used is 2-5 mL / mmol (preferably 2.5 mL / mmol) based on the amount of the compound shown in formula (6); the volume of dichloromethane used for dilution is 20-50 mL / mmol (preferably 25 mL / mmol) based on the amount of the compound shown in formula (6).

[0018] (4) Synthesis of the mTOR protein-targeted degradation chimera shown in formula (I): Intermediate 5 was dissolved in N,N-dimethylformamide, and the compound shown in formula (8) was added. After mixing, N,N-diisopropylethylamine (DIEA) was slowly added dropwise. The mixture was stirred at 90°C for 8 h and then cooled to 25°C. The reaction solution was diluted with ethyl acetate, washed once with water and once with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The concentrate was dissolved in dichloromethane:methanol = 8:1 (volume ratio) and then subjected to silica gel column chromatography. A dichloromethane / methanol mixture with a volume ratio of 30:1 was used as the eluent at a flow rate of 0.5 mL / s for 3 column volumes. Thin-layer chromatography was performed using a dichloromethane:methanol mixture with a volume ratio of 20:1 as the developing solvent. The eluent with an Rf value of 0.25-0.45 was collected and dried at 25°C. The mTOR protein-targeted degradation chimera shown in formula (I) was obtained; the molar ratio of intermediate 5 to intermediate 8 was 1:1-2 (preferably 1:1.1); the molar ratio of intermediate 5 to N,N-diisopropylethylamine was 1:1-5 (preferably 1:3); the volumetric amount of N,N-dimethylformamide was 10-15 mL / mmol (preferably 12.5 mL / mmol) based on the amount of intermediate 5; the volumetric amount of ethyl acetate was 50-100 mL / mmol (preferably 93 mL / mmol) based on the amount of intermediate 5.

[0019]

[0020] In formula (9), R is a C2-C11 straight-chain alkyl or alkoxy group, more preferably ethane, hexane, 3,6-dioxaoctane, or 3,6,9-trioxaundecanane; R in formulas (I), (4), and (5) is the same as R in formula (9).

[0021] Preferably, the compound shown in formula (3) is prepared by the following steps: cyanuric chloride shown in formula (1) is dissolved in tetrahydrofuran, piperazine shown in formula (2) is added under ice bath conditions, and N,N-diisopropylethylamine (DIEA) is slowly added dropwise. The mixture is stirred at room temperature for 3 hours. The reaction solution is diluted with ethyl acetate, washed once with water and once with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and the filtrate is evaporated to dryness by rotary evaporation to obtain the compound 4,4-(6-chloro-1,3,5-triazine) shown in formula (3). -2,4-dimethyl)dimorpholine, denoted as intermediate 3; the molar ratio of cyanuric chloride to piperazine is 1:2; the molar ratio of cyanuric chloride to N,N-diisopropylethylamine is 1:1-2 (preferably 1:1.5); the volumetric amount of tetrahydrofuran is 1-3 mL / mmol (preferably 1.5 mL / mmol) based on the amount of cyanuric chloride; the volumetric amount of ethyl acetate is 5-10 mL / mmol (preferably 7 mL / mmol) based on the amount of cyanuric chloride.

[0022]

[0023] The synthetic route for the mTOR protein-targeted degradation chimera shown in formula (I) of this invention is as follows:

[0024]

[0025] The present invention also provides the application of the mTOR protein-targeting degradation chimera in the preparation of a targeted mTOR degradation formulation, wherein the formulation can effectively reduce the expression of mTOR protein in cells.

[0026] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0027] This invention develops a novel mTOR protein-targeting degradation chimera based on the mTOR agonist MHY1485 and the E3 ubiquitin ligase CRBN protein ligand. This protein-targeting degradation chimera can bind to mTOR protein and induce effective degradation, significantly downregulating mTOR protein levels. In the HeLa cell line, PD-M6 molecules showed the highest mTOR degradation rate, reaching 90% (DC). 50 (PD-M2>10μM; PD-M4>10μM; PD-M6=4.8μM; PD-M2P>10μM; PD-M1P>10μM), providing new drug forms for mTOR inhibition. (iv) Description of the attached drawings

[0028] Figure 1 Intermediates and products of PDM molecules 1 H NMR spectra and13 C NMR spectrum; a represents intermediate 3. 1 HNMR spectrum; b is intermediate 4a 1 1H NMR spectrum, c represents intermediate 4a 13 C10 NMR spectrum; d represents intermediate 5a. 1 HNMR spectrum, e represents intermediate 5a 13 C10 NMR spectrum; f represents compound 8. 1 1H NMR spectrum; g is PD-M2 1 H NMR spectrum;

[0029] h is intermediate 4b 1 1H NMR spectrum; i represents intermediate 5b. 1 1H NMR spectrum; j represents PD-M4. 1 1H NMR spectrum; k represents intermediate 4c. 1 1H NMR spectrum, l represents intermediate 4C 13 C10 NMR spectrum; m represents intermediate 5C. 1 1H NMR spectrum; n is PD-M6 1 1H NMR spectrum; o represents intermediate 4d. 1 1H NMR spectrum; p represents the intermediate 4d 13 C10 NMR spectrum; q represents the 5d intermediate. 1 HNMR spectrum, r represents the intermediate at 5d. 13 C NMR spectrum; s represents PD-M1P 1 1H NMR spectrum; t represents intermediate 4e. 1 1H NMR spectrum, u represents intermediate 4e 13 C10 NMR spectrum; v represents intermediate 5e. 1 1H NMR spectrum, w represents intermediate 5e 13 C NMR spectrum; x represents PD-M2P 1 H NMR spectrum.

[0030] Figure 2 Immunoblot patterns of mTOR protein degradation in human cervical cancer cells HeLa by PD-M molecules. The mTOR activator MHY-1485 and the E3 ubiquitin ligand Pomalidomide (Poma) served as the control group, while PD-M2, PD-M4, PD-M6, PD-M1P, and PD-M2P served as the sample treatment groups.

[0031] Figure 3Immunoblot maps of the degradation of mTOR protein in human cervical cancer cells HeLa by PD-M molecules (PD-M2, PD-M4, PD-M6, PD-M1P, and PD-M2P). The concentration gradients of PD-M molecules in the experiment were set from left to right as follows: 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 5 μM, and 10 μM. GAPDH represents the abundance of intracellular actin; the mTOR band represents the abundance of intracellular mTOR protein. (V) Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0033] The technical and scientific terms used in the following embodiments have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains. The raw materials and reagents were obtained commercially and all had a purity of 97% or higher.

[0034] The following examples use QF-254 indicator for thin-layer chromatography (TLC) analysis on Huanghai thin-layer chromatography silica gel plates under ultraviolet spectroscopy. Rapid column chromatography separation is performed using silica gel 60 (silica gel, 200-300 mesh). 1 HNMR and 13 C10 NMR spectra were recorded on a Bruker 500MHz NMR spectrometer. The mTOR activator MHY-1485 (CAS No. 326914-06-1) was purchased from Aladdin, catalog number N303452. The E3 ubiquitin ligand ligand Pomalidomide was purchased from Anengji Chemical, catalog number B010937. Antibodies used for Western blotting (WB) were from the following suppliers: rabbit anti-mTOR (ET1608-5, Huaan Biotechnology), HRP-conjugated goat rabbit anti-IgG (H+L) secondary antibody (#31460, HRP), and HRP-conjugated mouse anti-GAPDH (HRP-60004, Proteintech). Immunoblotting results were recorded using an Invitrogeni Bright 1500 microscope. Cell fluorescence images were recorded using a Leica SP8 confocal microscope.

[0035] The room temperature described in this invention is 25-30℃.

[0036] The compound represented by formula (8) was prepared according to the procedure described in the literature (Guo WH, Qi X, Yu X, et al. Enhancing intracellular accumulation and target engagement of PROTACs with reversible covalent chemistry. Nat Commun. 2020; 11(1):4268).

[0037] Example 1: Synthetic route of PD-M2

[0038]

[0039] 1. Synthesis of intermediate 3

[0040]

[0041] The cyanuric chloride (1000 mg, 5.43 mmol) shown in formula (1) was dissolved in tetrahydrofuran (THF) (8 mL). Piperazine (954 mg, 11 mmol) shown in formula (2) was then added under ice bath conditions. N,N-diisopropylethylamine (DIEA) (1053 mg, 8.15 mmol) was slowly added dropwise. The mixture was stirred at room temperature for 3 h. The reaction solution was diluted with ethyl acetate (40 mL), washed once with water, then once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was rotary evaporated to dryness to obtain 1245 mg of the product (80% yield), which is the compound 4,4-(6-chloro-1,3,5-triazine-2,4-diyl)dimorpholine shown in formula (3), denoted as intermediate 3. Intermediate 3... 1 The H NMR spectrum is shown in [reference]. Figure 1 As shown in Figure a.

[0042] 1 H NMR (400MHz, Chloroform-d) δ3.80 (s, 8H), 3.75-3.70 (m, 8H). 13 C NMR(101MHz,Chloroform-d)δ169.70,164.48,66.55,43.86.MS calculated for C 13 H9FN2O4[M+H] + 285.10, found 285.98.

[0043] 2. Synthesis of intermediate 4a

[0044]

[0045] Intermediate 3 (200 mg, 0.70 mmol) prepared in step 1 was dissolved in 2 mL of N,N-dimethylformamide (DMF), followed by dropwise addition of N-Boc-ethylenediamine 9-1 (173 mg, 1.08 mmol), and then slowly dropwise addition of DIEA (192 mg, 1.49 mmol). The mixture was stirred at 90 °C for 6 h, and then cooled to 25 °C. The reaction solution was diluted with 40 mL of dichloromethane, washed once with water, then once with saturated sodium chloride aqueous solution, and dried over anhydrous sodium sulfate. The solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain a concentrate. The concentrate was dissolved in a small amount of ethyl acetate, and then methyl tert-butyl ether was added, precipitating a white solid. The solid was filtered, and the filter cake was dried under vacuum at 25 °C for 2 h to obtain 249 mg (87% yield) of white powder, which was intermediate 4a, and could be used directly in the next reaction without purification. 1 The H NMR spectrum is shown in [reference]. Figure 1 As shown in Figure b, intermediate 4a 13 The C NMR spectrum is shown below. Figure 1 As shown in c.

[0046] 1 H NMR (400MHz, Chloroform-d) δ5.52(s,1H),5.23(s,1H),3.71(d,J=9.7Hz,16H),3.47(q,J=5.6Hz,2H),3.36-3.21(m,2H),1.41(s,9H). 13 C NMR(101MHz,Chloroform-d)δ166.64,165.08,156.15,79.14,66.82,43.58,41.94,40.52,28.44.MS calculated forC 18 H 31 N7O4[M+H] + 409.24, found 409.34.

[0047] 3. Synthesis of intermediate 5a

[0048]

[0049] Intermediate 4a (100 mg, 0.24 mmol) prepared in step 2 was added to 200 μL of a 4.0 mol / L hydrochloric acid solution containing 1,4-dioxane. The mixture was stirred at room temperature for 1.5 hours, and then 20 mL of methyl tert-butyl ether was added, precipitating a yellow solid. The solid was filtered, washed with 20 mL of diethyl ether, and finally dried under vacuum at 25 °C to obtain 252 mg (85% yield) of a yellow solid, which was intermediate 5a. This intermediate could be used directly in the next reaction without purification.1 The H NMR spectrum is shown in [reference]. Figure 1 As shown in d, intermediate 5a 13 The C NMR spectrum is shown below. Figure 1 As shown in e.

[0050] 1 H NMR (500MHz, Methanol-d4) δ3.99-3.73 (m, 18H), 3.25 (t, J = 5.7Hz, 2H). 13 CNMR(126MHz,Methanol-d4)δ161.15,153.73,66.18,65.66,44.73,44.54,38.65,37.99.MScalculated for C 13 H 23 N7O2[M+H] + 309.19, found 309.23.

[0051] 13C NMR (126MHz, Methanol-d4) δ161.15,156.52,153.73,66.18,65.66,44.73,44.54,38.73,38.65,37.99.

[0052] 4. Preparation of Compound 8

[0053] Sodium acetate (NaOAc) (2.6 g, 27.0 mmol) was added to acetic acid (20 mL), followed by 3-amino-2,6-piperidinidone 6 (1.5 mg, 9.0 mmol) and 3-fluorophthalic anhydride 7 (1.5 mg, 10.0 mmol). The mixture was stirred at 90 °C for 8 h, then cooled to room temperature. The mixture was diluted with dichloromethane (200 mL), washed once with water, then once with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was rotary evaporated to dryness to give 2.1 g of a white solid product (yield 84%), which was compound 8. 1 The H NMR spectrum is shown in [reference]. Figure 1 As shown in f.

[0054] 1H NMR (600MHz, DMSO-d6) δ11.16(s,1H),7.95(td,J=8.2,4.4Hz,1H),7.80(d,J=7.3Hz,1H),7.74(t,J=8.9Hz,1H),5.17(dd,J=13.0,5.4Hz ,1H),2.90(ddd,J=17.3,14.0,5.5Hz,1H),2.64-2.59(m,1H),2.60-2.53(dd,J=13.0,4.4Hz,1H),2.07(dtd,J=12.9,5.4,2.3Hz,1H).MS calculated for C13H9FN2O4[M+H]+277.05, found 277.06.

[0055] 5. Synthesis of compound PD-M2

[0056]

[0057] The intermediate 5a (50 mg, 0.16 mmol) prepared in step 3 was dissolved in N,N-dimethylformamide (DMF) (2 mL), then compound 8 (50 mg, 0.18 mmol) was added, mixed well, and then DIEA (118 mg, 0.92 mmol) was slowly added dropwise. The mixture was stirred at 90℃ for 8 hours and then cooled to 25℃. The reaction solution was diluted with ethyl acetate (15 mL), washed once with water, then once with a saturated sodium chloride aqueous solution, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The concentrate was dissolved in a solvent of dichloromethane:methanol = 8:1 (v / v) and purified by silica gel column chromatography (silica gel column height 10 cm, diameter 305 mm, silica gel 200-300 mesh). A dichloromethane / methanol mixture of 30:1 (v / v) was used as the eluent at a flow rate of 0.5 mL / s for 3 column volumes. Thin-layer chromatography was performed using a dichloromethane:methanol mixture of 20:1 (v / v) as the developing solvent. The eluent with an Rf value of 0.25-0.45 was collected and dried at 25℃ to obtain 34 mg (yield 37%) of yellow solid, denoted as PD-M2. 1 The H NMR spectrum is shown in [reference]. Figure 1 As shown in g.

[0058] 1H NMR(500MHz,Chloroform-d)δ9.21(s,1H),7.46(t,J=7.4Hz,1H),7.09(d,J=6.7Hz,1H),6.92(d,J=8.2Hz,1H),6.43( s,1H),5.56(s,1H),5.07-4.73(m,1H),3.67(d,J=31.8Hz,18H),3.53-3.45(m,2H),2.92-2.72(m,3H),2.09(s,1H).MS calculated forC26H31N9O6[M+H]+565.24,found 565.36.

[0059] Example 2: Synthesis route of PD-M4

[0060]

[0061] 1. Synthesis of intermediate 4b

[0062] In step 2 of Example 1, N-Boc-ethylenediamine was replaced with N-BOC-1,4-butanediamine 9-2 (30 mg, 0.16 mmol), and all other operations were the same, yielding 46 mg of intermediate 4b. 1 The H NMR spectrum is shown in [reference]. Figure 1 As shown in h.

[0063] 1 H NMR(400MHz,Chloroform-d)δ4.90(s,1H),4.61(s,1H),3.72(d,J=9.4Hz,16H),3.38 (q,J=6.3Hz,2H),3.14(d,J=5.8Hz,2H),1.56(dt,J=14.7,6.1Hz,4H),1.44(s,9H).MS calculated for C 20 H 35 N7O4[M+H] + 437.28, found 443.34.

[0064] 2. Synthesis of intermediate 5b

[0065] By replacing intermediate 4a in step 3 of Example 1 with intermediate 4b, and performing the same other operations, 45 mg of intermediate 5b was obtained. 1 The H NMR spectrum is shown in [reference]. Figure 1 As shown in i.

[0066] 1H NMR(400MHz, DMSO-d6)δ8.05(s,3H),3.80-3.63(m,16H),3.42-3.36(m,2H),2.79(q,J=6.2Hz,2H),1.68-1.53(m,4H).MS calculated for C 15 H 27 N7O2[M+H] + 337.22, found 337.34.

[0067] 3. Synthesis of PDM4

[0068] In step 4 of Example 1, intermediate 5a was replaced with intermediate 5b, and all other operations were the same, yielding 8 mg of product, denoted as PD-M4. 1 The H NMR spectrum is shown in [reference]. Figure 1 As shown in j.

[0069] 1 H NMR (400MHz, DMSO-d6) δ11.10(s,1H),7.56(dd,J=8.4,7.2Hz,1H),7.05(dd,J=21 .3,7.8Hz,2H),6.85(t,J=5.8Hz,1H),6.54(t,J=5.9Hz,1H),5.05(dd,J=12.8,5. 4Hz,1H),3.59(d,J=17.9Hz,16H),3.32-3.25(m,4H),2.89(ddd,J=17.5,14.5,5. 4Hz,1H),2.63-2.53(m,2H),2.03(dtd,J=12.8,6.0,5.5,3.0Hz,1H),1.57(s,4H). 13 CNMR(101MHz,DMSO-d6)δ165.25,136.72,117.67,110.85,66.50,49.00,43.65,42.05,40.40,31.45,27.04,26.60,22.63.MS calculated for C 28 H 35 N9O6[M+H] + 593.27, found 593.54.

[0070] Example 3: Synthetic route of PD-M6

[0071]

[0072] 1. Synthesis of intermediate 4C

[0073] In step 2 of Example 1, N-Boc-ethylenediamine was replaced with N-BOC-1,6-hexanediamine 9-3 (30 mg, 0.14 mmol), and all other operations were the same, yielding 46 mg of intermediate 4c. 1 The H NMR spectrum is shown in [reference]. Figure 1 As shown in k, 13 The C NMR spectrum is shown below. Figure 1 As shown in Figure 1.

[0074] 1 H NMR(500MHz,Chloroform-d)δ8.19(s,1H),7.92(s,3H),3.74(dd,J=50.2,20.3Hz,17H),3.35(s,2H),2.96(s,2H),1.73(s,2H),1.56(s,2H),1.36(s,4H). 13 C NMR(101MHz,Chloroform-d)δ166.18,165.20,156.00,78.97,66.84,43.56,40.51,30.00,29.72,28.43,26.59,26.50.MS calculated for C 22 H 39 N7O4[M+H] + 465.31 found 465.54.

[0075] 13 C NMR (101MHz, Chloroform-d) δ166.18,165.20,156.00,78.97,66.84,43.56,40.51,30.00,29.72,28.43,26.59,26.50.

[0076] 2. Synthesis of intermediate 5c

[0077] By replacing intermediate 4a in step 3 of Example 1 with intermediate 4c, and performing the same other operations, 45 mg of intermediate 5c was obtained. 1 The H NMR spectrum is shown in [reference]. Figure 1 As shown in m.

[0078] 1 H NMR(500MHz,Chloroform-d)δ8.19(s,1H),7.92(s,3H),3.74(dd,J=50.2,20.3Hz,17H),3.35(s,2H),2.96(s,2H),1.73(s,2H),1.56(s,2H),1.36(s,4H). 13C NMR(126MHz,Chloroform-d)δ161.25,155.79,153.70,66.55,66.45,66.09,44.42,40.75,39.83,28.28,27.05,25.98,25.88.MS calculated for C 17 H 31 N7O2[M+H] + 365.25, found 365.43.

[0079] 3. Synthesis of PD-6

[0080] In step 4 of Example 1, intermediate 5a was replaced with intermediate 5c, and all other operations were the same to obtain 10 mg of product, denoted as PD-M6. 1 The H NMR spectrum is shown in [reference]. Figure 1 As shown in the figure.

[0081] 1 H NMR(500MHz,Chloroform-d)δ8.33(s,1H),7.50(t,J=7.5Hz,1H),7.10(d,J=6.8Hz,1H),6.88(d,J=8.3Hz,1H),6.23(s,1H),4.96-4.89(m,1 H),3.73(d,J=16.1Hz,16H),3.42-3.24(m,4H),2.95-2.70(m,3H),2.19-2.11(m,1H),1.72-1.65(m,2H),1.62-1.56(m,2H),1.44(s,4H).MS calculated for C 30 H 39 N9O6[M+H] + 621.30, found 621.54.

[0082] Example 4: Synthetic route of PD-M1P

[0083]

[0084] 1. Synthesis of intermediate 4d

[0085] In step 2 of Example 1, N-Boc-ethylenediamine was replaced with N-Boc-1,8-diamino-3,6-dioxane 9-4 (30 mg, 0.2 mmol), and all other procedures were the same, yielding 66 mg of the intermediate over 4 days. 1 The H NMR spectrum is shown in [reference]. Figure 1 As shown in o, 13 The C NMR spectrum is shown below. Figure 1 As shown in p.

[0086] 1 H NMR (400MHz, Chloroform-d) δ5.27 (d, J = 16.7Hz, 2H), 3.83-3.64 (m, 16H), 3.64-3.50 (m, 10H), 3.37-3.26 (m, 2H), 1.43 (s, 9H). 13 C NMR(101MHz,Chloroform-d)δ165.21,70.27,70.18,70.14,66.85,43.57,40.40,40.35,28.41.MS calculated forC 22 H 39 N7O6[M+H] + 497.30, found 497.45.

[0087] 13 C NMR (101MHz, Chloroform-d) δ166.14,165.21,156.03,79.19,70.27,70.18,70.14,66.85,43.57,40.40,40.35,28.41.

[0088] 2. Synthesis of intermediate 5d

[0089] Replacing intermediate 4a in step 3 of Example 1 with intermediate 4d, and performing the same other operations, yielded 65mg of intermediate 5d. 1 The H NMR spectrum is shown in [reference]. Figure 1 As shown in q. 13 The C NMR spectrum is shown below. Figure 1 As shown in r.

[0090] 1 H NMR (400MHz, Chloroform-d) δ8.30 (s, 1H), 8.12 (s, 2H), 4.10-3.98 (m, 2H), 3.87-3.60 (m, 24H), 3.19 (s, 2H), 1.96 (d, J = 4.0Hz, 1H), 1.21-1.13 (m, 1H). 13 C NMR(101MHz,Chloroform-d)δ171.14,161.13,155.97,153.65,70.37,70.07,68.68,66.71,66.54,66.46,66.08,60.34,44.43,40.77,39.76.MS calculated for C 17H 31 N7O4[M+H] + 397.24, found 397.43.

[0091] 13 C NMR(101MHz,Chloroform-d)δ171.14,161.13,155.97,153.65,70.37,70.07, 68.68,66.71,66.54,66.46,66.08,60.34,44.43,40.77,39.76,21.02,14.14.

[0092] 3. Synthesis of PD-M1P

[0093] In step 4 of Example 1, intermediate 5a was replaced with intermediate 5d, and all other operations were the same, yielding 13 mg of product, denoted as PD-M1P. 1 The H NMR spectrum is shown in [reference]. Figure 1 As shown in the middle s.

[0094] 1 H NMR(500MHz,Chloroform-d)δ8.69(s,1H),7.49(t,J=7.8Hz,1H),7.10(d,J=7.1Hz,1H),6.92(d,J=8.5Hz,1H),6.50(t,J=5.3Hz,1H),4.92(dd,J=12.2, 5.3Hz,1H),3.75-3.68(m,21H),3.63(dd,J=5.3,2.9Hz,2H),3.58(dt,J=9.7 ,4.3Hz,4H),3.48(q,J=5.2Hz,2H),2.92-2.68(m,3H),2.16-2.08(m,1H).MS calculated for C 30 H 39 N9O8[M+H] + 653.29, found 653.87.

[0095] Example 5: Synthetic route of PD-M2P

[0096]

[0097] 1. Synthesis of intermediate 4e

[0098] In step 2 of Example 1, N-Boc-ethylenediamine was replaced with N-Boc-1,11-diamino-3,6,9-trioxaundecan 9-5 (30 mg, 0.1 mmol), and all other procedures were the same to obtain 35 mg of intermediate 4e. 1The H NMR spectrum is shown in [reference]. Figure 1 As shown in t, 13 See the CNMR spectrum. Figure 1 As shown in the figure.

[0099] 1 H NMR (400MHz, Chloroform-d) δ5.25 (d, J = 36.4Hz, 2H), 3.80-3.66 (m, 16H), 3.66-3.49 (m, 14H), 3.38-3.23 (m, 2H), 1.42 (s, 9H). 13 C NMR(101MHz,Chloroform-d)δ166.14,165.21,156.03,70.27,70.18,70.14,66.85,43.57,40.40,40.35,28.41.MScalculated for C 24 H 43 N7O7[M+H] + 541.32, found 541.37.

[0100] 13 C NMR (101MHz, Chloroform-d) δ166.14,165.21,156.03,79.19,70.27,70.18,70.14,66.85,43.57,40.40,40.35,28.41.

[0101] 2. Synthesis of intermediate 5e

[0102] By replacing intermediate 4a in step 3 of Example 1 with intermediate 4e, and performing the same other operations, 34 mg of intermediate 5e was obtained. 1 The H NMR spectrum is shown in [reference]. Figure 1 As shown in v, 13 The C NMR spectrum is shown below. Figure 1 As shown in the middle w.

[0103] 1 H NMR (400MHz, Chloroform-d) δ12.48 (s, 1H), 8.30 (d, J = 28.7Hz, 3H), 3.88-3.56 (m, 29H), 3.34 (d, J = 6.5Hz, 2H), 3.15 (s, 2H). 13C NMR(101MHz,Chloroform-d)δ161.16,155.98,153.68,70.31,70.11,70.05,69.78,68.97,66.49,66.46,66.41,66.05,44.41,40.65,39.74.MS calculated for C 19 H 35 N7O5[M+H] + 441.27, found 441.45.

[0104] 13 C NMR (101MHz, Chloroform-d) δ161.16,155.98,153.68,70.31,70.11,70.05,69.78,68.97,66.49,66.46,66.41,66.05,44.41,40.65,39.74.

[0105] 3. Synthesis of PD-M2P

[0106] In step 4 of Example 1, intermediate 5a was replaced with intermediate 5e, and all other operations were the same, yielding 13 mg of product, denoted as PD-M2P. 1 The H NMR spectrum is shown in [reference]. Figure 1 As shown in the figure.

[0107] 1 H NMR(500MHz,Chloroform-d)δ8.85(s,1H),7.53–7.47(m,1H),7.10(d,J=7.1Hz,1H),6.91(d,J=8.5Hz,1H),6.54(t,J=5.3Hz,1H),4.92(dd,J=12.2,5.3 Hz,1H),3.77–3.65(m,24H),3.62–3.55(m,4H),3.48(t,J=5.2Hz,2H),2.76( dddd,J=48.8,29.2,14.4,3.8Hz,3H),2.10(dddd,J=12.6,5.8,3.7Hz,1H).MS calculated for C 32 H 43 N9O9[M+H] + 697.32, found 697.45.

[0108] Example 6: Verification of the degradation of intracellular mTOR by PD-M molecules

[0109] Cell culture medium: high-glucose DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin.

[0110] Phosphate buffer: pH 7.4, 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.76mM KH2PO4.

[0111] Immunoblot: HeLa cells (2×10⁻⁶) were used to bleach the cells. 5 Cells were seeded into 12-well Titan plates containing 1 mL of cell culture medium and cultured at 37°C for 24 h. After the cells reached 70% confluence, the original culture medium was discarded, and the wells were divided into 8 groups. Group 1 was added with 500 μL of cell culture medium containing 1% DMSO; Group 2 was added with 500 μL of cell culture medium containing 10 μM mTOR activator MHY-1485; Group 3 was added with 500 μL of cell culture medium containing 10 μM E3 ubiquitin ligand Poma; Groups 4-8 were each added with 500 μL of cell culture medium containing the same concentration (10 μM) of different PD-M molecules (PD-M2, PD-M4, PD-M6, PD-M1P, PD-M2P). After incubation at 37°C for 24 hours, the culture medium was discarded, and the cells in each well were washed twice with phosphate-buffered saline (PBS). The washings were discarded, and 80 μL of PBS containing 0.2% DMSO was added to each well. Cells were lysed in phosphate-buffered saline (PBPS) with Triton-X and 100 μM benzyl sulfonyl fluoride (PMSF) at room temperature. Cells were scraped off with a spatula and placed in a 1.5 mL EP tube. 20 μL of 5×SDS loading buffer was added to the EP tube, and the mixture was heated at 95 °C for 10 min. The sample was separated by 12% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 3% skim milk (in TBST buffer) at room temperature for 1.5 h. The membrane was then cut at approximately 40 kDa. The portion >40 kDa was incubated overnight at 4 °C with rabbit anti-mTOR (1:5000 dilution), followed by incubation with HRP-conjugated goat anti-rabbit IgG (1:7500 dilution) at room temperature for 1 h. The PVDF membrane <40 kDa was incubated overnight at 4 °C with HRP-conjugated mouse anti-GAPDH (1:7500 dilution). Western blots were recorded using an Invitrogen iBright 1500.

[0112] Immunoblotting results as follows Figure 2Using GAPDH as an internal control protein, the results showed that the PD-M molecules in this invention selectively degraded mTOR protein, with PD-M6 exhibiting the best degradation effect. At a concentration of 10 μM, approximately 90% of the mTOR protein level was successfully degraded, while the MHY-1485 treatment group alone did not show any effect on mTOR degradation. Other PROTAC molecules with different linker chains also showed varying degrees of mTOR degradation, with degradation efficiencies of 35% for PD-M2, 8% for PD-M4, and 37% for PD-M1P. No significant degradation of PD-M2P was observed under the above experimental conditions.

[0113] Example 7: Degradation of intracellular mTOR by PD-M series PROTACs at different concentrations

[0114] HeLa cells were loaded at 1.5 x 10⁻⁶. 5 Cells were seeded at a density of 500 μL of cell culture medium (same as in Example 5) in glass culture dishes. After overnight adhesion, the original culture medium was discarded, and 500 μL of cell culture medium containing different concentrations (0 μM, 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 5 μM, 10 μM) of PD-M molecules (PD-M2, PD-M4, PD-M6, PD-M1P, PD-M2P) was added to each well. The cells were incubated at 37°C for 24 hours. Subsequent procedures were the same as in Example 6. Blots were recorded using an Invitrogen iBright 1500.

[0115] Immunoblotting results as follows Figure 3 Using GAPDH as an internal control protein, the results showed that PD-M6 exhibited a dose-dependent downregulation effect on mTOR. Significant mTOR degradation was observed at a concentration of 1 μM, while the downregulation effect reached its maximum at a concentration of 10 μM, with a degradation rate of 90% (PD-M6 DC). 50 =4.8 μM). Similar to PD-M2P, PD-M2, PD-M4, and PD-M1P did not significantly downregulate mTOR at higher concentrations. This finding further confirms the significant impact of linker type on degradation efficiency.

Claims

1. A chimeric mTOR protein targeting degradation shown in formula (Ⅰ), characterized in that, （Ⅰ） In equation (Ⅰ), R is 、 、 。 2. A method for preparing the mTOR protein targeted degradation chimera according to claim 1, characterized by, The method is performed according to the following steps: (1) Synthesis of the compound shown in formula (4): The compound shown in formula (3) was added to N,N-dimethylformamide, and then the compound shown in formula (9) was added dropwise. Then N,N-diisopropylethylamine was added slowly. The mixture was stirred at 90°C for 6 hours and then cooled to 25°C. It was diluted with dichloromethane, washed once with water and once with saturated sodium chloride aqueous solution, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to dryness to obtain the concentrate. It was dissolved in ethyl acetate and then methyl tert-butyl ether was added to precipitate a white solid. It was filtered and the filter cake was dried under vacuum at 25°C. The white powder was collected to obtain the compound shown in formula (4), which was designated as intermediate 4. (2) Synthesis of the compound shown in formula (5): Intermediate 4 was added to a 1,4-dioxane solution with a hydrochloric acid concentration of 4.0 mol / L. The mixture was stirred at room temperature for 1.5 hours. Then methyl tert-butyl ether was added, and a yellow solid was precipitated. The solid was filtered, and the filter cake was washed with ether and dried under vacuum at 25°C to obtain the compound shown in formula (5), which is denoted as intermediate 5. (3) Synthesis of the compound shown in formula (8): Sodium acetate was added to acetic acid, and then the compound shown in formula (6) and the compound shown in formula (7) were added. The mixture was stirred at 90°C for 8 hours and then cooled to room temperature. It was diluted with dichloromethane, washed once with water and once with saturated sodium chloride aqueous solution, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to dryness to obtain a white solid product, which is the compound shown in formula (8), and is referred to as intermediate 8. (4) Synthesis of the mTOR protein targeted degradation chimera shown in formula (I): Intermediate 5 was dissolved in N,N-dimethylformamide, and the compound shown in formula (8) was added. After mixing, N,N-diisopropylethylamine was slowly added dropwise. The mixture was stirred at 90°C for 8 hours and then cooled to 25°C. The reaction solution was diluted with ethyl acetate, washed once with water and once with saturated sodium chloride aqueous solution, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The concentrate was dissolved in dichloromethane / methanol at a volume ratio of 8:1 and then subjected to silica gel column chromatography. A dichloromethane / methanol mixture at a volume ratio of 30:1 was used as the eluent, and the flow rate was 0.5 mL / s. Three column volumes were eluted. Thin-layer chromatography was performed using dichloromethane / methanol at a volume ratio of 20:1 as the developing solvent. The eluent with an Rf value of 0.25-0.45 was collected and dried at 25°C to obtain the mTOR protein targeted degradation chimera shown in formula (I). In equations (9), (4), and (5), R is the same as in claim 1.

3. The production method according to claim 2, wherein In step (1), the ratio of the amount of compound shown in formula (3) to the amount of compound shown in formula (9) is 1:1-2; the ratio of the amount of compound shown in formula (3) to the amount of N,N-diisopropylethylamine is 1:1-3; and the volume of N,N-dimethylformamide used is 2-5 mL / mmol based on the amount of compound shown in formula (3).

4. The production method according to claim 2, wherein In step (2), the volume of the 1,4-dioxane solution used is 0.6-2.5 mL / mmol, calculated based on the amount of intermediate 4.

5. The production method according to claim 2, wherein In step (3), the ratio of the amount of compound shown in formula (6) to the amount of compound shown in formula (7) is 1:1-2; the ratio of the amount of compound shown in formula (6) to the amount of sodium acetate is 1:1-5; and the volume of acetic acid used is 2-5 mL / mmol based on the amount of compound shown in formula (6).

6. The production method according to claim 2, wherein In step (4), the ratio of the amount of intermediate 5 to intermediate 8 is 1:1-2; the ratio of the amount of intermediate 5 to N,N-diisopropylethylamine is 1:5-7; and the volume of N,N-dimethylformamide used is 10-15 mL / mmol based on the amount of intermediate 5.

7. The production method according to claim 2, wherein The compound shown in formula (3) was prepared by the following steps: cyanuric chloride (1) was dissolved in tetrahydrofuran, morpholine (2) was added under ice bath conditions, and N,N-diisopropylethylamine was slowly added dropwise. The mixture was stirred at room temperature for 3 hours. The reaction solution was diluted with ethyl acetate, washed once with water and once with saturated sodium chloride aqueous solution, dried with anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness by rotary evaporation to obtain the compound shown in formula (3), which was designated as intermediate 3. The molar ratio of cyanuric chloride to morpholine was 1:

2. The molar ratio of cyanuric chloride to N,N-diisopropylethylamine was 1:1-2. The volume of tetrahydrofuran used was 1-3 mL / mmol based on the amount of cyanuric chloride. 。 8. The use of the mTOR protein-targeting degradation chimera of claim 1 in the preparation of a targeted mTOR degradation formulation.

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