Compounds targeting endoplasmic reticulum-associated autophagy, pharmaceutical compositions, and methods of preparation and use thereof

Abstract

The application provides a compound targeting endoplasmic reticulum autophagy, a pharmaceutical composition and a preparation method and application thereof, and belongs to the field of biological medicine. The application provides a compound with a structure shown in formula I or a pharmaceutically acceptable salt, a solvate, a tautomer, an isotopic compound, a metabolite thereof, and a preparation method and application thereof. The compound is covalently coupled by a connecting chain of a chemical structure capable of targeting and binding LC3 protein and a chemical structure capable of targeting and binding endoplasmic reticulum, to form a bifunctional molecule capable of synchronously targeting endoplasmic reticulum and LC3 protein. The compound selectively induces endoplasmic reticulum degradation through an autophagy-lysosome pathway, and has no significant influence on other cell organelles such as Golgi and mitochondria. The compound can effectively inhibit tumor cell proliferation, and has a wide application prospect in preparation of antitumor drugs. Formula I

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to compounds, pharmaceutical compositions, preparation methods, and applications of targeted induction of endoplasmic reticulum autophagy. Background Technology

[0002] Malignant tumors have become one of the leading causes of death worldwide. Currently, commonly used clinical treatments for tumors include surgery, radiotherapy, chemotherapy, molecular targeted therapy, and immunotherapy. Although these methods have achieved some success in treating certain types of tumors, they generally suffer from significant side effects, easy drug resistance, and limited efficacy against metastatic or refractory tumors. Therefore, there is an urgent clinical need to continuously develop anti-tumor drugs with novel mechanisms of action.

[0003] The endoplasmic reticulum (ER) is a vital cellular organelle responsible for protein folding, processing, and transport, lipid synthesis, and calcium ion storage. Tumor cells, due to their rapid proliferation and harsh microenvironment, often face the accumulation of unfolded or misfolded proteins within the ER, a condition known as ER stress. To restore homeostasis, cells initiate unfolded protein responses. However, when ER stress exceeds the cell's tolerance or persists for too long, it triggers programmed cell death. Therefore, intervening in the regulation of ER homeostasis is a highly promising anti-tumor strategy.

[0004] Autophagy is a highly conserved degradation system in eukaryotes, maintaining cellular homeostasis by forming autophagosomes and transporting cytoplasmic components or organelles to lysosomes for degradation. In recent years, selective organelle autophagy has attracted widespread attention, with endoplasmic reticulum (ER) autophagy referring to the selective targeting and clearance of damaged or excess ER regions to autophagosomes via the autophagic pathway. Studies have shown that regulating ER autophagy can profoundly affect tumor cell survival and apoptosis; in certain tumor types, inducing excessive ER autophagy has been proven to effectively lead to tumor cell death and overcome drug resistance. Therefore, specific induction of ER autophagy represents a novel direction in cancer therapy.

[0005] While some small molecule compounds capable of inducing autophagy exist in the prior art, such as rapamycin and its analogues, they primarily induce non-selective macrophagy, exhibiting extremely limited targeting and efficiency towards endoplasmic reticulum (ER) autophagy. Other reported ER stress inducers (such as tunicamycin and carotenoids) can also indirectly induce some degree of ER autophagy, but their mechanisms of action lack specificity and are accompanied by severe ER stress toxicity, thus limiting their clinical application. To date, there is a lack of small molecule compounds in this field capable of efficiently targeting and inducing ER autophagy, and there are no reports of such compounds being used as anti-tumor drugs. Therefore, designing and developing novel structural types of small molecule compounds that can directly and selectively activate the ER autophagy pathway is of significant scientific and practical value for providing novel anti-tumor therapeutics and overcoming existing therapeutic bottlenecks. Summary of the Invention

[0006] The purpose of this invention is to provide compounds, pharmaceutical compositions, preparation methods, and applications of targeted induction of endoplasmic reticulum autophagy.

[0007] This invention provides compounds of Formula I, or pharmaceutically acceptable salts thereof, or solvates thereof, or tautomers thereof, or isotopic compounds thereof, or metabolites thereof: Formula I R1 and R2 are each independently selected from OH or OR3; When R1 is OH, R2 is OR3; when R1 is OR3, R2 is OH; the two cannot both be OH or OR3 at the same time. R3 is -(CH2) n -O-(CH2)2-NHTs, where n is any integer from 3 to 11.

[0008] "Ts" represents the abbreviation for p-toluenesulfonyl, and its structure is represented as: .

[0009] Furthermore, n can be 3, 5, 7, 9, or 11.

[0010] Furthermore, the compound is selected from one of the following structures: .

[0011] The present invention also provides a method for preparing the above-mentioned compound, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a tautomer thereof, or an isotopic compound thereof, or a metabolite thereof, the method comprising the following steps: Compound 1a was reacted with p-toluenesulfonyl chloride to give compound 2a; compound 2a was then reacted with compound 3a to give compound 4a; compound 4a was then deprotected to give compound 5a; compound 5a was then reacted with p-toluenesulfonyl chloride to give compound 6a; and compound 6a was then reacted with compound 7a to give the compound shown in Formula I.

[0012] The present invention also provides a pharmaceutical composition, wherein the pharmaceutical composition is a formulation prepared by adding pharmaceutically acceptable excipients to the above-mentioned compound, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a tautomer thereof, or an isotopic compound thereof, or a metabolite thereof as the active ingredient.

[0013] The present invention also provides the use of the above-described compounds, or pharmaceutically acceptable salts thereof, or solvates thereof, or tautomers thereof, or isotopic compounds thereof, or metabolites thereof, or the above-described pharmaceutical compositions in the preparation of endoplasmic reticulum autophagy inducers.

[0014] Furthermore, the endoplasmic reticulum autophagy inducer is a drug for the prevention and / or treatment of tumors.

[0015] Furthermore, the tumor is selected from breast cancer, lung cancer, liver cancer, kidney cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, bladder cancer, myelodysplastic syndrome, lymphoma, esophageal cancer, gastrointestinal cancer, osteosarcoma, and tumors of the central or peripheral nervous system.

[0016] Furthermore, the tumor is breast cancer.

[0017] The present invention has achieved the following beneficial effects: The compound provided by this invention covalently couples the chemical structure targeting the LC3 protein with the chemical structure targeting the endoplasmic reticulum (ER) via a linker chain. This allows for the simultaneous recognition and binding of the LC3 protein, a key protein in both the ER and the autophagosome membrane, thereby specifically "binding" the ER to autophagosomes and achieving selective ER degradation via the autophagy-lysosome pathway. Mechanism of action verification shows that this compound effectively enriches and stably localizes in the ER, exhibiting high co-localization with LC3, and the degradation process strictly depends on the autophagy-lysosome pathway. Furthermore, the compound has no significant effect on other organelles such as the Golgi apparatus and mitochondria, demonstrating excellent ER-targeting specificity. Functionally, this compound can target and induce ER autophagic degradation, effectively reducing the expression level of the ER marker protein RAMP4, and exhibits significant inhibitory activity against the proliferation of triple-negative breast cancer 4T1 cells at the cellular level. In vivo efficacy evaluation further confirms that this compound effectively inhibits tumor growth in tumor-bearing mouse models without significantly affecting animal body weight, demonstrating good safety. In summary, the compounds of this invention, as a novel class of endoplasmic reticulum autophagy-targeting inducers, provide strong candidate molecules for the development of novel antitumor drugs and have significant potential for clinical application.

[0018] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0019] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0020] Figure 1Transmission electron microscopy (TEM) images of cells after treatment with different analytes. (A) Control group: nucleus (N), nucleolus (No), mitochondria (Mi), rough endoplasmic reticulum (RER), normal RER morphology (blue arrow), autolysosomes (dark blue arrow). (B) Magnified view of the selected area in Figure A of the control group: nucleus (N), mitochondria (Mi), rough endoplasmic reticulum (RER), normal mitochondrial morphology (yellow arrow), normal RER morphology (blue arrow). (C) E8 treatment group: nucleus (N), nucleolus (No), mitochondria (Mi), rough endoplasmic reticulum (RER), abnormal RER morphology (blue arrow), autolysosomes (dark blue arrow). (D) Magnified view of the selected area in Figure C of the E8 treatment group: mitochondria (Mi), rough endoplasmic reticulum (RER), abnormal RER morphology (blue arrow), autolysosomes (dark blue arrow), endoplasmic reticulum fragments in autolysosomes (red arrow).

[0021] Figure 2 To observe the endoplasmic reticulum fluorescence of cells after treatment with blank group (A) and different concentrations of E8 (BF) using laser confocal microscopy (endoplasmic reticulum: green fluorescence, cell nucleus: blue fluorescence, E8 concentrations in BF group were 0.05, 0.2, 0.5, 2, and 5 μM, respectively).

[0022] Figure 3 The tumor volume growth curve (A), mouse body weight change curve (B), and ex vivo tumor weight (C) of tumor-bearing mice after different treatment groups are shown.

[0023] Figure 4 To observe the colocalization of the endoplasmic reticulum (green fluorescence) and compound E8 (blue fluorescence) in blank group (A) and E8 group (B) using laser confocal microscopy; and the colocalization of LC3 (red fluorescence) and compound E8 (blue fluorescence) in blank group (C) and E8 group (D).

[0024] Figure 5 The changes in mCherry-EGFP-RAMP4 fluorescence in cells of the blank group (A) and the E8 group (B) were observed using laser confocal microscopy.

[0025] Figure 6 The co-localization of endoplasmic reticulum (green fluorescence) and lysosomes (red fluorescence) in cells of the blank group (A) and E8 group (B) was observed using laser confocal microscopy; the co-localization of Golgi apparatus (green fluorescence) and lysosomes (red fluorescence) in cells of the blank group (C) and E8 group (D); and the co-localization of mitochondria (green fluorescence) and lysosomes (red fluorescence) in cells of the blank group (E) and E8 group (F). Blue fluorescence represents the cell nucleus. Detailed Implementation

[0026] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0027] Example 1, Preparation of Compounds 1 and 2 Step 1: p-Toluenesulfonyl chloride (57.62 g, 302.24 mmol, 2.30 eq) was added to a pyridine (160 mL) solution of compound 1-1 (10.00 g, 131.41 mmol, 1.00 eq) at 0 °C, followed by stirring at 0 °C for 3 h. After the reaction was monitored by LC-MS, the mixture was filtered, and the filter cake was washed with dichloromethane. The filtrate was concentrated and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1~2 / 1) to obtain compound 1-2 (11.50 g, 29.90 mmol, 22.8% yield). [M+Na] + 407.0.

[0028] Step 2: At 0 °C, potassium hydroxide (1.39 g, 24.82 mmol, 0.80 eq) and tetrabutylammonium bromide (1.20 g, 3.72 mmol, 0.30 eq) were added to a 20 mL solution of tetrahydrofuran containing compounds 1-2 (3.82 g, 9.93 mmol, 0.80 eq) and compounds 1-3 (2.00 g, 12.41 mmol, 1.00 eq). The solution was then heated to 25 °C and stirred for 2 h. After the reaction of compounds 1-2 was complete as monitored by LC-MS, the solution was filtered and the filter cake was washed with dichloromethane. The filtrate was concentrated and then subjected to preparative liquid chromatography (PLC) using a Welch Ultimate XB-CN 250 column. 70 Compounds 1-4 were isolated and purified using a 10 μm elution system (n-hexane / ethanol; gradient: mobile phase B, 1%-40%, 15 min) to obtain 3.81 g, 10.2 mmol, 82.2% yield. [M+Na] + 396.1.

[0029] Step 3: Under nitrogen protection, compounds 1-4 (3.50 g, 9.37 mmol, 1.00 eq) were added to a hydrogen chloride / dioxane solution (2.00 M, 35.0 mL, 70 mmol, 7.47 eq), and the mixture was stirred at 25 °C for 0.5 h. After the reaction was completed as monitored by LC-MS, the crude product of compounds 1-5 (2.10 g) was concentrated under reduced pressure and used directly in the next reaction step.

[0030] Step 4: p-Toluenesulfonyl chloride (1.11 g, 5.81 mmol, 1.00 eq) and triethylamine (1.47 g, 14.53 mmol, 2.50 eq) were added to a dichloromethane (20 mL) solution of compounds 1-5 (1.59 g, 5.81 mmol, 1.00 eq) at 0 °C, followed by stirring at 0 °C for 2 h. After the reaction was monitored by LC-MS, 1N HCl solution (20 mL) was added for quenching, and the mixture was extracted three times with ethyl acetate (15 mL × 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, concentrated, and purified using a preparative liquid chromatography column (column model: Welch Ultimate XB-CN 250). 70 Compounds 1-6 were isolated and purified using a 10µm elution system (n-hexane / ethanol; gradient: mobile phase B, 30%-70%, 15 min) to obtain 1.05 g, 2.46 mmol, 42.3% yield. [M+H] + 428.0.

[0031] Step 5: Compounds 1-6 (1.70 g, 3.98 mmol, 1.00 eq) and potassium carbonate (550.1 mg, 3.98 mmol, 1.00 eq) were added to a DMF (20 mL) solution of compounds 1-7 (2.02 g, 7.96 mmol, 2.00 eq), and the reaction was carried out at 50 °C for 12 h. After the consumption of compounds 1-6 was monitored by LC-MS, the reaction system was cooled to room temperature, water (20 mL) was added, and the mixture was extracted three times with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, concentrated, and then subjected to preparative liquid chromatography (PLC) on a Welch Ultimate XB-CN 250 column. 70 10µm; elution system: n-hexane / ethanol; gradient: mobile phase B, 30%-70%, 15 min) and SFC column (column model: DAICEL CHIRALCEL OJ (250 mm)). Compound 1 and compound 2 were obtained by separation and purification using a 30 mm, 10 μm elution system (carbon dioxide-methanol (0.1% ammonia) and a gradient of 32% mobile phase B, constant elution).

[0032] Compound 1 (abbreviated as E2) (1.41 g, 2.77 mmol, 69.6% yield). 1 H-NMR (400 MHz, CDCl3,ppm) δ 7.71 (d,J = 8.0 Hz, 2H), 7.33-7.32 (m, 3H), 7.26 (d, J = 8.0 Hz, 2H),7.24-7.22 (m, 2H), 6.60 (d, J = 2.0 Hz, 1H), 6.34 (d, J = 2.0 Hz, 1H), 5.95 (s,1H), 5.11 (brs, 1H), 3.71 (t, J = 6.0 Hz, 2H), 3.30 (t, J = 5.2 Hz, 2H), 3.07 (q, J = 5.2 Hz, 2H), 2.91 (t, J = 6.0 Hz, 2H), 2.41 (s, 3H), 1.29–1.23 (m, 2H). MS (ESI) analysis shows that, based on the molecular formula C 27 H 28 NO7S's [M+H] + The theoretical value is 510.2, and the measured value is 510.0.

[0033] Compound 2 (abbreviated as E15) (280.3 mg, 0.55 mmol, 13.8% yield). 1 H-NMR (400 MHz, CDCl3, ppm) δ 7.72 (d, J = 8.0 Hz, 2H), 7.56-7.54 (m, 3H), 7.46-7.43 (m, 2H),7.30 (d, J = 8.0 Hz, 2H), 6.51 (d, J = 2.4 Hz, 1H), 6.28 (d, J = 2.4 Hz, 1H), 5.97(s, 1H), 5.42 (s, 1H), 4.79 (t, J = 6.0 Hz, 1H), 4.05 (t, J = 6.0 Hz, 2H), 3.53(t, J = 6.0 Hz, 2H), 3.46 (t, J = 5.2 Hz, 2H), 3.12 (q, J= 5.2 Hz, 2H), 2.42 (s, 3H), 2.03–1.97 (m, 2H). MS (ESI) analysis shows that, based on the molecular formula C 27 H 28 NO7S's [M+H] + The theoretical value is 510.2, and the measured value is 510.0.

[0034] Example 2, Preparation of compounds 3 and 4 Step 1: p-Toluenesulfonyl chloride (30.8 g, 161.39 mmol, 2.10 eq) was added to a pyridine (100 mL) solution of compound 3-1 (8.00 g, 76.85 mmol, 1.00 eq) at 0 °C, followed by stirring at 0 °C for 3 h. After the reaction was monitored by LC-MS, the mixture was filtered, and the filter cake was washed with dichloromethane. The filtrate was concentrated and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1~2 / 1) to obtain compound 3-2 (29.58 g, 71.70 mmol, 93.3% yield). [M+H] + 413.1.

[0035] Step 2: At 0 °C, potassium hydroxide (695.8 mg, 12.40 mmol, 1.00 eq) and tetrabutylammonium bromide (599.6 mg, 1.86 mmol, 0.30 eq) were added to a tetrahydrofuran (10 mL) solution containing compound 3-2 (2.55 g, 6.20 mmol, 1.00 eq) and compound 1-3 (0.80 g, 4.96 mmol, 0.80 eq), followed by heating to 25 °C and stirring for 2 h. After the reaction of compound 3-2 was complete as monitored by LC-MS, the mixture was diluted with water (20 mL) and extracted three times with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, concentrated, and subjected to preparative liquid chromatography (HPLC) using a Welch Ultimate XB-CN 250 column. 70 Compound 3-3 was purified by separation and purification using a mobile phase B (5%-45%, 15 min) at a concentration of 10 μm (2.10 g, 5.23 mmol, 84.4% yield). [M+Na] + 424.1.

[0036] Step 3: Under nitrogen protection, compound 3-3 (2.50 g, 6.23 mmol, 1.00 eq) was added to a hydrogen chloride / dioxane solution (2.00 M, 26.5 mL, 53 mmol, 8.51 eq), and the mixture was stirred at 25 °C for 3 h. After the reaction was completed as monitored by LC-MS, the crude product of compound 3-4 (1.81 g) was concentrated under reduced pressure and used directly in the next reaction step.

[0037] Step 4: p-Toluenesulfonyl chloride (810.3 mg, 4.25 mmol, 1.00 eq) and triethylamine (1.08 g, 10.63 mmol, 2.50 eq) were added to a dichloromethane (12 mL) solution of compound 3-4 (1.28 g, 4.25 mmol, 1.00 eq) at 0 °C, followed by stirring at 0 °C for 1 h. After the reaction was monitored by LC-MS, 1N HCl solution (50 mL) was added for quenching, and the mixture was extracted three times with ethyl acetate (30 mL × 3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, concentrated, and purified by preparative liquid chromatography (column model: Welch Ultimate XB-CN 250). 70 10 μm; elution system: n-hexane / ethanol; gradient: mobile phase B, 20%-50%, 15 min) to separate and purify compounds 3-5 (1.52 g, 3.34 mmol, 78.6% yield). [M+H] + 456.3.

[0038] Step 5: Compounds 3-5 (1.74 g, 3.83 mmol, 1.20 eq) and potassium carbonate (440.9 mg, 3.19 mmol, 1.00 eq) were added to a DMF (26 mL) solution of compounds 1-7 (810.9 mg, 3.19 mmol, 1.00 eq), and the reaction was carried out at 50 °C for 12 h. After the consumption of compounds 1-7 was monitored by LC-MS, the reaction system was cooled to room temperature, water (20 mL) was added, and the mixture was extracted three times with ethyl acetate (10 mL × 3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, concentrated, and then subjected to preparative liquid chromatography (PLC) on a Welch Ultimate XB-CN 250 column. 70 10µm; elution system: n-hexane / ethanol; gradient: mobile phase B, 25%-55%, 20 min) and SFC column (column model: DAICEL CHIRALCEL OJ (250mm)). Compounds 3 and 4 were obtained by separation and purification using a 30 mm, 10 μm elution system (carbon dioxide-methanol (0.1% ammonia) and a gradient of 30% mobile phase B, constant elution).

[0039] Compound 3 (abbreviated as E4) (930.0 mg, 1.73 mmol, 54.2% yield). 1 H-NMR (400 MHz, DMSO- d 6 , ppm) δ 10.65 (brs, 1H), 7.68 (d, J = 8.0 Hz, 2H), 7.59 (t, J = 6.0 Hz,1H), 7.39-7.35 (m, 5H), 7.28-7.27 (m, 2H), 6.39 (d, J = 2.0 Hz, 1H), 6.25 (d, J =2.0 Hz, 1H), 5.78 (s, 1H), 3.63 (t, J = 6.0 Hz, 2H), 3.30 (t, J = 6.0 Hz, 2H), 3.15 (t, J = 6.8 Hz, 2H), 2.86 (q, J = 6.0 Hz, 2H), 2.37 (s, 3H), 1.21–1.14 (m, 2H), 1.05–0.97 (m, 2H), 0.78–0.70 (m, 2H). MS(ESI) analysis shows that, based on the molecular formula C 29 H 32 NO7S's [M+H] + The theoretical value is 538.2, and the measured value is 538.0.

[0040] Compound 4 (abbreviated as E17) (301.1 mg, 0.56 mmol, 17.6% yield). 1 H-NMR (400 MHz, CDCl3, ppm) δ 7.74 (d, J = 8.0 Hz, 2H), 7.56-7.54 (m, 3H), 7.45-7.43 (m, 2H),7.31 (d, J = 8.0 Hz, 2H), 6.51 (d, J = 2.4 Hz, 1H), 6.27 (d, J= 2.4 Hz, 1H), 5.97(s, 1H), 5.36 (s, 1H), 4.77 (brs, 1H), 3.99 (t, J = 6.4 Hz, 2H), 3.44 (t, J = 5.2Hz, 2H), 3.38 (t, J = 6.4 Hz, 2H), 3.11 (q, J = 5.2 Hz, 2H), 2.42 (s, 3H), 1.83-1.76 (m, 2H), 1.62-1.59 (m, 2H), 1.50-1.46 (m, 2H). MS(ESI) analysis shows that, based on the molecular formula C 29 H 32 NO7S's [M+H] + The theoretical value is 538.2, and the measured value is 538.1.

[0041] Example 3, Preparation of compounds 5 and 6 Step 1: p-Toluenesulfonyl chloride (9.95 g, 52.19 mmol, 2.30 eq) was added to a pyridine (100 mL) solution of compound 5-1 (3.00 g, 22.69 mmol, 1.00 eq) at 0 °C, followed by stirring at 0 °C for 3 h. After the reaction was monitored by LC-MS, the mixture was filtered, and the filter cake was washed with dichloromethane. The filtrate was concentrated and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1 to 1 / 1) to obtain compound 5-2 (6.60 g, 14.98 mmol, 66.0% yield). [M+H] + 441.0.

[0042] Step 2: At 0 °C, potassium hydroxide (695.8 mg, 12.40 mmol, 2.00 eq) and tetrabutylammonium bromide (599.6 mg, 1.86 mmol, 0.30 eq) were added to a tetrahydrofuran (10 mL) solution containing compound 5-2 (2.19 g, 4.96 mmol, 0.80 eq) and compound 1-3 (1.00 g, 6.20 mmol, 1.00 eq), followed by heating to 25 °C and stirring for 1 h. After the reaction of compound 5-2 was complete as monitored by LC-MS, the mixture was diluted with water (20 mL) and extracted three times with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, concentrated, and subjected to preparative liquid chromatography (PLC) using a Welch Ultimate XB-CN 250 column. 70 Compound 5-3 was purified by separation and purification using a mobile phase B (8%-46%, 18 min) with a molecular weight of 10 μm; elution system: n-hexane / ethanol; gradient: mobile phase B, 8%-46%, 18 min (2.10 g, 4.89 mmol, 78.9% yield). [M+Na] + 452.0.

[0043] Step 3: Under nitrogen protection, compound 5-3 (5.00 g, 11.64 mmol, 1.00 eq) was added to a hydrogen chloride / dioxane solution (2.00 M, 40.0 mL, 80 mmol, 6.87 eq), and the mixture was stirred at 25 °C for 0.5 h. After the reaction was completed as monitored by LC-MS, the crude product of compound 5-4 (3.80 g) was concentrated under reduced pressure and used directly in the next reaction step.

[0044] Step 4: p-Toluenesulfonyl chloride (1.91 g, 10.02 mmol, 1.00 eq) and triethylamine (2.03 g, 20.04 mmol, 2.50 eq) were added to a dichloromethane (30 mL) solution of compound 5-4 (3.30 g, 10.02 mmol, 1.00 eq) at 0 °C, followed by stirring at 0 °C for 1 h. After the reaction was monitored by LC-MS, 1N HCl solution (20 mL) was added for quenching, and the mixture was extracted three times with ethyl acetate (15 mL × 3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, concentrated, and subjected to preparative liquid chromatography (YMC-Gel SiL-HG 250 mm column). 70 mm Compound 5-5 was isolated and purified using a 10 μm elution system (n-hexane / ethanol); gradient: mobile phase B, 1%-30%, 15 min (3.44 g, 7.12 mmol, 71.1% yield). [M+Na] + 506.1.

[0045] Step 5: Compound 5-5 (3.00 g, 6.20 mmol, 1.00 eq) and potassium carbonate (856.9 mg, 6.20 mmol, 1.00 eq) were added to a DMF (30 mL) solution of compounds 1-7 (3.15 g, 12.40 mmol, 2.00 eq), and the mixture was reacted at 50 °C for 12 h. After the consumption of compound 5-5 was monitored by LC-MS, the reaction mixture was cooled to room temperature, water (20 mL) was added, and the mixture was extracted three times with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, concentrated, and then subjected to preparative liquid chromatography (PLC) on a Welch Ultimate XB-CN 250 column. 50 10µm; elution system: n-hexane / ethanol; gradient: mobile phase B, 20%-50%, 20 min) and SFC column (column model: DAICEL CHIRALCEL OJ (250mm)). Compounds 5 and 6 were obtained by separation and purification using a 30 mm, 10 μm elution system (carbon dioxide-methanol (0.1% ammonia) and a gradient of 25% mobile phase B, constant elution).

[0046] Compound 5 (abbreviated as E6) (1.69 g, 2.99 mmol, 48.2% yield). 1 H-NMR (400 MHz, DMSO- d 6 ,ppm) δ 10.63 (brs, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.61 (t, J = 6.0 Hz, 1H), 7.39-7.35 (m, 5H), 7.29-7.27 (m, 2H), 6.39 (d, J = 2.0 Hz, 1H), 6.25 (d, J = 2.0 Hz,1H), 5.78 (s, 1H), 3.63 (t, J = 6.0 Hz, 2H), 3.32 (t, J= 6.0 Hz, 2H), 3.26 (t, J =6.4 Hz, 2H), 2.87 (q, J = 6.0 Hz, 2H), 2.37 (s, 3H), 1.40-1.32 (m, 2H), 1.14-1.05 (m, 2H), 1.00-0.95 (m, 4H), 0.78-0.71 (m, 2H). MS(ESI) analysis shows that, based on the molecular formula C 31 H 36 NO7S's [M+H] + The theoretical value is 566.2, and the measured value is 566.1.

[0047] Compound 6 (abbreviated as E19) (633.6 mg, 1.12 mmol, 18.1% yield). 1 H-NMR (400 MHz, CDCl3, ppm) δ 7.74 (d, J = 8.0 Hz, 2H), 7.57-7.55 (m, 3H), 7.46-7.43 (m, 2H),7.30 (d, J = 8.0 Hz, 2H), 6.52 (d, J = 2.8 Hz, 1H), 6.25 (d, J = 2.8 Hz, 1H), 5.96(s, 1H), 5.30 (s, 1H), 4.77 (t, J = 6.0 Hz, 1H), 3.99 (t, J = 6.4 Hz, 2H), 3.43(t, J = 5.2 Hz, 2H), 3.34 (t, J = 6.4 Hz, 2H), 3.10 (q, J = 5.2 Hz, 2H), 2.43 (s, 3H), 1.83–1.76 (m, 2H), 1.54–1.44 (m, 4H), 1.35–1.32 (m, 4H). MS (ESI) analysis shows that, based on the molecular formula C 31 H 36 NO7S's [M+H] + The theoretical value is 566.2, and the measured value is 566.1.

[0048] Example 4, Preparation of compounds 7 and 8 Step 1: p-Toluenesulfonyl chloride (13.68 g, 71.74 mmol, 2.30 eq) was added to a pyridine (5.00 g, 31.19 mmol, 1.00 eq) solution of compound 7-1 at 0 °C, followed by stirring at 0 °C for 2 h. After the reaction was monitored by LC-MS, the mixture was filtered, and the filter cake was washed with dichloromethane. The filtrate was concentrated and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1~1 / 1) to obtain compound 7-2 (12.26 g, 26.16 mmol, 83.9% yield). [M+NH4] + 486.2.

[0049] Step 2: At 0 °C, potassium hydroxide (2.39 g, 42.68 mmol, 2.00 eq) and tetrabutylammonium bromide (2.06 g, 6.40 mmol, 0.30 eq) were added to a tetrahydrofuran (150 mL) solution containing compound 7-2 (8.00 g, 17.07 mmol, 0.80 eq) and compound 1-3 (3.44 g, 21.34 mmol, 1.00 eq). The mixture was then heated to 25 °C and stirred for 3 h. After the reaction of compound 7-2 was complete as monitored by LC-MS, the mixture was diluted with water (80 mL) and extracted three times with ethyl acetate (80 mL × 3). The organic phases were combined, washed with saturated brine (80 mL), dried over anhydrous sodium sulfate, concentrated, and subjected to preparative liquid chromatography (PLC) using a Welch Ultimate XB-CN 250 column. 70 Compound 7-3 was isolated and purified using a 10 μm elution system (n-hexane / ethanol); gradient: mobile phase B, 8%-50%, 18 min (4.96 g, 10.85 mmol, 63.6% yield). [M-Boc+H] + 358.1.

[0050] Step 3: Under nitrogen protection, compound 7-3 (2.25 g, 4.92 mmol, 1.00 eq) was added to a hydrogen chloride / dioxane solution (2.00 M, 20.0 mL, 40 mmol, 8.13 eq), and the mixture was stirred at 25 °C for 8 h. After the reaction was completed as monitored by LC-MS, the mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain crude compound 7-4 (1.72 g), which was used directly in the next reaction step.

[0051] Step 4: p-Toluenesulfonyl chloride (650.1 mg, 3.41 mmol, 1.00 eq) and triethylamine (863.2 mg, 8.53 mmol, 2.50 eq) were added to a dichloromethane (12 mL) solution of compound 7-4 (1.22 g, 3.41 mmol, 1.00 eq) at 0 °C, followed by stirring at 0 °C for 5 h. After the reaction was monitored by LC-MS, 1N HCl solution (20 mL) was added for quenching, and the mixture was extracted three times with ethyl acetate (30 mL × 3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, concentrated, and purified by preparative liquid chromatography (column model: Welch Ultimate XB-CN 250). 70 10 μm; elution system: n-hexane / ethanol; gradient: mobile phase B, 20%-50%, 15 min) to separate and purify compounds 7-5 (1.45 g, 2.83 mmol, 83.0% yield). [M+H] + 512.2.

[0052] Step 5: Compound 7-5 (1.45 g, 2.83 mmol, 1.00 eq) and potassium carbonate (391.1 mg, 2.83 mmol, 1.00 eq) were added to a DMF (15 mL) solution of compounds 1-7 (1.44 g, 5.66 mmol, 2.00 eq), and the reaction was carried out at 60 °C for 16 h. After the consumption of compound 7-5 was monitored by LC-MS, the reaction system was cooled to room temperature, water (200 mL) was added, and the mixture was extracted three times with ethyl acetate (60 mL × 3). The organic phases were combined, washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, concentrated, and then subjected to preparative liquid chromatography (PLC) on a Welch Ultimate XB-CN 250 column. 70 10µm; elution system: n-hexane / ethanol; gradient: mobile phase B, 20%-60%, 20 min) and SFC column (column model: DAICEL CHIRALCEL OJ (250mm)). Compounds 7 and 8 were obtained by separation and purification using a 30 mm, 10 μm elution system (carbon dioxide-methanol (0.1% ammonia) and a gradient of 35% mobile phase B, constant elution).

[0053] Compound 7 (abbreviated as E8) (985.5 mg, 1.66 mmol, 58.7% yield). 1 H-NMR (400 MHz, DMSO- d6 , ppm) δ 10.63 (brs, 1H), 7.68 (d, J = 7.6 Hz, 2H), 7.60 (brs, 1H), 7.38-7.36 (m, 5H), 7.28 (s, 2H), 6.39 (s, 1H), 6.25 (s, 1H), 5.77 (s, 1H), 3.64-3.61 (m, 2H), 3.27-3.25 (m, 4H), 2.87-2.84 (m, 2H), 2.37 (s, 3H), 1.42-1.38 (m, 2H), 1.19-0.99 (m, 10H), 0.77-0.71 (m, 2H). MS(ESI) analysis shows that, based on the molecular formula C 33 H 40 NO7S's [M+H] + The theoretical value is 594.3, and the measured value is 594.2.

[0054] Compound 8 (abbreviated as E21) (267.1 mg, 0.45 mmol, 15.9% yield). 1 H-NMR (400 MHz, CDCl3, ppm) δ 7.74 (d, J = 8.0 Hz, 2H), 7.56-7.55 (m, 3H), 7.45-7.43 (m, 2H),7.30 (d, J = 8.0 Hz, 2H), 6.52 (d, J = 2.4 Hz, 1H), 6.26 (d, J = 2.4 Hz, 1H), 5.96(s, 1H), 5.32 (s, 1H), 4.78 (t, J = 6.0 Hz, 1H), 3.99 (t, J = 6.4 Hz, 2H), 3.42(t, J = 5.2 Hz, 2H), 3.33 (t, J = 6.4 Hz, 2H), 3.10 (q, J = 5.2 Hz, 2H), 2.42 (s, 3H), 1.82–1.75 (m, 2H), 1.52–1.41 (m, 4H), 1.37–1.26 (m, 8H). MS (ESI) analysis shows that, based on the molecular formula C 33 H 40 NO7S's [M+H]+ The theoretical value is 594.3, and the measured value is 594.1.

[0055] Example 5: Preparation of compounds 9 and 10 Step 1: p-Toluenesulfonyl chloride (4.66 g, 24.43 mmol, 2.30 eq) was added to a pyridine (20 mL) solution of compound 9-1 (2.00 g, 10.62 mmol, 1.00 eq) at 0 °C, followed by stirring at 0 °C for 3 h. After the reaction was monitored by LC-MS, the mixture was filtered, and the filter cake was washed with dichloromethane. The filtrate was concentrated and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1~1 / 1) to obtain compound 9-2 (5.02 g, 10.11 mmol, 95.2% yield). [M+H] + 497.2.

[0056] Step 2: At 0 °C, potassium hydroxide (988.7 mg, 17.62 mmol, 2.00 eq) and tetrabutylammonium bromide (851.1 mg, 2.64 mmol, 0.30 eq) were added to a tetrahydrofuran (35 mL) solution containing compound 9-2 (3.50 g, 7.05 mmol, 0.80 eq) and compound 1-3 (1.42 g, 8.81 mmol, 1.00 eq). The mixture was then heated to 25 °C and stirred for 3 h. After the reaction of compound 9-2 was complete as monitored by LC-MS, the mixture was diluted with water (50 mL) and extracted three times with ethyl acetate (50 mL × 3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, concentrated, and subjected to preparative liquid chromatography (PLC) using a Welch Ultimate XB-CN 250 column. 70 Compound 9-3 was isolated and purified using a 10 μm elution system (n-hexane / ethanol); gradient: mobile phase B, 5%-45%, 15 min (2.62 g, 5.39 mmol, 76.5% yield). [M-Boc+H] + 386.1.

[0057] Step 3: Under nitrogen protection, compound 9-3 (1.13 g, 2.33 mmol, 1.00 eq) was added to a hydrogen chloride / dioxane solution (2.00 M, 11.0 mL, 22 mmol, 9.44 eq), and the mixture was stirred at 25 °C for 5 h. After the reaction was completed as monitored by LC-MS, the mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain crude compound 9-4 (850 mg), which was used directly in the next reaction step.

[0058] Step 4: p-Toluenesulfonyl chloride (394.6 mg, 2.07 mmol, 1.00 eq) and triethylamine (524.2 mg, 5.18 mmol, 2.50 eq) were added to a dichloromethane (12 mL) solution of compound 9-4 (800 mg, 2.07 mmol, 1.00 eq) at 0 °C, followed by stirring at 0 °C for 3 h. After the reaction was monitored by LC-MS, 1N HCl solution (20 mL) was added for quenching, and the mixture was extracted three times with ethyl acetate (30 mL × 3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, concentrated, and subjected to preparative liquid chromatography (YMC-Gel SiL-HG 250 mm column). 70 mm 10 μm; elution system: n-hexane / ethanol; gradient: mobile phase B, 1%-30%, 15 min) to separate and purify compound 9-5 (653.0 mg, 1.21 mmol, 58.4% yield). [M+H] + 540.3.

[0059] Step 5: Compound 9-5 (600 mg, 1.11 mmol, 1.00 eq) and potassium carbonate (153.4 mg, 1.11 mmol, 1.00 eq) were added to a DMF (10 mL) solution of compounds 1-7 (564.3 mg, 2.22 mmol, 2.00 eq), and the mixture was reacted at 60 °C for 4 h. After the consumption of compound 9-5 was monitored by LC-MS, the reaction mixture was cooled to room temperature, water (100 mL) was added, and the mixture was extracted three times with ethyl acetate (150 mL × 3). The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, concentrated, and then subjected to preparative liquid chromatography (PLC) on a Welch Ultimate XB-CN 250 column. 70 10µm; elution system: n-hexane / ethanol; gradient: mobile phase B, 20%-65%, 17 min) and SFC column (column model: DAICEL CHIRALCEL OJ (250 mm)). Compounds 9 and 10 were obtained by separation and purification using a 30 mm, 10 μm elution system (carbon dioxide-methanol (0.1% ammonia) and a gradient of 45% mobile phase B, constant elution).

[0060] Compound 9 (abbreviated as E10) (292.2 mg, 0.47 mmol, 42.3% yield). 1 H-NMR (400 MHz, DMSO- d 6 , ppm) δ 10.64 (brs, 1H), 7.67 (d, J = 8.0 Hz, 2H), 7.60 (t, J = 6.0 Hz,1H), 7.38-7.35 (m, 5H), 7.29-7.27 (m, 2H), 6.39 (d, J = 2.0 Hz, 1H), 6.25 (d, J =2.0 Hz, 1H), 5.77 (s, 1H), 3.63 (t, J = 6.0 Hz, 2H), 3.31 (t, J = 6.0 Hz, 2H), 3.26 (t, J = 6.4 Hz, 2H), 2.86 (q, J = 6.0 Hz, 2H), 2.37 (s, 3H), 1.43-1.40 (m, 2H), 1.23-1.18 (m, 8H), 1.13-1.01 (m, 2H), 1.05-0.96 (m, 4H), 0.79-0.71 (m, 2H). MS(ESI) analysis shows that, based on the molecular formula C 35 H 44 NO7S's [M+H] + The theoretical value is 622.3, and the measured value is 622.1.

[0061] Compound 10 (abbreviated as E23) (87.0 mg, 0.14 mmol, 12.6% yield). 1 H-NMR (400 MHz, CDCl3, ppm) δ 7.74 (d, J = 8.0 Hz, 2H), 7.56-7.55 (m, 3H), 7.45-7.43 (m, 2H),7.30 (d, J = 8.0 Hz, 2H), 6.52 (d,J = 2.8 Hz, 1H), 6.24 (d, J = 2.8 Hz, 1H), 5.96(s, 1H), 5.32 (s, 1H), 4.78 (t, J = 6.0 Hz, 1H), 3.99 (t, J = 6.4 Hz, 2H), 3.41(t, J = 5.2 Hz, 2H), 3.32 (t, J = 6.8 Hz, 2H), 3.10 (q, J = 5.2 Hz, 2H), 2.42 (s, 3H), 1.82–1.75 (m, 2H), 1.51–1.41 (m, 4H), 1.32–1.27 (m, 12H). MS (ESI) analysis shows that, based on the molecular formula C 35 H 44 NO7S's [M+H] + The theoretical value is 622.3, and the measured value is 622.1.

[0062] The following experimental examples demonstrate the beneficial effects of the present invention.

[0063] Experimental Example 1: Test of the ability of the compound of this invention to target and induce endoplasmic reticulum autophagy (Western Blot) 1. Experimental Methods This experiment used triple-negative breast cancer 4T1 cells as the test vector and RAMP4 (endoplasmic reticulum membrane-associated protein 4) as an endoplasmic reticulum marker. The expression level of RAMP4 protein was detected by Western blotting to evaluate the ability of the compounds of this invention to induce endoplasmic reticulum autophagy and degradation. The specific methods are as follows: 4T1 cells were seeded into 6-well plates and cultured adherently for 24 h. A specific concentration (2 μM) of the compound of this invention and a corresponding solvent control (blank group) were added for 24 h. After treatment, cells were collected, and pre-chilled RIPA lysis buffer (containing protease inhibitors and PMSF) was added to the cell pellet. After resuspending, the cells were sonicated on ice, and the supernatant was collected by centrifugation. Protein concentration was determined using the BCA method. Based on the determination results, each group of samples was diluted to the same final concentration with lysis buffer, and SDS-PAGE loading buffer was added. The samples were then denatured at 95°C to obtain the protein samples to be tested. SDS-PAGE electrophoresis was performed, and the samples were transferred to a 0.45 μM PVDF membrane. The PVDF membrane was blocked in 5% skim milk powder blocking solution prepared with TBST for 2 h. Then, the membrane was immersed in the primary antibody working solution diluted according to the recommended ratio (anti-SERP1 antibody dilution ratio of 1:1000) and incubated overnight in a shaker at 4 degrees Celsius. The next day, the membrane was washed 3 times with TBST. Secondary antibody was added and incubated in a shaker at room temperature for 2 h. The membrane was then washed 3 times with TBST. Equal volumes of ECL chemiluminescence substrate A and B were mixed and uniformly applied to the membrane in the dark. The membrane was then exposed and imaged in a fully automated chemiluminescence imaging system. The grayscale of the target band was analyzed using image analysis software. The relative expression level of the target protein was represented by the ratio of the grayscale value of the target protein band to that of the internal control β-actin band.

[0064] 2. Experimental Results Table 1. Effects of relative RAMP4 protein expression levels in each group of cells. The experimental results are shown in Table 1. Compared with the control group, the expression level of RAMP4 protein in 4T1 cells decreased significantly after treatment with the compounds of this invention, indicating that the compounds of this invention can effectively induce endoplasmic reticulum autophagy degradation. Among them, E8 showed the strongest effect.

[0065] Experimental Example 2: Test of the inhibitory activity of the compound of the present invention on the proliferation of tumor cells 1. Experimental Methods This experiment used the CCK-8 assay to evaluate the inhibitory activity of the compound on the proliferation of triple-negative breast cancer 4T1 cells. The specific method is as follows: 4T1 cells were seeded into 96-well plates and cultured adherently for 24 h. The compound of this invention was added at specific concentration gradients (0.1, 1, 1.5, 2.5, 5, 10, 25, 50, 100 μM) and treated for 24 h. The drug-containing medium was discarded, and medium containing 10% CCK-8 was added to each well. Incubation was continued for 1-2 h, and the absorbance (A) at 450 nm was measured using a microplate reader. 样品 The absorbance value A of DMSO 空白As a blank, the absorbance value A of the cell pores without drug treatment is used. 对照 As a control, the formula for calculating cell viability in the administration wells is: Viability (%) = (A 样品 –A 空白 ) / (A 对照 –A 空白 The half-maximal inhibitory concentration (IC50) of each compound was calculated using GraphpadPrism software, representing 100% of the total concentration. 50 ).

[0066] 2. Experimental Results Table 2. Inhibitory activity of compounds against the proliferation of 4T1 tumor cells The experimental results are shown in Table 2. Most compounds exhibited moderate to excellent antitumor activity. Among them, E8 showed the most significant activity, with an IC50 value of [missing value]. 50 The value reached 1.8 μM; E6 and E21 also showed strong inhibitory activity, IC50... 50 The values ​​were 2.5 μM and 2.6 μM, respectively; the compounds of the present invention show good potential as antitumor active substances.

[0067] Experimental Example 3: Test of the ability of the compound of this invention to target and induce endoplasmic reticulum autophagy (transmission electron microscopy) 1. Experimental Methods This experiment was conducted in triple-negative breast cancer 4T1 cells. Transmission electron microscopy (TEM) is the standard method for identifying morphological changes in autophagy; therefore, TEM was used to observe the cell microstructure to investigate the ability of compound E8 to induce endoplasmic reticulum degradation in 4T1 cells. The specific method is as follows: 4T1 cells were seeded in culture dishes and cultured adherently in an incubator for 24 h. Then, a certain concentration (2 μM) of E8 was added for 24 h. The drug-containing culture medium was discarded, and the cells were collected and pre-fixed with 2.5% glutaraldehyde. Subsequently, they were dehydrated stepwise with acetone, infiltrated, and embedded in pure Epon-812 embedding medium. Afterward, sections were stained, and images of copper mesh were acquired using a transmission electron microscope.

[0068] 2. Experimental Results The results are as follows Figure 1 As shown in AD, the rough endoplasmic reticulum (RER) in the control group (4T1 cells) had a normal morphology and structure (parallel sac-like structure with narrow intervesicular spaces and ribosomes attached to the cytoplasmic surface), with a few autolysosomes occasionally observed in the cytoplasm, and no RER fragments were found. In E8-treated cells, obvious vacuoles and autolysosomes were observed, and the RER morphology was abnormal (irregular sac-like structure with widened intervesicular spaces). RER fragments were clearly observed in some autolysosomes, confirming that compound E8 successfully targeted and induced autophagic degradation of the RER.

[0069] Experimental Example 4: Test of the ability of the compound of this invention to target and induce endoplasmic reticulum autophagy (fluorescence method) 1. Experimental Methods This experiment was conducted in triple-negative breast cancer 4T1 cells. The effect of compound E8 on the endoplasmic reticulum (ER) was detected using the ER-Tracker Green fluorescent probe staining method. ER-Tracker Green is a cell membrane-permeable green fluorescent probe for the ER, exhibiting high targeting specificity to the ER. It can be used for live-cell ER-specific fluorescence imaging, and its fluorescence intensity changes can reflect the integrity of the ER and the degree of autophagic degradation. The specific method is as follows: 4T1 cells were seeded in 6-well plates and cultured adherently for 24 h. After treatment with different concentrations of E8 (0.05, 0.2, 0.5, 2, 5 μM) for 24 h, ER-Tracker Green staining was performed, followed by fixation and mounting. The cells were then observed under a confocal microscope. The ability of compound E8 to target and induce ER autophagic degradation was evaluated by comparing the fluorescence (green) intensity of the ER in different groups.

[0070] 2. Experimental Results The results are as follows Figure 2 As shown in AF, compared with the blank group, the green fluorescence of each E8 treatment group was significantly weakened, and the intensity of green fluorescence decreased sequentially with the increase of E8 concentration, confirming that compound E8 can effectively target and induce endoplasmic reticulum autophagy degradation in a concentration-dependent manner.

[0071] Experimental Example 5: In vivo efficacy test in animals 1. Experimental Methods SPF-grade female BALB / c mice (weighing 18±2 g) were used for the experiment. Logarithmic growth phase 4T1 cells were collected, resuspended in sterile PBS, and each BALB / c mouse was injected with 1×10⁻⁶ cells in the right dorsal region. 6 The tumor, consisting of 4 T1 cells, grew to approximately 100 mm in size. 3 The experiment begins at [time]. The formula for calculating tumor volume is as follows: Tumor volume (mm²) 3 = Major axis (mm) × Minor axis (mm) 2 / 2. Tumor-bearing mice were randomly divided into two groups (blank group and E8 group), with 5 mice in each group. The blank solvent or E8 (10 mg / kg) was administered via tail vein on days 1, 4, 7, 10, and 13, respectively. Starting from day 1, mouse body weight and tumor volume were monitored every two days, and tumor volume growth curves and mouse body weight curves were plotted. After the experiment, the tumor-bearing mice were euthanized by cervical dislocation, and the tumor tissue was immediately separated, washed with PBS, blotted dry with filter paper, photographed, and weighed for recording.

[0072] 2. Experimental Results The results are as follows Figure 3As shown in A and 3C, compound E8 exhibited significant antitumor efficacy compared to the control group. There was no significant decrease in mouse body weight. Figure 3 B), demonstrating the E8's superior security.

[0073] Experimental Example 6: Fluorescence experiment to investigate the mechanism of action of the compound of the present invention 1. Colocalization with endoplasmic reticulum and LC3 Compound E8 exhibits autofluorescence (maximum emission wavelength: 470 nm), allowing direct observation of its co-localization with the key endoplasmic reticulum and autophagosome protein LC3. The specific method is as follows: 4T1 cells were seeded in 6-well plates and cultured adherently for 24 h. After treatment with E8 for 6 h, the cells were stained with ER-Tracker Green and LC3 antibodies, respectively. The cells were then fixed, mounted, and observed under a confocal microscope to detect fluorescence co-localization.

[0074] like Figure 4 As shown in Figure A, only the endoplasmic reticulum showed green fluorescence in the blank group cells, while the E8-treated group ( Figure 4 B) A distinct cyan fluorescence, resulting from the superposition of green and blue, was observed, exhibiting endoplasmic reticulum (ER) localization characteristics rather than diffuse distribution. This confirms that E8, after entering the cell, can effectively accumulate and stably localize on the ER, demonstrating excellent ER targeting ability. Similarly, Figure 4 CD analysis revealed a high degree of co-localization between E8 and LC3, with a distinct magenta fluorescence resulting from the superposition of red and blue fluorescence. This experiment successfully confirmed that compound E8 can simultaneously target and bind to the endoplasmic reticulum and LC3, and this spatial localization evidence provides important morphological support for its mechanism of action as an endoplasmic reticulum autophagy-targeting inducer.

[0075] 2. Degradation is completed via the autophagy-lysosome pathway. 4T1 cells were infected with the mCherry-EGFP-RAMP4 adenovirus, which can be localized to the endoplasmic reticulum membrane. Under normal circumstances, the red fluorescence of mCherry and the green fluorescence of EGFP coexist, overlapping to form a yellow color. When the endoplasmic reticulum is degraded via the autophagy-lysosomal pathway, the green fluorescence of EGFP is quenched in the acidic environment of the lysosome, leaving only the red fluorescence signal of mCherry. Therefore, the degradation of the endoplasmic reticulum via the autophagy-lysosomal pathway can be assessed by detecting intracellular fluorescence. The specific method is as follows: 4T1 cells were seeded in 6-well plates and cultured until the cell confluence reached 50%-70%. Viral infection was then performed, and stable fluorescence expression was confirmed under a fluorescence microscope. Compound E8 was then administered. After 24 h, the cells were fixed and mounted, and the fluorescence changes (the quantity and ratio of yellow and red fluorescence) were observed under a confocal microscope.

[0076] The results are as follows Figure 5 As shown in AB, the blank group exhibited obvious yellow fluorescence, while the green fluorescence of the E8-treated group was significantly reduced, and a large amount of red fluorescence was observed, indicating that E8 successfully targeted and induced the degradation of the endoplasmic reticulum via the autophagy-lysosome pathway.

[0077] 3. No effect on other organelles 4T1 cells were seeded into 6-well plates and cultured adherently for 24 h. After treatment with E8 for 24 h, the cells were divided into three groups. Staining was performed using ER-Tracker Green and Lyso-Tracker Red, Golgi-Tracker Green and Lyso-Tracker Red, and Mito-Tracker Green and Lyso-Tracker Red, respectively. The cells were then fixed and mounted, and the co-localization of the endoplasmic reticulum and lysosomes, Golgi apparatus and lysosomes, and mitochondria and lysosomes in the cells after E8 treatment was observed using confocal microscopy.

[0078] like Figure 6 As shown in Figure AB, after treatment with compound E8, a large amount of yellow fluorescence, formed by the superposition of green fluorescence from the endoplasmic reticulum and red fluorescence from lysosomes, was observed in the cells, suggesting that E8 can effectively promote the co-localization of the endoplasmic reticulum and lysosomes, successfully inducing endoplasmic reticulum autophagy. Meanwhile, in the Golgi apparatus and lysosomes (… Figure 6 CD), mitochondria and lysosomes ( Figure 6 In the colocalization experiment of E8, no yellow fluorescence was observed in either the blank group or the E8 group, indicating that compound E8 can specifically target and induce endoplasmic reticulum autophagy, without having a significant effect on the Golgi apparatus and mitochondria.

[0079] In summary, the above three experiments successfully demonstrated that the compound of the present invention can directly "bind" the endoplasmic reticulum to the autophagosome by simultaneously targeting and binding to the key protein LC3 on the endoplasmic reticulum and autophagosome membranes, and degrade it through the autophagy-lysosome pathway, without affecting other organelles.

Claims

1. The compound of formula I, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a tautomer thereof, or an isotopic compound thereof, or a metabolite thereof: Formula I in, R1 and R2 are each independently selected from OH or OR3; When R1 is OH, R2 is OR3; when R1 is OR3, R2 is OH; the two cannot both be OH or OR3 at the same time. R3 is -(CH2) n -O-(CH2)2-NHTs, where n is any integer from 3 to 11.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a tautomer thereof, or an isotopic compound thereof, or a metabolite thereof, characterized in that: n is 3, 5, 7, 9 or 11.

3. The compound according to any one of claims 1 or 2, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a tautomer thereof, or an isotopic compound thereof, or a metabolite thereof, characterized in that: The compound is selected from one of the following structures: 。 4. A method for preparing the compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a tautomer thereof, or an isotopic compound thereof, or a metabolite thereof, characterized in that: The method includes the following steps: Compound 1a was reacted with p-toluenesulfonyl chloride to give compound 2a; compound 2a was then reacted with compound 3a to give compound 4a; compound 4a was then deprotected to give compound 5a; compound 5a was then reacted with p-toluenesulfonyl chloride to give compound 6a; and compound 6a was then reacted with compound 7a to give the compound shown in Formula I.

5. A pharmaceutical composition, characterized in that: The pharmaceutical composition is a formulation prepared by adding pharmaceutically acceptable excipients to a compound as described in any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a tautomer thereof, or an isotopic compound thereof, or a metabolite thereof as the active ingredient.

6. Use of the compound of any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a tautomer thereof, or an isotopic compound thereof, or a metabolite thereof, or the pharmaceutical composition of claim 5, in the preparation of an endoplasmic reticulum autophagy inducer.

7. The use according to claim 6, characterized in that: The endoplasmic reticulum autophagy inducer is a drug for the prevention and / or treatment of tumors.

8. The use according to claim 7, characterized in that: The tumor is selected from breast cancer, lung cancer, liver cancer, kidney cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, bladder cancer, myelodysplastic syndrome, lymphoma, esophageal cancer, gastrointestinal cancer, osteosarcoma, and tumors of the central or peripheral nervous system.

9. The use according to claim 8, characterized in that: The tumor is breast cancer.

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