A compound with endoplasmic reticulum targeting and its application

Abstract

The present invention discloses a compound with endoplasmic reticulum targeting and its application. The compound has the following chemical formula: #imgabs0# The compound provided by the present invention has good endoplasmic reticulum targeting and the ability to induce the production of type I reactive oxygen species. In addition, the compound can induce time-dependent depletion of endoplasmic reticulum calcium in cancer cells under light conditions. More importantly, the existing anti-diabetic drug glibenclamide closes the ATP-dependent potassium ion channel (K) on the endoplasmic reticulum membrane. + ATP ), the compound-induced loss of endoplasmic reticulum calcium into the cytoplasm will lead to severe depolarization of the endoplasmic reticulum membrane potential, and ultimately activate the downstream endoplasmic reticulum and mitochondria-dependent apoptosis pathways, leading to cancer cell death, while also embodying the new use of glibenclamide.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a compound with endoplasmic reticulum targeting and application thereof. Background Art

[0002] Cancer cell resistance to anticancer drugs severely reduces the effectiveness of chemotherapy and shortens patient life expectancy. Cancer cells exploit diverse pathways to evade drugs, allowing them to survive treatment and acquire resistance. However, existing diagnostic and therapeutic approaches are unable to predict and fully characterize these resistance pathways. Drug combination therapy is a key strategy to mitigate the emergence of cancer cell resistance. While the emergence of cancer cell resistance is generally believed to be driven by genetic alterations, the use of a single anticancer drug can sometimes promote the emergence and proliferation of a more adaptable subpopulation within a tumor cell population. However, the combination of two or more drugs with different targets creates a differential selective pressure environment, making it difficult for tumor cells to overcome these barriers and ultimately effectively suppressing tumor growth. While anticancer drug combinations can help combat drug-resistant cancer cells, the limited selection of available drugs and the potential for increased toxicity and side effects associated with combination therapy still present numerous challenges. Therefore, developing novel, safe, and effective anticancer molecules and exploring their combination with existing drugs will not only facilitate the repurposing of established drugs but also provide new avenues for cancer treatment.

[0003] Aggregation-induced emission (AIE) is a unique activity possessed by a class of fluorescent molecules. These compounds exhibit a significantly enhanced photochemical effect upon aggregation, rapidly inducing the accumulation of reactive oxygen species and heat. Current research has found that AIE molecules generally possess good biocompatibility and exhibit excellent biological activity in the tracing and treatment of tumors and pathogenic microorganisms. In particular, some AIE molecules can not only specifically label key organelles of tumor cells, such as the nucleus, mitochondria, lysosomes, endoplasmic reticulum, and Golgi apparatus, but can also rapidly increase tumor tissue temperature and reactive oxygen species production under illumination, thereby achieving effective cancer cell killing. Furthermore, some studies have found that AIE molecules can regulate physiological processes such as autophagy and apoptosis, thereby inducing cancer cell death and ablation of cancerous tissue in animals. Despite this, the incomplete anti-cancer effect of AIE molecules remains.

[0004] Therefore, the existing technology needs to be further developed and improved. Summary of the Invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a compound with endoplasmic reticulum targeting and its application, aiming to solve the problem in the prior art that AIE molecules are not able to kill cancer cells completely when used to kill cancer cells.

[0006] The present invention provides a compound with endoplasmic reticulum targeting, wherein the compound has the following chemical structure:

[0007]

[0008] Where: R 1 is one of hydrogen, alkyl, alkynyl or heteroaryl; X is one of oxygen, sulfur or selenium; n is one of 0, 1 or 2; R 2 is one of hydrogen, alkyl, alkynyl, pyridyl or heteroaryl.

[0009] Preferably, R 1 is hydrogen; X is sulfur; n is 1; R 2 is methyl;

[0010]

[0011] A drug combination comprising the above-mentioned compound and the anti-diabetic drug glibenclamide.

[0012] A nanoparticle comprising the above-mentioned drug combination.

[0013] A use of the above-mentioned drug combination and nanoparticles in the preparation of anticancer drugs.

[0014] A method for preparing the above-mentioned compound having endoplasmic reticulum targeting, characterized by comprising:

[0015] 4-(5-bromothiophene)phenyldiphenylamine, 2,6-dimethylpyridine-4-boric acid, [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride and potassium carbonate were added to a three-necked reaction flask, and methanol and toluene were added respectively under nitrogen protection. After heating and reflux, the mixture was spin-dried and extracted with dichloromethane. After passing through a silica gel column, the endoplasmic reticulum-targeted compound (TPA-DMPy) was isolated.

[0016] Beneficial effects: Compared with the prior art, the compound provided by the present invention has good endoplasmic reticulum targeting and induction of type I reactive oxygen species production. In addition, the compound can induce time-dependent depletion of endoplasmic reticulum calcium in cancer cells under light conditions. More importantly, the existing anti-diabetic drug glibenclamide closes the ATP-dependent potassium ion channel (K + ATP), the compound-induced loss of endoplasmic reticulum calcium into the cytoplasm will lead to severe depolarization of the endoplasmic reticulum membrane potential, and ultimately activate the downstream endoplasmic reticulum and mitochondria-dependent apoptosis pathways, leading to cancer cell death, while also embodying the new use of glibenclamide. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 TPA-DMPy molecular formula and superoxide anion, singlet oxygen, and hydroxyl radical generation assays. (B) TPA-DMPy molecular absorption and emission; (C, D) TPA-DMPy emission wavelength curves in toluene / water solutions at different ratios; (E) Fluorescent dye absorption peak change detection of TPA-DMPy singlet oxygen generation; (F, G) Fluorescent dye emission peak change detection of TPA-DMPy hydroxyl radical and superoxide anion generation.

[0018] Figure 2 TPA-DMPy alone or co-labeled with a commercial dye to label the endoplasmic reticulum. (A) TPA-DMPy alone or co-labeled with the commercial dye ER Tracker Red. (B) Co-administration of TPA-DMPy and ER Tracker Red induces vacuolization of cancer cell membranes.

[0019] Figure 3 In vitro anticancer effects of TPA-DMPy when used alone or in combination with glibenclamide. (A) Changes in the viability of A549 cancer cells when TPA-DMPy was used alone or in combination with glibenclamide under light exposure; (B) Changes in the viability of Hela cancer cells when TPA-DMPy was used alone or in combination with glibenclamide under light exposure; (C) Effects of glibenclamide on the viability of Hela cancer cells; (D) Changes in the viability of Hela cancer cells when TPA-DMPy was used alone or in combination with glibenclamide under dark conditions; (E) Morphological changes in the endoplasmic reticulum of Hela cells were observed by confocal fluorescence microscopy after treatment with TPA-DMPy alone or in combination with glibenclamide; (F) Morphological changes in the endoplasmic reticulum and cell apoptosis in Hela cells were observed by high-resolution transmission electron microscopy after treatment with TPA-DMPy alone or in combination with glibenclamide.

[0020] Figure 4 In vitro anticancer effects of TPA-DMPy alone or in combination with glibenclamide. (A) Effects of TPA-DMPy alone or in combination with glibenclamide on the expression of autophagy- and apoptosis-related proteins in Hela cells under dark conditions; (B) Effects of TPA-DMPy alone or in combination with glibenclamide on the expression of autophagy- and apoptosis-related proteins in Hela cells under light conditions.

[0021] Figure 5TPA-DMPy combined with glibenclamide disrupts ER calcium potential. (A) Effects of TPA-DMPy alone or in combination with glibenclamide on ER calcium leakage in Hela cells under light conditions for 1 hour; (B) Effects of TPA-DMPy alone or in combination with glibenclamide on ER calcium leakage in Hela cells under dark conditions for 12 hours; (C) Effects of TPA-DMPy treatment on ER calcium leakage in Hela cells at different times under dark conditions; (D) Effects of TPA-DMPy and other drugs on ER calcium leakage in Hela cells under 12 hours.

[0022] Figure 6 The combination of TPA-DMPy and glibenclamide promotes subcutaneous tumor ablation in mice. (A) Comparison of subcutaneous tumor size among different treatment groups after mouse sacrifice; (B) Time course of subcutaneous tumor size changes among different treatment groups; (C) Comparison of subcutaneous tumor weight among different treatment groups after mouse sacrifice; (D, E, F) Serum analysis of changes in liver and kidney function in mice after treatment with TPA-DMPy and glibenclamide alone or in combination. DETAILED DESCRIPTION

[0023] The present invention is further illustrated below by way of examples, but the invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the commercial specifications. It should also be understood that after reading the contents of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims appended hereto.

[0024] Example 1 Preparation of compounds with endoplasmic reticulum targeting

[0025] 1. The preparation method of the compound with endoplasmic reticulum targeting comprises the following steps:

[0026] (1) 4-(5-Bromothiophene)phenyldiphenylamine (288 mg, 0.7 mmol), 2,6-dimethylpyridine-4-boric acid (134 mg, 0.875 mmol), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (51 mg, 0.07 mmol) and potassium carbonate (484 mg, 3.5 mmol) were added to a three-necked reaction flask. 5 mL of methanol (MeOH) and 5 mL of toluene were added under nitrogen protection, and the mixture was heated to reflux for 24 h.

[0027] (2) The product was dried by rotation, extracted with dichloromethane (DCM), and separated by silica gel column (DCM: MeOH = 200:1) to obtain TPA-DMPy.

[0028] 2. Preparation steps of nanoparticles:

[0029] 1 mg of TPA-DMPy, 1 mg of glibenclamide, and 5 mg of DSPE-mPEG2000 were added to 1 mL of tetrahydrofuran, and then added to 9 mL of deionized water. The mixture was sonicated for 2 min using a sonicator. The mixture was then transferred to a dialysis tube (MWCO 1000 Da) and dialyzed against deionized water to obtain a nanoparticle solution.

[0030] Example 2 Characterization of the Photodynamic Properties of TPA-DMPy Molecules

[0031] like Figure 1 As shown in the figure, by measuring the absorption and emission of the molecule in toluene / water solutions with different ratios, TPA-DMPy was confirmed to be a photosensitizer with AIE properties. Using specific fluorescent probes ABDA, HPF, and DHR123 to detect the production of reactive oxygen species under light, it was found that the ABDA absorption peak did not show a significant decrease, while the fluorescence of HPF and DHR123 increased slowly over 10 minutes, indicating that the molecule can slowly induce the production of superoxide anions and hydroxyl radicals (i.e., type I reactive oxygen species).

[0032] Example 3 Characterization of TPA-DMPy molecular organelle labeling function

[0033] Cancerous epithelial cells Hela were inoculated into a confocal microplate. After culturing for 24 hours, 5 μM AIEs molecules TPA-DMPy were added. After culturing for another 15 minutes, the cells were observed using a confocal microscope. Figure 2 As shown, TPA-DMPy molecules bind to perinuclear organelles in Hela cells. Co-staining with the commercial endoplasmic reticulum fluorescent dye ER Tracker Red further demonstrated that TPA-DMPy can bind to the endoplasmic reticulum of cancer cells, emitting bright green fluorescence. Furthermore, when TPA-DMPy and ER Tracker Red were co-labeled in Hela cells, rapid membrane vacuolation was observed, suggesting that the combination of these two molecules can strongly induce apoptosis. Since the commercial dye ER Tracker Red is conjugated to the drug glibenclamide and the fluorescent molecule BODIPYTR, we hypothesized that TPA-DMPy and glibenclamide may similarly accelerate the induction of cancer cell death.

[0034] Characterization of the cancer cell-killing function of TPA-DMPy molecules

[0035] Example 4 Figure 3As shown, even under illumination, 10 μM TPA-DMPy showed no significant toxicity to two cancerous epithelial cell lines, A549 and Hela. However, when 10 μM TPA-DMPy and 10 μM glibenclamide were added to the cell culture medium simultaneously, a large number of cancer cells died under illumination, with nearly 80% of the cells dying after only 1 hour of drug incubation and 10 minutes of illumination. More importantly, glibenclamide itself had no significant effect on cancer cell growth, and even in the dark, glibenclamide could still enhance the cytotoxicity of TPA-DMPy to Hela cells. Furthermore, confocal fluorescence microscopy and high-resolution transmission electron microscopy further confirmed that the combined application of TPA-DMPy and glibenclamide disrupted endoplasmic reticulum morphology and caused cell apoptosis.

[0036] Example 5: TPA-DMPy molecules combined with glibenclamide activate tumor cell apoptosis pathway

[0037] After confirming that Hela cells will undergo rapid and strong apoptosis after TPA-DMPy molecules are combined with glibenclamide, we lysed the cells and collected total proteins for Western Blot analysis. Figure 4 As shown, the combined use of TPA-DMPy and glibenclamide significantly increased the expression of the autophagy marker LC3 and the apoptosis-promoting proteins caspase-3 and caspase-12, while TPA-DMPy alone had no significant effect. These results indicate that the combined use of TPA-DMPy and glibenclamide strongly induces damage to the endoplasmic reticulum (ER). When autophagy fails to rescue cells, cancer cells ultimately undergo apoptosis through both the ER and mitochondrial pathways.

[0038] Example 6: Mechanism of apoptosis induction by TPA-DMPy molecules combined with glibenclamide

[0039] Glibenclamide is an ATP-dependent potassium channel (K + ATP) inhibitors can effectively inhibit the transport of potassium ions from the cytoplasm to organelles, causing depolarization of the organelle membrane potential. Furthermore, ER stress is often accompanied by ER calcium loss, and loss of ER membrane potential can lead to apoptosis. Based on the above background, we examined changes in ER calcium under the action of TPA-DMPy and found that TPA-DMPy significantly enhanced the outflow of ER calcium from cancer cells to the cytoplasm in a time-dependent manner, and this effect was enhanced under light. The combination of TPA-DMPy and glibenclamide did not further enhance ER calcium loss. Therefore, it is speculated that the combination therapy promotes cell apoptosis because, in the context of TPA-DMPy-induced ER calcium loss, glibenclamide prevents the influx of cytoplasmic potassium ions that can balance the membrane potential, ultimately leading to severe ER membrane potential depolarization and subsequent initiation of cell apoptosis.

[0040] Example 7: Combination of TPA-DMPy and Glibenclamide Promotes Ablation of Subcutaneous Tumors in Mice

[0041] Balb / c nude mice were subcutaneously inoculated with 3×10 6 All mice were divided into 4 groups: 1. blank group; 2. TPA-DMPy alone treatment group; 3. glibenclamide alone treatment group; 4. TPA-DMPy and glibenclamide combined treatment group. The size of the subcutaneous tumor was measured daily. When the tumor grew to 150 mm 3 The drug was injected into the tumor at about 1 hour, once every 4 days, for a total of 3 treatments. After each administration, the subcutaneous tumor site was illuminated for 20 minutes. The mice were killed 14 days after the first administration. The statistical results are as follows Figure 6 As shown, intratumoral injection of TPA-DMPy alone slowed the growth of subcutaneous tumors, while intratumoral injection of a premixed solution of TPA-DMPy and glibenclamide gradually reduced the size of subcutaneous tumors. Furthermore, TPA-DMPy and glibenclamide, alone or in combination, had no significant effect on liver and kidney function in mice, suggesting the high biosafety of AIE molecules.

[0042] In summary, the present invention provides a novel ER-targeting AIE molecule for use in combination with existing drugs for the treatment of cancer. Specifically, the present invention explores how the existing drug glibenclamide enhances the ability of an AIE photosensitizer, which induces ER calcium loss, to kill cancer cells, and the specific mechanism by which the AIE molecule rapidly induces cancer cell apoptosis when used in combination with the drug. This molecule exhibits excellent stability, high-resolution ER imaging, and photodynamic cancer therapy capabilities, enabling the rapid elimination of cancer cells in animals.

[0043] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A compound with endoplasmic reticulum targeting, characterized in that The compound is: 。 2. A drug combination, characterized in that The invention comprises the compound according to claim 1 and the antidiabetic drug glibenclamide.

3. A nanoparticle, characterized in that: The pharmaceutical combination according to claim 2.

4. Use of the pharmaceutical combination of claim 2 or the nanoparticles of claim 3 in the preparation of an anticancer drug; wherein the anticancer drug is an anticancer cell A549 or Hela.

5. A method for preparing the endoplasmic reticulum-targeting compound according to claim 1, characterized in that: include: 4-(5-bromothiophene)phenyldiphenylamine, 2,6-dimethylpyridine-4-boric acid, [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride and potassium carbonate were added to a three-necked reaction flask, and methanol and toluene were added respectively under nitrogen protection. After heating and reflux, the mixture was spin-dried and extracted with dichloromethane. The endoplasmic reticulum-targeted compound was separated after passing through a silica gel column.

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