Copper hyperplasia inhibitor for tumor chemoimmunotherapy and application of copper hyperplasia inhibitor

Abstract

The invention belongs to the technical field of medicines, and discloses a copper hyperplasia inhibitor for tumor chemoimmunotherapy and application of the copper hyperplasia inhibitor. The structure of the copper hyperplasia inhibitor is shown in the specification, wherein R is a copper chelating coordination group. The copper hyperplasia inhibitor can interfere with a copper hyperplasia signal channel, break the redox balance of tumor cells, trigger endoplasmic reticulum and mitochondrial stress and cause melanoma cell death. Meanwhile, the expression of matrix metalloproteinase can be down-regulated, and diffusion and metastasis of tumor cells are effectively inhibited. In addition, the copper hyperplasia inhibitor can also inhibit PHGDH expression to remodel macrophage subpopulation, promote immune stimulatory cell maturation, remodel tumor microenvironment and activate immune response to indirectly kill tumor cells, thereby enhancing the anti-tumor immune response effect. The copper hyperplasia inhibitor, namely CC, provided by the invention simultaneously has chemical treatment and immunoregulation functions, so that the problems of large action dose, poor immunotherapy effect and the like of a pure chelating agent are solved. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to a copper proliferation inhibitor for tumor chemoimmunotherapy and application thereof. Background Art

[0002] Copper (Cu) is an essential element for maintaining normal physiological functions and cell growth and metabolism, but its homeostasis imbalance (excess or deficiency) is associated with a variety of diseases, including tumor occurrence and development (Y. Yang, Coord. Chem. Rev. 2023, 495, 215395). In the tumor microenvironment, abnormal copper accumulation induced by mutation or drugs can induce copper death (cuproptosis) (P. Tsvetkov, Science 2022, 375, 1254-1261). Excessive copper promotes the growth and proliferation of tumor cells by regulating the activity of key proteins in tumors. This dependent growth and proliferation is called copper hyperplasia (cuproplasia) regulating cell growth and proliferation (EJ Ge, Nat. Rev. Cancer, 2022, 22, 102-113). The copper hyperplasia effect is partly due to the biological function of copper as a cofactor of mitochondrial cytochrome c oxidase, which is essential for the energy supply of rapidly proliferating cells. Many tumors, such as melanoma and triple-negative breast cancer, are highly invasive and lethal, and their progression is driven by multiple pro-oncogenic signaling pathways. Furthermore, extracellular matrix (ECM) remodeling can regulate the biological behavior of tumor cells, promoting their proliferation, migration, and invasion. Studies have shown that copper ion concentrations are significantly elevated in tumor tissues with active oxidative phosphorylation metabolism; copper promotes tumor malignancy by regulating the activity of key oncogenic proteins involved in tumor survival, proliferation, angiogenesis, and metastasis (N. Chrzan, Redox Biol., 2025, 81, 103552). Existing copper chelators, such as trientine (TETA) and tetrathiomolybdate (TTM), can inhibit tumor angiogenesis by chelating copper. However, their clinical application is limited by their limited antitumor efficacy and lack of direct cytotoxicity when used alone at high doses. Therefore, developing novel antitumor strategies that precisely inhibit the pro-proliferative effects of copper at low doses is of great clinical significance.

[0003] Tumors are susceptible to reactive oxygen and nitrogen species, and overactivation can overcome drug resistance in tumor cells (LQ Wang, Cell, 2018, 173, 1413-1425). Nitric oxide (NO) is an important gaseous signaling molecule that has attracted widespread attention in recent years due to its multiple roles in regulating cytotoxicity and immune function. The anti-cancer effect of NO is not limited to its own role, but can also be combined with other active molecules such as O2 ·- The reaction generates peroxynitrite (ONOO- ) and other reactive nitrogen (RNS) synergistic effects. - It can induce more severe cell damage and death, including DNA damage, protein nitration and lipid peroxidation, and ultimately lead to programmed cell death. ·- They all have short half-lives and are highly reactive and easily quenched by biomolecules.

[0004] Chemoimmunotherapy is a new combined anti-tumor strategy that integrates chemotherapy drugs and immune regulatory factors. This therapy exerts a synergistic effect through a dual mechanism of action: its chemotherapy component directly induces tumor cell death, while the immunomodulatory component can stimulate the body's immune response and reshape the tumor microenvironment to inhibit recurrence and metastasis, thereby reducing the dosage and improving the therapeutic index. However, current regimens mostly use a sequential combination of immune cell therapy and chemotherapy, or a combined infusion of regulators such as immune checkpoint inhibitors and chemotherapy drugs. In this traditional combination strategy, the components have different pharmacokinetic characteristics and distribution in the body, coupled with heterogeneity of the target sites and asynchronous time effects, resulting in difficulty in accurately controlling the local drug concentration ratio in the tumor, which can easily induce systemic toxic and side effects and weaken the synergistic anti-tumor efficacy. Summary of the Invention

[0005] In response to the problems existing in the prior art, the primary purpose of the present invention is to provide a copper proliferation inhibitor for tumor chemoimmunotherapy to solve the technical problems that the treatment dose of single copper chelators is large, the immunotherapy anti-tumor effect is poor, and there are many toxic side effects, while the existing anti-tumor immunotherapy methods are cumbersome and lack a single small molecule chemoimmunotherapy agent.

[0006] Another object of the present invention is to provide a method for preparing the copper proliferation inhibitor for tumor chemoimmunotherapy.

[0007] Another object of the present invention is to provide the use of the above copper proliferation inhibitor for tumor chemoimmunotherapy.

[0008] A copper proliferation inhibitor for tumor chemoimmunotherapy is obtained by connecting a chelating ligand to a benzenesulfonylfuran-based coumarin derivative capable of releasing NO. The general structural formula of the copper proliferation inhibitor is shown in the following formula (1):

[0009]

[0010] Wherein, R is a copper chelating ligand group.

[0011] Furthermore, the copper chelate ligand is selected from one of the groups having the following structural formula:

[0012]

[0013] Furthermore, the copper growth inhibitor comprises one of the following structures:

[0014]

[0015] A method for preparing the copper proliferation inhibitor for tumor chemoimmunotherapy comprises the following steps:

[0016] S1. Dissolving 3-carboxy-6-hydroxycoumarin in a solvent, adding 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) and a chelating agent ligand RH to react, removing the solvent after the reaction, and purifying to obtain a chelating agent precursor;

[0017] S2, dissolving the chelating agent precursor and K2CO3 in a solvent, adding 2-bromoethanol and heating to react, after the reaction is completed, adding the mixture into ice water, and continuing to stir until a precipitate is precipitated;

[0018] S3. Adding the precipitate from step S2 and 3,4-diphenylsulfonyl-1,2,5-oxadiazole 2-oxide into a solvent for reaction, and purifying after the reaction to obtain a copper proliferation inhibitor for tumor chemoimmunotherapy.

[0019] The molar ratio of 3-carboxy-6-hydroxycoumarin, 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) and chelating agent ligand RH described in step S1 satisfies the following requirements: 1 to 3:1 to 3:1.

[0020] The solvent in step S1 is DMF; the amount of the solvent used is sufficient to ensure sufficient reaction between the raw materials.

[0021] The reaction in step S1 is stirred at room temperature overnight.

[0022] In order to promote the condensation reaction to produce the target product, an acid binding agent may be added in step S1. The acid binding agent is at least one of N,N-diisopropylethylamine, triethylamine, pyridine, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]undecene-7, N-methylmorpholine, sodium carbonate, and potassium carbonate. The amount of the acid binding agent is 1 to 3 times the molar amount of the chelating agent ligand RH.

[0023] In step S2, the amounts of the chelating agent precursor, K2CO3 and 2-bromoethanol in S1 satisfy a molar ratio of 1:1-3:1-3.

[0024] The solvent in step S2 is DMF; the amount of the solvent is sufficient to ensure sufficient reaction between the raw materials;

[0025] The heating reaction in step S2 refers to heating to 65-80° C. and stirring the reaction for 10-24 hours.

[0026] In step S3 , the molar ratio of the precipitate of S2 to 3,4-diphenylsulfonyl-1,2,5-oxadiazole 2-oxide satisfies 1:1.2-5.

[0027] An acid binding agent may also be added in step S3, wherein the acid binding agent is at least one of N,N-diisopropylethylamine, triethylamine, pyridine, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]undecene-7, N-methylmorpholine, sodium carbonate, and potassium carbonate, and the amount of the acid binding agent is 1 to 3 times the molar amount of 3,4-diphenylsulfonyl-1,2,5-oxadiazole 2-oxide.

[0028] The solvent in step S3 is DCM. Preferably, 3,4-diphenylsulfonyl-1,2,5-oxadiazole 2-oxide is first added to DCM to form a DCM solution of 3,4-diphenylsulfonyl-1,2,5-oxadiazole 2-oxide, and then the precipitate from step S2 is added to the DCM solution of 3,4-diphenylsulfonyl-1,2,5-oxadiazole 2-oxide. The amount of solvent used is sufficient to ensure sufficient reaction between the raw materials.

[0029] The reaction in step S3 is stirred at room temperature for 10 to 24 hours.

[0030] The purification described in step S3 refers to washing with saturated brine after the reaction is completed, combining the organic layers, then adding anhydrous Na2SO4 for dehumidification, rotary evaporation to remove the organic solvent, and then adding methanol for precipitation. After filtering, the filter residue is recrystallized and purified with ethanol to obtain a copper proliferation inhibitor for tumor chemoimmunotherapy.

[0031] One object of the present invention is to provide the use of the copper proliferation inhibitor for tumor chemoimmunotherapy in the preparation of anti-tumor drugs.

[0032] Preferably, in the above application, the tumor is a primary and metastatic tumor, including but not limited to melanoma, breast cancer, lung cancer, colon cancer, pancreatic cancer, breast cancer, lung cancer, liver cancer, head and neck squamous cell carcinoma, osteosarcoma, thyroid cancer, salivary gland cancer, esophageal cancer, glioma, prostate cancer, thymoma, endometrial cancer, etc.

[0033] The copper proliferation inhibitor CC has the following effects: (1) it can chelate copper ions to release NO; (2) it can interfere with the intracellular redox balance; (3) it can cause endoplasmic reticulum stress and mitochondrial stress; and (4) it can inhibit the activity of matrix metalloproteinases and inhibit the spread and metastasis of tumor cells.

[0034] Another object of the present invention is to provide the use of the copper proliferation inhibitor for tumor chemoimmunotherapy in the preparation of a drug for enhancing immune response, wherein the copper proliferation inhibitor has the following effects: (1) inhibiting the expression of 3-phosphoglycerate dehydrogenase (PHGDH) in immune cell macrophages; (2) promoting macrophage metabolic reprogramming; (3) inducing macrophages in the tumor microenvironment to polarize toward the anti-tumor M1 phenotype; (4) increasing CD4 + and CD8 + T lymphocyte function and number; (5) can downregulate the proportion of regulatory T cells in tumors; (6) can reshape the tumor immune microenvironment.

[0035] The present invention has the following beneficial effects:

[0036] The present invention provides a copper proliferation inhibitor for intervening in copper proliferation, that is, a copper proliferation inhibitor is obtained by connecting a copper chelating ligand to a coumarin derivative based on benzenesulfonylfuran that can release NO. Studies have found that copper proliferation inhibitors can interfere with the copper proliferation signaling pathway, disrupt the redox balance of tumor cells, trigger endoplasmic reticulum and mitochondrial stress, and induce para-apoptosis in melanoma cells. Copper proliferation inhibitors effectively inhibit the spread and metastasis of tumor cells and also show significant anti-tumor effects in mouse organoid models. In addition, copper proliferation inhibitors can also reshape tumor-associated macrophages, promote the maturation of immunostimulatory cells, reshape the tumor microenvironment and activate immune responses through copper chelation, indirectly killing tumor cells, thereby enhancing anti-tumor immune response. The copper proliferation inhibitor provided by the present invention has both chemotherapeutic and immunomodulatory functions, thereby solving the problems of large dosage of simple chelating agents and poor immunotherapy effects. The present invention provides a theoretical and experimental basis for the design and preparation of copper proliferation inhibitors for enhancing anti-tumor immune responses. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0038] Figure 1 The chemical structural formula of the copper proliferation inhibitor CC of the present invention is:

[0039] Figure 2 is CC in Example 1 1H NMR (600 MHz, CDCl 3 ) spectrum;

[0040] Figure 3 HR-ESI-MS (CH3OH) spectrum of CC in Example 1;

[0041] Figure 4 is the ability of CC to chelate copper ions in solution (A) and the resulting copper complex (B);

[0042] Figure 5 The results of the copper content determination in cells (A) and cell supernatant (B) after A375 cells were co-incubated with CC (4 μM) for 12 h;

[0043] Figure 6 CC interference redox change image: GSH / GSSG ratio (A); intracellular O2 detected by flow cytometry ·- (B) and NO (C) as well as total intracellular ROS / RNS (D) detected by laser confocal microscopy;

[0044] Figure 7 Figure 3 shows the killing effect of CC in melanoma organoids (48 h): Bright field microscope image and H&E image (A); Confocal microscopy imaging (B);

[0045] Figure 8 The cell survival rate of A375 cells after incubation with CC (4 μM) and different metal ions for 36 h;

[0046] Figure 9 CC (15 mg kg -1 or 30 mg kg -1 ) on the growth of B16-F10 melanoma transplanted tumors in C57BL / 6 mice (10 days). Effect diagram: tumor images of mice (A), tumor weight and inhibition rate (B), and body weight changes (C).

[0047] Figure 10 CC (15 mg kg -1 or 30 mg kg -1 ) on the effect of macrophages in tumors of C57BL / 6 mice bearing B16-F10 melanoma for 10 days: Flow cytometry results (A), immunosuppressive cell subset CD206 + Quantitative analysis (B), and its effect on immunostimulatory CD86 + Quantitative analysis (C);

[0048] Figure 11 CC (15 mg kg -1 or 30 mg kg -1) Effect of T cells in tumors of C57BL / 6 mice bearing B16-F10 melanoma for 10 days: Flow cytometry analysis of CD4 + and its quantitative analysis (A), flow cytometry determination of CD8 + and its quantitative analysis (B), immunofluorescence detection of regulatory T cells (C);

[0049] Figure 12 CC (15 mg kg -1 or 30 mg kg -1 ) Quantification of cytokine secretion in mouse serum after 10-day treatment of C57BL / 6 mice bearing B16-F10 melanoma: IFN-γ (A), TNF-α (B), IL-1β (C), and TGF-β1 (D);

[0050] Figure 13 Transmission electron microscopy morphological images of A375 cells before and after treatment with CC (4 μM) for 6 h;

[0051] Figure 14 Images showing changes in intracellular matrix metalloproteinase activity after co-incubation of CC with A375 melanoma cells: Western blotting (A), protein quantification analysis (B), and organoid immunofluorescence (C) and (D);

[0052] Figure 15 Images showing changes in intracellular PHGDH expression after co-incubation of CC with tumor-associated macrophages: RT-qPCR quantitative analysis (A), and immunoblotting (B). DETAILED DESCRIPTION

[0053] In the following description, specific details such as specific system structures and technologies are provided for the purpose of illustration rather than limitation to facilitate a thorough understanding of the embodiments of the present invention. However, it should be clear to those skilled in the art that the present invention may also be implemented in other embodiments without these specific details.

[0054] Example 1. Preparation of CC and RC

[0055] The synthesis route of copper proliferation inhibitor CC is as follows:

[0056]

[0057] S1. 3-carboxy-6-hydroxycoumarin (247.20 mg, 1.20 mmol) was dissolved in anhydrous DMF (15 mL) solution, and 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU, 467.70 mg, 1.23 mmol) and 3,6,12,15-tetrathio-9-amino-heptadecane (313.6 mg, 1.00 mmol) were added, followed by N,N-diisopropylethylamine (161.55 mg, 1.25 mmol) and stirred at room temperature overnight. After the reaction was completed, DMF was removed under reduced pressure. Purification by rapid column chromatography was performed by gradient elution with a mixed solution of DCM and MeOH to obtain a chelating agent precursor;

[0058] S2. Dissolve the chelating agent precursor (250.00 mg, 0.58 mmol) and K2CO3 (172.20 mg, 1.25 mmol) in anhydrous DMF (10 mL). Add 2-bromoethanol (124.50 mg, 1.00 mmol) and stir at 80°C for 12 h. After the reaction is complete, add the mixture dropwise to ice water and continue stirring for 30 min to precipitate a precipitate.

[0059] S3. 1,8-Diazacyclo[5,4,0]undecene-7 (DBU) (87.90 mg, 0.58 mmol) was added to a solution of dichloromethane (20 mL) containing the precipitate of S2 (150.00 mg, 0.28 mmol) and benzenesulfonylfuran N-oxide (201.40 mg, 0.55 mmol, cas: 66074-00-8). The mixture was stirred at room temperature for 12 h and then washed with saturated brine (3 × 10 mL). The organic layers after extraction were combined and dehumidified with anhydrous Na2SO4. The organic solvent was removed by rotary evaporation to obtain a crude product. Methanol was added for precipitation. After filtration, the residue was purified by recrystallization from ethanol to obtain CC as a white powdery solid with a yield of approximately 56.1%. 1 H NMR (600MHz, CDCl3): δ (ppm) 8.03-8.05 (d, 2H) 7.95-7.86 (s, 1H) 7.78-7.7 2(t,1H)7.62-7.55(t,2H)7.52-7.48(d,2H)6.98-6.93(d,1H)6.90-6.86(d ,1H)4.84-4.80(d,2H)4.52-4.42(d,2H)3.72-3.62(t,2H)3.56-3.48(t,2 H)2.92-2.78(m,8H)2.68-2.62(m,4H)2.60-2.45(m,4H)1.28-1.18(m,6H). C 32 H39 ESI-MS analysis of N3O9S5 (positive ion mode, methanol solution) showed the following characteristic ion peaks (calculated values ​​in brackets): m / z 770.1337 (770.1363), [M+H] + ;792.5930(792.1182),[M+Na] + .

[0060] The control compound RC was synthesized by a similar method to that of CC, except that the 3,6,12,15-tetrathio-9-amino-heptadecane in step S1 was replaced with an equal amount of dioctylamine. The other parameters were the same as those in Example 1.

[0061] The synthetic route of the control compound RC is as follows:

[0062]

[0063] Example 2. CC chelating ability

[0064] The bathocuproin disulphonate disodium salt (BCS) method was used to evaluate the Cu + Chelating ability. Prepare 10μM [Cu(MeCN)4][PF6] copper ion solution, 5μM CC solution and 10μM BCS solution respectively. Take 100μL of the prepared BCS solution and mix it with the following groups: deionized water; 8μL copper ion solution; 8μL CC solution; 8μL CC solution and 8μL copper ion mixed solution. The total volume of the mixed samples is 600μL, and the rest is made up with deionized water. The sample is placed at room temperature to react for 30 minutes, and then the mixed solution is detected by UV-spectrophotometer to read the absorbance of the copper-BCS complex at 480nm. The test results are shown in Figure 4 .Depend on Figure 4 The results show that CC has a stronger chelating effect on Cu than BCS. + ability and could serve as a potential copper proliferation inhibitor.

[0065] The copper concentration in cells and cell supernatant was detected by inductively coupled plasma mass spectrometry (ICP-MS). 5 The cells were seeded at a density of 100 μg / mL in 6-well plates and placed in a 37°C, 5% The cells were cultured in an incubator for 12 to 24 hours. Subsequently, fresh DMEM medium containing CC (4 μM) was replaced and incubated for another 12 hours. The supernatant was collected and the cells were washed twice with cold PBS. Subsequently, the cells were digested with trypsin and collected by centrifugation at 1000 rpm for 4 minutes. The cell pellet and supernatant were digested with aqua regia and analyzed by ICP-MS. The results are shown in Figure 5 .Depend on Figure 5The results showed that the intracellular copper content of A375 cells was significantly reduced, while the extracellular copper content was significantly increased. This suggests that CC may excrete intracellular copper outside the cell by chelating intracellular copper and promoting its metabolism, further demonstrating that the copper proliferation inhibitor CC can interfere with copper homeostasis.

[0066] Example 3. CC interferes with redox balance

[0067] GSH and GSSG detection kits were used to detect intracellular GSH / GSSG. A375 cells were cultured at 6×10 5 Cells were seeded into 6-well plates at a density of 10 cells / well and cultured for 12–24 hours. After removing the culture medium, fresh DMEM containing CC (4 μM) was added and incubated for an additional 24 hours. Attached cells were washed twice with PBS, trypsinized, harvested, and washed again with 1 mL of PBS. The harvested cells were then processed according to the instructions for the GSH and GSSG detection kits.

[0068] The generation of intracellular ROS / RNS was detected by confocal laser scanning microscopy (CLSM). A375 cells were seeded in a confocal dish (1×10 5 / dish), after culturing for 12 to 24 hours, the cells were treated with control solution (PBS, step S1 product CSD (4 μM) and compound RC (4 μM)) or CC (4 μM) for 10 hours. Subsequently, they were incubated with fresh culture medium containing dihydroethidium (DHE), 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM-DA) and dichlorodihydrofluorescein-acetoacetate (DCFH-DA) for 30 minutes, washed 3 times with PBS, and then washed three times with PBS and imaged under CLSM. At the same time, O2 was measured using flow cytometry. ·- and NO levels were quantitatively detected.

[0069] See the results Figure 6 .Depend on Figure 6 The results showed that the GSH / GSSG ratio decreased from 7.22 to 1.78 after CC treatment, indicating a weakened antioxidant capacity of the cells. Furthermore, intracellular ROS / RNS levels increased significantly, potentially damaging the endoplasmic reticulum or mitochondria of cancer cells. CC exhibits significant GSH responsiveness, triggering a burst of RNS and causing oxidative stress. This property offers significant potential for effectively altering the reducing microenvironment of tumors and achieving tumorigenic effects.

[0070] Example 4. Comparison of the anticancer activity of the copper proliferation inhibitor CC in vivo and in vitro

[0071] The cytotoxicity was assessed using a CCK-8 kit. Human melanoma A375, mouse melanoma B16-F10, human breast cancer MDA-MB-435, mouse breast cancer 4T1, human colon cancer SW480, and human renal cortical proximal tubule epithelial cells HK-2 were cultured at 5×10 cells per well. 3 The cells were seeded into 96-well plates at a density of 100 μL. After culturing for 12 to 24 hours, the cells were treated with PBS, fresh DMEM culture medium with different concentrations of CC, fresh DMEM culture medium with different concentrations of RC, and fresh DMEM culture medium with different concentrations of TETA (TETA is a commercial chelating agent, trientine). After 36 hours of co-incubation, 10 μL of CCK-8 solution was added to each well, incubated in the dark for 1 hour, and then the absorbance was measured at a wavelength of 450 nm. Three parallel samples were made under each experimental condition and the standard deviation (± SD) was calculated. The stock solution of the compound was prepared in DMSO and diluted to different concentrations (DMSO concentration was less than 0.1%). The results are shown in Table 1. CC has a high selectivity and a strong killing effect on human melanoma A375 cells, and a low toxicity to normal cells. By comparing the half inhibitory concentration (IC 50 ), reflecting the great advantage of CC as a new copper proliferation inhibitor.

[0072] Table 1 IC of different cell lines after treatment with CC, RC, CSD and TETA for 36 h 50 (μM). Data are expressed as mean ± standard deviation (SD, n=3).

[0073]

[0074] In addition, the anti-cancer effect of CC was evaluated using an organoid model (PDOs). Organoids grown to a certain size were evenly divided into 96-well ultra-low adhesion cell culture plates and CC was added according to experimental requirements. After 48 hours of drug treatment, the activity of the organoids was evaluated using CellCounting-Lite 3D reagent according to the manufacturer's instructions. The test results are shown in Figure 7 .Depend on Figure 7 The results showed that CC significantly reduced the proliferation of PDOs and disintegrated many organoids after treatment, as observed by brightfield (BF) imaging. The effect gradually increased with increasing CC concentration. Live / dead staining also confirmed CC's cytotoxicity, consistent with H&E staining results. These results further confirm CC's significant cytotoxicity against melanoma.

[0075] The “metal ion remediation experiment” was used to test whether the effect of CC was related to copper: Cu 2+ (10μM), Zn 2+ (10μM), Fe2+ (10 μM), Mg 2+ (10μM), Mn 2+ (10 μM) and Ca 2+ A375 cells were pre-incubated with CC (10 μM) for 12 h, and then incubated with CC (4 μM) for 36 h before measuring cell viability. Figure 8 .Depend on Figure 8 The results showed that 4 μM CC alone could significantly kill tumor cells after 36 h of incubation; 2+ After that, the cell-killing effect of CC was successfully offset, indicating that Cu 2+ The presence of copper can effectively prevent the cytotoxic effect of CC, and the consumption of copper is crucial for the anti-proliferative activity of CC.

[0076] The present invention uses a B16-F10 melanoma mouse model to test the inhibitory effect of CC on tumor growth. The specific implementation method is as follows: C57BL / 6 mice (n=24, 5-6 weeks old, female) were injected subcutaneously on the right side with 5×10 5 B16-F10 cells. When the tumor volume reaches 50-60mm 3 The patients were randomly divided into four groups. They were given normal saline, trientine (TETA, dose 30 mg kg -1 ) or CC (dose 15 mg kg -1 and 30 mg kg -1 ) were injected intratumorally every two days, and the tumor size and body weight were recorded. Tumor volume was calculated according to the following formula: 0.5 × length × width 2 To monitor tumor growth, the results are shown in Figure 9 .Depend on Figure 9 The results showed that low-dose CC (15 mg kg -1 ) showed a moderate tumor inhibition effect, with a tumor inhibition rate of 64.0%. High-dose CC (30 mg kg -1 ) reached an inhibition rate of 79.2%. There was no significant difference in tumor growth rate between the TETA-treated and saline-treated mice, indicating that CC can effectively inhibit tumor growth.

[0077] Example 5. Copper proliferation inhibitor reprograms macrophage phenotype and its effect on the tumor immune microenvironment in mice

[0078] Immune cells from C57BL / 6 mice were stained with different antibodies and analyzed by flow cytometry. Female C57BL / 6 mice (4-8 weeks old) were purchased. 2×10 6 B16-F10 cells were suspended in 100 μL PBS. The mixture was injected subcutaneously into the right hind leg of the mouse. When the tumor volume reached about 60 mm3 PBS, TETA (30 mg kg -1 ) and CC (15 or 30 mg kg -1 ) treatment (4 mice per group), once every two days. On the 10th day, blood was collected from the mouse eyeball vein, serum was collected and the mice were euthanized. Tumor tissue was removed from mice in different groups. The tumor tissue was mechanically ground to prepare a single cell suspension. Then, the cell suspension was collected by centrifugation at 1500rpm for 5 minutes, and then treated with red blood cell lysis buffer at room temperature for 5 minutes to remove red blood cells, and washed twice with cold PBS. The cells were stained with the corresponding markers, including macrophages (CD45 + CD11b + f / 4 / 80 + CD86 + / CD206 + ) and T cells (CD45 + CD3 + CD4 + CD8 + ) were immediately analyzed by flow cytometry. Serum was collected and used to measure immune cytokines. IFN-γ, TNF-α, TGF-β1, IL-1β, IL-10, and IL-6 secretion in mice was measured by ELISA according to the manufacturer's instructions.

[0079] See the results Figure 10-12 .Depend on Figure 10-12 The results showed that after treatment with different doses of CC, CD11b + f / 4 / 80 + CD86 + The percentages of M1 were 10.6% and 14.8%, respectively, while CD11b + f / 4 / 80 + CD206 + The percentages of M1 and M2 TAMs were 25.8% and 17.7%, respectively. This suggests that CC can promote the generation of M1 phenotype TAMs and reduce the expression of M2 phenotype, thereby reshaping the tumor microenvironment. This effect also promotes the increase in the immune function and number of effector T cells and reduces the number of immunosuppressive regulatory T cells (Tregs).

[0080] Example 6. CC causes endoplasmic reticulum stress and mitochondrial stress

[0081] Endoplasmic reticulum stress and mitochondrial stress were assessed by observing cell morphology under transmission electron microscopy. 6) After pre-incubation with 2μM cycloheximide (CHX) for 1 hour, the cell samples were incubated with CC (4μM) for 6 hours to collect. At 4°C, the cell pellets were fixed with 2.5% electron microscopy fixative for 2 hours, then washed three times with PBS, and fixed with 1% OsO4. The samples were dehydrated by gradually soaking in acetone solutions of different concentrations (50%, 75%, 90%, 100%), and then soaked in resin acetone solutions of different concentrations (25%, 50%, 75%, 100%) for 24 hours. Finally, the samples were collected on copper grids and TEM images were obtained by HitachiTEM system. The results are shown in Figure 13 .Depend on Figure 13 The results showed that after CC treatment, the mitochondria in the cells were significantly swollen (indicated by arrows) and the cristae structure was damaged. The endoplasmic reticulum structure was blurred (indicated by arrows). At the same time, extensive vacuolation was observed in the cytoplasm (indicated by asterisks), indicating that the copper proliferation inhibitor CC caused endoplasmic reticulum stress and mitochondrial stress.

[0082] Example 7. CC inhibits metalloproteinase expression

[0083] Metalloproteinase expression was assessed by immunoblotting and immunofluorescence. A375 cells were plated at 1 × 10 5 Cells were seeded at a density of 100 μg / mL in a 6-well plate and cultured for 12 to 24 hours. The cells were then treated with PBS and CC (4 μM) for 36 hours, respectively. The "metal ion remediation experiment" followed the same method as in Example 4. The cells were then collected and lysed, and the cell supernatant was collected by centrifugation. A certain amount of the supernatant was diluted and diluted, and the protein concentration at a wavelength of 590 nm was measured by adding Coomassie Brilliant Blue. The remaining supernatant was supplemented with deionized water and loading buffer at the same protein concentration, and then placed in a 95°C metal bath for protein denaturation. An equal amount of protein sample was added to each well and separated on an SDS-PAGE gel. The protein bands were transferred to a PVDF membrane by membrane transfer. After blocking, the samples were incubated with primary and secondary antibodies in sequence to detect the expression of FOSL1, MMP2, and MMP9. Protein bands were observed using an enhanced chemiluminescence system, and relative grayscale values ​​were calculated using ImageJ.

[0084] After administration of various compounds, organoid samples were collected and fixed in 4% PFA. Fixed PDOs were cryoprotected and paraffin-embedded tumor tissue sections were prepared. Sections were washed twice in xylene (10 minutes each) and then deparaffinized by sequential washes in anhydrous ethanol, 95% ethanol / H2O, 85% ethanol / H2O, 75% ethanol / H2O, 50% ethanol / H2O, and deionized water (5 minutes each). Sections were then heated in antigen retrieval buffer for 15 minutes, cooled, and washed three times with PBS. After briefly drying the sections, a water-blocking ring was drawn around the tissue using a histochemical pen and permeabilized with 0.5% Triton X-100 in PBS for 15 minutes. The sections were then washed with cold PBS for 5 minutes and blocked within the ring with 0.5% BSA in PBS for 30 minutes. Primary antibodies against MMP2 and MMP9 (antibodies at a 1:100 dilution) were prepared in PBS and incubated overnight at 4°C. The sections were then washed with PBS and incubated with fluorescent secondary antibodies (1:100 dilution) for 1 hour at room temperature. After removing the secondary antibody, anti-fluorescence quenching mounting solution containing DAPI was added to the tissue sections, covered with a coverslip, and incubated in the dark for 15 minutes. CLSM was then used for immediate observation and analysis.

[0085] See the results Figure 14 .Depend on Figure 14 Results showed that compared with the control group, CC significantly reduced the expression levels of FOSL1, MMP2, and MMP9 in A375 cells. The effect of CC was attenuated after copper remediation, demonstrating that this process is related to copper chelation. Inhibition of this matrix metalloproteinase may effectively inhibit melanoma invasion and metastasis.

[0086] Example 8. Effect of copper proliferation inhibitors on 3-phosphoglycerate dehydrogenase

[0087] The expression of PHGDH gene and protein was detected by real-time qPCR (RT-qPCR) and immunoblotting. The attached human mononuclear macrophage THP-1 cells were seeded into 6-well plates and 20 ng mL -1 IL-4 and 20 ng mL -1Cells were cultured in fresh 1640 medium containing IL-13 for 48 h to induce cell polarization. Subsequently, fresh medium containing 1% FBS and 4 μM CC was used and incubated for another 48 h. The medium was discarded, and THP-1 cells under different treatment conditions were collected and rinsed with PBS. Total RNA was extracted using a cell / tissue genomic RNA extraction kit (RC112-01, Vazyme, Nanjing, Jiangsu). The extracted RNA was reverse transcribed into cDNA using a HiScript III RTSuperMix (R323-01, Vazyme, Nanjing, Jiangsu). Subsequently, a 20 μL reaction mixture was prepared: 10 μL SoFast EvaGreen Supermix (Bio-Rad, Hercules, CA, USA), 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), 1 μL DNA template (1 μg), and 7 μL deionized water. The amplification curve of the PCR product was measured using a Bio-Rad real-time PCR system. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference gene and 2 -ΔΔCt The RT-qPCR results were analyzed by immunoblotting. The specific implementation of immunoblotting was the same as in Example 8. The results are shown in Figure 15 .Depend on Figure 15 The results showed that CC treatment significantly downregulated both PHGDH gene and protein expression levels. Furthermore, under the action of CC-chelated copper, downregulation of PHGDH not only reduced the expression of arginase 1 (ARG1), a marker specific for M2 macrophages, but also upregulated the expression of iNOS, a marker specific for M1 macrophages. This suggests that CC can affect the phenotype of macrophages and promote polarization from M2 to M1 by inhibiting PHGDH in the serine biosynthesis pathway.

[0088] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A copper proliferation inhibitor for tumor chemoimmunotherapy, characterized in that The general structural formula of the copper growth inhibitor is shown in the following formula (1): Wherein, R is a copper chelate ligand group, and the copper chelate ligand is selected from one of the groups having the following structural formula:

2. The copper proliferation inhibitor for tumor chemoimmunotherapy according to claim 1, characterized in that The structure of the copper proliferation inhibitor is shown below:

3. A method for preparing a copper proliferation inhibitor for tumor chemoimmunotherapy according to any one of claims 1 to 2, characterized in that The following steps are involved: S1. Dissolving 3-carboxy-6-hydroxycoumarin in a solvent, adding 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate and a chelating agent ligand RH to react, removing the solvent after the reaction, and purifying to obtain a chelating agent precursor; S2, dissolving the chelating agent precursor and K2CO3 in a solvent, adding 2-bromoethanol and heating to react, after the reaction is completed, adding the mixture into ice water, and continuing to stir until a precipitate is precipitated; S3. Adding the precipitate from step S2 and 3,4-diphenylsulfonyl-1,2,5-oxadiazole 2-oxide into a solvent for reaction, and purifying after the reaction to obtain a copper proliferation inhibitor for tumor chemoimmunotherapy.

4. The method for preparing the copper proliferation inhibitor for tumor chemoimmunotherapy according to claim 3, characterized in that: The molar ratio of the 3-carboxy-6-hydroxycoumarin, 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate and the chelating agent ligand RH described in step S1 is 1 to 3:1 to 3:1; The solvent described in step S1 is DMF; The reaction in step S1 is stirred at room temperature overnight; In step S1, an acid binding agent is further added, wherein the acid binding agent is at least one of N,N-diisopropylethylamine, triethylamine, pyridine, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]undecene-7, N-methylmorpholine, sodium carbonate, and potassium carbonate. The amount of the acid binding agent is 1 to 3 times the molar amount of the chelating agent ligand RH.

5. The method for preparing the copper proliferation inhibitor for tumor chemoimmunotherapy according to claim 3, characterized in that: In step S2, the amounts of the chelating agent precursor, K2CO3 and 2-bromoethanol in S1 satisfy the molar ratio of 1:1-3:1-3; The solvent described in step S2 is DMF; The heating reaction in step S2 refers to heating to 65-80° C. and stirring the reaction for 10-24 hours.

6. The method for preparing the copper proliferation inhibitor for tumor chemoimmunotherapy according to claim 3, characterized in that: In step S3, the molar ratio of the precipitate of S2 to 3,4-diphenylsulfonyl-1,2,5-oxadiazole 2-oxide satisfies: 1:1.2-5; In step S3, an acid binding agent is further added, wherein the acid binding agent is at least one of N,N-diisopropylethylamine, triethylamine, pyridine, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]undecene-7, N-methylmorpholine, sodium carbonate, and potassium carbonate, and the amount of the acid binding agent is 1 to 3 times the molar amount of 3,4-diphenylsulfonyl-1,2,5-oxadiazole 2-oxide. The solvent described in step S3 is DCM; The reaction in step S3 is stirred at room temperature for 10 to 24 hours.

7. Use of the copper proliferation inhibitor for tumor chemoimmunotherapy according to any one of claims 1 to 2 in the preparation of antitumor drugs.

8. Use of the copper proliferation inhibitor for tumor chemoimmunotherapy according to claim 7 in the preparation of anti-tumor drugs, characterized in that: The tumor is a primary and metastatic tumor, including but not limited to at least one of melanoma, breast cancer, lung cancer, colon cancer, pancreatic cancer, breast cancer, lung cancer, liver cancer, head and neck squamous cell carcinoma, osteosarcoma, thyroid cancer, salivary gland cancer, esophageal cancer, glioma, prostate cancer, thymoma, and endometrial cancer.

9. Use of the copper proliferation inhibitor for tumor chemoimmunotherapy according to any one of claims 1 to 2 in the preparation of a drug for enhancing immune response.

10. A single-molecule chemoimmunotherapeutic agent for enhancing anti-tumor immune response, characterized in that: The active ingredient is the copper proliferation inhibitor according to claim 1 or 2.

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