An osthole compound, a metal complex, and its preparation method and application

Abstract

The present invention discloses an osthole compound, a metal complex, a preparation method, and applications thereof. The osthole compound (OST-Alkyne) has significant anti-tumor biological activity. OST is further linked to a cyclometallated iridium precursor via a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, exhibiting stronger anti-tumor activity and targeting mitochondria to kill tumors by reducing mitochondrial membrane potential and inducing cell autophagy and necroptosis. The present invention combines natural products with metals, not only achieving good selectivity and anti-proliferation ability for brain glioma cells, but also overcoming toxic side effects on normal cells. This provides a new design concept for novel metallodrugs for treating brain gliomas and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to an osthole compound, and also relates to a metal complex of the osthole compound, as well as a preparation method and application of the compound and the metal complex. Background Art

[0002] Osthole is a natural coumarin derivative isolated from the fruit of Cnidium monnieri. Studies have shown that osthole exerts its effects by inhibiting cancer cell proliferation, inducing apoptosis, inhibiting invasion and migration, and inhibiting tumor angiogenesis. Researchers have modified the osthole skeleton to obtain a series of osthole derivatives, which also have excellent anti-tumor activity. Existing technologies for the study and improvement of the osthole skeleton include modification of the coumarin lactone ring, modification of the coumarin 7-substituent, modification of the coumarin 8-substituent, and modification of multiple sites of coumarin. Farooq et al. have shown that the coumarin lactone ring-modified derivatives have significant anti-tumor activity in human colon cancer cell lines such as Colo-205 and human non-small cell lung cancer cell lines A549. They can induce apoptosis in human colon cancer cell lines Colo-205 by inducing a decrease in mitochondrial membrane potential. Zhang et al. reported that the introduction of piperazine, tetrahydropyrrole, and aromatic amine structures into the 7-substituent of coumarin can enhance the neuroprotective properties of osthole. However, these compounds lack specificity in cancer treatment, and further research is needed to identify specific compounds for specific treatments.

[0003] Compared to traditional platinum-based anti-tumor metallodrugs, iridium complexes offer advantages such as high stability, good water solubility, excellent luminescence, multiple coordination sites, ease of conversion, and high catalytic activity. They can form stable complexes with O^O, C^N, and N^N bidentate ligands and are therefore widely used in fields such as organic electroluminescence, bioluminescent probes, chemical sensors, and catalytic synthesis. Cyclometallated iridium(III) complexes possess unique properties such as DNA binding, inhibition of protein-protein interactions, catalytic oxidation, and inhibition of protein kinase activity, making them a significant anti-cancer agent. However, cyclometallated iridium(III) complexes often exhibit significant cytotoxicity, harming normal cells while simultaneously combating cancer. Therefore, there is an urgent need to optimize the structure of these metal complexes, reduce their toxic side effects, improve their efficacy, and explore new therapeutic strategies. Summary of the Invention

[0004] Purpose of the invention: The purpose of the present invention is to provide an osthole compound with significant anti-tumor biological activity, and also to provide an osthole compound metal complex with enhanced anti-cancer activity and reduced toxic side effects, as well as the preparation method and application of the compound and its metal complex.

[0005] Technical solution: The present invention discloses an osthole compound (OST-Alkyne), the structure of which is shown in Formula I:

[0006]

[0007] The method for preparing the above-mentioned osthole-related compounds comprises the following steps: dissolving cysteine ​​(Cys), sodium hydride (NaH), and osthole (OST) in an organic solvent under an inert atmosphere, separating and purifying the crude product OST-OH generated by the reaction, adding propidium bromide and dissolving it in an organic solvent, and separating and purifying the product OST-Alkyne after the reaction to obtain the osthole-related compounds:

[0008]

[0009] The molar ratio of cysteine, sodium hydride and osthole is 2-10:1-5:1, and the molar ratio of OST-OH and propyne bromide is 2-10:1.

[0010] The organic solvent is N,N-dimethylformamide or acetamide, the separation and purification adopts column chromatography, and the reaction temperature is 25 to 150° C. and the reaction time is 2 to 32 hours.

[0011] The above-mentioned osthole compounds can also be used in the preparation of anti-tumor drugs.

[0012] Wherein, the tumor is a glioma.

[0013] The metal complexes of the above-mentioned osthole compounds, Ir-OST and Ru-OST, have structures shown in Formula II or Formula III, respectively:

[0014]

[0015] The method for preparing the metal complex comprises the following steps: dissolving an osthole compound, a metal precursor, CuSO4·5H2O and sodium ascorbate in an organic solvent under an inert atmosphere, and separating and purifying the mixture after the reaction to obtain the metal complex.

[0016] The molar ratio of the osthole compound, the metal precursor, CuSO4·5H2O and sodium ascorbate is 2-10:1-5:1; and the metal precursor is Ir-N3 or Ru-N3.

[0017] The organic solvent is a mixed solution of dichloromethane and methanol, and the reaction temperature is 25-150° C. and the reaction time is 1 to 48 hours.

[0018] Wherein, the metal precursors Ir-N3 and Ru-N3 have the following structural formulas:

[0019]

[0020] The metal precursor preparation method is as follows: under an inert atmosphere, the bridging ligand and the metal dimer are dissolved in an organic solvent, and the crude product generated by the reaction is separated and purified by column chromatography to obtain the metal precursor.

[0021] Wherein, the bridging ligand bpy-N3 is 4-azidomethyl-4'-methyl-2,2'-bipyridine, and its structural formula is:

[0022]

[0023] Wherein, the metal iridium dimer is Ir(ppy)2Cl]2 or [Ru(cym)2Cl2]2, and the structural formula is as follows:

[0024]

[0025] The molar ratio of the bridging ligand bpy-N3 to the metal dimer is 2:1, the reaction temperature is 25-150° C., the reaction time is 2-48 h, and the organic solvent is a mixed solution of dichloromethane and methanol.

[0026] Application of the metal complex of the above-mentioned osthole compound in the preparation of anti-tumor drugs.

[0027] Wherein, the tumor is a glioma.

[0028] Principle of the invention: The present invention specifically modifies the small molecule osthole (OST) with special activity against brain glioma and coordinates it with a metal to obtain the complex Ir-OST with good anti-cancer activity. The complex Ir-OST is formed by connecting osthole (OST) with a cyclometallated iridium precursor through a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Compared with the metal precursor Ir-N3, Ir-OST exhibits stronger anti-tumor activity against human brain glioma (U87-MG) cells and is almost non-toxic to normal cells. At the same time, it has excellent fluorescence properties, can achieve the integration of anti-cancer and luminescent imaging, and can well enter cells and localize in mitochondria, showing organelle targeting. Among multiple cell lines, it only exhibits excellent anti-tumor activity against brain glioma cells, which indicates that it has cell selectivity and specific anti-cancer effects.

[0029] The complex of the present invention introduces a molecule with biological activity into cyclometallated iridium, regulates the charge, lipophilicity and solubility, and can regulate its physicochemical properties and biological activity, so that the cyclometallated iridium is better positioned, which helps to achieve localization in subcellular organelles, achieve the effect of mitochondrial accumulation, cause abnormal mitochondrial reactions, and produce anti-cancer effects; at the same time, the cyclometallated iridium can provide the characteristics of luminescent imaging, which is helpful for tracking testing, improves organelle targeting, and reduces the toxic side effects of the therapeutic agent while enhancing the anti-cancer activity.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The osthole compounds and metal complexes thereof of the present invention have significant anti-tumor biological activity, and the metal complex Ir-OST has targeting properties, can specifically target mitochondria, and significantly reduce the adverse effects on normal cells, and has excellent activity in treating glioma cells U87MG; (2) The osthole compounds and metal complexes thereof of the present invention have simple structures, few by-products in the synthesis process, are suitable for large-scale production, and have application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the uptake diagram of cyclometallated iridium complex Ir-OST in U87MG cells;

[0032] Figure 2 This is the localization map of the cyclometallated iridium complex Ir-OST in U87MG cells;

[0033] Figure 3 Laser confocal imaging of the decrease in mitochondrial membrane potential caused by the cyclometallated iridium complex Ir-OST in U87MG cells;

[0034] Figure 4 Flow cytometric plots generated for the decrease in mitochondrial membrane potential induced by the cyclometallated iridium complex Ir-OST in U87MG cells;

[0035] Figure 5 This is a flow cytometric graph showing that cyclometallated iridium complex Ir-OST induces apoptosis in U87MG cells;

[0036] Figure 6 This is a laser confocal micrograph of the cyclometallated iridium complex Ir-OST inducing autophagy in U87MG cells;

[0037] Figure 7 This is a Western blotting image of the cyclometallated iridium complex Ir-OST inducing autophagy in U87MG cells;

[0038] Figure 8 This is a test diagram of the lipid solubility of the cyclometallated iridium complex Ir-OST in U87MG cells;

[0039] Figure 9 This is a protein immunoblot image of the cyclometallated iridium complex Ir-OST passing through the blood-brain barrier in U87MG cells;

[0040] Figure 10 Figure 2 shows the inhibition of 3D cell spheroid growth by cyclometallated iridium complex Ir-OST in U87MG cells.

[0041] Figure 11 This is the live-dead staining image of the cyclometallated iridium complex Ir-OST in U87MG cells. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further described below with reference to the examples. The test materials used in the examples can all be purchased through conventional channels.

[0043] Example 1

[0044] The preparation method of the osthole compound OST-Alkyne of the present invention is specifically as follows:

[0045]

[0046] (1) Preparation of osthole-modified organic compound OST-alkyne: Cys (2 mmol, 242.3 mg), NaH (6 mmol, 144 mg), and OST (1 mmol, 244.3 mg) were dissolved in anhydrous DMF (100 mL) under an inert atmosphere. The mixture was condensed and refluxed for 10 hours, cooled to room temperature, and the pH was adjusted to acidic. The organic phases were extracted with ethyl acetate (3 × 50 mL), combined, washed with brine, and dried over anhydrous sodium sulfate. Finally, it was purified by flash chromatography (petroleum ether / ethyl acetate, 15:1) to obtain a brown solid OST-OH. 1 H NMR(400MHz,Chloroform-d)δ7.66(d,J=9.4Hz,1H),7.26(d,J=8.5Hz,1H),6.83(d,J=8.5Hz,1H),6.27(d, J=9.4Hz,1H),5.30(tdd,J=1.5,3.0,5.9Hz,1H),3.64(d,J=7.2Hz,2H),1.88(s,3H),1.78(d,J=1.5Hz,3H).

[0047] (2) In an inert atmosphere, brown solid OST-OH (1 mmol, 230.3 mg), propargyl bromide (3 mmol, 356.8 mg) and K2CO3 (1 mmol, 138 mg) were dissolved in anhydrous DMF and reacted at 50°C for 4-18 hours. The reaction was monitored by TLC thin-layer chromatography until the end of the reaction. The mixture was cooled to room temperature and extracted with ethyl acetate (3×10 mL). Finally, it was purified by flash chromatography (petroleum ether / ethyl acetate, 50:1) to obtain a light yellow solid OST-Alkyne (126.1 mg, 47%). 1 H NMR(400MHz,Chloroform-d)δ7.65(d,J=9.5Hz,1H),7.32(d,J=8.6Hz,1H),6.97(d,J=8.6Hz,1H),6.29(d,J=9.5Hz,1 H),5.35-5.14(m,1H),4.83(d,J=2.4Hz,2H),3.58(d,J=7.3Hz,2H),2.56(t,J=2.4Hz,1H),1.87(s,3H),1.69(s,3H).

[0048] Example 2

[0049] The preparation method of the metal precursors Ir-N3 and Ru-N3 of the present invention is specifically as follows:

[0050]

[0051] (1) Preparation of metal precursor Ir-N3: White solid bpy-N3 (0.2 mmol, 52.6 mg) and [Ir(ppy)2Cl]2 (0.1 mmol, 112.6 mg) were dissolved in a mixture of dichloromethane and methanol (20 mL v / v, 3 / 1) under an inert atmosphere and stirred at room temperature for 48 h. The solvent was removed by rotary evaporation, and a saturated methanolic solution of NH4PF6 was added. Finally, pure Ir-N3 (64 mg, 84%) was obtained by flash chromatography (dichloromethane / methanol, 10:1). 1 H NMR (400MHz, DMSO-d6) δ8.81 (s, 2H), 8.26 (d, J = 8.1Hz, 2H), 7.92 (s, 4H), 7.82 (s, 1H), 7.65 (d, J = 17. 2Hz,4H),7.53(s,1H),7.15(s,2H),7.01(s,2H),6.89(s,2H),6.18(s,2H),4.83(s,2H),2.53(s,3H).

[0052] (2) Preparation of metal precursor Ru-N3: White solid bpy-N3 (0.2 mmol, 52.6 mg) and [Ru(cym)2Cl2]2 (0.1 mmol, 61.33 mg) were dissolved in a mixture of dichloromethane and methanol (20 mL v / v, 3 / 1) under an inert atmosphere and stirred at room temperature for 48 h. The solvent was removed by rotary evaporation, and a saturated methanolic solution of NH4PF6 was added. Finally, pure Ru-N3 (86.58 mg, 76%) was obtained by flash chromatography (dichloromethane / methanol, 15:1). 1 H NMR (400MHz, DMSO-d6) δ9.50(dd,J=1.9,5.9Hz,1H),9.36(dd,J=1.9,5.8Hz,1H),8.58(s,2H),7.74(d,J=6.4Hz,1H),7.65(d,J=5.9 Hz,1H),6.24-6.16(m,2H),6.02-5.92(m,2H),4.88(s,2H),2.58(s,3H),2.51(s,1H),2.18(d,J=1.9Hz,3H),0.94(d,J=6.9Hz,6H).

[0053] Example 3

[0054] The preparation method of the metal complexes Ir-OST and Ru-OST of the present invention is specifically as follows:

[0055]

[0056] (1) Preparation of Ir-OST: Under an inert atmosphere, OST-Alkyne (0.1 mmol, 26.8 mg), Ir-N (0.1 mmol, 87.1 mg), CuSO·5H O (25 mg, 0.1 mmol), and sodium ascorbate (5 mg, 0.1 mmol) were dissolved in anhydrous DMF. The mixture was reacted in the dark for 24 h, and the solvent was removed by rotary evaporation. The crude product was further purified by flash chromatography (dichloromethane / methanol, 100:1) to give an orange solid (89.9 mg, 79%). 1H NMR (400MHz, Chloroform-d) δ8.58(s,1H),8.46(s,1H),8.19(s,1H),7.95-7.87(m,3H),7.81-7.74(m,3H),7.69(ddd,J=1.4,4. 1,7.8Hz,2H),7.63(d,J=9.5Hz,1H),7.53(d,J=5.6Hz,1H),7.48(d,J=5.6Hz,1H),7.34(d,J=8.6Hz,1H),7.23(dd,J=5.6,12.5H z,2H),7.11-6.98(m,5H),6.93(tt,J=1.5,7.3Hz,2H),6.32-6.22(m,3H),5.84(s,2H),5.30(s,2H),5.18(t,J=7.4Hz,1H),3.48 (d,J=7.4Hz,2H),2.59(s,3H),1.61(d,J=1.3Hz,3H),1.55(d,J=1.4Hz,3H).ESI-MS(positivemode,m / z):Calcd.994.17,found 994.4

[0057] (2) Preparation of Ru-OST: Under an inert atmosphere, OST-Alkyne (0.1 mmol, 26.8 mg), Ru-N3 (0.1 mmol, 64.1 mg), CuSO4·5H2O (25 mg, 0.02 mmol), and sodium ascorbate (20 mg, 0.02 mmol) were dissolved in anhydrous DMF. The mixture was reacted in the dark for 24 h, and the solvent was removed by rotary evaporation. The crude product was further purified by flash chromatography (dichloromethane / methanol, 100:1) to give an orange solid (64.18 mg, 84%). 1H NMR (400MHz, DMSO-d6) δ9.50(d,J=5.9Hz,1H),9.37(d,J=5.8Hz,1H),8.62(s,1H),8.50(s,1H),8.47(s,1H),8.32( d,J=0.8Hz,1H),7.99(d,J=9.6Hz,1H),7.67(d,J=5.9Hz,1H),7.58(d,J=8.6Hz,1H),7.48(d,J=5.9Hz,1H),7.28(d, J=8.7Hz,1H),6.30(d,J=9.5Hz,1H),6.19(dd,J=6.2,14.0Hz,2H),5.97-5.93(m,3H),5.35(s,2H),5.10(t,J=7.4Hz ,1H),2.58(s,3H),2.53(s,1H),2.16(s,3H),1.54(d,J=13.1Hz,6H),0.94(dd,J=4.5,6.9Hz,6H).ESI-MS(positive mode,m / z):Calcd.764.51,found 764.3

[0058] Example 4

[0059] Application of the cytotoxicity of the cyclometallated iridium complex Ir-OST prepared in Example 3:

[0060] Methods: The in vitro cytotoxicity of osthole, the ligand OST-Alkyne, the metal precursors Ir-N3 and Ru-N3, and the complexes Ir-OST and Ru-OST against human glioma cells (U87MG) was investigated using the MTT assay. U87MG cells were seeded in 96-well cell culture plates. After the cells reached a density of 70%, the test drug was added and incubated for 48 hours. MTT solution (20 μL, 5 mg / mL) was added and incubated for another 4 hours. The culture medium was aspirated, DMSO (150 μL) was added, and the absorbance at 490 nm was measured using a LabServ K3 microplate reader. The IC50 value, indicating cytotoxicity, was calculated using SPSS software.

[0061] The results showed that, as shown in Table 1, neither osthole nor its ligand, OST-Alkyne, exhibited significant cytotoxicity against both cancer and normal cell lines (IC50 > 100 μM), whereas the metal precursor Ir-N3 exhibited high cytotoxicity against both cancer and normal cell lines. The complex, Ir-OST, exhibited varying degrees of cytotoxicity against cancer cells, with the best anticancer activity against U87MG cells. It was virtually nontoxic to HLF cells, demonstrating a certain selectivity for U87MG. Therefore, Ir-OST was selected for further biological mechanism studies on U87MG cells.

[0062] Table 1. IC values ​​of precursors and complexes for different cell lines 50 value

[0063]

[0064]

[0065] Example 5

[0066] Example 3 Application of the synthesized cyclometallated iridium complex Ir-OST in U87MG cell uptake:

[0067] Methods: U87MG cells were seeded in a confocal laser microscope culture dish. When the cell density reached 70%, Ir-OST-containing culture medium was added in a time-dependent manner and incubated for the indicated time periods. Images were captured using a confocal laser microscope.

[0068] The results show that: Figure 1 As shown, the intracellular fluorescence of the complex gradually increased within 4 hours and finally reached the highest value at 4 hours, indicating that the complex can quickly enter the cell and provide conditions for interacting with intracellular small molecules.

[0069] Example 6

[0070] Example 3: Application of the synthesized cyclometallated iridium complex Ir-OST in localization in U87MG cells:

[0071] Methods: U87MG cells were seeded in a confocal laser scanning microscope dish. After reaching a cell density of 70%, medium containing Ir-OST was added and incubated for an additional 6 hours. The medium was aspirated, and the cells were washed three times with PBS. Serum-free medium containing MitoGreen was then added, the medium was removed, and the cells were washed with PBS. Fluorescence imaging was performed using a confocal laser scanning microscope.

[0072] The results show that: Figure 2 As shown, after 8 hours of drug treatment, the drug in the Ir-OST treatment group accumulated in the mitochondria, showing good mitochondrial targeting.

[0073] Example 7

[0074] Example 3 Application of the synthesized cyclometallated iridium complex Ir-OST in inducing a decrease in mitochondrial membrane potential in U87MG cells:

[0075] Method 1: U87MG cells were seeded in six-well plates and grown to a cell density of 70%. The different test drugs were then added, mixed with culture medium, and cultured for 24 hours. The cells were then digested, harvested, and resuspended three times in PBS. 1× JC-1 working solution (2 mL, prepared according to the JC-1 kit instructions) was added to the cell sample and incubated in the dark at 37°C for 20 minutes. The cells were then washed two to three times with PBS to obtain the stained cells. Flow cytometry was used, and data were analyzed using FlowJo V10 software.

[0076] Method 2: In a laser confocal culture dish, U87MG cells were inoculated and the cell density was grown to 70%. The drug to be tested was added after mixing with the culture medium and cultured for 24 hours. 1× JC-1 working solution (500 μL, prepared according to the instructions of the JC-1 kit) was added to the cell sample, and the cells were incubated in the dark at 37°C for 20 minutes. The cells were washed 2-3 times with PBS to obtain the stained cells and imaged using a laser confocal microscope.

[0077] The results showed that: the results of laser confocal microscopy were as follows: Figure 3 As shown, at the same time, the results of the flow cytometer test are as follows Figure 4 The Ir-OST complex treatment group induced a decrease in mitochondrial membrane potential, which may lead to mitochondrial dysfunction.

[0078] Example 8

[0079] Example 3 Application of the synthesized cyclometallated iridium complex Ir-OST in inducing apoptosis in U87MG cells:

[0080] Methods: U87MG cells were seeded in six-well plates to a cell density of 70%. Test drugs were added after mixing with culture medium and cultured for 24 hours. Cells were then digested, harvested, and resuspended three times in PBS. Annexin V-FITC and PI were then added, and the cells were incubated in the dark for 15 minutes. Flow cytometry was used, and data were analyzed using FlowJo V10 software.

[0081] The results show that: Figure 5 As shown in the figure, the cell population induced by Ir-OST complex shifted to the Q1 region, indicating that Ir-OST complex can induce necroptosis of U87MG cells to a certain extent.

[0082] Example 9

[0083] Example 3 Application of the synthesized cyclometallated iridium complex Ir-OST in inducing autophagy in U87MG cells:

[0084] Method 1: U87MG cells were seeded in a confocal microplate dish to a cell density of 70%. The test drug was then added and cultured for 24 hours. The cells were then washed three times with PBS and fixed with 4% paraformaldehyde (750 μL / well) for 15 minutes. The cells were then stimulated with 0.2% Triton X-100 permeabilization buffer for 15 minutes and blocked with 3% BSA blocking buffer for 30 minutes. The cells were then incubated with the primary antibody and incubated overnight at 4°C. The primary antibody dilution was removed, the cells were washed three times with PBS, and then incubated with the secondary antibody working solution for 1 hour in the dark. After removing the secondary antibody working solution and washing three times with PBS, the cells were stained with DAPI for 30 minutes, washed three times with PBS, and imaged using a laser confocal microscope.

[0085] Method 2: Immunoblotting was used to detect LC3 and P62 protein expression, with GAPDH used as a control protein. U87MG cells were seeded in 10 cm culture dishes overnight. After the cells adhered, the old culture medium was removed and culture medium containing the different test drugs was added. The cells were incubated for another 24 hours. The cells were harvested and lysed in RIPA lysis buffer containing 100 μg / mL phenylmethylsulfonyl fluoride (PMSF) for 30 minutes, with shaking every 5 minutes. The samples were then resuspended and centrifuged at 14,000 rpm for 20 minutes. The protein supernatant was collected and quantified using a BCA protein quantification kit to ensure consistent protein concentration across the samples. SDS-PAGE loading buffer was then added to the samples, boiled at 100°C for 20 minutes, and stored at -80°C until use. The PVDF membrane was washed five times with PBST (10 mM, 0.05% Tween-20), incubated with FITC-conjugated secondary antibody at room temperature for 1 h, and then washed five times with PBST. The immunoblot signal was enhanced with Tanon High-sig ECL Western Blotting Substrate.

[0086] The results showed that: the results of laser confocal test were as follows: Figure 6 As shown in Figure 2, cells treated with Ir-OST complex and Ir-N3 ligand produced obvious green fluorescence. Figure 7 As shown in the results, Ir-OST significantly upregulated LC3-II protein expression in a concentration-dependent manner, indicating that the drug can effectively induce autophagy in U87MG cells, leading to cell death.

[0087] Example 10

[0088] Example 3 Application of the synthesized cyclometallated iridium complex Ir-OST in its ability to penetrate the blood-brain barrier in U87MG cells:

[0089] Method 1: Prepare a PBS solution presaturated with n-octanol, add the different test drugs, and then add an equal volume of n-octanol solution. Mix thoroughly for 8 hours. Centrifuge to separate the organic and aqueous phases. Measure the absorbance of the compounds in the n-octanol and PBS phases by UV-Vis spectroscopy. Calculate the log Po / w (the logarithmic ratio of the concentrations in the organic and aqueous phases) to determine the lipid-water partition coefficient.

[0090] Method 2: Immunoblotting was used to detect the expression of MMP-9 and Claudin5 proteins, with GAPDH used as a control protein. U87MG cells were seeded in 10 cm culture dishes overnight. After the cells adhered, the old culture medium was removed and replaced with culture medium containing the different drugs. The cells were incubated for an additional 24 hours. The cells were harvested and lysed in RIPA lysis buffer containing 100 μg / mL phenylmethylsulfonyl fluoride (PMSF) for 30 minutes, with shaking every 5 minutes. The samples were then resuspended and centrifuged at 14,000 rpm for 20 minutes. The protein supernatant was collected and quantified using a BCA protein quantification kit to ensure consistent protein concentration across the samples. SDS-PAGE loading buffer was then added to the samples, boiled at 100°C for 20 minutes, and stored at -80°C until use. The PVDF membrane was washed five times with PBST (10 mM, 0.05% Tween-20), incubated with FITC-conjugated secondary antibody at room temperature for 1 hour, and then washed five times with PBST. The immunoblot signal was enhanced with Tanon High-sig ECL Western Blotting Substrate.

[0091] The results showed that: the results of protein immunoblotting were as follows: Figure 8 As shown in Figure 2, Ir-OST can effectively induce the upregulation of MMP-9 and the downregulation of Claudin5. Figure 9 As shown, the complex Ir-OST has a stronger lipophilicity compared with its precursor, which indicates that it has a greater potential to penetrate the blood-brain barrier.

[0092] Example 11

[0093] Example 3: Application of the synthesized cyclometallated iridium complex Ir-OST in inhibiting the growth of 3D cell spheroids in U87MG cells:

[0094] Methods: U87MG cells were seeded in ultra-low-adhesion 96-well plates. Three days later, the formed tumor spheroids were incubated with drug-containing medium, and the size of the tumor spheroids was monitored by phase contrast microscopy.

[0095] The results show that: Figure 10 As shown, the Ir-OST treatment group could effectively inhibit the growth of tumor volume within 7 days.

[0096] Example 12

[0097] Example 3 Application of the synthesized cyclometallated iridium complex Ir-OST in live-dead staining of U87MG cells:

[0098] Methods: U87MG cells were seeded in ultra-low-adhesion 96-well plates. Three days later, formed tumor spheroids were incubated with the drug in culture medium. Seven days later, the spheroids were washed three times with PBS, stained with Calcein AM / PI, and fixed with 4% paraformaldehyde. Fluorescence imaging was performed on a confocal laser microscope.

[0099] The results show that: Figure 11 As shown in the live-dead staining experiment, the control group showed green fluorescence, indicating that the cells were in good condition. In contrast, the fluorescence results of the Ir-OST-treated group were red, indicating that the cells were dead.

[0100] Therefore, the osthole compounds and metal complexes of the present invention combine natural products with metals, can exhibit stronger anti-tumor activity, and target mitochondria, killing tumors by reducing mitochondrial membrane potential, inducing cell autophagy and necroptosis; not only do they achieve good selectivity and anti-proliferation ability for brain glioma cells, but they also overcome the toxic side effects on normal cells, providing a new design idea for the treatment of brain glioma with new metal drugs, and have broad application prospects.

Claims

1. An osthole compound, characterized in that: The structure of the compound is shown in Formula I:

2. A method for preparing the osthole compound according to claim 1, characterized in that: The following steps are involved: In an inert atmosphere, cysteine, sodium hydride, and osthole are dissolved in an organic solvent. The crude product OST-OH generated by the reaction is separated and purified. Propylene bromide is then added and dissolved in an organic solvent. After the reaction, the product OST-Alkyne is separated and purified to obtain an osthole compound:

3. The method according to claim 2, characterized in that: The molar ratio of cysteine, sodium hydride and osthole is 2-10:1-5:1, and the molar ratio of OST-OH and propyne bromide is 2-10:

1.

4. The method according to claim 2, wherein: The organic solvent is N,N-dimethylformamide or acetamide. The reaction temperature is 25 to 150° C. and the reaction time is 2 to 32 hours.

5. Use of the osthole compound according to claim 1 in the preparation of anti-tumor drugs.

6. A metal complex of an osthole compound according to claim 1, characterized in that: The structure of the complex is shown in Formula II or Formula III:

7. A method for preparing the metal complex according to claim 6, characterized in that: The following steps are involved: Under an inert atmosphere, an osthole compound, a metal precursor, CuSO4·5H2O and sodium ascorbate are dissolved in an organic solvent, and after reaction, the metal complex is separated and purified.

8. The method according to claim 7, characterized in that: The molar ratio of the osthole compound, the metal precursor, CuSO4.5H2O and sodium ascorbate is 2-10:1-5:1; and the metal precursor is Ir-N3 or Ru-N3.

9. The method according to claim 7, characterized in that: The organic solvent is a mixed solution of dichloromethane and methanol. The reaction temperature is 25-150° C. and the reaction time is 1-48 hours.

10. Use of the metal complex of the osthole compound according to claim 6 in the preparation of anti-tumor drugs.

Images