Osthole compound, metal complex and preparation method and application thereof

Abstract

The invention discloses an osthole compound, a metal complex and a preparation method and application of the osthole compound, the osthole compound (OST-Alkyne) has remarkable anti-tumor biological activity, osthole (OST) and a cyclometalated iridium precursor are further connected through a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, higher anti-tumor activity is shown, and the osthole compound has a good application prospect. Mitochondria is targeted, and tumor is killed by reducing mitochondrial membrane potential and inducing cell autophagy and necrotic apoptosis; according to the invention, natural products are combined with metals, so that not only are good selectivity and anti-proliferation ability to brain glioma cells realized, but also toxic and side effects to normal cells are overcome, a new design idea is provided for novel metal drugs to treat brain glioma, and the application prospect is wide.

Description

Technical Field

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

[0002] Osthole is a natural coumarin derivative isolated from Cnidium monnieri. Studies have shown that osthole works by inhibiting cancer cell proliferation, inducing apoptosis, inhibiting invasion and migration, and inhibiting tumor angiogenesis. Researchers have obtained a series of osthole derivatives by modifying the osthole skeleton, which also have excellent anti-tumor activity. The existing technology for the study and improvement of the osthole skeleton includes the modification of the coumarin lactone ring, the modification of the coumarin 7-substituent, the modification of the coumarin 8-substituent, and the modification of coumarin multi-sites. Farooq et al. showed that the derivatives modified with the coumarin lactone ring have significant anti-tumor activity in human colon cancer cell Colo-205, human non-small cell lung cancer cell A549 and other cell lines, and can induce apoptosis in human colon cancer cell 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 is beneficial to enhance the neuroprotective properties of osthole. However, the above compounds lack specificity in cancer treatment, and further research is still needed on specific compounds corresponding to specific treatments.

[0003] Compared with traditional platinum anti-tumor metal drugs, iridium complexes have the advantages of high stability, good water solubility, excellent luminescence, multiple coordination sites, easy 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 organic electroluminescence, bioluminescent probes, chemical sensors and catalytic synthesis. Cyclometallated iridium (III) complexes have unique properties such as DNA binding, protein-protein interaction inhibition, catalytic oxidation, and protein kinase activity inhibition, and have had an important impact in the field of anti-cancer. However, cyclometallated iridium (III) complexes usually produce greater cytotoxicity, causing damage to normal cells while anti-cancer. Therefore, it is urgent to optimize the structure of metal complexes, reduce 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 preparation method of the above-mentioned osthole compound comprises the following steps: dissolving cysteine ​​(Cys), sodium hydride (NaH) and osthole (OST) in an organic solvent in an inert atmosphere, separating and purifying the crude product OST-OH generated by the reaction, adding propyne bromide and dissolving it in an organic solvent, separating and purifying the product OST-Alkyne after the reaction, and obtaining the osthole compound:

[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] Wherein, the organic solvent is N,N-dimethylformamide or acetamide solvent, the separation and purification adopts column chromatography, and the reaction temperature is 25-150° C. and the reaction time is 2-32 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 osthole compounds, Ir-OST and Ru-OST, have structures as shown in Formula II or Formula III, respectively:

[0014]

[0015] The method for preparing the metal complex comprises the following steps: dissolving osthole compounds, metal precursors, CuSO4·5H2O and sodium ascorbate in an organic solvent under an inert atmosphere, and separating and purifying 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] Wherein, 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 method for preparing the metal precursor 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 the 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 to 150° C., the reaction time is 2 to 48 hours, and the organic solvent is a mixed solution of dichloromethane and methanol.

[0026] Application of the metal complex of the above 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 metal to obtain the complex Ir-OST with good anti-cancer activity. The complex Ir-OST is obtained 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 locate 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 biologically active molecules into cyclometal iridium, adjusts the charge, lipophilicity and solubility, and can adjust its physical and chemical properties and biological activity, so that the cyclometal iridium is better positioned to help achieve positioning in subcellular organelles, achieve the effect of mitochondrial accumulation, cause abnormal mitochondrial reactions, and produce anti-cancer effects; at the same time, the cyclometal iridium can provide the characteristics of luminescent imaging, which is helpful for tracking tests, improves organelle targeting, and reduces the toxic and side effects of therapeutic agents while enhancing 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 property, 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 This is the uptake diagram of the cyclometalated iridium complex Ir-OST in U87MG cells;

[0032] Figure 2 This is the localization map of the cyclometalated 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 graphs generated for the decrease in mitochondrial membrane potential caused by the cyclometallated iridium complex Ir-OST in U87MG cells;

[0035] Figure 5 The figure is the flow cytometry graph of apoptosis induced by cyclometallated iridium complex Ir-OST in U87MG cells;

[0036] Figure 6 This is a laser confocal image 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] Fig. 9 This is a protein immunoblot image of the cyclometallated iridium complex Ir-OST passing through the blood-brain barrier in U87MG cells;

[0040] Fig.10 This is a graph showing that the cyclometalated iridium complex Ir-OST inhibits 3D cell spheroid growth in U87MG cells;

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

[0042] The technical scheme of the present invention is further described below in conjunction with 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: In an inert atmosphere, 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). The mixture was condensed and refluxed for 10 hours, cooled to room temperature, and the pH was adjusted to acidic. The organic phases were combined by ethyl acetate extraction (3×50 mL), 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 reaction was completed. The mixture was cooled to room temperature, extracted with ethyl acetate (3×10 mL) and finally 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: In an inert atmosphere, 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 mixed solution of dichloromethane and methanol (20 mL v / v, 3 / 1) and stirred at room temperature for 48 h. After the solvent was removed by rotary evaporation, a saturated methanol solution of NH4PF6 was added. Finally, it was purified by flash chromatography (dichloromethane / methanol, 10:1) to obtain pure Ir-N3 (64 mg, 84%). 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: In an inert atmosphere, 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 mixed solution of dichloromethane and methanol (20 mL v / v, 3 / 1) and stirred at room temperature for 48 h. After the solvent was removed by rotary evaporation, a saturated methanol solution of NH4PF6 was added. Finally, it was purified by flash chromatography (dichloromethane / methanol, 15:1) to obtain pure Ru-N3 (86.58 mg, 76%). 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 complex Ir-OST and Ru-OST of the present invention is specifically as follows:

[0055]

[0056] (1) Preparation of metal complex Ir-OST: Under an inert atmosphere, OST-Alkyne (0.1 mmol, 26.8 mg), Ir-N3 (0.1 mmol, 87.1 mg), CuSO4·5H2O (25 mg, 0.1 mmol) and sodium ascorbate (5 mg, 0.1 mmol) were dissolved in anhydrous DMF solution, 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 obtain 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 metal complex 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 solution, 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 obtain 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 obtained in Example 3:

[0060] Methods: The MTT method was used to study the in vitro cytotoxicity of the compound osthole, ligand OST-Alkyne, metal precursors Ir-N3, Ru-N3 and complexes Ir-OST, Ru-OST on human glioma cells (U87MG). U87MG cells were seeded in a 96-well cell culture plate. When the cell density reached 70%, the drug to be tested was added and incubated for 48 hours. MTT solution (20μL, 5mg / mL) was added and cultured for 4 hours. The culture medium was precipitated, DMSO (150μL) was added, and the absorbance at 490nm was detected by LabServ K3 microplate reader. The IC50 value was calculated by SPSS software to show the cytotoxicity.

[0061] The results showed that: as shown in Table 1, osthole OST and ligand OST-Alkyne did not show obvious cytotoxicity to cancer cells and normal cell lines (IC50>100μM), while the metal precursor Ir-N3 showed high cytotoxicity to both cancer cells and normal cell lines. The complex Ir-OST showed different degrees of cytotoxicity to cancer cells, especially to U87MG cells, with the best anticancer activity, and was almost non-toxic to HLF cells, showing a certain selectivity for U87MG. Therefore, Ir-OST was selected for further biological mechanism studies on U87MG cells.

[0062] Table 1. IC 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 inoculated in a laser confocal culture dish. When the cell density reached 70%, the medium containing Ir-OST was added according to the time gradient and incubated for the corresponding time. The images were taken using a laser confocal 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 maximum 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 inoculated in a laser confocal culture dish. When the cell density reached 70%, medium containing Ir-OST was added and incubated for 6 hours. The medium was aspirated, the cells were washed 3 times with PBS, serum-free medium containing MitoGreen was added, the medium was removed, the cells were washed with PBS, and fluorescence imaging was performed using a laser confocal 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: In a six-well plate, inoculate U87MG cells, grow the cell density to 70%, add different drugs to be tested mixed with culture medium, culture for 24 hours, digest and collect cells, and resuspend cells three times with PBS. Add 1×JC-1 working solution (2mL, configured according to the instructions of JC-1 kit) to the cell sample, incubate at 37℃ in the dark for 20 minutes, wash with PBS 2-3 times to obtain stained cells. Detection was performed by flow cytometry, and the data was analyzed by 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 mixed with the culture medium was added 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, incubated at 37°C in the dark for 20 minutes, washed 2-3 times with PBS, and the stained cells were obtained 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 As shown. 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 a six-well plate and grown to a cell density of 70%. The drug to be tested was added and cultured for 24 hours. The cells were digested and collected, and resuspended in PBS three times. Annexin V-FITC and PI were then added and incubated in the dark for 15 minutes. The detection was performed by flow cytometry, and the data were analyzed by FlowJo V10 software.

[0081] The results show that: Figure 5 As shown in the figure, the cell colony induced by Ir-OST complex shifted to the Q1 region, which indicated that Ir-OST complex could induce necroptosis of U87MG cells to some 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: In a confocal dish, U87MG cells were inoculated, the cell density was grown to 70%, the drug to be tested was added, and the culture was continued for 24 hours. Then, after washing with PBS three times and fixing with 4% paraformaldehyde (750μL / well) for 15 minutes, 0.2% TritonX-100 permeabilization solution was used for stimulation for 15 minutes, and then 3% BSA blocking solution was added for blocking for 30 minutes, and the cell samples were incubated with the primary antibody, and the primary antibody dilution was removed after overnight at 4°C, and the cells were washed with PBS three times, and then incubated with the secondary antibody working solution in the dark for 1 hour. After removing the secondary antibody working solution and washing with PBS three times, the nucleus was stained with DAPI dye for 30 minutes, and then washed with PBS three times, and imaging was performed by laser confocal microscopy.

[0085] Method 2: Immunoblotting was used to detect the expression of LC3 and P62 proteins, and GAPDH was used as a control protein. U87MG cells were inoculated in a 10 cm culture dish overnight. After the cells adhered to the wall, the old culture medium was removed, and the culture medium containing different drugs to be tested was added, and the incubation continued for 24 hours. The cells were digested and collected and lysed with RIPA lysis buffer containing 100 μg / mL phenylmethylsulfonyl fluoride (PMSF) for 30 minutes, and shaken every 5 minutes. Then the sample was resuspended and centrifuged at 14000 rpm for 20 minutes, and the protein supernatant was collected. Protein quantification was performed according to the BCA protein quantitative detection kit to ensure that the sample protein concentration was consistent. Then SDS-PAGE loading buffer was added to the sample, boiled at 100°C for 20 minutes, and stored in a -80°C refrigerator for use. The PVDF membrane was washed 5 times with PBST (10 mM, 0.05% Tween-20), incubated with FITC-conjugated secondary antibody at room temperature for 1 h, and then washed 5 times with PBST. The immunoblot signal was enhanced by Tanon High-sig ECL Western Blotting Substrate.

[0086] The results show that: the results of laser confocal test are as follows: Figure 6 As shown in Figure 2, cells treated with Ir-OST complex and Ir-N3 ligand produced obvious green fluorescence. The results of Western blotting test are shown in Figure 2. Figure 7 As shown in the figure, Ir-OST can significantly increase the expression level of LC3-Ⅱ protein, and this increase is concentration-dependent. This indicates 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 the ability to penetrate the blood-brain barrier in U87MG cells:

[0089] Method 1: Prepare a PBS solution pre-saturated with n-octanol, add different drugs to be tested, and then add an equal volume of n-octanol solution. Mix well and continue for 8 hours. Centrifuge to obtain an organic phase and an aqueous phase, and test the absorbance of the compounds in the n-octanol and PBS solution phases by UV-Vis spectroscopy. The lipid-water partition coefficient is obtained by calculating log Po / w (logarithmic ratio of concentration in organic phase and aqueous phase).

[0090] Method 2: Immunoblotting was used to detect the expression of MMP-9 and Claudin5 proteins, and GAPDH was used as a control protein. U87MG cells were inoculated in a 10 cm culture dish overnight. After the cells adhered to the wall, the old culture medium was removed, and the culture medium containing different drugs was added and incubated for another 24 h. The cells were digested and collected and lysed for 30 min with RIPA lysis buffer containing 100 μg / mL phenylmethylsulfonyl fluoride (PMSF), and shaken every 5 min. The sample was then resuspended and centrifuged at 14000 rpm for 20 min, the protein supernatant was collected, and protein was quantified according to the BCA protein quantitative detection kit to ensure that the sample protein concentration was consistent. Then SDS-PAGE loading buffer was added to the sample, boiled at 100 ° C for 20 min, and stored in a -80 ° C refrigerator for use. The PVDF membrane was washed 5 times with PBST (10 mM, 0.05% Tween-20), and incubated with FITC-conjugated secondary antibody at room temperature for 1 h. After that, it was washed 5 times with PBST, and the immunoblot signal was enhanced by 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. Fig. 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] Embodiment 11

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

[0094] Methods: U87MG cells were seeded in ultra-low-adhesion 96-well plates. After 3 days, the formed tumor spheroids were incubated with drugs in medium, and the size of tumor cell spheroids was monitored by phase contrast microscopy.

[0095] The results show that: Fig.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 adsorption 96-well plates. After 3 days, the formed tumor spheroids were incubated with drugs in culture medium. After 7 days, the cell spheroids were washed with PBS for 3 times, stained with Calcein AM / PI, fixed with 4% paraformaldehyde, and placed on a laser confocal microscope for fluorescence imaging.

[0099] The results show that: Fig.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 thereof 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, and then propyne bromide is 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, characterized in that: The organic solvent is N,N-dimethylformamide or acetamide solvent, and the reaction temperature is 25-150° C. and the reaction time is 2-32 hours.

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

6. A metal complex of the 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, osthole compounds, metal precursors, CuSO4·5H2O and sodium ascorbate are dissolved in an organic solvent, and after reaction, they are separated and purified to obtain a metal complex.

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