Ruthenium complex for inducing mitochondrial DNA polycondensation to overcome tumor drug resistance, anti-tumor drug and preparation method and application thereof

Abstract

The invention discloses a ruthenium complex for inducing mitochondrial DNA polycondensation to overcome tumor drug resistance, an anti-tumor drug and a preparation method and application of the ruthenium complex and the anti-tumor drug. The ruthenium complex is connected with a triphenylphosphine structure-modified bipyridine diazo-anthracene ligand through an alkyl chain, so that the fat solubility of the complex is improved, and the complex can enter cells more easily; the complex has a mitochondrial enrichment function and can be enriched in mitochondria; the complex induces mitochondrial DNA polycondensation so as to cause cancer cell death; the targeting property on tumor cells is effectively improved; the IC50 of the compound on human non-small cell lung cancer cell strains and cis-platinum drug-resistant strains thereof is far lower than that of cis-platinum, and the compound can be used as an excellent anti-tumor drug. The complex takes a ruthenium-coordinated ruthenium precursor compound as a raw material, and the raw material is economical and easy to obtain; the selectivity of the ruthenium complex can be improved, and the cost is saved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of metal organic complex functional materials, and particularly relates to a ruthenium complex for inducing mitochondrial DNA condensation to overcome tumor drug resistance, an anti-tumor drug and a preparation method and application thereof. BACKGROUND

[0002] Cancer has posed a great threat to human health worldwide. With the continuous progress of modern medicine, remarkable achievements have been made in the field of cancer treatment. At present, the main means of cancer treatment include surgical treatment, targeted therapy, immunotherapy, radiotherapy and chemotherapy. However, traditional treatment methods such as chemotherapy have some limitations and side effects, although they have achieved good effects in many cases. Chemotherapy drugs represented by cisplatin have poor targeting and low selectivity, and are highly toxic to normal tissues. Therefore, it is of great prospect to develop new anti-tumor chemotherapy reagents with high tumor selectivity and low toxic side effects.

[0003] Mitochondria are the energy factories of cells, and their functions are highly related to the occurrence, development and metastasis of tumors. In addition, due to the vigorous division of tumor cells, the demand for energy is higher than that of normal cells, and the mitochondrial content in tumor tissue cells is much higher than that in normal cells. Therefore, mitochondria are important anti-tumor targets. Since mitochondrial DNA lacks histone protection and is extremely susceptible to drug damage, and mitochondrial DNA lacks damage repair mechanisms, targeting mitochondrial DNA is beneficial to improve the sensitivity of tumor cells to drugs.

[0004] Therefore, anti-tumor drugs with low cytotoxicity, low genotoxicity, high biological activity, low toxic side effects, the ability to target mitochondrial DNA and activity on some platinum-resistant cancer cell lines are particularly important. SUMMARY

[0005] Based on the above reasons, the first object of the present application is to provide a ruthenium complex for inducing mitochondrial DNA condensation to overcome tumor drug resistance, which has a triphenylphosphine structure modified dipyridyl diazine heteroanthracene ligand. The modified triphenylphosphine structure enables the complex to be enriched in mitochondria, the complex induces mitochondrial DNA condensation, thereby causing cancer cell death, and the in vitro anticancer activity is better than that of cisplatin.

[0006] The second object of the present application is to provide a preparation method of the ruthenium complex for inducing mitochondrial DNA condensation to overcome tumor drug resistance. The triphenylphosphine structure modified dipyridyl diazine heteroanthracene ligand is directly coordinated and connected to a fixed position using a ruthenium precursor compound as a raw material, which improves selectivity and saves cost.

[0007] The third object of the present application is to provide the use of the ruthenium complex for inducing mitochondrial DNA condensation to overcome tumor drug resistance in the preparation of an antitumor drug.

[0008] The first object of the present application can be achieved by adopting the following technical solution:

[0009] A ruthenium complex for inducing mitochondrial DNA condensation to overcome tumor drug resistance, the structural formula of which is shown in formula I:

[0010]

[0011] wherein n is an integer of 1-7.

[0012] Further, X is an inorganic salt anion; the inorganic salt anion is chloride ion, perchlorate ion, nitrate ion or hexafluorophosphate ion.

[0013] The second object of the present application can be achieved by adopting the following technical solution:

[0014] A preparation method of a ruthenium complex for inducing mitochondrial DNA condensation to overcome tumor drug resistance,

[0015] The ligand shown in formula II and the ruthenium precursor compound shown in formula III are heated in an alcohol solvent under a protective gas atmosphere, after the reaction is completed, an aqueous solution of ammonium hexafluorophosphate inorganic salt is added after cooling, to obtain the ruthenium complex for inducing mitochondrial DNA condensation to overcome tumor drug resistance shown in formula I;

[0016]

[0017]

[0018] Further, the mass ratio of the ligand shown in formula II to the ruthenium precursor compound shown in formula III is 1:1-1.5.

[0019] Further, the alcohol solvent is ethylene glycol.

[0020] Further, the reaction conditions are 100-130℃ for 6-8h.

[0021] Further, the ruthenium precursor compound shown in formula III is prepared by dissolving 4,7-diphenyl-1,10-phenanthroline and a ruthenium inorganic salt in a solvent and heating.

[0022] Furthermore, the solvent is N,N-dimethylformamide.

[0023] Furthermore, the reaction conditions are: in a dark and protective atmosphere, at 130-150° C. for 6-8 hours;

[0024] Furthermore, the ruthenium inorganic salt is RuCl3.

[0025] Furthermore, the molar ratio of 4,7-diphenyl-1,10-phenanthroline to the ruthenium inorganic salt is 1:2-2.5.

[0026] Furthermore, the preparation path of the ligand of the structure shown in Formula II includes:

[0027]

[0028] The third object of the present invention can be achieved by adopting the following technical solutions:

[0029] The ruthenium complex that induces mitochondrial DNA condensation to overcome tumor drug resistance or the ruthenium complex that induces mitochondrial DNA condensation to overcome tumor drug resistance prepared by the preparation method of the ruthenium complex that induces mitochondrial DNA condensation to overcome tumor drug resistance is used in the preparation of anti-tumor drugs.

[0030] The fourth object of the present invention can be achieved by adopting the following technical solutions:

[0031] An anti-tumor drug, the active ingredient of which comprises the ruthenium complex that induces mitochondrial DNA condensation to overcome tumor resistance, or the ruthenium complex that induces mitochondrial DNA condensation to overcome tumor resistance prepared by the preparation method of the ruthenium complex that induces mitochondrial DNA condensation to overcome tumor resistance.

[0032] Furthermore, the tumor is lung cancer; preferably, the tumor is non-small cell lung cancer; more preferably, the tumor is human non-small cell lung cancer; more preferably, the human non-small cell lung cancer cells are cisplatin-resistant strain A549R cells.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. The ruthenium complex of the present invention, which induces mitochondrial DNA condensation to overcome tumor drug resistance, comprises a bipyridyl dinitrogen anthracene ligand and a phenanthroline ligand modified with a triphenylphosphine structure, wherein the bipyridyl dinitrogen anthracene ligand is modified by connecting the triphenylphosphine structure with an alkyl chain. The alkyl chain improves the lipid solubility of the complex, making it easier for the complex to enter cells, while the triphenylphosphine structure gives the complex a mitochondrial enrichment function, enabling it to be enriched in mitochondria. The complex induces mitochondrial DNA condensation, thereby causing cancer cell death, and effectively improving the targeting of tumor cells.

[0035] 2. A second object of the present application is to provide a preparation method of a ruthenium complex for inducing mitochondrial DNA condensation to overcome tumor drug resistance, using a ruthenium precursor compound coordinated with phenanthroline di-ligand as a raw material, which is economical and easy to obtain; the raw material reserves a coordination position, which can directly coordinate a triphenylphosphine-structured bipyridine diazine heteroanthracene ligand at the reserved position, improving the selectivity of the ruthenium complex and saving costs.

[0036] 3. The ruthenium complex for inducing mitochondrial DNA condensation to overcome tumor drug resistance of the present application can be enriched in mitochondria, induce mitochondrial DNA condensation, and kill cancer cells; the IC 50 of the ruthenium complex for human non-small cell lung cancer cell lines and their cisplatin-resistant strains is much lower than that of cisplatin, and the cancer cell killing effect is higher than that of cisplatin, which is expected to be used as an excellent anti-tumor drug. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 ESI-MS diagram of the ruthenium complex;

[0038] Figure 2 NMR hydrogen spectrum diagram (DMSO-d6) of the ruthenium complex;

[0039] Figure 3 UV-visible absorption spectrum diagram of the ruthenium complex;

[0040] Figure 4 Phosphorescence spectrum diagram of the ruthenium complex;

[0041] Figure 5 Fluorescence spectrum diagram of the ruthenium complex of the examples and comparative example 1 in response to biological macromolecules;

[0042] Figure 6 DNA titration diagram of the ruthenium complex;

[0043] Figure 7 Effect of the ruthenium complex on the relative viscosity of pBR322 DNA at 30±0.1℃;

[0044] Figure 8 Fluorescence spectrum diagram of the competitive test of the ruthenium complex and Hoechst 33342;

[0045] Figure 9 Gel electrophoresis diagram of the ruthenium complex and DNA;

[0046] Figure 10 Dynamic light scattering particle size diagram of DNA;

[0047] Figure 11 Dynamic light scattering particle size diagram of the ruthenium complex and DNA after co-incubation;

[0048] Figure 12Zeta potential diagram of the ruthenium complex after co-incubation with DNA;

[0049] Figure 13 Structure diagram of the ruthenium complex prepared in the embodiment of the present application;

[0050] Figure 14 Mass spectrum diagram of the ruthenium complex prepared in Comparative Example 1;

[0051] Figure 15 Nuclear magnetic hydrogen spectrum diagram of the ruthenium complex prepared in Comparative Example 1. DETAILED DESCRIPTION

[0052] The technical solutions of the present application will be clearly and completely described below in combination with specific embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0053] Targeting mitochondria DNA is beneficial to improve the sensitivity of tumor cells to drugs; therefore, it is particularly important to synthesize anti-tumor drugs with low cytotoxicity, low genetic toxicity, high biological activity, low toxic side effects, the ability to target mitochondria DNA, and activity on some platinum-resistant cancer cell lines, etc. by regulating the structure of ligands.

[0054] A ruthenium complex for inducing mitochondria DNA condensation to overcome tumor drug resistance, the structural formula of which is shown as formula I:

[0055]

[0056] wherein n is an integer of 1-7.

[0057] The ruthenium complex for inducing mitochondria DNA condensation to overcome tumor drug resistance of the present application has six coordination with six nitrogen atoms, and the nitrogen atoms are provided by two phenanthroline ligands and one dipyridyl diazine heteroanthracene ligand. The dipyridyl diazine heteroanthracene is modified by a peralkyl chain connected to a triphenylphosphine structure, the alkyl chain improves the liposolubility of the complex so that the complex is more easily entered into cells, and the triphenylphosphine structure increases the charge amount of the complex, so that the complex has a mitochondria enrichment function and can be enriched in mitochondria; the complex induces mitochondria DNA condensation, thereby causing cancer cell death; and the targeting of tumor cells is effectively improved.

[0058] In the present embodiment, n is 1, 2, 3, 4, 5, 6, 7; corresponding to ethyl, propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl alkyl chain. Preferably, n in the present application is 5, corresponding to n-hexyl alkyl chain.

[0059] As one of the embodiments, X is an inorganic salt anion; the inorganic salt anion is any one of chloride ion, perchlorate ion, nitrate ion or hexafluorophosphate ion.

[0060] The application also provides a preparation method of the ruthenium complex for inducing mitochondrial DNA condensation to overcome tumor drug resistance. The ligand shown in formula II and the ruthenium precursor compound shown in formula III are heated to react in an alcohol solvent under a protective gas atmosphere. After the reaction is completed, an aqueous solution of ammonium hexafluorophosphate inorganic salt is added after cooling to obtain the ruthenium complex for inducing mitochondrial DNA condensation to overcome tumor drug resistance shown in formula I. The reaction process is as follows:

[0061]

[0062] One of the raw materials of the application is a ruthenium precursor compound shown in formula III, in which the phenanthroline ligand is coordinated with ruthenium, and the coordination position of the ligand shown in formula II is reserved, so that the ligand shown in formula II is coordinated and connected with the ruthenium precursor compound shown in formula III during the reaction, thereby improving the selectivity of the ruthenium complex.

[0063] As one of the embodiments, the protective atmosphere is an argon atmosphere.

[0064] As one of the embodiments, the molar ratio of the ligand shown in formula II to the ruthenium precursor compound shown in formula III is 1:1-1.5; preferably 1:1.

[0065] As one of the embodiments, the alcohol solvent is ethylene glycol.

[0066] As one of the embodiments, the reaction conditions are 100-130℃ for 6-8h. Preferably, the reaction is carried out at 120℃ for 8h.

[0067] As one of the embodiments, the ruthenium precursor compound shown in formula III is prepared by dissolving 4,7-diphenyl-1,10-phenanthroline and a ruthenium inorganic salt in a solvent and heating to react; the reaction process is as follows:

[0068]

[0069] As one of the embodiments, the solvent is N,N-dimethylformamide.

[0070] As one of the embodiments, the reaction conditions are 130-150℃ for 6-8h under light protection and a protective atmosphere; preferably, the protective atmosphere is an argon atmosphere. Preferably, the reaction is carried out at 140℃ for 8h.

[0071] As one of the embodiments, the ruthenium inorganic salt is RuCl3.

[0072] As one of the embodiments, the molar ratio of 4,7-diphenyl-1,10-phenanthroline to the inorganic ruthenium salt is 1:2-2.5; preferably 1:2.5.

[0073] As one of the embodiments, the preparation route of the ligand with the structure shown in formula II comprises:

[0074]

[0075] As one of the embodiments, the ligand with the structure shown in formula II is prepared by heating reaction of the compound with the structure shown in formula IV and triphenylphosphine in a solvent; the reaction process is as follows:

[0076]

[0077] As one of the embodiments, the solvent is N,N-dimethylformamide.

[0078] As one of the embodiments, the reaction is carried out at 100-120℃; preferably the reaction temperature is 110-105℃; more preferably 110℃; the reaction time is 24-26h; more preferably 24h.

[0079] As one of the embodiments, the molar ratio of the compound with the structure shown in formula IV to triphenylphosphine is 1:3-4; preferably 1:4.

[0080] As one of the embodiments, the compound with the structure shown in formula IV is prepared by reaction of the compound with the structure shown in formula V and carbon-terminated dihaloalkane in a solvent; preferably, the carbon-terminated dihaloalkane is wherein n is an integer of 1-7; the reaction process is as follows:

[0081]

[0082] As one of the embodiments, the solvent is a ketone solvent; preferably a lower ketone; more preferably acetone.

[0083] As one of the embodiments, the reaction is carried out at 50-80℃ under reflux; preferably the reaction temperature is 60-70℃; more preferably 65℃; the reaction time is 12-14h; preferably 14h.

[0084] As one of the embodiments, the molar ratio of the compound with the structure shown in formula V and is 1:8-10; preferably 1:10.

[0085] As one of the embodiments, the compound of formula V is prepared by demethylation of the compound of formula VI; the reaction process is as follows:

[0086]

[0087] As one of the embodiments, the reaction is carried out in an acid system; preferably, the acid system is a mixture of glacial acetic acid and hydrobromic acid; more preferably, the volume ratio of glacial acetic acid to hydrobromic acid is 2:1.

[0088] As one of the embodiments, the reaction is carried out at 110-140℃; preferably, the reaction temperature is 120-130℃; more preferably, the reaction temperature is 126℃; the reaction time is 24-26h; more preferably, the reaction time is 24h.

[0089] As one of the embodiments, the compound of formula VI is prepared by heating reaction of 1,10-phenanthroline-5,6-dione and 4-methoxy-ortho-phenylenediamine in a solvent.

[0090] As one of the embodiments, the reaction is carried out in an alcohol solvent system; preferably, the alcohol is a lower alcohol; more preferably, the lower alcohol is a C1-C6 alcohol; more preferably, the lower alcohol is ethanol.

[0091] As one of the embodiments, the reaction is carried out at 70-90℃ under reflux; preferably, the reaction temperature is 75-85℃; more preferably, the reaction temperature is 80℃; the reaction time is 12-14h; more preferably, the reaction time is 14h.

[0092] As one of the embodiments, ammonium acetate is also present in the reaction; the molar ratio of 1,10-phenanthroline-5,6-dione, ammonium acetate and 4-methoxy-ortho-phenylenediamine is 1:20:2-2.5; more preferably, the molar ratio is 1:20:2.5.

[0093] The ruthenium complex for inducing mitochondrial DNA polycondensation to overcome tumor drug resistance or the ruthenium complex for inducing mitochondrial DNA polycondensation to overcome tumor drug resistance prepared by the preparation method of the ruthenium complex for inducing mitochondrial DNA polycondensation to overcome tumor drug resistance is used in the preparation of an antitumor drug.

[0094] An antitumor drug, wherein the active ingredient of the antitumor drug comprises the ruthenium complex for inducing mitochondrial DNA polycondensation to overcome tumor drug resistance or the ruthenium complex for inducing mitochondrial DNA polycondensation to overcome tumor drug resistance prepared by the preparation method of the ruthenium complex for inducing mitochondrial DNA polycondensation to overcome tumor drug resistance.

[0095] As one of the embodiments, the tumor is lung cancer; preferably, the tumor is non-small cell lung cancer; more preferably, the tumor is human non-small cell lung cancer; more preferably, the human non-small cell lung cancer cell is cisplatin-resistant strain A549R cell.

[0096] The following is further illustrated with specific examples.

[0097] Embodiment:

[0098] (1) Synthesis of organic ligand with structure shown in formula VI

[0099] 1,10-phenanthroline-5,6-dione (1 g, 4.76 mmol), 4-methoxy-ortho-phenylenediamine (1.38 g, 10 mmol) and ammonium acetate (1.8760 g, 25 mmol) were uniformly mixed in 20 mL of ethanol, refluxed at 80°C for 14 h, a large amount of black precipitate was generated, the precipitate was collected by suction filtration, and the product was washed with deionized water and anhydrous ethanol during the process; dried in a vacuum drying box at 50°C for 12 h to obtain 1.231 g of organic ligand with structure shown in formula VI, named dppz-OCH3; the yield was 82.8%.

[0100] (2) Synthesis of organic ligand with structure shown in formula V

[0101] dppz-OCH3 (1.231 g, 3.94 mmol) was refluxed in 90 mL of a mixed solvent of glacial acetic acid and hydrobromic acid (volume ratio 2:1) at 126°C for 24 h, a dark green precipitate was generated, 50 mL of deionized water was added after the solution cooled to room temperature, transferred into a beaker, neutralized to pH 6 with ammonia water, and placed for 30 min, then the precipitate was collected by suction filtration, and the product was washed with deionized water during the process; dried in a vacuum drying box at 50°C for 12 h to obtain 0.884 g of organic ligand with structure shown in formula V, named dppz-OH, with a yield of 75.3%.

[0102] (3) Synthesis of organic ligand with structure shown in formula IV

[0103] dppz-OH (0.298 g, 1 mmol), 1,6-dibromohexane (2.4397 g, 10 mmol) and potassium carbonate (0.82 g, 6 mmol) were placed in 100 mL of acetone, refluxed at 65°C for 14 h, the filtrate was collected by suction filtration after the solution cooled to room temperature, the solvent was removed from the filtrate under reduced pressure, and the solid was collected, then the product was washed with diethyl ether; dried in a vacuum drying box at 50°C for 12 h to obtain 0.393 g of organic ligand with structure shown in the following formula, named dppz-OBr, with a yield of 85.4%;

[0104]

[0105] (4) Synthesis of the organic ligand with the structure shown in Formula II

[0106] dppz-OBr (0.280 g, 0.61 mmol) and triphenylphosphine (0.632 g, 2.41 mmol) were dissolved in 10 mL of N,N-dimethylformamide and refluxed at 110°C for 24 h. After the solution cooled, the solvent was removed under reduced pressure, the solid was collected, and the product was washed with ether. The product was dried in a vacuum oven at 50°C for 12 h to obtain 0.299 g of the organic ligand with the structure shown below; it was named dppztpp; the yield was 76.2%.

[0107]

[0108] (5) Synthesis of the Ruthenium Precursor Compound Represented by Formula III

[0109] Ruthenium trichloride (0.4149 g, 2 mmol), 4,7-diphenyl-1,10-phenanthroline (1.328 g, 4 mmol), and lithium chloride (0.5595 g, 13.2 mmol) were dissolved in 4 mL of N,N-dimethylformamide and refluxed at 140°C for 8 h. After the solution cooled, it was added to 20 mL of acetone and frozen at -20°C overnight. The precipitate was collected by filtration, and the filter cake was washed with acetone and ether. The filter cake was vacuum-dried at 50°C for 12 h to obtain 1.32 g of the ruthenium precursor compound represented by formula III; the yield was 78.9%;

[0110]

[0111] (4) Synthesis of ruthenium complexes

[0112] The ruthenium precursor compound represented by the structure of formula III (0.0335 g, 0.04 mmol) and the organic ligand dppztpp (0.030 g, 0.04 mmol) were dissolved in 3 mL of ethylene glycol and refluxed at 120° C. in the dark for 8 h under an inert atmosphere. After the solution was cooled to room temperature, a saturated aqueous solution of ammonium hexafluorophosphate was added to the solution to obtain a solid precipitate. The precipitate was filtered and collected. The filter cake was washed with water and ether during the process. The filter cake was vacuum dried at 50° C. for 12 h to obtain a ruthenium complex represented by the structure shown below ( Figure 13 The product was purified by silica gel chromatography using dichloromethane:acetonitrile (v / v=20 / 1) as eluent to obtain an orange-red solid.

[0113]

[0114] The mass spectrum of ruthenium complex is shown in Figure 1 As shown, ESI-MS [CH2Cl2, m / z]: 469.34 [M-3PF6 - ] 3+ ;

[0115] The1H NMR of the ruthenium complex is shown in Figure 2 1 H NMR (400 MHz, DMSO) δ 9.63 (d, J = 8.2 Hz, 2H), 8.45 - 8.38 (m, 2H), 8.38 - 8.33 (m, 4H),

[0116] δ 9.63 (d, J = 8.2 Hz, 2H), 8.45 - 8.38 (m, 2H), 8.38 - 8.33 (m, 4H),

[0117] 8.33 - 8.28 (m, 5H), 8.04 - 7.99 (m, 2H), 7.93 - 7.89 (m, 3H), 7.86 - 7.82 (m, 6H), 7.81 - 7.79 (m, 6H), 7.77 (dd, J = 7.8, 4.0 Hz, 5H), 7.69 (dd, J = 6.8, 5.1 Hz, 7H), 7.65 (d, J = 3.1 Hz, 9H), 7.64 - 7.61 (m, 3H),

[0118] 4.31 (t, J = 6.1 Hz, 2H), 3.64 - 3.57 (m, 2H), 1.87 - 1.81 (m, 2H), 1.68 - 1.50 (m, 7H), 1.23 (s, 1H).

[0119] Comparative Example 1

[0120] The ligand of structure shown in Formula IV was synthesized using 1-bromohexane and the ruthenium complex was synthesized directly with the ruthenium precursor compound of structure shown in Formula III following the preparation method of the example;

[0121] Mass spectrometry data of Comparative Example 1 ESI-MS (CH3OH): m / z = 573.98 ([M-2PF6] 2+ ); the spectrum is shown in Figure 14

[0122] NMR data 1 H NMR (400 MHz, CD3CN) δ 9.63 (dd, J = 8.2, 1.2 Hz, 2H), 8.34 - 8.29 (m, 4H), 8.28 - 8.25 (m, 3H), 8.22 (s, 4H), 7.85 (dt, J = 8.3, 5.4 Hz, 2H), 7.73 (dd, J = 9.3, 2.7 Hz, 1H), 7.69 - 7.67 (m, 2H), 7.65 (s, 2H), 7.64 (d, J = 1.5 Hz, 7H), 7.62 (d, J = 3.2 Hz, 3H), 7.60 (t, J = 2.5 Hz, 8H), 7.58 (d, J = 5.6 Hz, 1H), 4.30 (t, J = 6.5 Hz, 2H), 1.65 - 1.46 (m, 3H), 1.41 (dd, J = 6.9, 3.0 Hz, 2H), 1.37 - 1.33 (m, 2H), 1.32 - 1.28 (m, 2H).​​

[0123] 7.2, 3.6 Hz, 5H), 0.94 (t, J = 7.1 Hz, 4H); spectrum as shown. Figure 15

[0124] The structural formula is as follows:

[0125]

[0126] Performance test of the ruthenium complex

[0127] 1. Absorbance of the ruthenium complex in a buffer solution

[0128] A 5 μM sample solution of the ruthenium complex prepared in the example was prepared using Tris as a solvent, and then the ultraviolet absorption spectrum of the ruthenium complex was recorded using a UV-Vis spectrophotometer, and the absorbance in the solvent is shown in Figure 3

[0129] As can be seen from Figure 3 , the ruthenium complex has good light absorption capacity in an organic solvent.

[0130] 2. Phosphorescence emission of the ruthenium complex in different solvents

[0131] A 5 μM sample solution of the ruthenium complex prepared in the example was prepared using Tris and methanol (MeOH) as solvents, and then the emission spectrum of the ruthenium complex was recorded using a fluorescence spectrometer with 450 nm as the excitation wavelength of the complex, and the phosphorescence spectrum is shown in Figure 4

[0132] As can be seen from Figure 4 , the complex has a high emission intensity in methanol, and the emission wavelength is 600 nm. In Tris, the emission wavelength is red-shifted, and the emission wavelength is 611 nm.

[0133] 3. Fluorescence emission of the ruthenium complex in response to biological macromolecules

[0134] The source of DNA used is pBR 322 DNA, the source of RNA used is yeast RNA (Yeast RNA), and the source of protein used is bovine serum albumin (BSA).

[0135] ​​​The complex solution was diluted in a centrifuge tube with Tris-HCl buffer (50 mM Tris-HCl, 18 mM NaCl, pH 7.3) to a volume of 3 mL at a concentration of 5 μM. pBR 322 DNA, yeast RNA, and BSA were each prepared into a 9 mM stock solution with ultrapure water and stored in a 4 °C refrigerator. In a centrifuge tube, 1.0 μM of each biomacromolecule solution was added to a solution of 5 μM of the ruthenium complex prepared in the example and the ruthenium complex prepared in Comparative Example 1, and incubated at 37 °C for 30 min in a water bath, then transferred to a 3.5 mL quartz cuvette, and the emission spectrum was measured on a fluorescence spectrometer with 450 nm as the excitation wavelength of the complex. The fluorescence spectrum is shown in Figure 5 , where the left graph is the example, and the right graph is Comparative Example 1.

[0136] As shown in Figure 5 , in the Tris-HCl solution, the ruthenium complex has a weak response to RNA and BSA, but a clear response to DNA. After incubation with DNA for 5 min, the luminescence intensity increased by 5.4 times. The luminescence intensity of Comparative Example 1 did not change substantially. This indicates that the introduction of triphenylphosphine plays an important role in the DNA recognition of the complex.

[0137] 4. UV titration of the ruthenium complex with pBR 322 DNA

[0138] The ruthenium complex prepared in the example was prepared into a 5 μM sample solution with Tris-HCl buffer (Tris) as the solvent, and pBR 322 DNA was prepared into a 9 mM stock solution. The absorbance at 250 nm-600 nm was tested using a UV-visible spectrophotometer. To the prepared 5 μM sample solution, 1.0 μM of pBR 322 DNA was added, and after each addition of DNA, the solution was incubated for 5 min and 3 sets of data were repeatedly tested. The average value was taken, and the above operation was repeated until the absorbance peak intensity at 450 nm no longer changed. The results are shown in Figure 6 .

[0139] As can be seen from Figure 6 , the characteristic absorbance peak intensity of the ruthenium complex at 450 nm gradually decreases with increasing DNA concentration, indicating that the complex interacts with DNA.

[0140] 5. Relative viscosity analysis of the ruthenium complex after co-incubation with pBR 322 DNA

[0141] pBR322 DNA was dissolved in Tris-HCl buffer to obtain a working solution of 6 mg / mL. Then different concentrations of Ru(II) complex (0-25 μM) were added and incubated at 30 °C for 5 min, respectively. After incubation, the viscosity of the mixture was detected using an Ubbelohde viscometer (30 °C), and the results are shown inFigure 7 shown.

[0142] from Figure 7 It can be concluded that with the increase of complex concentration, the solution viscosity decreased by 11.09%, which indicates that the complex binds to DNA through a different interaction mode from insertion, groove binding and electrostatic interaction.

[0143] 6. Fluorescence emission of ruthenium complexes in competitive assay with Hoechst 33342

[0144] 10 μM Hoechst 33342 was prepared in dimethyl sulfoxide, and 10 μM pBR322 DNA was added and incubated in a 37°C water bath for 10 min. Different doses of the complex (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μM) were added and the emission spectrum was measured in a fluorescence spectrometer with an excitation wavelength of 405 nm. The results are shown in Figure 2. Figure 8 shown.

[0145] Figure 8 In the experiment, as the concentration of the complex increased, the fluorescence intensity of DNA-Hoechst 33342 at a wavelength of 490nm gradually weakened. When the concentration of the complex increased to 10μM, the fluorescence intensity decreased by 82.58%. Since the possibility of the complex inserting into DNA has been ruled out in the UV experiment and viscosity experiment, it is inferred that Ru3 extrude the Hoechst 33342 dye through a new mechanism of action.

[0146] 7. Gel electrophoresis experiment of ruthenium complex and pBR 322 DNA

[0147] Weigh 0.18g of agarose and dissolve it in TBE buffer solution (18mM Tris-boric acid, 0.4mM EDTA, pH 8.3) to make a gel. Insert a comb to form a sample well. Prepare a DNA concentration of 10μM and add ruthenium complexes at concentrations of 0, 5, 10, 15, 20, 25, and 30μM, respectively. Incubate at 37°C for 15min. Add 2μL of bromophenol blue dye to each sample and pipette it evenly. Then take 10μL and add it to the sample well. Electrophoresis is carried out at 80V for 15min. Stain the gel with nucleic acid dye for 20min. Observe and record the results directly on a chemiluminescence imager. The results are as follows. Figure 9 shown.

[0148] from Figure 9 It can be seen that as the concentration of the ruthenium complex increases, the amount of DNA remaining in the loading well gradually increases. When the concentration of the ruthenium complex reaches 15 μM, the pBR 322 DNA basically remains in the loading well, indicating that the ruthenium complex has the ability to induce DNA condensation.

[0149] 8. Dynamic light scattering particle size detection of complex and pBR 322 DNA co-incubation

[0150] 10 μM pBR 322 DNA was incubated with different concentrations of complex (0-10 μM) in Tris-HCl buffer at 37 °C for 30 min. The mixture was transferred to 3 mL cuvette and the particle size of the mixture was measured by particle size analyzer; usually, 3 measurements were taken for each solution and the average value was taken; the results are shown in Figure 10 and Figure 11 .

[0151] Figure 10 In the absence of complex, DNA has no obvious particle size, and the average particle size is less than 1 nm, because DNA is well soluble in water and completely dissolved in Tris-HCl to form a DNA solution. Figure 11 In the presence of complex, the particle size of DNA increased to 164.3 nm when the concentration of complex increased to 5.0 μM. When the concentration of complex increased to 10 μM, the average particle size of DNA increased to 255.5 nm. This indicates that the complex can effectively induce DNA condensation, and has the potential to develop into a DNA condensation reagent; these results are consistent with gel electrophoresis.

[0152] 9. Zeta potential detection of ruthenium complex and pBR 322 DNA co-incubation

[0153] 10 μM pBR 322 DNA was incubated with different concentrations of complex (0-15 μM) in Tris-HCl buffer at 37 °C for 30 min; the mixture was transferred to 3 mL cuvette and the Zeta potential of the mixture was measured by particle size analyzer, and the results are shown in Figure 12 .

[0154] Figure 12 In the presence of complex, the particle size of DNA increased to 164.3 nm when the concentration of complex increased to 5.0 μM. When the concentration of complex increased to 10 μM, the average particle size of DNA increased to 255.5 nm. This indicates that the complex can effectively induce DNA condensation, and has the potential to develop into a DNA condensation reagent; these results are consistent with gel electrophoresis.

[0155] 10. Treatment effect of ruthenium complex on human non-small cell lung cancer cell line (A549 cells) and its corresponding cisplatin-resistant strain (A549R cells)

[0156] MTT method is also called MTT colorimetric method, which is a method for detecting cell survival and growth. The detection principle is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple formazan and deposit in cells, while dead cells have no such function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and the light absorption value is measured at 595 nm wavelength by enzyme-linked immunoassay instrument, which can indirectly reflect the number of living cells.

[0157] The experimental steps of MTT method are as follows:

[0158] (1) Prepare MTT: weigh 0.25 g thiazole blue and dissolve it in 50 mL sterile phosphate buffer solution (PBS) to prepare 5 mg / mL MTT;

[0159] (2) Revive 1 tube of A549 cells and 1 tube of A549R cells, A549 cells are cultured with DMEM medium, and A549R cells are cultured with F-12K medium, and 10% (by volume) fetal bovine serum is added to the medium, and the cells are subcultured twice before starting the experiment;

[0160] (3) When the cells reach the logarithmic growth phase, seed them into a 96-well plate at a cell density of 5000 cells per well (100 μL of culture medium is used to culture cells per well), and place them in a 37°C, 5vol% CO2 incubator for culture;

[0161] (4) After adhering, add 100 μL of ruthenium complex at 20, 10, 5, 2.5, 1.25, 0.625, and 0.3125 μM per well to A549 cells, respectively, and use commercial drug cisplatin as a control group, add 100 μL of cisplatin at 60, 30, 15, 7.5, 3.75, and 1.875 μM per well, respectively. Add 100 μL of ruthenium complex at 20, 10, 5, 2.5, 1.25, 0.625, and 0.3125 μM per well to A549R cells, respectively, and use commercial drug cisplatin as a control group, add 100 μL of cisplatin at 150, 75, 37.5, 18.75, 9.375, and 4.6875 μM per well, respectively. Gently shake the 96-well plate and place it in a carbon dioxide incubator (37°C, 5vol% CO2) for further incubation for 72 h;

[0162] (5) Add 20 μL of 5 mg / ml MTT to each well, shake gently, and place in a carbon dioxide incubator (37°C, 5vol% CO2) for 2-4 h;

[0163] (6) Take the 96-well plate out of the incubator, discard the solution in the plate, add 150 μL of dimethyl sulfoxide (DMSO) to dissolve the generated formazan, and measure the light absorption value at 595 nm wavelength by enzyme-linked immunoassay instrument, calculate the cell proliferation inhibition rate, and calculate the IC50 The results are shown in Table 1.

[0164] Table 1 IC values ​​of ruthenium complexes and cisplatin on A549 and A549R cells 50 Value (μM, 72h)

[0165]

[0166] It can be seen that the IC of ruthenium complexes on A549 cells 50 =2.13 μM, IC for A549R cells 50 =4.87μM, IC of commercial drug cisplatin for A549 cells 50 =5.42 μM, IC for A549R cells 50 =46.69 μM, indicating that the ruthenium complex of the present invention has a strong potential to overcome tumor resistance.

[0167] The above embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and replacements made by technicians in this field on the basis of the present invention fall within the scope of protection required by the present invention.

Claims

1. A ruthenium complex that induces mitochondrial DNA condensation to overcome tumor drug resistance, characterized in that: The structural formula is shown in Formula I: Wherein n is an integer from 1 to 7.

2. The ruthenium complex for inducing mitochondrial DNA condensation and overcoming tumor drug resistance according to claim 1, characterized in that: X is an inorganic salt anion; the inorganic salt anion is any one of chloride ion, perchlorate ion, nitrate ion or hexafluorophosphate ion.

3. A method for preparing a ruthenium complex that induces mitochondrial DNA condensation to overcome tumor drug resistance, characterized in that: The ligand of formula II and the ruthenium precursor compound of formula III are heated to react in an alcohol solvent under a protective gas atmosphere. After the reaction is completed and cooled, an aqueous solution of ammonium hexafluorophosphate inorganic salt is added to obtain a ruthenium complex of formula I that induces mitochondrial DNA condensation and overcomes tumor resistance.

4. The method for preparing a ruthenium complex for inducing mitochondrial DNA condensation and overcoming tumor drug resistance according to claim 3, characterized in that: The molar ratio of the ligand of formula II to the ruthenium precursor compound of formula III is 1:1-1.5; The alcohol solvent is ethylene glycol; The reaction conditions are: 100-130°C for 6-8 hours.

5. The method for preparing a ruthenium complex for inducing mitochondrial DNA condensation and overcoming tumor drug resistance according to claim 3, characterized in that: The ruthenium precursor compound with a structure shown in formula III is prepared by dissolving 4,7-diphenyl-1,10-phenanthroline and a ruthenium inorganic salt in a solvent and subjecting the mixture to a heating reaction.

6. The method for preparing a ruthenium complex for inducing mitochondrial DNA condensation and overcoming tumor drug resistance according to claim 3, characterized in that: The solvent is N,N-dimethylformamide; The reaction conditions are: in a dark and protective atmosphere, at 130-150°C for 6-8 hours; The ruthenium inorganic salt is RuCl3; the molar ratio of 4,7-diphenyl-1,10-phenanthroline to the ruthenium inorganic salt is 1:2-2.

5.

7. The method for preparing a ruthenium complex for inducing mitochondrial DNA condensation and overcoming tumor drug resistance according to claim 3, characterized in that: The preparation path of the ligand of the structure shown in Formula II includes:

8. Use of the ruthenium complex that induces mitochondrial DNA condensation to overcome tumor drug resistance as described in claim 1 or 2, or the ruthenium complex that induces mitochondrial DNA condensation to overcome tumor drug resistance as described in any one of claims 3-7 in the preparation of anti-tumor drugs.

9. An anti-tumor drug, characterized in that: The active ingredient of the anti-tumor drug comprises the ruthenium complex that induces mitochondrial DNA condensation to overcome tumor resistance as described in claim 1 or 2, or the ruthenium complex that induces mitochondrial DNA condensation to overcome tumor resistance prepared by the preparation method of the ruthenium complex that induces mitochondrial DNA condensation to overcome tumor resistance as described in any one of claims 3-7.

10. The antitumor drug according to claim 9, characterized in that The tumor is lung cancer; preferably, the tumor is non-small cell lung cancer; more preferably, the tumor is human non-small cell lung cancer; more preferably, the human non-small cell lung cancer cells are cisplatin-resistant strain A549R cells.

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