A ruthenium polypyridine complex and its preparation method and anti-tumor application

Abstract

The present invention relates to the field of metal complexes, and in particular to a ruthenium polypyridine complex, a preparation method thereof, and anti-tumor applications. The absorption wavelength of the ruthenium polypyridine complex can be extended to 750 nm, and singlet oxygen can be generated under illumination conditions. The singlet oxygen quantum yield thereof in acetonitrile solution was measured to be 0.54. Compared with the chemotherapy drug cisplatin, the ruthenium polypyridine complex exhibits stronger cytotoxicity, and its toxicity to tumor cells after illumination is greater than that under dark conditions, and has the potential for photodynamic therapy (PDT). The content of the present invention has good clinical application prospects and can be used to further prepare photodynamic anti-tumor drugs.

Description

Technical Field

[0001] The present invention relates to the field of metal complexes, and in particular to a ruthenium polypyridine complex, a preparation method thereof, and anti-tumor applications thereof. Background Art

[0002] Cisplatin is one of the most effective chemotherapy drugs in clinical use to date, but its application is limited by significant side effects and the development of drug resistance. Transition metal complexes, particularly ruthenium complexes, have become the most promising alternatives to platinum drugs due to their low toxicity, ease of absorption and metabolism, and selectivity for certain tumor cells.

[0003] Photodynamic therapy (PDT) activates photosensitizers with light of a specific wavelength, generating cytotoxic singlet oxygen, which kills tumor cells. This therapy avoids the systemic toxicity of traditional chemotherapy drugs and offers excellent therapeutic selectivity. Ruthenium(II) complexes show promising application prospects in PDT due to their unique photophysical properties, including long-lived excited states and tunable emission wavelengths.

[0004] The ideal phototherapy window range is between 600 and 900 nm. How to shift the photoactivation wavelength of Ru(Ⅱ)-based phototherapy preparations to the phototherapy window range to achieve deeper tissue penetration is a huge challenge currently faced. The photophysical properties of Ru(Ⅱ) polypyridine complexes (including absorption, emission, and excited state lifetime) depend on the ligands bound to the Ru center, so their photophysical properties can be adjusted by selecting suitable auxiliary ligands. By introducing dip ligands with large conjugated systems, the absorption wavelength of ruthenium complexes can be red-shifted to approach the phototherapy window range. At the same time, dip ligands are highly hydrophobic and can be effectively taken up by cells. Therefore, complexes of the Ru-dip system usually also show efficient cellular uptake. Summary of the Invention

[0005] The first object of the present invention is to provide a ruthenium polypyridine complex whose absorption wavelength can be extended to 750nm.

[0006] The second object of the present invention is to provide a method for preparing a ruthenium polypyridine complex.

[0007] The third object of the present invention is to provide a ruthenium polypyridine complex for use in photodynamic anti-tumor therapy.

[0008] The present invention is achieved through the following technical solutions:

[0009] The structural formula of a ruthenium polypyridine complex is shown in Formula 1:

[0010]

[0011] Abbreviated as [Ru(dip)2(dpb)](Cl2).

[0012] The preparation method of the ruthenium polypyridine complex comprises the following steps:

[0013] S1. Dissolve RuCl3·xH2O, dip, and LiCl in 15 mL of N-N-dimethylformamide (DMF) solution, heat under reflux for 24 h, cool to room temperature, add an appropriate amount of acetone, and place in a -20°C refrigerator for 4 h to precipitate a large amount of purple-brown precipitate. Filter to obtain [Ru(dip)2(Cl)2];

[0014] S2. 2,2'-pyridone and 2,3-diaminonaphthalene were dissolved in anhydrous ethanol and refluxed for 3 h. After cooling, a large amount of yellow-brown needle-like solids precipitated, which were filtered to obtain the bidentate ligand dpb;

[0015] S3. [Ru(dip)2(Cl)2] and dpb were weighed in a stoichiometric ratio (1:1) and placed in a three-necked flask. A mixed solution of ethanol and water (volume ratio of 1:1) was added and heated under reflux for 3 h. The mixture was cooled to room temperature, filtered, and the filtrate was dried to obtain a reddish-brown solid crude product.

[0016] S4. Add a small amount of dichloromethane to dissolve the solid, add petroleum ether dropwise and shake, solid precipitates, filter and wash the precipitate with water and ether, and finally dry in vacuum to obtain the ruthenium polypyridine complex.

[0017] Preferably, steps S1, S3 and S4 are all performed under light-proof conditions.

[0018] Preferably, steps S1 and S3 are performed under the protection of an inert gas, nitrogen.

[0019] The synthetic route of the present invention is as follows:

[0020]

[0021] The present invention has the following technical effects:

[0022] The complex of the present invention can produce 1 CCK8 experiments showed that compared with the chemotherapy drug cisplatin, the complex has stronger toxicity to human lung cancer cells A549 and human cervical cancer cells Hela, and the phototoxicity index (PI*) for the two cell lines is 17.6 and 9.3, respectively, indicating that it has a good photodynamic therapy effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Attachment Figure 1 This is a UV-visible absorption spectrum of the ruthenium polypyridine complex obtained in Example 1 of the present invention in acetonitrile.

[0024] Attachment Figure 2 This is an absorption quenching graph of 1,3-diphenylisobenzofuran (DPBF) at 410 nm under irradiation with emitted light (λ=522 nm) of the ruthenium polypyridine complex obtained in Example 1 of the present invention.

[0025] Attachment Figure 3 This is a diagram showing the generation of reactive oxygen species (ROS) in cells by the ruthenium polypyridine complex obtained in Example 1 of the present invention.

[0026] Attachment Figure 4 These are comparison graphs of the cell viability of the ruthenium polypyridine complex obtained in Example 1 of the present invention and the reference compound cisplatin under different conditions. (a) and (b) are comparison graphs of the viability of the ruthenium polypyridine complex on A549 cells under light (λ = 470 nm) and dark conditions, respectively; (c) and (d) are comparison graphs of the viability of the ruthenium polypyridine complex on Hela cells under light and dark conditions, respectively.

[0027] Attachment Figure 5 This is a graph showing the live-dead cell staining results of human lung cancer cells (A549) using the ruthenium polypyridyl complex obtained in Example 1 of the present invention under dark and light (λ=470 nm) conditions.

[0028] Attachment Figure 6 This is a graph showing the cell apoptosis rate of the ruthenium polypyridine complex obtained in Example 1 of the present invention stained with Annexin V-FITC / PI. DETAILED DESCRIPTION

[0029] The technical solutions of the present invention are further described below with reference to the accompanying drawings and specific examples. It should be noted that the descriptions of these embodiments are intended to facilitate understanding of the present invention and do not constitute limitations of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0030] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0031] Example 1, a method for synthesizing the complex, the steps are as follows:

[0032] S1. Weigh 196.5 mg of RuCl₃·xH₂O, 511.9 mg of dip ligand, and 223.2 mg of LiCl and dissolve them in 15 mL of DMF. Heat under reflux at 150°C for 24 hours under nitrogen. Cool to room temperature, add excess acetone, and place in a -20°C refrigerator for 4 hours. A large amount of purple-brown crystals will precipitate. Filter, wash with water, and dry the solid to obtain [Ru(dip)₂(Cl)₂]. The reaction equation is as follows:

[0033]

[0034] S2. Dissolve 848.2 mg of 2,2'-pyridone and 633.4 mg of 2,3-diaminonaphthalene in 30 mL of anhydrous ethanol and heat under reflux at 75°C for 3 hours. Cool to precipitate a large amount of yellow-brown needle-like solids, which are filtered and dried to obtain the bidentate ligand dpb. The above reaction equation is as follows:

[0035]

[0036] S3. Accurately weigh 166.4 mg of [Ru(dip)2(Cl)2] and 132.8 mg of dpb and dissolve them in a 1:1 volume ratio of ethanol and water. Heat under reflux at 90°C for 3 h under nitrogen. After cooling, filter, and spin dry to obtain a reddish-brown crude product.

[0037] S4. Dissolve the crude solid product in a small amount of dichloromethane, add a small amount of petroleum ether dropwise to precipitate, filter, and wash with water and ether. Dry the solid to obtain the ruthenium polypyridine complex. After drying, weigh and calculate the yield.

[0038] The structural formula of the ruthenium polypyridine complex is as follows:

[0039]

[0040] It is abbreviated as [Ru(dip)2(dpb)](Cl2), and the yield is 32.2%.

[0041] H NMR spectrum: 1H NMR(600MHz,in[D6]DMSO)δ8.89(d,J=6.0Hz,1H),8.87(s,1H),8.81(dd,J=1.2,4.8Hz,1H),8.73(d,J=5.4Hz,1H) ,8.54(d,J=6.0Hz,1H),8.41(d,J=9.0Hz,1H),8.35(dd,J=9.6,7.8Hz,2H),8.31(s,1H),8.27(m,1H),8.20(t,J=8. 4Hz,2H),8.13(d,J=5.4Hz,1H),8.05(d,J=4.2Hz,1H),7.98(dd,J=9.6,6.0Hz,2H),7.91(dd,J=6.0,1.2Hz,2H),7. 76(m,4H),7.67(m,10H),7.60–7.54(m,9H),7.49(dd,J=3.0,1.2Hz,3H),7.46(d,J=8.4Hz,1H),7.41–7.37(m,1H).

[0042] Example 2, UV-visible absorption spectrum measurement of the ruthenium polypyridine complex obtained in Example 1

[0043] The UV-visible absorption spectrum of the ruthenium polypyridine complex was tested, and the results showed that its absorption wavelength can be extended to 750nm, which is in the phototherapy window range. Figure 1 .

[0044] Example 3, Determination of Singlet Oxygen Quantum Yield of Ruthenium Polypyridine Complex Obtained in Example 1

[0045] The DPBF method was used to determine the concentration of the complex in acetonitrile solution. 1 O2 quantum yield. DPBF is a highly efficient singlet oxygen scavenger that can be used with 1 O2 reacts in acetonitrile solution, causing the absorbance of DPBF at 410nm to decrease. Select [Ru(bpy)3] 2+ As a standard substance, its 1 The quantum yield of O2 is 0.57. 2+ Adjust the absorption at 522nm to the same value, add DPBF, mix well, irradiate with 522nm excitation light, and monitor the change of DPBF absorbance at λ=410nm. Figure 2 As shown, the ruthenium polypyridine complex was measured 1 The O2 quantum yield is 0.54.

[0046] Example 4, ROS experiment of the ruthenium polypyridine complex obtained in Example 1 in cells

[0047] 2'7'-dichlorofluorescein diacetate (DCFH-DA) was used as a fluorescent probe to detect the production of ROS in cells by the ruthenium polypyridine complex. A549 cells were seeded in six-well plates and divided into light group and dark group, and cultured in a constant temperature incubator (37°C, 5% CO2) for 24 hours. Ruthenium polypyridine complex (2μM) was added, incubated for 4 hours, the drug solution was discarded and washed twice with PBS, 15μM DCFH-DA probe was added, cultured in a constant temperature incubator for 30 minutes, washed 3 times with PBS, and the light group was illuminated with an LED dual-channel controller (λ=470nm) for 20 minutes, and the production of ROS was observed with an inverted fluorescence microscope. The results are shown in Figure 2. Figure 3 As shown, after being irradiated with light, the complex showed obvious green fluorescence, indicating that it produced ROS in the cells.

[0048] Example 5, Cytotoxicity Test of Ruthenium Polypyridine Complex Obtained in Example 1

[0049] The phototoxicity and dark toxicity of the complex to A549 cells and Hela cells were investigated using the CCK8 experiment, and the chemotherapy drug cisplatin was used as a reference. The specific steps are as follows: take well-grown A549 cells and Hela cells, digest and centrifuge them, and then plant them in 96-well plates at a density of 5000 to 10000 per well, incubate them in a constant temperature incubator (37°C, 5% CO2) for 24 hours, discard the old culture medium, add different concentrations of ruthenium polypyridine complex [Ru(dip)2(dpb)](Cl2) and cisplatin, and continue to culture for 4 hours. The light group was illuminated with an LED dual-channel controller (λ=470nm) for 15 minutes, and then continued to culture for 20 hours. The dark group was not illuminated, and the other conditions were the same as the light group. After adding the CCK8 reagent and culturing for 1 hour, the OD value at 450nm was detected with an enzyme marker to obtain the cell survival rate under different conditions (see Figure 4 ), and calculate IC 50 The results are shown in Table 1 below. Compared with the chemotherapy drug cisplatin, the ruthenium polypyridine complex is more toxic, and the complex shows light-enhanced cytotoxicity to both cells. Under light conditions, the IC value of the ruthenium polypyridine complex on A549 cells is 50 The value is 0.7μmol / L, and the PI* value is 17.6; for Hela cells, the IC 50 The PI* value was 1.1 μmol / L and 9.3, indicating that the complex has a good photodynamic therapy effect. In addition, the ruthenium polypyridine complex has a high lipophilicity and is easily taken up by cells, which may be the reason for its strong cytotoxicity.

[0050] Table 1 IC values ​​of the complexes with cisplatin on A549 and Hela cells under light and dark conditions 50 value

[0051]

[0052] PI*=Dark IC 50 / Light IC 50

[0053] Example 6, Live and Dead Cell Staining Experiment of Ruthenium Polypyridine Complex Obtained in Example 1

[0054] In this experiment, human lung cancer cells A549 were selected as model tumor cells. The well-grown A549 cells were digested and centrifuged, and seeded into six-well plates at a density of 20,000 cells per well. The cells were divided into two groups, light and dark. The cells were cultured in a constant temperature incubator (37°C, 5% CO2) for 24 hours. The old culture medium was discarded, and the ruthenium polypyridine complex (5μM) was added and incubated with the cells for 4 hours. The light group was then illuminated for 15 minutes using a LED dual-channel controller (λ=470nm) and cultured for another 20 hours. The dark group had the same conditions as the light group except that no illumination was used. After discarding the drug solution and washing with PBS, the diluted Meilun Biological Cell Viability Detection Reagent (Calcein AM concentration was 1μM, PI concentration was 4μM, live cells appeared green and dead cells appeared red) was added and incubated at 37°C in the dark for 30 minutes. Finally, the live and dead cell staining results were detected using an inverted fluorescence microscope. The results are shown in the figure below. Figure 5 As shown in the figure, after drug treatment, the number of dead cells in the light-irradiated group was significantly higher than that in the dark, indicating that the cytotoxicity of the ruthenium polypyridine complex after light irradiation was greater than that produced in the dark.

[0055] Example 7, apoptosis experiment of the ruthenium polypyridine complex obtained in Example 1

[0056] The cell death mechanism includes necrosis and apoptosis, which are usually stained with Annexin V-FITC / PI (propidium iodide) and detected using a flow cytometer. First, take the well-grown A549 cells, digest and centrifuge them to make a cell suspension, and seed them in a six-well plate at a density of 50,000 per well. They are divided into a light group and a dark group, and placed in a constant temperature incubator (37°C, 5% CO2) for 24 hours. Then add 1mL (1.2μM) of the ruthenium polypyridine complex, and continue to culture for 4 hours. The light group is illuminated for 15 minutes using an LED dual-channel controller (λ=470nm), and then cultured for 20 hours. Except for no illumination, the other conditions of the dark group are the same as those of the light group. After staining with the AnnexinV-FITC / PI cell apoptosis detection kit, the cells are detected using a flow cytometer. The results are as follows Figure 6 As shown, after drug treatment, the sum of the upper right and lower right quadrants of the light group was higher than that of the dark group and other control groups, indicating that the ruthenium complex mainly caused cell death through the apoptosis pathway, and the drug toxicity was greater under light conditions.

[0057] The embodiments described in the present invention are merely examples for the purpose of clearly illustrating the present invention, and are not intended to limit the scope of the present invention. Based on the above description, there may be other different forms of changes, and it is impossible to list all the changes here. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solution of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A ruthenium polypyridine complex, characterized in that: The molecular formula is [(Ru(dip)2(dpb)](Cl2); wherein dip is 4,7-diphenyl-1,10-phenanthroline, and dpb is a bidentate ligand synthesized from 2,2'-pyridone and 2,3-diaminonaphthalene; it has the structure described in the following formula 1:

2. The method for preparing a ruthenium polypyridine complex according to claim 1, wherein The method comprises the following preparation steps: S1. Dissolve RuCl3·xH2O, dip, and LiCl in 15 mL of NN-dimethylformamide (DMF) solution, heat under reflux for 24 h, cool to room temperature, add an appropriate amount of acetone, and place in a -20°C refrigerator for 4 h. A large amount of purple-brown precipitate will precipitate, which is filtered to obtain [Ru(dip)2(Cl)2]; S2. 2,2'-pyridone and 2,3-diaminonaphthalene were dissolved in anhydrous ethanol and refluxed for 3 h. After cooling, a large amount of yellow-brown needle-like solids precipitated, which were filtered to obtain the bidentate ligand dpb; S3. [Ru(dip)2(Cl)2] and dpb were weighed in a stoichiometric ratio of 1:1 and placed in a three-necked flask. An equal volume of a mixed solution of ethanol and water was added, heated under reflux for 3 h, cooled to room temperature, filtered, and the filtrate was dried to obtain a reddish-brown solid crude product; S4. Add a small amount of dichloromethane to dissolve the solid, add petroleum ether dropwise and shake, solid precipitates, filter and wash the precipitate with water and ether, and finally dry in vacuum to obtain the ruthenium polypyridine complex.

3. The method for preparing a ruthenium polypyridine complex according to claim 2, wherein: Steps S1, S3 and S4 are all performed under light-proof conditions.

4. The method for preparing a ruthenium polypyridine complex according to claim 2, wherein: Steps S1 and S3 are performed under the protection of inert gas nitrogen.

5. Use of the ruthenium polypyridine complex according to claim 1 in the preparation of photodynamic anti-tumor drugs.

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