Ruthenium polypyridine complex with multiple phototherapy mechanisms as well as preparation method and anti-lung cancer application thereof

Abstract

The invention relates to the field of metal complexes, in particular to a ruthenium polypyridine complex with multiple phototherapy mechanisms as well as a preparation method and anti-lung cancer application of the ruthenium polypyridine complex. The absorption wavelength of the ruthenium polypyridine complex is prolonged to 700nm, and the ruthenium polypyridine complex has good light and dark stability. The complex not only can efficiently generate singlet oxygen after light activation, but also can effectively destroy NADH / NAD < + > redox balance, and shows PDT and photocatalytic cancer treatment dual activities. The complex has relatively low dark toxicity and extremely strong phototoxicity to non-small cell lung cancer cells A549, and after light activation, cell apoptosis can be induced, so that A549 cell death can be caused. The content of the invention is expected to bring new breakthrough to lung cancer treatment.

Description

Technical Field

[0001] The present invention relates to a ruthenium metal complex, in particular to a ruthenium polypyridine complex with multiple phototherapy mechanisms, a preparation method and an application in treating lung cancer. Background Art

[0002] Lung cancer is currently one of the most commonly diagnosed cancers and the second leading cause of cancer death worldwide. Traditional treatments for lung cancer primarily include surgery, radiotherapy, and chemotherapy. While surgery is effective, it also carries disadvantages such as high trauma, high risk, and potential complications. Radiotherapy and chemotherapy also have poor tumor selectivity and are prone to significant toxic side effects. Therefore, the development of new and effective treatments for lung cancer is crucial.

[0003] Photodynamic therapy (PDT) is a new cancer treatment method developed based on the concept of light-activated prodrugs. Its mechanism of action is to activate photosensitizer drugs to produce reactive oxygen species (ROS) by using light of a specific wavelength. When ROS levels are above the physiological range, they damage components such as nucleic acids, lipids, and proteins, oxidize cells and biological substances, and cause oxidative damage and redox imbalance. By inducing mechanisms such as apoptosis, necrosis, and autophagy, PDT leads to tumor cell destruction and death. Compared with traditional cancer treatments, PDT has significant advantages in terms of treatment selectivity, minimal invasiveness, and control of systemic toxicity, and has been successfully used in the clinical treatment of lung cancer.

[0004] Ruthenium polypyridine complexes have become an ideal molecular platform for the development of new photosensitizers due to their excellent photophysical and photochemical properties (such as strong luminescence, high reactive oxygen species production and significant photostability). However, most of the reported Ru(II)-based photosensitizers have a dominant type II mechanism when exerting photodynamic anti-tumor effects, that is, they generate singlet oxygen ( 1 O2), its PDT efficacy will be limited by the hypoxic microenvironment of the tumor.

[0005] Photocatalytic cancer therapy is a potential non-invasive alternative therapy for treating malignant tumors proposed in recent years. In the development of metal-based photocatalytic anticancer drugs, NADH is an important target molecule that is overexpressed in tumor cells and participates in regulating cell proliferation and maintaining intracellular redox balance. After being activated by light of a specific wavelength, photocatalytic anticancer drugs can catalyze NADH to generate NAD. + , inhibiting ATP synthesis and destroying the energy supply required for DNA replication. It can exert self-oxygen supply capacity when acting simultaneously with PDT, enhance PDT capacity under hypoxic conditions, and more effectively induce tumor cell death.

[0006] Therefore, the development of Ru(II)-based photosensitizers with dual activity in PDT and photocatalytic cancer therapy can not only enhance the anti-tumor effect, but also overcome the oxygen-dependent limitation of PDT, which is expected to bring new breakthroughs in the treatment of lung cancer. Summary of the Invention

[0007] The first object of the present invention is to provide a ruthenium polypyridine complex whose absorption wavelength can be extended to 700nm.

[0008] The second object of the present invention is to provide a method for preparing a ruthenium polypyridine complex.

[0009] The third object of the present invention is to provide an application of a ruthenium polypyridine complex in light-activated anti-lung cancer treatment.

[0010] The present invention is achieved through the following technical solutions:

[0011] The structural formula of a ruthenium polypyridine complex is shown in Formula 1:

[0012]

[0013] Abbreviated as [Ru(dip)2(dppn)](PF6)2.

[0014] The preparation method of the ruthenium polypyridine complex comprises the following steps:

[0015] S1. Dissolve 1,10-phenanthroline-5,6-dione and 2,3-diaminonaphthalene in anhydrous ethanol, reflux for 3 h, cool, and precipitate an orange-red solid, which is filtered to obtain a bidentate ligand dppn;

[0016] S2. Dissolve RuCl3·xH2O, dip ligand, and anhydrous LiCl in 15 mL of N-N-dimethylformamide (DMF) solution and heat under reflux for 24 h under N2 protection. After cooling to room temperature, add excess acetone and place in a -20°C refrigerator overnight to precipitate a large amount of purple-black precipitate. Filter and spin-dry to obtain a purple-black solid crude product. Separate and purify by silica gel column chromatography, eluent: acetonitrile: water: saturated potassium nitrate aqueous solution = 40:4:1. After the separated solution is spin-dried, methanol is added to dissolve the solid, and the solid is precipitated with petroleum ether, filtered, and dried in vacuo to obtain [Ru(dip)2(Cl)2];

[0017] S3. Weigh [Ru(dip)2(Cl)2] and dppn ligand in a three-necked flask with a stoichiometric ratio of 1:1, dissolve them in anhydrous ethanol, heat under reflux for 5 hours, cool to room temperature, filter, and spin-dry the filtrate to obtain a reddish-brown solid crude product; separate and purify it by silica gel column chromatography, spin-dry, add a small amount of methanol to dissolve the solid, and then precipitate the solid with a saturated ammonium hexafluorophosphate aqueous solution, filter, and vacuum dry to obtain a ruthenium polypyridine complex.

[0018] Preferably, steps S2 and S3 are both performed under light-proof conditions and under the protection of 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] Under light conditions, the ruthenium polypyridine complex of the present invention can, on the one hand, exert the PDT therapeutic effect to generate reactive oxygen species, with a singlet oxygen quantum yield of 0.58; on the other hand, it can catalyze the conversion of NADH into NAD. + , play a photocatalytic oxidation role, and its conversion frequency (TOF) reaches 163.79h -1 Cell experiments showed that the complex showed low dark toxicity and strong phototoxicity to human non-small cell lung cancer A549 cells. 50 The value reached 0.3±0.1μM, the phototoxicity index value was 235.3, and it was able to cause lung cancer cell death by inducing cell apoptosis. 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 PBS buffer solution (pH = 7.2-7.4).

[0024] Attachment Figure 2 These are UV-visible absorption spectra of the ruthenium polypyridine complex obtained in Example 1 of the present invention in a PBS solution, after being placed in the dark (a) for 24 hours and irradiated with light (b) for 45 minutes.

[0025] Attachment Figure 3 This is an absorption quenching graph of 1,3-diphenylisobenzofuran (DPBF) at 410 nm under irradiation with emitted light (λ=489 nm) of the ruthenium polypyridine complex obtained in Example 1 of the present invention.

[0026] Attachment Figure 4 Schematic diagram of the catalytic oxidation of NADH by the ruthenium polypyridine complex obtained in Example 1 of the present invention in the dark (a) and under irradiation of a solar simulator (λ>450nm) (b).

[0027] Attachment Figure 5 This is a graph showing the live-dead cell staining results of A549 cells using Calcein-AM / PI double staining experiments with the ruthenium polypyridine complex obtained in Example 1 of the present invention under light (λ=470 nm) and dark conditions.

[0028] Attachment Figure 6 Figure 1 is a chart showing the results of Annexin V-FITC / PI staining of the ruthenium polypyridyl complex obtained in Example 1. DETAILED DESCRIPTION

[0029] The technical solutions of the present application will be further described below in conjunction with the drawings and specific examples. It should be noted that the descriptions of these embodiments are used to help understand the present application and do not constitute a limitation of the present application. In addition, the technical features involved in the various embodiments of the present application described below can be combined with each other as long as they do not conflict with each other.

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

[0031] Example 1

[0032] The synthesis method of the complex is as follows:

[0033] S1. Dissolve 421.39 mg of 1,10-phenanthroline-5,6-dione and 323.21 mg of 2,3-diaminonaphthalene in 20 mL of anhydrous ethanol, heat to reflux at 75°C for 3 h. Cool to obtain orange-red solid, filter and dry to obtain the bidentate ligand dppn. The above reaction equation is shown below:

[0034]

[0035] S2. Weigh 201.89 mg of RuCl3xH2O, 625.15 mg of dip ligand and 402.69 mg of anhydrous LiCl and dissolve them in 15 mL of DMF solution. Heat to reflux under N2 protection at 150°C for 24 h. After cooling to room temperature, add excess acetone and place in a -20°C refrigerator overnight. Filter and wash with water to obtain a large amount of purple-black crystals. Purify and separate the crude product by silica gel column chromatography. Eluent: acetonitrile: water: saturated potassium nitrate aqueous solution = 40:4:1. Dissolve the separated liquid in a small amount of methanol, precipitate with petroleum ether, filter and vacuum dry to obtain [Ru(dip)2(Cl)2]. The above reaction equation is shown below:

[0036]

[0037] S3. Accurately weigh 311.00 mg of [Ru(dip)2(Cl)2] and 126.6 mg of dppn and dissolve them in anhydrous ethanol. Heat under reflux at 75°C for 5 h under N2 protection. Cool to room temperature, filter, and spin dry to obtain a reddish-brown crude product. After separation and purification by silica gel column chromatography, dissolve in a small amount of methanol and add aqueous ammonium hexafluorophosphate dropwise until no further precipitation occurs. Filter and dry under vacuum to obtain [Ru(dip)2(dppn)](PF6)2.

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

[0039]

[0040] It is abbreviated as [Ru(dip)2(dppn)](PF6)2, and the yield is 32.5%.

[0041] H NMR (600 MHz, in [D6]DMSO): δ 9.64 (d, J = 7.8 Hz, 2H), 9.25 (s, 2H), 8.48-8.41 (m, 4H), 8.32 (dd, J = 10.8, 4.8 Hz, 4H), 8.29-8.25 (m, 4H), 8.03-7.97 (m, 2H), 7.82 (d, J = 5.4 Hz, 4H), 7.79 (dd, J = 6.6, 3.0 Hz, 2H), 7.70-7.59 (m, 20H).

[0042] Mass spectrometry: Q-TOF MS: [CH3CN, m / z]: 549.1365 [M-2PF6] 2+ ,1243.2369[M-PF6] + .

[0043] Example 2

[0044] UV-visible absorption spectrum of the ruthenium polypyridine complex obtained in Example 1

[0045] The UV-visible absorption spectrum of the ruthenium polypyridine complex was tested, and the results showed that its absorption wavelength can be extended to 700nm, which is in the ideal phototherapy window range. Figure 1 .

[0046] Example 3

[0047] Stability study of the ruthenium polypyridine complex obtained in Example 1

[0048] The stability of the ruthenium polypyridine complex was investigated, and it maintained good stability after being placed in the dark for 24 hours and irradiated with a solar simulator (λ>450nm) for 45 minutes. Figure 2 .

[0049] Example 4

[0050] In vitro ROS generation ability of the ruthenium polypyridine complex obtained in Example 1

[0051] Determination of the concentration of the complex in acetonitrile by DPBF 1 O2 quantum yield. DPBF can be compared with 1 O2 undergoes epoxidation in acetonitrile, causing the characteristic absorption peak of DPBF at 410 nm to attenuate, and the reduction level is similar to 1 O2 level is positively correlated. [Ru(bpy)3](Cl)2 (bpy = 2,2'-bipyridine) was selected as a reference. The absorbance values ​​of the complex to be tested and [Ru(bpy)3](Cl)2 at 489nm were adjusted to the same value. DPBF was added and mixed. The absorbance value of DPBF at λ = 410nm was monitored using 489nm excitation light. Figure 3 As shown, the singlet oxygen quantum yield of the ruthenium polypyridine complex in acetonitrile was measured to be 0.58.

[0052] Example 5

[0053] Photocatalytic oxidation ability of NADH by the ruthenium polypyridine complex obtained in Example 1

[0054] NADH was used to test the photocatalytic oxidation ability of ruthenium polypyridine complexes in PBS solution. Under light, NADH can be catalytically oxidized by the complex to NAD + , which weakens the characteristic absorption peak of NADH at 339nm, and NAD + The absorbance at 259nm increased. The changes in the absorbance of NADH at 339nm in the dark and after illumination with a solar simulator (λ>450nm) were quantitatively detected using an ultraviolet spectrophotometer. There was no significant change in the dark condition, but after light activation, a significant decrease occurred at 339nm and a significant increase occurred at 259nm. Figure 4 As shown, (a) UV-visible absorption spectrum under dark conditions, (b) UV-visible absorption spectrum after illumination, the conversion frequency TOF of ruthenium polypyridine complex was measured to be 163.79h -1 .

[0055] Example 6

[0056] Cytotoxicity test of ruthenium polypyridine complex obtained in Example 1

[0057] The phototoxicity and dark toxicity of the complex to non-small cell lung cancer A549 cells were investigated using CCK8 assay, and [Ru(bpy)3](Cl)2 was used as a reference. The specific steps are as follows: well-grown A549 cells were taken, digested and centrifuged, and 5×10 3Cells were seeded at a density of 100 μg / mL in a 96-well plate and incubated in a constant temperature incubator (37°C, 5% CO2) for 24 hours. The old culture medium was discarded and culture medium containing the complex at an equal gradient concentration was added. The culture was continued for 4 hours. The light group was irradiated with an LED dual-channel controller (λ = 470 nm) for 15 minutes and then cultured for 20 hours. The dark group was treated synchronously with the light group except that no light was applied. After incubation for 1 hour with culture medium containing CCK8 reagent, the OD value at 450 nm was measured with a microplate reader and the IC was calculated. 50 The results are shown in Table 1. Compared with [Ru(bpy)3](Cl)2, the IC 50 The values ​​are similar, but after exposure to light, the phototoxicity of the ruthenium polypyridine complex to A549 cells increased significantly, and its IC 50 The value was 0.3±0.1μM, and the phototoxicity index (PI) value reached 235.3, which was significantly increased compared with the reference complex. Analysis showed that the excellent phototoxicity of the ruthenium polypyridine complex to A549 cells may be attributed to its higher 1 O2 quantum yield and strong photocatalytic oxidation ability of NADH.

[0058] Table 1 Phototoxicity and dark toxicity of the complexes to A549 cells

[0059]

[0060] PI = Dark IC 50 / Light IC 50

[0061] Example 7

[0062] Live and dead cell staining experiment of ruthenium polypyridine complex obtained in Example 1

[0063] Non-small cell lung cancer A549 cells were selected as experimental cells and [Ru(bpy)3](Cl)2 was used as reference. A549 cells with good growth were digested and centrifuged, and 2×10 4The cells were seeded at a density of 100 cells / mL in a 24-well plate and divided into light and dark groups. They were cultured in a constant temperature incubator (37°C, 5% CO2) for 24 hours, the old culture medium was discarded, and the culture medium containing ruthenium polypyridine complex (2μM) was added to incubate 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. Except for the dark group, the rest were carried out simultaneously with the light group. After discarding the drug solution and washing with PBS, Calcein AM / PI (calcein / propidium iodide) reagent was added (the concentration of Calcein AM was 1μM, the concentration of PI was 4μM, live cells appeared green and dead cells appeared red), and the cells were 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 Figure 2. Figure 5 As shown, cells in all groups grew normally under dark conditions, but under light conditions, the experimental group with the complex showed a significant increase in red fluorescence and a large number of cells died, indicating that the complex is highly phototoxic, consistent with the conclusions of the CCK-8 experiment.

[0064] Example 8

[0065] Apoptosis experiment of ruthenium polypyridine complex obtained in Example 1

[0066] Annexin V-FITC / PI double staining was used, [Ru(bpy)3](Cl)2 was used as a reference, and flow cytometry was used to detect the mechanism by which the ruthenium polypyridine complex caused lung cancer cell death. Well-grown A549 cells were digested and centrifuged, and 5×10 4 The cells were seeded at a density of per well in a six-well plate and divided into a light group and a dark group. They were placed in a constant temperature incubator (37°C, 5% CO2) and cultured for 24 hours. The old culture medium was discarded, and a culture medium containing a ruthenium polypyridine complex (2μM) was added. The culture was continued for 4 hours. The light group was illuminated for 15 minutes using a LED dual-channel controller (λ=470nm) and then cultured for another 20 hours. The dark group was synchronized with the light group except that it was not illuminated. The cells were stained with the Annexin V-FITC / PI cell apoptosis detection kit and detected using a flow cytometer. The results are shown in Figure 2. Figure 6 As shown in the data, after drug treatment, the Annexin V+ / PI+ in the first quadrant and the Annexin V+ / PI- in the fourth quadrant of the light group were significantly higher than those in the dark group, and the Annexin V- / PI- in the third quadrant was lower than that in the dark group. The cell apoptosis rate reached 52.88%, indicating that the ruthenium complex mainly causes cell death through the apoptosis pathway, and the drug toxicity is greater under light conditions.

[0067] 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 having the molecular formula [Ru(dip)2(dppn)](PF6)2; wherein dip is 4,7-diphenyl-1,10-phenanthroline, and dppn is a bidentate ligand synthesized from 1,10-phenanthroline-5,6-dione and 2,3-diaminonaphthalene; and having the structure described in Formula 1 below:

2. The method for preparing a ruthenium polypyridine complex according to claim 1, wherein The method comprises the following preparation steps: S1. 1,10-phenanthroline-5,6-dione and 2,3-diaminonaphthalene were dissolved in anhydrous ethanol and refluxed for 3 h. The mixture was cooled to room temperature to precipitate an orange-red solid, which was filtered and dried to obtain a bidentate ligand dppn. S2. RuCl3·xH2O, dip, and anhydrous LiCl were dissolved in 15 mL of N-N-dimethylformamide (DMF) solution, heated under reflux for 24 h, cooled to room temperature, added with an appropriate amount of acetone, and placed in a -20°C refrigerator overnight. Filtered to obtain a purple-black crude product, which was separated and purified by silica gel column chromatography (eluent: acetonitrile: water: saturated potassium nitrate = 40:4:1). The separated solution was spin-dried and dissolved in a small amount of methanol. The precipitate was precipitated with petroleum ether, filtered, and dried in vacuo to obtain [Ru(dip)2(Cl)2]. S3. Weigh [Ru(dip)2(Cl)2] and dppn in a three-necked flask with a stoichiometric ratio of 1:1, add anhydrous ethanol to dissolve, heat under reflux for 5 hours, cool to room temperature, filter, and spin-dry to obtain a reddish-brown crude product, which is separated and purified by silica gel column chromatography. The eluent is acetonitrile: water: saturated potassium nitrate = 40:4:

1. The separated liquid is spin-dried and dissolved in a small amount of methanol. Saturated aqueous ammonium hexafluorophosphate solution is added dropwise until no more precipitate is precipitated. Filter and vacuum dry to obtain a reddish-brown product.

3. The method for preparing a ruthenium polypyridine complex according to claim 2, wherein: The steps S2 and S3 are both carried out in the dark and under the protection of inert gas nitrogen.

4. Use of the ruthenium polypyridine complex according to claim 1 in the preparation of light-activated anti-lung cancer drugs.

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