Ligand, osmium complex and preparation method and application thereof

Abstract

The invention discloses a ligand, an osmium complex and a preparation method and application thereof. A metal osmium complex formed by the ligand can efficiently generate various active oxygen species such as singlet oxygen (1O2), superoxide anions (O2.-) and hydroxyl radicals (. OH) under the irradiation of near-infrared laser, NADH / NADPH metabolic coenzyme in tumor cells is selectively oxidized through a photocatalytic reaction, and the redox steady state of the cells is effectively destroyed. In-vitro experiments show that the complex shows remarkable photodynamic anti-proliferative activity on mouse breast cancer cells (4T1), and the IC50 value (0.43 mu M) of the complex is reduced by about 28 times compared with that of a clinical chemotherapeutic drug cis-platinum (IC50 is equal to 12.39 mu M). The scheme provided by the invention has important significance for researching the antitumor of the metal osmium complex, and provides a new thought for clinically developing deep tumor photodynamic therapy.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and in particular to a ligand, an osmium complex, and a preparation method and application thereof. Background Art

[0002] Photodynamic therapy (PDT) is a non-invasive tumor treatment method, and its mechanism of action relies on the ternary synergistic effect of photosensitizer, excitation light source and molecular oxygen in tissue. In clinical practice, PDT has become an important treatment for superficial tumors (such as skin cancer and oral cancer) due to its advantages such as high spatial selectivity, low systemic toxicity and repeatability. However, the existing PDT technology faces significant technical bottlenecks in the treatment of deep solid tumors (such as liver cancer and pancreatic cancer):

[0003] 1. Insufficient penetration depth of the excitation light source: Traditional photosensitizers (such as hematoporphyrin derivatives and phthalocyanine compounds) rely on visible light (400-700nm) for excitation, but the high scattering / absorption characteristics of visible light in biological tissues limit the effective treatment depth to within 2-3mm, and cannot cover centimeter-level deep lesions;

[0004] 2. Aggregation inactivation of photosensitizers: Commonly used photosensitizers in clinical practice are prone to molecular aggregation in physiological environments, triggering aggregation-induced fluorescence quenching (ACQ) effect, resulting in a significant decrease in the quantum yield of singlet oxygen (1O2), seriously weakening the therapeutic effect.

[0005] In order to break through the limitation of penetration depth, near-infrared (NIR, 700-1700nm) responsive photosensitizers have become a research hotspot. Such photosensitizers (such as IR-780 and Cypate) can increase the tissue penetration depth to 1-2cm in the NIR-I region (700-950nm), and further extend to the NIR-II region (1000-1700nm) through two-photon absorption (TPA) technology, achieving three-dimensional space selective activation. However, existing NIR photosensitizers still have two major defects: I) Imbalance between photostability and ROS yield: long-wavelength absorption is usually accompanied by excited-state energy decay, and the 1O2 quantum yield of NIR-II photosensitizers (such as BODIPY-based derivatives) under 1064nm excitation is generally lower than 0.3, which is difficult to meet the needs of deep tumor ablation; II) Inhibition of aggregated ROS generation: When administered at high concentrations, the aggregation of photosensitizers at the tumor site leads to non-radiative dissipation of excited-state energy through π-π stacking, further reducing the ROS yield.

[0006] Therefore, the development of a new metal-organic complex that can achieve synergistic optimization of NIR excitation efficiency and ROS quantum yield will be expected to fundamentally resolve the contradiction between light energy transfer efficiency and reactive oxygen generation capacity in deep tumor PDT, and provide a disruptive technical solution for the treatment of solid tumors. Summary of the invention

[0007] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a ligand, and a complex prepared by using the ligand can efficiently generate various ROS under NIR excitation.

[0008] The invention also provides a method for preparing the ligand.

[0009] The present invention also provides a complex prepared by using the above ligand.

[0010] The invention also provides a method for preparing the complex.

[0011] The invention also provides the application of the complex.

[0012] According to the ligand of the first embodiment of the present invention, the ligand has the following structural formula:

[0013] The ligand according to the embodiment of the present invention has at least the following beneficial effects: the terthiophene pyridine dipyrimidine ligand of the present invention has a unique molecular structure, which has significant advantages in constructing near-infrared photosensitizing metal complexes.

[0014] The ligand molecule cleverly integrates the terthiophene unit, pyrimidine ring and pyridine ring. The terthiophene unit, with its unique extended π conjugated system, effectively promotes the red shift of the absorption spectrum of the complex, thereby better absorbing and utilizing near-infrared light. The pyrimidine ring, with its electron-rich characteristics, can effectively regulate the electron cloud distribution of the metal center. This regulation promotes the transfer of excited state energy to the triplet-singlet state (T1→S0) transition of molecular oxygen, thereby significantly improving the singlet oxygen ( 1 At the same time, the nitrogen atoms in the pyridine and pyrimidine rings give them strong coordination ability, allowing the ligands to bind tightly to metal ions to form stable and structurally diverse multi-dentate coordination metal complexes. This highly stable ligand structure not only significantly enhances the stability of the overall structure of the complex, but also ensures that the complex can maintain its structural and functional integrity under various conditions.

[0015] The method for preparing a ligand according to the second aspect of the present invention comprises the following steps:

[0016] The product is obtained by reacting 1-(pyrimidin-2-yl)ethan-1-one with 2,2':5',2"-terthiophene-5-carboxaldehyde to undergo nucleophilic substitution reaction.

[0017] The method for preparing the ligand according to the embodiment of the present invention has at least the following beneficial effects: the method for preparing the terthiophene pyridine dipyrimidine ligand according to the scheme of the present invention is easy to operate and has good industrial application prospects.

[0018] In some embodiments of the present invention, the method for preparing the ligand specifically comprises the following steps: 1-(pyrimidin-2-yl)ethan-1-one and 2,2':5',2"-terthiophene-5-carboxaldehyde undergo a nucleophilic substitution reaction in the presence of a protective atmosphere and a nucleophilic reagent to obtain the ligand.

[0019] In some embodiments of the invention, the nucleophile comprises a tert-butoxide, such as potassium tert-butoxide, sodium tert-butoxide, or lithium tert-butoxide.

[0020] In some embodiments of the present invention, the nucleophilic substitution reaction is carried out in a solution state, and the solvent used in the solution state includes at least one of tetrahydrofuran (THF), dioxane (1,4-dioxane) or diethyl ether (Diethyl Ether).

[0021] In some embodiments of the present invention, the nucleophilic substitution reaction temperature is 25±5°C.

[0022] In some embodiments of the present invention, the nucleophilic substitution reaction time is 18 to 36 hours.

[0023] In some embodiments of the present invention, the nucleophilic substitution reaction temperature is 25° C. and the reaction time is 18 h.

[0024] In some embodiments of the present invention, the protective atmosphere includes an inert gas atmosphere or a nitrogen atmosphere.

[0025] In some embodiments of the present invention, the inert gas comprises argon.

[0026] In some embodiments of the present invention, the preparation method further comprises a purification step after the reaction is completed.

[0027] In some embodiments of the present invention, the purification step comprises adding ammonium acetate to a mixed solution of ethanol / acetic acid (preferably, the volume ratio is 1 to 3:1; more preferably about 2:1), heating the reaction mixture to reflux for 4 to 6 hours (preferably 5 hours), cooling the reaction mixture to room temperature and pouring it onto ice, allowing it to stand for 2 to 4 hours (preferably 3 hours), filtering to obtain a solid precipitate, and purifying the crude product by alumina column chromatography.

[0028] In some embodiments of the present invention, the heating reflux temperature is 80-120°C.

[0029] In some embodiments of the present invention, the heating reflux temperature is 100° C. and the time is 5 hours.

[0030] In some embodiments of the present invention, the reaction formula involved in the preparation method is as follows:

[0031]

[0032] In some embodiments of the present invention, the molar ratio of 1-(pyrimidin-2-yl)ethan-1-one, 2,2':5',2"-terthiophene-5-carboxaldehyde, tert-butoxide and ammonium acetate is 2:0.8-1.2:2-3:30-34. For example, it is 2:1:2.2:32.

[0033] According to the osmium complex of the third aspect of the present invention, the structural formula of the osmium complex is as follows:

[0034]

[0035] Where, X - Represents anion.

[0036] According to a preferred embodiment of the present invention, at least the following beneficial effects are achieved: the metal osmium complex of the present invention can efficiently generate multiple reactive oxygen species such as singlet oxygen (1O2), superoxide anion (O2·-) and hydroxyl radical (·OH) under near-infrared laser irradiation, which selectively oxidizes NADH / NADPH metabolic coenzymes in tumor cells through photocatalytic reactions, effectively destroying the redox homeostasis of cells. In vitro experiments have shown that the complex exhibits significant photodynamic antiproliferative activity against mouse breast cancer cells (4T1) and its IC 50 The value (0.43 μM) is higher than that of the clinical chemotherapy drug cisplatin (IC 50 =12.39μM) is reduced by about 28 times. The scheme of the present invention is of great significance for the study of metal osmium complexes for anti-tumor, and provides a new idea for the clinical development of photodynamic therapy for deep tumors.

[0037] In the complex of the present invention, the donor and the acceptor are covalently attached, the covalent interaction is more stable, and the distance is closer, so a higher transfer efficiency can be achieved.

[0038] In some embodiments of the present invention, X - Represents PF6 - At least one of halogen ions (preferably chloride ions), tetrafluoroboric acid, tetraphenylboric acid, trifluoromethanesulfonic acid, or tetrakis(3,5-bis(trifluoromethyl)phenyl)boric acid. Anions have little effect on the generation of active oxygen.

[0039] According to the fourth aspect of the present invention, the method for preparing the above-mentioned osmium complex comprises the following steps:

[0040] Allowing the ligand and the osmium source to undergo a coordination reaction to obtain the osmium complex;

[0041] Wherein, the structural formula of the ligand is as follows:

[0042] The preparation method according to a preferred embodiment of the present invention has at least the following beneficial effects: the preparation method of the present invention is easy to operate and has good industrial application prospects.

[0043] In some embodiments of the present invention, the method for preparing the complex comprises: mixing the ligand and the osmium source in a solution state, and stirring at 180-220° C. (preferably 200° C.) for 1-5 h (preferably 3 h).

[0044] In some embodiments of the present invention, the coordination reaction system is in ethylene glycol, dimethyl sulfoxide (DMSO) or N-methylpyrrolidone (NMP).

[0045] In some embodiments of the present invention, the coordination reaction temperature is 200° C. and the time is 3 hours.

[0046] In some embodiments of the present invention, the osmium source includes ammonium chloroosmate. Other osmium inorganic salts or osmium-containing organic compounds may also be used.

[0047] In some embodiments of the present invention, the molar ratio of the ligand to ammonium chloroosmate is 1:1.8-2.2.

[0048] In some embodiments of the present invention, the molar ratio of the ligand to ammonium chloroosmate is 1:2.

[0049] In some embodiments of the present invention, the preparation method of the complex further comprises: cooling after the reaction is completed, adding excess tetrahydrofuran and standing overnight, filtering, washing with tetrahydrofuran to obtain a black precipitate, and purifying the crude product by alumina column chromatography to obtain the metal osmium complex. The reaction formula of this step is as follows:

[0050]

[0051] According to the application of the fifth aspect of the present invention, the application of the above-mentioned osmium complex in the preparation of anti-tumor drugs is proposed.

[0052] According to the application of a preferred embodiment of the present invention, at least the following beneficial effects are achieved: the complex of the present invention can generate superoxide anions under near-infrared light, and superoxide anions are usually associated with oxidative stress in the body and can be used as signal molecules to participate in a variety of physiological and pathological processes. Therefore, the complex can kill tumor cells or inhibit their growth by inducing oxidative stress, and has good anti-tumor potential, which indicates that it has good application prospects in the field of anti-tumor drugs.

[0053] According to the application of the fifth aspect of the present invention, an anti-tumor drug is also provided, wherein the anti-tumor drug contains the above-mentioned osmium complex.

[0054] According to the application of the fifth aspect of the present invention, an anti-tumor metal photosensitizer is also provided, wherein the active ingredient of the anti-tumor metal photosensitizer includes the above-mentioned osmium complex and may also include other ingredients.

[0055] In some embodiments of the present invention, the tumor is breast cancer. The complex of the present invention has a strong inhibitory effect on the proliferation of adenocarcinoma cells and has a good application prospect in the fight against breast cancer.

[0056] In some embodiments of the present invention, the breast cancer cell line is a mouse breast cancer cell line (4T1).

[0057] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable carrier and / or excipient. That is, the drug or photosensitizer uses the metal osmium complex as the main active ingredient, is mixed with a pharmaceutically acceptable carrier and / or excipient to prepare a composition, and is prepared into a clinically acceptable dosage form.

[0058] In some embodiments of the present invention, the excipient refers to diluents, binders, lubricants, disintegrants, solubilizers, stabilizers and other pharmaceutical matrices that can be used in the pharmaceutical field.

[0059] In some embodiments of the invention, different pharmaceutical excipients used in pharmaceutical dosage forms may vary depending on the specific medical application. Pharmaceutical excipients can be used to adjust the solubility and bioavailability of the drug, increase its stability, adjust the host's immunogenic response and act as emulsifiers, antioxidants, aerosol propellants, tablet binders, tablet disintegrants. Preferred pharmaceutical excipients include but are not limited to binders / fillers, coating agents, disintegrants, lubricants and sweeteners that are compatible with the use of the active pharmaceutical ingredient.

[0060] In some embodiments of the present invention, the carrier is a functional pharmaceutical excipient acceptable in the pharmaceutical field, including surfactants, suspending agents, emulsifiers and some pharmaceutical polymer materials prepared in embodiments of the present invention, such as cyclodextrin, chitosan, polylactic acid (PLA), polyglycolic acid-polylactic acid copolymer (PLGA), hyaluronic acid, etc.

[0061] In some embodiments of the present invention, the present invention has no particular restrictions on the dosage form of the above-mentioned drugs, and the active substance can be administered together with an assimilable edible carrier, an inert diluent, or directly combined with food. Drug dosage forms include, but are not limited to, hard-shell or soft-shell gelatin capsules, tablets, pills, powder injections, solutions, suspensions, elixirs, syrups, wafers, gels, buccal or sublingual tablets, films, suppositories, and enemas.

[0062] In other embodiments of the present invention, the complexes of the present invention can be prepared without any formulation adjuvants or using other drug delivery systems known in the prior art, such as liposome-forming components, vectored and non-vectored proteins, organic and inorganic nanoparticles, nanoemulsions and microemulsions, nanocrystals, single solvents or appropriate solvent mixtures, components such as lactose, polyvinylpyrrolidone (PVP), etc.

[0063] In some embodiments of the present invention, the prepared drug can be administered orally, involving any part of the gastrointestinal tract (such as mouth, pharynx, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum) and anus) or parenterally (such as intravenous, subcutaneous, intraperitoneal or local). If certain drugs are unstable under gastric conditions, they can be prepared into enteric-coated tablets.

[0064] According to the application of the fifth aspect of the present invention, a reagent or kit for biomedical imaging is also provided, comprising the above-mentioned osmium complex.

[0065] According to the application of the fifth aspect of the present invention, a chemical sensor is also provided, comprising the above-mentioned osmium complex. It is used as a chemical sensor for detecting hydrogen peroxide or other related chemicals in the environment. These sensors have high sensitivity and high selectivity, and can accurately and quickly respond to changes in the concentration of the target substance in a complex environment.

[0066] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1This is the hydrogen nuclear magnetic resonance spectrum of the ligand prepared in Example 1 of the present invention;

[0068] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of the metal osmium complex prepared in Example 2 of the present invention;

[0069] Figure 3 The UV-visible absorption spectra of the metal osmium complex prepared in Example 2 of the present invention in different solvents;

[0070] Figure 4 The fluorescence excitation spectrum and fluorescence emission spectrum of the metal osmium complex prepared in Example 2 of the present invention;

[0071] Figure 5 The fluorescence emission spectra of the metal osmium complex prepared in Example 2 of the present invention in different solvents are shown in FIG.

[0072] Figure 6 This is a graph showing the test results of the ability of the metal osmium complex prepared in Example 2 of the present invention to photocatalytically generate singlet oxygen;

[0073] Figure 7 This is a graph showing the test results of the ability of the metal osmium complex prepared in Example 2 of the present invention to photocatalytically generate superoxide anions;

[0074] Figure 8 This is a graph showing the test results of the ability of the metal osmium complex prepared in Example 2 of the present invention to photocatalytically generate hydroxyl radicals;

[0075] Fig. 9 This is a graph showing the test results of the ability of the metal osmium complex prepared in Example 2 of the present invention to photocatalytically oxidize NADH and NADPH;

[0076] Fig.10 This is a graph showing the test results of the photometal osmium complex prepared in Example 2 of the present invention against the cytotoxicity of a mouse breast cancer cell line (4T1) under light and dark conditions. DETAILED DESCRIPTION

[0077] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of them. Based on the embodiments of the present invention, other embodiments obtained by technicians in this field without creative work are all within the scope of protection of the present invention. The test methods used in the embodiments are conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can be reagents and materials obtained from commercial channels. Unless otherwise specified, the same parameter in each embodiment has the same value. The embodiments described below are exemplary and are only used to explain the present invention, and cannot be understood as limitations on the present invention.

[0078] In the description of the present invention, the description with reference to the term "some embodiments" or the like means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0079] The "room temperature" referred to in the present invention refers to 25±5°C, and is specifically 25°C in the embodiments.

[0080] Example 1

[0081] In this example, a ligand was prepared, and the structural formula of the ligand is as follows:

[0082] The specific preparation process is as follows:

[0083] The ligand was generated from 2,2':5',2"-trithiophene-5-carboxaldehyde and 1-(pyrimidin-2-yl)ethan-1-one. 1-(pyrimidin-2-yl)ethan-1-one (488 mg, 4 mmol), 2,2':5',2"-trithiophene-5-carboxaldehyde (552 mg, 2 mmol), and potassium tert-butoxide (488 mg, 4.4 mmol) were dissolved in 30 mL of anhydrous tetrahydrofuran under an argon atmosphere (other inert gases or nitrogen may also be used) and stirred at 25°C for 18 h. After the reaction at room temperature, a 60 ml mixed solution of ammonium acetate (2500 mg, 32 mmol) in ethanol / acetic acid (2:1) was added, and the reaction mixture was heated to reflux at 100°C for 5 hours. After the reaction mixture was cooled to room temperature, it was poured onto ice and allowed to stand for 3 hours. A solid precipitate was obtained by filtration, and the crude product was purified by alumina column chromatography to obtain the ligand (277 mg, 29%).

[0084] The chemical reaction equation above is as follows:

[0085]

[0086] The H NMR spectrum of the product is Figure 1 Specifically, the measured nuclear magnetic hydrogen spectrum data is:

[0087] 1H NMR (500MHz, Chloroform-d) δ8.99 (d, J = 4.8 Hz, 4H), 8.81 (s, 2H), 7.71 (d, J = 3.9 Hz, 1H), 7.38 (t, J = 4.8 Hz, 2H), 7.25 (dd, J = 4.4, 2.4 Hz, 2H), 7.22-7.19 (m, 2H), 7.13 (d, J = 3.8 Hz, 1H), 7.05 (dd, J = 5.1, 3.6 Hz, 1H). This shows that the above operation prepared the target compound with the correct structure.

[0088] Example 2

[0089] In this example, a metal osmium complex with high efficiency in generating various active oxygen species was prepared. The structural formula of the complex is as follows:

[0090]

[0091] The specific preparation process is as follows: the ligand prepared in Example 1 above is reacted with ammonium chloroosmate to generate an osmium complex. The ligand (119.3 mg, 0.4 mmol) and ammonium chloroosmate (88 mg, 0.2 mmol) are dissolved in 3 mL of ethylene glycol and stirred at 200°C for 3 hours. After the reaction is completed, the mixture is cooled to room temperature, and an excess of tetrahydrofuran is added to precipitate overnight, and the mixture is filtered, the filter cake is washed with tetrahydrofuran, and dried under vacuum. The crude product is purified by alumina column chromatography to obtain an osmium complex (123 mg, 26%).

[0092] The chemical reaction equation above is as follows:

[0093]

[0094] The H NMR spectrum of the product is Figure 2 Specifically, the measured H NMR spectrum data is:

[0095] 1 H NMR (500MHz, DMSO-d6) δ9.11 (s, 4H), 8.91 (dd, J = 4.7, 2.0 Hz, 4H), 8.44 (d, J = 3.9 Hz, 2H), 8.02 (dd, J = 5.9, 2.0 Hz, 4H), 7.68 (d, J = 3.9 Hz, 2H), 7.61 (dd, J = 5.1, 1.2 Hz, 2H), 7.59 (d, J = 3.8 Hz, 2H), 7.45–7.42 (m, 4H), 7.38–7.35 (m, 4H), 7.17 (dd, J = 5.1, 3.6 Hz, 2H). This indicates that the above operation produced the target compound with the correct structure.

[0096] Test example

[0097] In order to verify the application effect of the above complex, its performance was tested, as follows:

[0098] 1. UV-visible absorption and fluorescence spectra of metal osmium complexes in different solvents

[0099] (1) UV-visible absorption spectra of metal osmium complexes in different solvents

[0100] The metal osmium complex prepared by the above operation was prepared into a 10 μM sample solution using water (H2O), dichloromethane (DCM), and methanol (MeOH) as solvents, and then the UV-visible absorption spectrum of the metal osmium complex was recorded using a double-beam UV-visible spectrophotometer to characterize its absorbance in different solvents. The results are as follows Figure 3 The results in the figure show that the maximum absorption of the compound is around 520nm, and the absorption wavelength in dichloromethane can be extended to 750nm.

[0101] (2) Fluorescence excitation and emission spectra of metal osmium complexes

[0102] Using dichloromethane as solvent, the metal osmium complex prepared by the above operation was prepared into a 10 μM sample solution, and the fluorescence emission spectrum of the metal osmium complex was recorded using a fluorescence spectrophotometer with 550 nm as the EX fixed wavelength. Similarly, the fluorescence excitation spectrum of the metal osmium complex was recorded with 800 nm as the EM fixed wavelength to characterize its excitation spectrum and emission spectrum in dichloromethane. The results are as follows: Figure 4 As shown in the figure, it can be seen that the best excitation wavelength in dichloromethane is 550nm and the best emission wavelength is 800nm.

[0103] (3) Fluorescence emission spectra of metal osmium complexes in different solvents

[0104] The metal osmium complex prepared by the above operation was prepared into a 10 μM sample solution using water (H2O), dichloromethane (DCM), and methanol (MeOH) as solvents, and the fluorescence emission spectrum of the metal osmium complex was recorded using a fluorescence spectrophotometer with 550 nm as the EX fixed wavelength to characterize its fluorescence emission intensity in different solvents. The results are as follows: Figure 5 The results in the figure show that the fluorescence intensity of the compound is stronger in dichloromethane and weaker in water.

[0105] 2. Determination of the ability of metal osmium complexes to generate singlet oxygen

[0106] In order to detect the ability of the synthesized metal osmium complex prepared by the above operation to photocatalytically generate singlet oxygen, the singlet oxygen probe 9,10-Anthracenediyl-bis(methylene)dimalonic Acid (ABDA) was used to measure the ability of the new metal osmium complex to generate singlet oxygen. When singlet oxygen is generated in the solution, ABDA will immediately capture the singlet oxygen in the solution to generate ABDA endoperoxide. The change in the ultraviolet absorption spectrum of the sample to be tested and the ABDA mixed solution under different illumination times can reflect the ability to generate singlet oxygen.

[0107] A PBS solution containing metal osmium complex (5 μM) and ABDA reagent (100 μM) was placed in a cuvette and its singlet oxygen generation capacity was measured under light conditions. The results are as follows: Figure 6 As shown in the figure, it can be seen that the metal osmium complex has the ability to produce singlet oxygen after light exposure.

[0108] 3. Determination of the ability of metal osmium complexes to generate superoxide anions

[0109] In order to detect the ability of the synthesized metal osmium complex prepared by the above operation to photocatalytically generate superoxide anions, dihydrorhodamine 123 (abbreviated as DHR123) is used to measure the ability of the new metal osmium complex to generate superoxide anions. When superoxide anions are generated in the solution, DHR123 will immediately capture the superoxide anions in the solution, and DHR123 is oxidized to generate the fluorescent derivative rhodamine 123, so that bright green fluorescence (Ex / Em=500 / 536nm) is emitted, wherein the increase in fluorescence intensity can indicate that superoxide anions are generated in the solution. The change in the fluorescence spectrum of the sample to be tested and the mixed solution of DHR123 under different illumination times can reflect the ability to generate superoxide anions.

[0110] A PBS solution containing metal osmium complex (5 μM) and DHR123 reagent (10 μM) was placed in a cuvette and its superoxide anion generation capacity under light conditions was measured. The results are as follows: Figure 7 As shown in the figure, it can be seen that the metal osmium complex has the ability to generate superoxide anions after light exposure.

[0111] 4. Determination of the ability of metal osmium complexes to generate hydroxyl radicals

[0112] In order to detect the ability of the synthesized metal osmium complex prepared by the above operation to photocatalytically generate hydroxyl radicals, hydroxyphenyl fluorescein (abbreviated as HPF) was used to determine the ability of the new metal osmium complex to generate hydroxyl radicals. HPF itself has no fluorescence. When hydroxyl radicals are generated in the solution, it reacts with the hydroxyl radicals to generate strong green fluorescence (Ex / Em=490 / 515nm). The fluorescence intensity is proportional to the concentration of hydroxyl radicals. The change of the fluorescence spectrum of the sample to be tested and the HPF mixed solution under different illumination time can reflect the ability to generate hydroxyl radicals.

[0113] A PBS solution containing metal osmium complex (5 μM) and HPF reagent (10 μM) was placed in a cuvette and its superoxide anion generation capacity under light conditions was measured. The results are as follows: Figure 8 As shown in the figure, it can be seen that the metal osmium complex has the ability to generate superoxide anions after light exposure.

[0114] 5. Determination of the photocatalytic oxidation ability of NADH / NADPH by metal osmium complexes

[0115] Under light irradiation, the metal complex can oxidize reduced coenzyme I (NADH) and reduced coenzyme II (NADPH) into their oxidized states NADPH and NADPH. + and NADP + Therefore, metal osmium complex (5 μM) and NADH or NADPH (A 339nm =1.0) in a cuvette to determine their ability to oxidize NADH / NADPH under light conditions. Fig. 9 As shown in the figure, it can be seen that the metal osmium complex has photocatalytic oxidation ability for NADH and NADPH.

[0116] 6. Therapeutic effect of metal osmium complexes on human and mouse breast cancer cell lines

[0117] The resazurin method was used to analyze the antiproliferative effect of metal osmium complexes on mouse breast cancer cell lines (4T1 cells). Resazurin is a non-toxic, membrane-permeable blue dye with weak fluorescence. Living cells absorb the resazurin dye into the cell through endocytosis, where it is reduced to red resorufin. The red fluorescence of resorufin can be detected by fluorescence microscopy at an excitation wavelength of 540nm and an emission wavelength of 590nm, or by measuring the change in absorbance with a photometer. This increase in fluorescence signal is related to the metabolic activity and proliferation capacity of the cells. By measuring the amount of resorufin in the cell or the intensity of the fluorescence signal, the viability and proliferation of the cells can be quantitatively evaluated.

[0118] The steps of the Resazurin experiment are as follows:

[0119] (1) First, revive one tube of 4T1 tumor cells and culture them with fresh complete culture medium (DMEM medium + 10 vol% fetal bovine serum + 1 vol% penicillin-streptomycin mixture). After two passages, start the experiment.

[0120] (2) When the cells reached the logarithmic growth phase, they were inoculated into two 96-well plates at a cell density of 40,000 cells / mL. Each well was cultured with 100 μL of culture medium. One plate was the light group and the other was the dark group. Both groups were cultured in a 37°C, 5% CO2 incubator.

[0121] (3) After the cells adhere to the wall, add 10 μL of metal osmium complexes at six concentrations, namely 10, 5, 1, 0.5, 0.1, and 0.01 μM, to each well, shake gently, and continue incubating in the dark for 4 h under the corresponding conditions.

[0122] (4) After 4 h of incubation, the dark group continued to culture without changing the conditions, and the light group was illuminated with a light source of 635 nm for 30 min (33.12 J / cm 2 ), and then continue to incubate in a 37°C incubator for 48 hours, remove the culture medium, add 80 μL of resazurin (1 mg / mL) to each well, and continue to incubate in a 37°C incubator for 4 hours. 540 , EM 590 , calculate the cell proliferation inhibition rate and find the IC 50 value (drug concentration when the inhibition rate is equal to 50%).

[0123] The results are as follows Fig.10 As shown in the figure, it can be seen that the resazurin method detected different concentrations of metal osmium complexes under light and dark conditions to kill human mouse breast cancer cell line (4T1 cells) with different effects. Under light conditions, the IC 50 0.43μM, which is better than cisplatin (IC 50 This indicates that the complex of the present invention has a strong tumor proliferation inhibitory effect.

[0124] The embodiments of the present invention are described in detail above, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A ligand, characterized in that: The ligand has the following structural formula:

2. A method for preparing the ligand according to claim 1, characterized in that: The steps include: The product is obtained by reacting 1-(pyrimidin-2-yl)ethan-1-one with 2,2':5',2"-terthiophene-5-carboxaldehyde to undergo nucleophilic substitution reaction.

3. An osmium complex, characterized in that: The structural formula of the osmium complex is as follows: Where, X - Represents anion.

4. The osmium complex according to claim 3, characterized in that: X - Represents PF6 - , at least one of a tetrafluoroborate, a tetraphenylborate, a trifluoromethanesulfonate or a tetrakis(3,5-bis(trifluoromethyl)phenyl)borate; preferably, the halogen ion is a chloride ion.

5. A method for preparing an osmium complex as claimed in claim 3 or 4, comprising the following steps: causing a ligand and an osmium source to undergo a coordination reaction to obtain the osmium complex; in, The structural formula of the ligand is as follows:

6. Use of the osmium complex as claimed in claim 3 or 4 in the preparation of antitumor drugs.

7. An anti-tumor drug, characterized in that: The anti-tumor drug contains the osmium complex as described in claim 3 or 4; preferably, the tumor is breast cancer.

8. An anti-tumor metal photosensitizer, characterized in that: The active ingredient of the anti-tumor metal photosensitizer includes the osmium complex as described in claim 3 or 4.

9. A reagent or kit for biomedical imaging, characterized in that: Contains the osmium complex as claimed in claim 3 or 4.

10. A chemical sensor, characterized in that: Contains the osmium complex as claimed in claim 3 or 4.

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