Osmium complex as well as preparation method and application thereof

Abstract

The invention discloses an osmium complex as well as a preparation method and application thereof, and the osmium complex has a structure as shown in the following formula: in the formula # imgabs0, X-represents an anion. According to the scheme, the compound has the aggregation-induced emission (AIE) characteristic in an aqueous environment, superoxide anions and singlet oxygen can be generated, and the compound has catalytic oxidation capacity on NADH / NADPH in tumor cells. The complex has good proliferation inhibition ability (IC50 is 15.61 [mu] M) on mouse breast cancer cells (4T1) under the condition of 635 nm red light illumination, almost has no cytotoxicity (IC50 is larger than 100 [mu] M) under the non-illumination condition, and 635 nm red light has high tissue penetrating ability and can go deep into a focus position, a photosensitizer is effectively activated, and the treatment effect is improved. The metal osmium complex has important significance in studying the antitumor effect of the metal osmium complex, and a new thought is provided for clinically developing deep tumor photodynamic therapy.

Description

Technical Field

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

[0002] Photodynamic therapy (PDT) is a new treatment method that is non-invasive, spatiotemporally selective and not prone to drug resistance. It has shown important clinical application value in the field of tumor treatment and can be used as an effective supplement to traditional therapies such as surgical resection, radiotherapy and chemotherapy. Its treatment mechanism is based on the photoactivation reaction of photosensitizers (PS) with molecular oxygen under specific wavelength light, which produces cytotoxic reactive oxygen species (ROS), achieving a synergistic therapeutic effect of targeted killing of tumor cells, destruction of the vascular system and regulation of the immune microenvironment.

[0003] The core challenge facing the current clinical transformation of PDT is that traditional photosensitizers generally have problems such as short excitation wavelength (<600nm) and insufficient excited state energy conversion efficiency, which leads to limited tissue penetration depth and low ROS yield, making it difficult to meet the needs of deep tumor treatment. This is mainly because conventional photosensitizers need to achieve singlet to triplet transitions through the spin-forbidden intersystem crossing (ISC) process, and this process is accompanied by significant energy loss, which seriously restricts the utilization efficiency of long-wavelength light energy. Therefore, it is of great significance to develop a new deep tissue phototherapy method that can be activated by deep red light or near-infrared region (600-800 nanometers) for photodynamic therapy of deep tissue lesions.

[0004] In recent years, the triplet direct excitation strategy based on the heavy atom effect has provided an innovative idea for long-wavelength PDT. Transition metal complexes represented by osmium (Os), with their strong spin-orbit coupling (SOC) effect, can directly reach the triplet excited state through spin-allowed electron transitions, effectively avoiding the energy loss of the traditional intersystem crossing process. Experiments have confirmed that osmium (II) complexes containing polypyridine ligands exhibit significant triplet direct absorption characteristics in the deep red / near-infrared region (600-800nm). Compared with conventional photosensitizers, their light energy utilization efficiency is improved by 2-3 orders of magnitude, and long-wavelength excitation significantly improves tissue penetration depth.

[0005] However, the osmium complexes currently developed face problems such as insufficient stability and limited penetration depth, making them difficult to use in the treatment of deep tumors. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a novel osmium complex, which has a stable structure and has the potential for photodynamic therapy of deep tumors.

[0007] The invention also provides a method for preparing the complex.

[0008] The invention also provides the application of the complex.

[0009] The osmium complex according to the first embodiment of the present invention has a structure as shown in the following formula:

[0010]

[0011] Where, X - Represents anion.

[0012] According to a preferred embodiment of the present invention, at least the following beneficial effects are achieved: a multidentate coordination structure is formed between the ligands of the complex of the present invention and the metal atoms, so that the structure has high stability, and the ligand has aggregation-induced emission (AIE) characteristics and can emit light spontaneously, so that it is expected to be applied to deep tumor treatment. In the design of the complex in the complex of the present invention, the donor and the acceptor are closely linked by covalent bonds. This covalent attachment not only enhances the stability of the interaction, but also shortens the distance between the donor and the acceptor, thereby achieving a higher energy transfer efficiency. In addition, the formation of covalent bonds also promotes the interaction within the molecule, further improving the structural stability of the entire complex. The complex has aggregation-induced emission (AIE) characteristics in an aqueous environment, can produce superoxide anions, singlet oxygen and hydroxyl radicals under illumination conditions, and has catalytic oxidation ability for NADH / NADPH in tumor cells. The complex has good proliferation inhibition ability (IC 50 15.61 μM), and had almost no cytotoxicity under non-illumination conditions (IC 50 >100μM), 635nm red light has strong tissue penetration ability, can penetrate deep into the lesion, effectively activate photosensitizers, and improve the therapeutic effect. This is of great significance for the study of metal osmium complexes against tumors, and provides a new idea for the clinical development of photodynamic therapy for deep tumors.

[0013] The AIE property enables the simultaneous improvement of aggregated luminescence and ROS yield. The complex of the present invention realizes the characteristics of "low background interference in the dispersed state and high-brightness luminescence in the aggregated state" through molecular structure innovation, which significantly improves the signal-to-noise ratio and photodynamic efficiency in the treatment of deep tumors.

[0014] Aggregation-induced emission (AIE) materials provide a breakthrough direction for the development of a new generation of photosensitizers. In the single-molecule state, AIE molecules have a dominant non-radiative attenuation due to intramolecular motion (RIM), while in the aggregated state, molecular vibration is inhibited by steric hindrance, significantly improving the fluorescence quantum yield and photostability. Studies have shown that AIE-type photosensitizers not only have the characteristics of large Stokes shift and excellent biocompatibility, but their tightly packed solid structure is more conducive to enhancing the efficiency of ROS generation through intermolecular electronic coupling. The scheme of the present invention has important research value by constructing a molecular engineering strategy of covalently integrating AIE elements and osmium metal complexes: on the one hand, the AIE property can solve the problem of fluorescence quenching (ACQ) caused by aggregation of traditional metal complexes, and realize real-time fluorescence navigation of the lesion during treatment; on the other hand, the long-wavelength response characteristics of osmium complexes can break through the light transmission limitation of deep tissues, and with the efficient ROS generation ability of the AIE aggregated state, it is expected to establish a new paradigm for the precise diagnosis and treatment of deep tumors. This research direction will provide a theoretical basis and technical support for the development of efficient and safe deep tissue PDT systems.

[0015] According to some embodiments of the present invention, X - Representing PF 6 - 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.

[0016] According to some embodiments of the present invention, the X - Represents an inorganic anion.

[0017] According to an embodiment of the second aspect of the present invention, a method for preparing the above-mentioned complex is proposed, comprising the following steps:

[0018] S1, causing 4'-bromo-2,2':6',2"-terpyridine to undergo coordination reaction with an osmium source to obtain a brominated bitridentate osmium complex;

[0019] S2, reacting the brominated bitridentate osmium complex with [1-(4-boronic acid phenyl)-1,2,2-triphenyl]ethylene to obtain the osmium complex.

[0020] 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.

[0021] According to some embodiments of the present invention, step S1 specifically comprises: 4'-bromo-2,2':6',2"-terpyridine and an osmium source undergo coordination reaction in the presence of solvent I to obtain a brominated bitridentate osmium complex.

[0022] According to 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.

[0023] According to some embodiments of the present invention, the solvent I includes at least one of ethylene glycol, diethylene glycol, ethylene glycol monomethyl ether, etc.

[0024] According to some embodiments of the present invention, step S1 specifically comprises: dissolving 4'-bromo-2,2':6',2"-terpyridine and ammonium chloroosmate in solvent I, and stirring at 180-220°C for 1-5h.

[0025] According to some embodiments of the present invention, step S1 specifically comprises: dissolving 4'-bromo-2,2':6',2"-terpyridine and ammonium chloroosmate in solvent I, and stirring at 200°C for 3h.

[0026] According to some embodiments of the present invention, step S1 further comprises: after the reaction is completed, adding excess tetrahydrofuran, filtering under reduced pressure to obtain a solid, and purifying the crude product by alumina column chromatography to obtain a brominated bitridentate osmium complex.

[0027] According to some embodiments of the present invention, the reaction formula of step S1 is as follows:

[0028]

[0029] According to some embodiments of the present invention, in step S1, the molar ratio of ammonium chloroosmate to 4'-bromo-2,2':6',2"-terpyridine is 1:2-3.

[0030] According to some embodiments of the present invention, in step S1, the molar ratio of ammonium chloroosmate to 4'-bromo-2,2':6',2"-terpyridine is 1:2.5.

[0031] According to some embodiments of the present invention, in step S1, the coordination reaction is carried out under heating reflux conditions.

[0032] According to some embodiments of the present invention, the heating reflux temperature is 200° C. and the reaction time is 3 h.

[0033] According to some embodiments of the present invention, step S2 specifically comprises: dissolving a brominated bitridentate osmium complex, [1-(4-boronic acid phenyl)-1,2,2-triphenyl]ethylene, tetrakis(triphenylphosphine)palladium and anhydrous potassium carbonate in solvent II under a protective atmosphere, and stirring at 110-120° C. for 12-18 hours.

[0034] According to some embodiments of the present invention, the protective atmosphere includes nitrogen or an inert gas atmosphere (such as argon, etc.).

[0035] According to some embodiments of the present invention, the solvent II in step S2 includes a mixed solvent of toluene, water and methanol, and the ratio of the mixed solvent is 3:1:1 to 4:1:1.

[0036] According to some embodiments of the present invention, the molar ratio of the brominated bitridentate osmium complex, [1-(4-boronic acid phenyl)-1,2,2-triphenyl]ethylene, tetrakis(triphenylphosphine)palladium and anhydrous potassium carbonate in step S2 is 1:1.5-2.5:0.05-0.15:5-7.

[0037] According to some embodiments of the present invention, the molar ratio of the brominated bitridentate osmium complex, [1-(4-boronic acid phenyl)-1,2,2-triphenyl]ethylene, tetrakis(triphenylphosphine)palladium and anhydrous potassium carbonate in step S2 is 1:2:0.1:6.

[0038] According to some embodiments of the present invention, the reaction temperature in step S2 is 110-120° C. and the reaction time is 12 h.

[0039] According to some embodiments of the present invention, step S2 specifically includes: adding a brominated bitridentate osmium complex, [1-(4-boronic acid phenyl)-1,2,2-triphenyl]ethylene, tetrakis(triphenylphosphine)palladium and anhydrous potassium carbonate to a reaction bottle, adding solvent II under a protective atmosphere, and reflux at 110-120°C for 12-18h. After the reaction is completed and the solvent is dried, the crude product is purified by alumina column chromatography to obtain an osmium complex. The reaction formula of this step is as follows:

[0040]

[0041] According to the application of the third aspect of the present invention, the application of the above-mentioned osmium complex in the preparation of anti-tumor drugs is proposed.

[0042] 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 light irradiation, 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, it can kill tumor cells or inhibit their growth by inducing oxidative stress. The complex of the present invention has a strong ability to generate superoxide anions after light irradiation, and can cause oxidative stress in tumor cells. Therefore, it has good anti-tumor potential, which shows that it has good application prospects in the field of anti-tumor drugs.

[0043] According to the application of the third aspect of the present invention, an anti-tumor drug is also provided, wherein the anti-tumor drug comprises the above-mentioned osmium complex.

[0044] According to the application of the third aspect of the present invention, an anti-tumor metal photosensitizer is also proposed, wherein the active ingredient of the anti-tumor metal photosensitizer includes the above-mentioned osmium complex. Other ingredients may also be included. The donor and the acceptor of the present invention are covalently linked to prepare a photosensitizer for photodynamic therapy of deep tumors, which is of great significance.

[0045] According to some embodiments of the present invention, the tumor is breast cancer. The complex of the present invention has a strong effect of inhibiting the proliferation of breast cancer cells and has a good application prospect in the fight against breast cancer.

[0046] According to some embodiments of the present invention, the breast cancer cell line is a mouse breast cancer cell line (4T1).

[0047] According to 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.

[0048] According to 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.

[0049] According to some embodiments of the present invention, in the preparation of pharmaceutical dosage forms, the types of pharmaceutical excipients selected will vary according to the specific medical application requirements. These excipients are intended to adjust the solubility and bioavailability of the photocatalyst, improve its stability, adjust the host's immune response, and play multiple functions such as emulsification, anti-oxidation, aerosol propulsion, tablet bonding, and disintegration. The preferred pharmaceutical excipients are broad in scope, including but not limited to adhesives / filling materials with good compatibility with photosensitizers, coating materials, disintegration aids, lubricants, and flavoring sweeteners, but are not limited thereto.

[0050] According to some embodiments of the present invention, the carrier is an acceptable functional pharmaceutical excipient 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.

[0051] According to some embodiments of the present invention, the present invention has no particular restrictions on the dosage form of the above-mentioned drug, and the active substance can be administered together with an assimilable edible carrier, an inert diluent, or directly combined with food. The drug dosage form includes, but is 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.

[0052] According to other embodiments of the present invention, the osmium complex of the scheme of the present invention can be prepared into a pharmaceutical preparation without any formulation additives or in the form of other drug delivery systems known in the prior art, including but not limited to combining with liposome constituents, carrying through carrier and non-carrier protein, integrating organic and inorganic nanoparticles, nanoemulsions and microemulsions, nanocrystals, or simply using solvents and suitable solvent mixtures, and may also contain auxiliary components such as lactose, polyvinyl pyrrolidone (PVP), etc.

[0053] According to some embodiments of the present invention, the pharmaceutical preparation of the scheme of the present invention is suitable for administration through a variety of routes of administration, including but not limited to absorption through various parts of the gastrointestinal tract (such as the mouth, throat, esophagus, stomach, small intestine area including duodenum, jejunum and ileum, and large intestine part including cecum, colon to rectum, and anus) after oral ingestion, and parenteral routes such as intravenous injection, subcutaneous injection, intraperitoneal injection or local administration. For drug ingredients with poor stability in the gastric environment, they can be prepared in the form of enteric coated tablets to ensure that they are released in the intestine after passing through the stomach.

[0054] According to the application of the third aspect of the present invention, a reagent or kit for biomedical imaging is also provided, comprising the above-mentioned osmium complex. By introducing these complexes into cells, the concentration change of hydrogen peroxide in the cells can be monitored in real time, thereby understanding the physiological state of the cells or the disease process.

[0055] According to the application of the third 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 target substances in complex environments.

[0056] According to the application of the third aspect of the embodiment of the present invention, a method for detecting hydrogen peroxide is also proposed, in which the above-mentioned osmium complex is added to the object to be detected.

[0057] 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

[0058] Figure 1 The nuclear magnetic resonance hydrogen spectrum of the brominated bitridentate osmium complex prepared in the embodiment of the present invention;

[0059] Figure 2 The hydrogen nuclear magnetic resonance spectrum of the tetraphenylethylene osmium complex prepared in the embodiment of the present invention;

[0060] Figure 3 The UV-visible absorption spectrum of the metal osmium complex prepared in the embodiment of the present invention;

[0061] Figure 4 The fluorescence excitation spectrum and emission spectrum of the metal osmium complex prepared in the embodiment of the present invention;

[0062] Figure 5 The fluorescence emission spectra of the metal osmium complex prepared in the embodiment of the present invention in different solvents;

[0063] Figure 6 The fluorescence spectrum of the aggregation-induced emission (AIE) phenomenon of the metal osmium complex prepared in the embodiment of the present invention;

[0064] Figure 7 This is a graph showing the test results of the ability of the metal osmium complex prepared in an embodiment of the present invention to photocatalytically generate superoxide anions;

[0065] Figure 8 This is a graph showing the test results of the ability of the metal osmium complex prepared in an embodiment of the present invention to photocatalytically generate hydroxyl radicals;

[0066] Fig. 9 This is a graph showing the test results of the ability of the metal osmium complex prepared in an embodiment of the present invention to photocatalytically oxidize NADH and NADPH;

[0067] Fig.10 This is a graph showing the test results of the ability of the metal osmium complex prepared in an embodiment of the present invention to photocatalytically generate singlet oxygen;

[0068] Fig.11 This is a graph showing the cytotoxicity test results of the metal osmium complex prepared in an embodiment of the present invention against mouse breast cancer cells (4T1) under light and dark treatment conditions. DETAILED DESCRIPTION

[0069] 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.

[0070] 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.

[0071] In the description of the present invention, if there is a description of I, II, etc., it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the sequence of the indicated technical features.

[0072] The "room temperature" referred to in the present invention refers to 25±5°C, and is specifically 25°C in the embodiments.

[0073] Example

[0074] In this example, a metal osmium complex with self-luminous properties was prepared. The structural formula of the complex is as follows:

[0075]

[0076] The specific preparation process is as follows:

[0077] (1) Formation of a brominated tridentate osmium complex from 4'-bromo-2,2':6',2"-terpyridine and ammonium chloroosmate

[0078] 4'-Bromo-2,2':6',2"-terpyridine (825 mg, 2.64 mmol) and ammonium chloroosmate (528 mg, 1.2 mol) were dissolved in 12 mL of ethylene glycol and stirred at 200 °C for 3 h. After the reaction was completed, the mixture was cooled to room temperature, and an excess of tetrahydrofuran was added to precipitate. The crude product was filtered off under reduced pressure and purified by alumina column chromatography to obtain a black powder, which was a brominated bitridentate osmium complex (755 mg, 71%).

[0079] The chemical reaction equation above is as follows:

[0080]

[0081] The H NMR spectrum of the product is Figure 1 Specifically, the measured nuclear magnetic hydrogen spectrum data is: 1 H NMR (500 MHz, DMSO-d 6 )δ9.49(d,J=39.8Hz,1H),8.97(d,J=8.0Hz,1H),7.90(t,J=6.2Hz,1H),7.47(d,J=5.3Hz,1H),7.20(t,J=6.9Hz,1H). This indicates that the above operation produced the target compound with the correct structure.

[0082] (2) Formation of tetraphenylethylene osmium complex from brominated bitridentate osmium complex and [1-(4-borylphenyl)-1,2,2-triphenyl]ethylene

[0083] Bromobistridentate osmium complex (177 mg, 0.2 mmol), [1-(4-boronic acid phenyl)-1,2,2-triphenyl]ethylene (188 mg, 0.5 mmol), tetrakis(triphenylphosphine)palladium (23 mg, 10% mol), anhydrous potassium carbonate (85 mg, 0.4 mmol), 6 mL toluene, 2 mL water, 1 mL methanol were added under argon (other inert gases or nitrogen can also be used) atmosphere, and stirred at 120°C for 12 hours. After the reaction was completed, the solvent was dried, and the crude product was purified by alumina column chromatography to obtain a brown tetraphenylethylene osmium complex (58 mg, 21%).

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

[0085]

[0086] The H NMR spectrum of the product is Figure 2 Specifically, the measured nuclear magnetic hydrogen spectrum data is: 1 H NMR (500 MHz, DMSO-d 6)δ9.42(s,2H),9.06(d,J=8.2Hz,2H),8.19(d,J=8.2Hz,2H),7.92–7.85(m,2H),7.38(d,J=5.3Hz,2H),7.33(d,J=8.1Hz,2H),7.25–7.14(m,13H),7.10(d,J=6.8Hz,2H),7.05(d,J=6.6Hz,2H). This indicates that the above operation produced the target compound with the correct structure.

[0087] Test example

[0088] In order to verify the application effect of the above complex, its performance was tested, as follows:

[0089] 1. UV-visible absorption and fluorescence spectra of metal osmium complexes

[0090] (1) UV-visible absorption spectra of metal osmium complexes

[0091] Water (H 2 O) as solvent, the metal osmium complex prepared by the above operation was prepared into a 10 μM sample solution, and then a double-beam UV-visible spectrophotometer was used to record the UV-visible absorption spectrum of the metal osmium complex 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 510 nanometers, and there is also good absorption around 700 nanometers.

[0092] (2) Fluorescence excitation and emission spectra of metal osmium complexes

[0093] Using water 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 510 nm as the EX fixed wavelength. Similarly, the fluorescence excitation spectrum of the metal osmium complex was recorded with 744 nm as the EM fixed wavelength to characterize its excitation spectrum and emission spectrum in water. The results are as follows: Figure 4 As shown in the figure, it can be seen that the best excitation wavelength in water is 510nm and the best emission wavelength is 744nm.

[0094] (3) Fluorescence emission spectra of metal osmium complexes in different solvents

[0095] Water (H 2 O) phosphate buffered saline (PBS), dichloromethane (DCM), methanol (MeOH), acetonitrile (CH 3CN) 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 635 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 compound has a strong fluorescence intensity in water and almost no fluorescence in organic solvents.

[0096] 2. Aggregation-induced emission (AIE) detection of metal osmium complexes

[0097] The metal osmium complex synthesized by the above operation has a tetraphenylethylene structure. In a good solvent, the tetraphenylethylene molecules have relatively free intramolecular motion, and the excited state energy is rapidly dissipated mainly through the non-radiative relaxation process, resulting in weak luminescence. In a poor solvent, the intramolecular motion is restricted, resulting in the inhibition of the non-radiative relaxation process, and the excited state energy can only be released through radiation transition, thereby producing strong fluorescence.

[0098] The metal osmium complex was dissolved in a mixed solvent of a good solvent (methanol) and a poor solvent (water) in different proportions, and the fluorescence emission spectrum of the metal osmium complex was recorded at 510 nm as the EX fixed wavelength. Figure 6 As shown, as the proportion of the poor solvent increases, the fluorescence of the metal osmium complex gradually increases, that is, the aggregation-induced emission phenomenon occurs.

[0099] 3. Determination of the ability of metal osmium complexes to generate superoxide anions

[0100] 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.

[0101] 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.

[0102] 4. Determination of the ability of metal osmium complexes to generate hydroxyl radicals

[0103] 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.

[0104] 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.

[0105] 5. Determination of the photocatalytic oxidation ability of NADH / NADPH by metal osmium complexes

[0106] 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.

[0107] 6. Determination of the ability of metal osmium complexes to generate singlet oxygen

[0108] 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)dicarboxylic acid (ABDA for short) 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 and photobleach to generate the corresponding endoperoxide. The generation of singlet oxygen can be monitored by measuring the change in its absorbance at 400nm, which can reflect the ability to generate singlet oxygen.

[0109] An aqueous solution containing a metal osmium complex (5 μM) and an ABDA reagent (30 μM) was placed in a cuvette and its singlet oxygen generation capacity was measured under light conditions. The results are as follows: Fig.10As shown in the figure, it can be seen that the metal osmium complex has the ability to produce singlet oxygen after light exposure.

[0110] 7. The therapeutic effect of metal osmium complexes on breast cancer

[0111] The resazurin colorimetric method was used to analyze the antiproliferative effect of metal osmium complexes on mouse breast cancer cells (4T1 cells). Resazurin, also known as sodium azurite or resin azurin, is a phenoxazine dye that is weakly fluorescent, non-toxic, cell permeable, and redox sensitive. Resazurin is metabolized and reduced in cells, and its oxidized state is purple-blue and essentially non-fluorescent, while the reduced product resorufin turns pink and is highly fluorescent. This color change can be detected by a fluorescence photometer to detect fluorescence changes, with an excitation maximum wavelength of 530-570nm and an emission maximum wavelength of 580-590nm. The resulting fluorescence intensity is proportional to the number of respiring live cells, thereby analyzing cell proliferation.

[0112] The experimental steps for resazurin detection are as follows:

[0113] (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.

[0114] (2) When the cells reached the logarithmic growth phase, they were inoculated into two 96-well plates at a cell density of 80,000 cells / mL. Each well was cultured with 90 μL of culture medium. One plate was the light control group and the other was the dark control group. Both plates were placed in a 37°C, 5% CO 2 Culture in an incubator.

[0115] (3) After the cells adhere to the wall, prepare 1 mM osmium complex stock solution and perform gradient dilution to dilute the cells into 6 concentrations of 1000, 500, 100, 10, 1, and 0.1 μM osmium complex. Pipette 10 μL of each concentration into a 96-well plate, so that the final drug concentrations are 100, 50, 10, 1, 0.1, and 0.01 μM, respectively. Set up three parallel tests for each concentration. Gently shake the plates and continue incubation for 4 h under the corresponding conditions.

[0116] (4) After 4 h of incubation, the illuminated plates were illuminated at 635 nm for 30 min, while the dark plates were left untreated. After the illumination period, the plates were incubated for another 24 h under the corresponding conditions.

[0117] (5) After incubation for 24 h, the supernatant was removed and 80 μL of resazurin was added to each well. The cells were incubated in a 37°C incubator for another 4 h. The fluorescence intensity was detected using an enzyme-linked immunosorbent assay (ELISA) and the cell proliferation inhibition rate was calculated to obtain the IC 50 value (drug concentration when the inhibition rate is equal to 50%).

[0118] The results are as follows Fig.11 As shown in the figure, it can be seen that the killing effects of different concentrations of metal osmium complexes on mouse breast cancer cells (4T1 cells) under dark treatment and light treatment conditions are different when detected by the resazurin method. Under dark treatment, the killing effects on mouse breast cancer cells IC 50 >100μM, that is, almost no dark toxicity. IC of mouse breast cancer cells under light conditions 50 It is 15.61 μM. This shows that the complex of the present invention has a strong tumor proliferation inhibition effect.

[0119] 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. An osmium complex, characterized in that: The structural formula of the osmium complex is as follows: Where, X - Represents anion.

2. The osmium complex according to claim 1, 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.

3. A method for preparing an osmium complex as claimed in claim 1 or 2, characterized in that: The steps include: S1, causing 4'-bromo-2,2':6',2"-terpyridine to undergo coordination reaction with an osmium source to obtain a brominated bitridentate osmium complex; S2, reacting the brominated bitridentate osmium complex with [1-(4-boronic acid phenyl)-1,2,2-triphenyl]ethylene to obtain the osmium complex.

4. The method for preparing the osmium complex according to claim 3, characterized in that: The osmium source includes ammonium chloroosmate.

5. Use of the osmium complex as claimed in claim 1 or 2 in the preparation of antitumor drugs.

6. An anti-tumor drug, characterized in that: The anti-tumor drug contains the osmium complex as described in claim 1 or 2; preferably, the tumor is breast cancer.

7. 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 1 or 2.

8. A reagent or kit for biomedical imaging, characterized in that: Contains the osmium complex as claimed in claim 1 or 2.

9. A chemical sensor, characterized in that: Contains the osmium complex as claimed in claim 1 or 2.

10. A method for detecting hydrogen peroxide, characterized in that: Add the osmium complex as claimed in claim 1 or 2 to the object to be detected.

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