An osmium complex, its preparation method and application

Abstract

This invention discloses an osmium complex, its preparation method, and its application, having the structure shown in the following formula: where X ‑ Representing anions. The compounds of this invention exhibit aggregation-induced emission (AIE) properties in an aqueous environment, generating superoxide anions and singlet oxygen, and possessing catalytic oxidation capabilities for both NADH and NADPH in tumor cells. This complex demonstrates good inhibitory activity against the proliferation of mouse breast cancer cells (4T1) under 635 nm red light irradiation (IC50). 50 The concentration was 15.61 μM, while it showed almost no cytotoxicity under non-light conditions (IC50). 50 Red light at 635nm (>100μM) has strong tissue penetration, enabling it to reach deep into lesions, effectively activate photosensitizers, and improve treatment efficacy. This is of great significance for the study of antitumor effects of osmium metal complexes and provides a new approach for the clinical development of photodynamic therapy for deep tumors.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to an osmium complex, its preparation method, and its application. Background Technology

[0002] Photodynamic therapy (PDT), as a non-invasive, spatiotemporally selective, and less likely to induce drug resistance novel treatment, has demonstrated significant clinical value in the field of cancer treatment, serving as an effective supplement to traditional therapies such as surgical resection, radiotherapy, and chemotherapy. Its therapeutic mechanism is based on the photoactivation reaction between a photosensitizer (PS) and molecular oxygen under specific wavelengths of light. This generates cytotoxic reactive oxygen species (ROS), achieving a synergistic therapeutic effect of targeted killing of tumor cells, disruption of the vascular system, and regulation of the immune microenvironment.

[0003] The core challenge currently facing the clinical translation of phototherapy (PDT) lies in the fact that traditional photosensitizers generally suffer from short excitation wavelengths (<600nm) and insufficient excited-state energy conversion efficiency, resulting in 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 require a spin-forbidden intersystem crossing (ISC) process to achieve the transition from singlet to triplet states, a process accompanied by significant energy loss, which severely restricts the utilization efficiency of long-wavelength light energy. Therefore, developing a novel deep tissue phototherapy method that can be activated in the deep red or near-infrared region (600-800 nm) for photodynamic therapy of deep tissue lesions is of great significance.

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

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

[0006] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention proposes a novel osmium complex that is structurally stable and has the potential for photodynamic therapy of deep tumors.

[0007] The present invention also proposes a method for preparing the above-mentioned complex.

[0008] The present invention also proposes applications of the above-mentioned complexes.

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

[0010]

[0011] In the formula, X - Represents anions.

[0012] According to a preferred embodiment of the present invention, at least the following beneficial effects are achieved: the complex ligands of the present invention form a polydentate coordination structure with the metal atoms, resulting in high structural stability. Simultaneously, the ligands possess aggregation-induced emission (AIE) properties, enabling self-luminescence, making it a promising candidate for deep tumor treatment. In the design of the complex of the present invention, the donor and acceptor are tightly linked by covalent bonds. This covalent attachment not only enhances the stability of the interaction but also shortens the distance between the donor and acceptor, thereby achieving higher energy transfer efficiency. Furthermore, the formation of covalent bonds promotes intramolecular interactions, further enhancing the structural stability of the entire complex. This complex exhibits aggregation-induced emission (AIE) properties in an aqueous environment and can generate superoxide anions, singlet oxygen, and hydroxyl radicals under light irradiation, catalyzing the oxidation of NADH / NADPH in tumor cells. This complex demonstrates good inhibitory activity against mouse breast cancer cells (4T1) under 635nm red light irradiation (IC50). 50 The concentration was 15.61 μM, while it showed almost no cytotoxicity under non-light conditions (IC50). 50 Red light at 635nm (>100μM) has strong tissue penetration, enabling it to reach deep into lesions, effectively activate photosensitizers, and improve treatment efficacy. This is of great significance for the study of antitumor effects of osmium metal complexes and provides a new approach for the clinical development of photodynamic therapy for deep tumors.

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

[0014] Aggregation-induced emission (AIE) materials offer a breakthrough direction for the development of next-generation photosensitizers. In the single-molecule state, AIE molecules exhibit dominant non-radiative decay due to intramolecular motion (RIM), while in the aggregated state, steric hindrance suppresses molecular vibrations, significantly enhancing fluorescence quantum yield and photostability. Studies have shown that AIE-type photosensitizers not only possess characteristics such as large Stokes shift and excellent biocompatibility, but their tightly packed solid-state structure is also more conducive to enhancing ROS generation efficiency through intermolecular electronic coupling. The molecular engineering strategy of constructing covalently integrated AIE motifs with osmium metal complexes has significant research value: on the one hand, the AIE properties can solve the problem of fluorescence quenching (ACQ) caused by the aggregation of traditional metal complexes, enabling real-time fluorescence navigation of lesion sites during treatment; on the other hand, the long-wavelength response characteristics of osmium complexes can overcome the light transmission limitations of deep tissues, and combined with the efficient ROS generation capability of the AIE aggregated state, it is expected to establish a new paradigm for precision diagnosis and treatment of deep tumors. This research direction will provide 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 - Represents PF6 - At least one of the following: halide ions (preferably chloride ions), tetrafluoroborate group, tetraphenylborate group, trifluoromethanesulfonic acid group, or tetra(3,5-bis(trifluoromethyl)phenyl)borate group. Anions have a relatively small impact on the reactive oxygen species generation process.

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

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

[0018] S1. A coordination reaction is carried out between 4'-bromo-2,2':6',2”-terpyridine and an osmium source to obtain a brominated bitridentate osmium complex;

[0019] S2. The brominated bitridentate osmium complex is reacted with [1-(4-boronicophenyl)-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 simple to operate and has good prospects for industrial application.

[0021] According to some embodiments of the present invention, step S1 specifically includes: 4'-bromo-2,2':6',2”-terpyridine undergoes a coordination reaction with an osmium source 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 osmium chloride. Other inorganic osmium salts or osmium-containing organic compounds may also be used.

[0023] According to some embodiments of the present invention, 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 includes: dissolving 4'-bromo-2,2':6',2”-terpyridine and ammonium chloroosmium tetroxide in solvent I, and stirring at 180-220°C for 1-5 hours.

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

[0026] According to some embodiments of the present invention, step S1 further includes: 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 bis-tridentate osmium complex.

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

[0028]

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

[0030] According to some embodiments of the present invention, in step S1, the molar ratio of ammonium chloroosmium tetroxide and 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 hours.

[0033] According to some embodiments of the present invention, step S2 specifically includes: dissolving the brominated bis-tridentate osmium complex, [1-(4-boratephenyl)-1,2,2-triphenyl]ethylene, tetra(triphenylphosphine)palladium and anhydrous potassium carbonate in solvent II under a protective atmosphere, and stirring at 110-120°C for 12-18 h.

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

[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 bitertentine osmium complex, [1-(4-boratephenyl)-1,2,2-triphenyl]ethylene, tetra(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 bitertentine osmium complex, [1-(4-boratephenyl)-1,2,2-triphenyl]ethylene, tetra(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 hours.

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

[0040]

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

[0042] According to a preferred embodiment of the present invention, the application has at least the following beneficial effects: the complex of the present invention can generate superoxide anions under light irradiation. Superoxide anions are generally associated with oxidative stress in organisms and can participate in various physiological and pathological processes as signaling molecules. 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, which can induce oxidative stress in tumor cells. Therefore, it has good anti-tumor potential, thus indicating its promising application prospects in the field of anti-tumor drugs.

[0043] According to an application of a third aspect of the present invention, an antitumor drug is also proposed, the antitumor drug comprising the above-mentioned osmium complex.

[0044] According to an application of a third aspect of the present invention, an antitumor metal photosensitizer is also proposed, wherein the active ingredient of the antitumor metal photosensitizer includes the aforementioned osmium complex. Other components may also be included. The present invention's method, with the donor and recipient covalently linked, is of great significance for preparing a photosensitizer for photodynamic therapy of deep tumors.

[0045] According to 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 breast cancer cells and shows good application prospects in the treatment of breast cancer.

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

[0047] According to some embodiments of the present invention, the drug further includes a pharmaceutically acceptable carrier and / or excipient. That is, the drug or photosensitizer uses an osmium metal complex as the main active ingredient, is mixed with a pharmaceutically acceptable carrier and / or excipient to prepare a composition, and is then formulated into a clinically acceptable dosage form.

[0048] According to some embodiments of the present invention, the excipient refers to a diluent, binder, lubricant, disintegrant, solubilizer, stabilizer, and other pharmaceutical matrix that can be used in the pharmaceutical field.

[0049] According to some embodiments of the present invention, the types of pharmaceutical excipients selected in the preparation of pharmaceutical dosage forms may vary depending on the specific medical application requirements. These excipients are designed to modulate the solubility and bioavailability of photocatalysts, enhance their stability, adjust the host's immune response, and perform multiple functions such as emulsification, antioxidation, aerosol propulsion, tablet adhesion, and disintegration. The preferred range of pharmaceutical excipients is broad, including but not limited to binders / fillers, coating materials, disintegration aids, lubricants, and flavoring sweeteners that are compatible with photosensitizers.

[0050] According to 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 polymers prepared in the 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, there are no particular limitations on the dosage form of the above-mentioned drugs. The active substance can be administered together with an assimilated edible carrier or 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 for injection, 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 complexes of the present invention can be prepared into pharmaceutical formulations without any formulation additives or using other drug delivery systems known in the prior art. Specifically, these include, but are not limited to, binding with liposome components, carrying proteins via carriers and non-carriers, integrating organic and inorganic nanoparticles, nanoemulsions and microemulsions, nanocrystals, or simply using solvents and suitable solvent mixtures. They may also contain auxiliary components such as lactose and polyvinylpyrrolidone (PVP).

[0053] According to some embodiments of the present invention, the pharmaceutical formulations of the present invention are suitable for administration via various routes, including but not limited to oral ingestion followed by absorption through various parts of the gastrointestinal tract (such as the mouth, pharynx, esophagus, stomach, small intestine including the duodenum, jejunum, and ileum, and large intestine including the cecum, colon, rectum, and anus), as well as parenteral routes such as intravenous injection, subcutaneous injection, intraperitoneal injection, or local administration. For pharmaceutical components with poor stability in the gastric environment, they can be prepared as enteric-coated tablets to ensure release into the intestines after passing through the stomach.

[0054] According to an application of a third aspect of the present invention, a reagent or kit for biomedical imaging is also provided, comprising the aforementioned osmium complex. By introducing these complexes into cells, changes in intracellular hydrogen peroxide concentration can be monitored in real time, thereby providing insight into the physiological state of the cells or the progression of diseases.

[0055] According to an application of a third aspect of the present invention, a chemical sensor comprising the aforementioned osmium complex is also proposed. It is used as a chemical sensor for detecting hydrogen peroxide or other relevant chemical substances in the environment. These sensors exhibit high sensitivity and selectivity, enabling them to accurately and rapidly respond to changes in the concentration of target substances in complex environments.

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

[0057] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0058] Figure 1 The above is the 1H NMR spectrum of the brominated bitridentate osmium complex prepared in an embodiment of the present invention.

[0059] Figure 2 The above is the 1H NMR spectrum of the tetraphenylethylene osmium complex prepared in an embodiment of the present invention.

[0060] Figure 3 The image shows the ultraviolet-visible absorption spectrum of the osmium metal complex obtained in an embodiment of the present invention.

[0061] Figure 4 The fluorescence excitation and emission spectra of the osmium metal complexes obtained in the embodiments of the present invention are shown below.

[0062] Figure 5 The following are fluorescence emission spectra of the osmium metal complexes prepared according to embodiments 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 an embodiment of the present invention is shown.

[0064] Figure 7 The graph shows the test results of the photocatalytic ability of the metal osmium complex prepared in the embodiments of the present invention to generate superoxide anions;

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

[0066] Figure 9 The graph shows the test results of the photocatalytic oxidation of NADH and NADPH by the metal osmium complexes prepared in the embodiments of the present invention.

[0067] Figure 10 The graph shows the test results of the photocatalytic ability of the metal osmium complex prepared in the embodiments of the present invention to generate singlet oxygen.

[0068] Figure 11 The figure shows the cytotoxicity test results of the metal osmium complex prepared in the embodiment of the present invention against mouse breast cancer cells (4T1) under light and dark treatment conditions. Detailed Implementation

[0069] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available. Unless otherwise specified, the same parameter value is the same in all embodiments. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0070] In the description of this invention, references to terms such as "some embodiments" indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0071] In the description of this invention, the use of I, II, etc., is only for the purpose of distinguishing technical features and should not be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated or the order of the technical features indicated.

[0072] The term "room temperature" as used in this invention refers to any temperature between 25 and 5°C, and specifically 25°C in the embodiments.

[0073] Example

[0074] This example demonstrates the preparation of a metal osmium complex with self-luminous properties. The structural formula of this complex is as follows:

[0075]

[0076] The specific preparation process is as follows:

[0077] (1) A brominated bitridentate osmium complex is generated by reacting 4'-bromo-2,2':6',2”-terpyridine with ammonium chloroosmium.

[0078] 4'-Bromo-2,2':6',2”-terpyridine (825 mg, 2.64 mmol) and ammonium chloroosmium tetroxide (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 excess tetrahydrofuran was added to precipitate the product. The precipitate was obtained by vacuum filtration 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 equations for the above reactions are shown below:

[0080]

[0081] The 1H NMR spectrum of the product is as follows Figure 1 As shown. Specifically, the measured 1H NMR data are as follows: 1 ¹H NMR (500MHz, DMSO-d⁶) δ 9.49 (d, J = 39.8 Hz, 1H), 8.97 (d, J = 8.0 Hz, 1H), 7.90 (t, J = 6.2 Hz, 1H), 7.47 (d, J = 5.3 Hz, 1H), 7.20 (t, J = 6.9 Hz, 1H). This indicates that the above operation yielded the target compound with the correct structure.

[0082] (2) A tetrastyrene-based osmium complex is formed by reacting the brominated bitridentate osmium complex with [1-(4-boronophenyl)-1,2,2-triphenyl]ethylene.

[0083] A brominated tridentate osmium complex (177 mg, 0.2 mmol), [1-(4-boratephenyl)-1,2,2-triphenyl]ethylene (188 mg, 0.5 mmol), tetrakis(triphenylphosphine)palladium (23 mg, 10% mol), and anhydrous potassium carbonate (85 mg, 0.4 mmol) were reacted with 6 mL of toluene, 2 mL of water, and 1 mL of methanol under an argon atmosphere (other inert gases or nitrogen can also be used). The mixture was stirred at 120 °C for 12 h. After the reaction was complete, the solvent was evaporated, and the crude product was purified by alumina column chromatography to obtain a brown tetraphenylphenyl osmium complex (58 mg, 21%).

[0084] The chemical reaction equations for the above reactions are shown below:

[0085]

[0086] The 1H NMR spectrum of the product is as follows Figure 2 As shown. Specifically, the measured 1H NMR data are as follows: 1¹H NMR (500MHz, DMSO-d⁶) δ 9.42 (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 operations yielded the target compound with the correct structure.

[0087] Test case

[0088] To verify the application effect of the above-mentioned complex, its performance was tested, as follows:

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

[0090] (1) Ultraviolet-visible absorption spectrum of metal osmium complex

[0091] Using water (H₂O) as the solvent, a 10 μM sample solution of the osmium metal complex prepared in the above operation was prepared. Then, a double-beam UV-Vis spectrophotometer was used to record the UV-Vis absorption spectra of the osmium metal complex to characterize its absorbance in different solvents. The results are as follows: Figure 3 As shown in the figure, the results indicate that the compound exhibits maximum absorption around 510 nm, with good absorption also observed around 700 nm.

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

[0093] Using water as a solvent, the osmium metal complex prepared in the above operation was prepared into a 10 μM sample solution. The fluorescence emission spectrum of the osmium metal complex was recorded using a fluorescence spectrophotometer at a fixed excitation wavelength of 510 nm. Similarly, the fluorescence excitation spectrum of the osmium metal complex in water was recorded at a fixed excitation wavelength of 744 nm. The results are as follows: Figure 4 As shown in the figure, the optimal excitation wavelength in water is 510 nm, and the optimal emission wavelength is 744 nm.

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

[0095] The metal osmium complexes prepared in the above procedure were prepared into 10 μM sample solutions using water (H2O), phosphate buffered saline (PBS), dichloromethane (DCM), methanol (MeOH), and acetonitrile (CH3CN) as solvents, respectively. The fluorescence emission spectra of the metal osmium complexes in different solvents were recorded using a fluorescence spectrophotometer at a fixed EX wavelength of 635 nm to characterize their fluorescence intensity. The results are as follows: Figure 5 As shown in the figure, the results indicate that the compound exhibits strong fluorescence in water but almost no fluorescence in organic solvents.

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

[0097] The osmium metal complex synthesized using the above methods exhibits a tetraphenylethylene structure. In good solvents, the tetraphenylethylene molecule exhibits relatively free intramolecular motion, and the excited-state energy is rapidly dissipated primarily through nonradiative relaxation processes, resulting in weak luminescence. However, in poor solvents, intramolecular motion is restricted, suppressing nonradiative relaxation processes, and the excited-state energy can only be released through radiative transitions, thus producing stronger fluorescence.

[0098] Osmium metal complexes were dissolved in mixed solvents containing varying proportions of a good solvent (methanol) and a poor solvent (water), and their fluorescence emission spectra were recorded at a fixed excitation wavelength of 510 nm. Figure 6 As shown, the fluorescence of the metal osmium complex gradually increases with the increase of the proportion of poor solvent, i.e., aggregation-induced emission occurs.

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

[0100] To assess the photocatalytic ability of the synthesized osmium metal complex to generate superoxide anions, dihydrorhodamine 123 (DHR123) was used to determine this ability. When superoxide anions are generated in the solution, DHR123 immediately captures them and is oxidized to form the fluorescent derivative rhodamine 123, emitting bright green fluorescence (Ex / Em = 500 / 536 nm). An increase in fluorescence intensity indicates the generation of superoxide anions in the solution. The superoxide anion generation ability can be reflected by monitoring the changes in the fluorescence spectra of the test sample and the DHR123 mixture under different illumination times using a fluorescence spectrophotometer.

[0101] A PBS solution containing osmium metal 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, this osmium metal complex has the ability to generate superoxide anions after illumination.

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

[0103] To assess the photocatalytic ability of the synthesized osmium metal complex to generate hydroxyl radicals, hydroxyphenyl fluorescein (HPF) was used to determine this ability. HPF itself is non-fluorescent, but it reacts with hydroxyl radicals in solution to produce strong green fluorescence (Ex / Em = 490 / 515 nm), with the fluorescence intensity directly proportional to the concentration of hydroxyl radicals. The ability to generate hydroxyl radicals was reflected by monitoring the changes in the fluorescence spectra of the test sample and the HPF mixture under different illumination times using a fluorescence spectrophotometer.

[0104] A PBS solution containing a 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, this osmium metal complex has the ability to generate superoxide anions after illumination.

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

[0106] Because under light irradiation, metal complexes can oxidize reduced coenzyme I (NADH) and reduced coenzyme II (NADPH) to their oxidized forms, NAD. + and NADP + Therefore, the osmium-containing complex (5 μM) and NADH or NADPH (A 339nm =1.0) mixed in a cuvette, the ability of it to oxidize NADH / NADPH under light conditions can be determined, and the results are as follows. Figure 9 As shown in the figure, the osmium metal complex exhibits photocatalytic oxidation capabilities for NADH and NADPH.

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

[0108] To assess the photocatalytic ability of the synthesized osmium metal complex to generate singlet oxygen, the singlet oxygen probe 9,10-anthratrium-bis(methylene)dicarboxylic acid (ABDA) was used. When singlet oxygen is generated in solution, ABDA immediately captures it, photobleaching it to form the corresponding internal peroxide. The generation ability of singlet oxygen can be reflected by measuring the change in absorbance at 400 nm.

[0109] An aqueous solution containing a metal osmium complex (5 μM) and ABDA reagent (30 μM) was placed in a cuvette, and its singlet oxygen generation capacity under illumination was measured. The results are as follows: Figure 10 As shown in the figure, this osmium complex has the ability to generate singlet oxygen after illumination.

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

[0111] The antiproliferative effect of osmium metalloenzene complexes on mouse breast cancer cells (4T1 cells) was analyzed using a resazurite colorimetric assay. Resazurite, also known as sodium resazurite or resin azurite, is a phenoxazine dye with weak fluorescence, non-toxicity, cell permeability, and redox sensitivity. Resazurite is metabolized and reduced within cells; its oxidized state is purplish-blue and essentially non-fluorescent, while the reduced product, resorufin, turns pink and highly fluorescent. This color change can be detected using a fluorophotometer, with a maximum excitation wavelength of 530-570 nm and a maximum emission wavelength of 580-590 nm. The fluorescence intensity is directly proportional to the number of respiring viable cells, thus allowing analysis of cell proliferation.

[0112] The experimental steps for detecting razor blue are as follows:

[0113] (1) First, revive one tube of 4T1 tumor cells and culture them in fresh complete culture medium (DMEM medium + 10 vol% fetal bovine serum + 1 vol% penicillin-streptomycin mixture). After passage twice, start the experiment.

[0114] (2) When the cells reach the logarithmic growth phase, they are seeded into two 96-well plates at a cell density of 80,000 cells / mL. Each well is filled with 90 μL of culture medium. One plate is the light group and the other is the dark group. Both are placed in a 37°C, 5% CO2 incubator for culture.

[0115] (3) After the osmium complex adheres to the plate, prepare a 1 mM stock solution and perform serial dilutions to obtain six concentrations of osmium complex: 1000, 500, 100, 10, 1, and 0.1 μM. Add 10 μL of each concentration to a 96-well plate, resulting in final drug concentrations of 100, 50, 10, 1, 0.1, and 0.01 μM. Set up three parallel experiments for each concentration. Gently shake well and incubate for 4 hours under the corresponding conditions.

[0116] (4) After incubation for 4 hours, the light-illuminated plate is illuminated under a 635nm light source for 30 minutes, while the dark plate is left untreated. After the illumination is completed, continue incubation for 24 hours under the corresponding conditions.

[0117] (5) After incubation for 24 hours, the supernatant was removed, and 80 μL of rezin was added to each well. The cells were then incubated at 37°C for another 4 hours. The fluorescence intensity was detected using an enzyme-linked immunosorbent assay (ELISA) reader, and the cell proliferation inhibition rate was calculated to determine the IC50 value. 50 Value (drug concentration when the inhibition rate is equal to 50%).

[0118] The results are as follows Figure 11 As shown in the figure, the cytotoxic effects of different concentrations of osmium metal complexes on mouse breast cancer cells (4T1 cells) under dark and light treatment conditions, as detected by the resazurite assay, are different. Under dark treatment, the IC50 value of the cytotoxic effect on mouse breast cancer cells is significantly lower. 50 >100 μM, meaning almost no dark toxicity. IC50 in mouse breast cancer cells under light conditions. 50 The concentration was 15.61 μM. This indicates that the complexes with the structure of the present invention have a strong inhibitory effect on tumor proliferation.

[0119] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. An osmium complex, characterized in that, The structural formula of the osmium complex is as follows: In the formula, X - Represents anions.

2. The osmium complex according to claim 1, characterized in that, X - Represents PF6 - At least one of the following: halide ions, tetrafluoroborate group, tetraphenylborate group, trifluoromethanesulfonic acid group or tetra(3,5-bis(trifluoromethyl)phenyl)borate group.

3. The osmium complex according to claim 2, characterized in that, The halide ion is a chloride ion.

4. A method for preparing an osmium complex as described in any one of claims 1 to 3, characterized in that, Includes the following steps: S1. A coordination reaction is carried out between 4'-bromo-2,2':6',2''-terpyridine and an osmium source to obtain a brominated bitridentate osmium complex; S2. The brominated bitridentate osmium complex is reacted with [1-(4-boronicophenyl)-1,2,2-triphenyl]ethylene to obtain the osmium complex.

5. The method for preparing the osmium complex according to claim 4, characterized in that, The osmium source is selected from ammonium chloroosmium.

6. The use of the osmium complex according to any one of claims 1 to 3 in the preparation of antitumor drugs, characterized in that, The tumor is breast cancer.

7. An antitumor drug, characterized in that, The antitumor drug contains an osmium complex as described in any one of claims 1 to 3.

8. An antitumor metal photosensitizer, characterized in that, The active ingredient of the antitumor metal photosensitizer includes the osmium complex as described in any one of claims 1 to 3.

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