A ligand, osmium complex and preparation method and application thereof

Abstract

The application discloses a ligand, an osmium complex and a preparation method and application thereof. The ligand has the following structural formula: the metal osmium complex formed by the ligand can efficiently generate various active oxygen species such as singlet oxygen (1O2), superoxide anion (O2·-) and hydroxyl radical (·OH) under near-infrared laser irradiation, and the active oxygen species selectively oxidize NADH / NADPH metabolic coenzymes in tumor cells through a photocatalytic reaction, thereby effectively destroying the cell redox homeostasis. In-vitro experiments show that the complex exhibits significant photodynamic anti-proliferation activity on mouse breast cancer cells (4T1), and the IC 50 value (0.43 muM) is about 28 times lower than that of a clinical chemotherapy drug cisplatin (IC 50 = 12.39 muM). The scheme has important significance for studying the metal osmium complex anti-tumor, and provides a new idea for the clinical development of deep tumor photodynamic therapy.

Description

Technical Field

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

[0002] Photodynamic therapy (PDT), as a non-invasive tumor treatment, relies on the synergistic effect of a photosensitizer, an excitation light source, and intracellular molecular oxygen. In clinical practice, PDT has become an important treatment for superficial tumors (such as skin cancer and oral cancer) due to its advantages of high spatial selectivity, low systemic toxicity, and reproducibility. However, current 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 excitation light source: Traditional photosensitizers (such as hematoporphyrin derivatives and phthalocyanine compounds) require visible light (400-700nm) for excitation, but the high scattering / absorption characteristics of biological tissues to visible light limit the effective treatment depth to within 2-3mm, which cannot cover deep lesions at the centimeter level.

[0004] 2. Aggregation-induced inactivation of photosensitizers: Clinically commonly used photosensitizers are prone to molecular aggregation in physiological environments, which can trigger aggregation-caused quenching (ACQ) effects, resulting in a significant decrease in the quantum yield of singlet oxygen (1O2) and severely weakening the therapeutic effect.

[0005] To overcome the limitations of penetration depth, near-infrared (NIR, 700-1700nm) responsive photosensitizers have become a research hotspot. These photosensitizers (such as IR-780 and Cypate) can enhance tissue penetration to 1-2cm in the NIR-I region (700-950nm), and can further extend to the NIR-II region (1000-1700nm) through two-photon absorption (TPA) technology, achieving three-dimensional spatial selective activation. However, existing NIR photosensitizers still have two major drawbacks: I) Imbalance between photostability and ROS yield: Long-wavelength absorption is usually accompanied by the decay of excited-state energy. NIR-II photosensitizers (such as BODIPY-based derivatives) generally have an 1O2 quantum yield of less than 0.3 under 1064nm excitation, which is difficult to meet the requirements 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 the non-radiative dissipation of excited-state energy through π-π stacking, which further reduces the ROS yield.

[0006] Therefore, developing a novel organometallic complex that can synergistically optimize NIR excitation efficiency and ROS quantum yield is expected to fundamentally resolve the contradiction between light energy transfer efficiency and reactive oxygen generation capacity in deep tumor PDT, providing a disruptive technological solution for solid tumor treatment. Summary of the Invention

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

[0008] The present invention also proposes a method for preparing the above-mentioned ligands.

[0009] The present invention also proposes complexes prepared using the above-mentioned ligands.

[0010] The present invention also proposes a method for preparing the above-mentioned complex.

[0011] The present invention also proposes applications of the above-mentioned complexes.

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

[0013] The ligands according to embodiments of the present invention have at least the following beneficial effects: the trithiophenepyridine dipyrimidine ligand of the present invention has a unique molecular structure, which has significant advantages in constructing near-infrared photosensitive metal complexes.

[0014] This ligand molecule cleverly integrates a triple thiophene unit, a pyrimidine ring, and a pyridine ring. The triple thiophene unit, with its unique extended π-conjugated system, effectively promotes a redshift in the absorption spectrum of the complex, thus enabling better absorption and utilization of near-infrared light. The pyrimidine ring, with its electron-rich properties, can effectively regulate the electron cloud distribution of the metal center. This regulation promotes the transition from the excited state energy to the triplet-singlet state (T1→S0) of molecular oxygen, thereby significantly increasing the singlet oxygen (… 1 The quantum yield of O2 is high. Simultaneously, the nitrogen atoms in the pyridine and pyrimidine rings endow them with strong coordination capabilities, enabling the ligands to bind tightly to metal ions, forming stable and structurally diverse polydentate coordination metal complexes. This highly stable ligand structure not only significantly enhances the overall structural robustness of the complex but also ensures that the complex maintains its structural and functional integrity under various conditions.

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

[0016] The product is obtained by nucleophilic substitution of 1-(pyrimidin-2-yl)ethyl-1-one with 2,2':5',2”-trithiophene-5-carboxaldehyde.

[0017] The ligand preparation method according to the embodiments of the present invention has at least the following beneficial effects: the preparation method of the triple thiophenepyridine dipyrimidine ligand of the present invention is simple to operate and has good prospects for industrial application.

[0018] In some embodiments of the present invention, the preparation method of the ligand specifically includes the following steps: 1-(pyrimidin-2-yl)ethyl-1-one and 2,2':5',2”-trithiophene-5-carboxaldehyde undergo a nucleophilic substitution reaction under a protective atmosphere and in the presence of a nucleophilic reagent to obtain the ligand.

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

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

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

[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 is carried out at a temperature of 25°C for a time of 18 hours.

[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 includes argon.

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

[0027] In some embodiments of the present invention, the purification step includes adding ammonium acetate to an ethanol / acetic acid (preferably, a volume ratio of 1 to 3:1; more preferably about 2:1) mixed solution, heating the reaction mixture under reflux for 4 to 6 hours (preferably 5 hours), cooling the reaction mixture to room temperature and then pouring it onto ice, allowing it to stand for 2 to 4 hours (preferably 3 hours), obtaining a solid precipitate by filtration, 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 preparation method involves the following reaction formulas:

[0031]

[0032] In some embodiments of the present invention, the molar ratio of 1-(pyrimidin-2-yl)ethyl-1-one, 2,2':5',2”-trithiophene-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 a third aspect embodiment of the present invention, the osmium complex has the following structural formula:

[0034]

[0035] In the formula, X - Represents anions.

[0036] According to a preferred embodiment of the present invention, at least the following beneficial effects are achieved: the osmium metal complex of the present invention can efficiently generate various reactive oxygen species such as singlet oxygen (1O2), superoxide anion (O2·-), and hydroxyl radical (·OH) under near-infrared laser irradiation. These reactive oxygen species selectively oxidize NADH / NADPH metabolic coenzymes in tumor cells through photocatalytic reactions, effectively disrupting cellular redox homeostasis. In vitro experiments show that this complex exhibits significant photodynamic antiproliferative activity against mouse breast cancer cells (4T1) with an IC50 value of [missing information]. 50 The value (0.43 μM) was lower than that of the clinical chemotherapy drug cisplatin (IC50). 50 The concentration (12.39 μM) was reduced by approximately 28-fold. This invention is significant for researching the antitumor effects of osmium metal complexes and provides new insights for the clinical development of photodynamic therapy for deep tumors.

[0037] In the complex of the present invention, the donor and acceptor are covalently attached, resulting in more stable covalent interactions and a closer distance, thus enabling higher transfer efficiency.

[0038] In 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.

[0039] The method for preparing the above-described osmium complex according to a fourth aspect embodiment of the present invention includes the following steps:

[0040] The ligand and the osmium source undergo a coordination reaction to obtain the osmium complex.

[0041] 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 simple to operate and has good prospects for industrial application.

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

[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 osmium chloride. Other inorganic osmium 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 chloroosmium tetroxide is 1:1.8 to 2.2.

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

[0049] In some embodiments of the present invention, the preparation method of the complex further includes: after the reaction is completed, cooling, adding excess tetrahydrofuran, allowing it to stand 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 for this step is as follows:

[0050]

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

[0052] 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 near-infrared 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, this complex can kill tumor cells or inhibit their growth by inducing oxidative stress, and has good anti-tumor potential, thus indicating its promising application prospects in the field of anti-tumor drugs.

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

[0054] According to an application of a fifth aspect of the present invention, an antitumor metal photosensitizer is also provided, wherein the active ingredient of the antitumor metal photosensitizer comprises the above-mentioned osmium complex. Other ingredients may also be included.

[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 shows good application prospects in the treatment of breast cancer.

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

[0057] In 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.

[0058] In 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.

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

[0061] In 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, 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.

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

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

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

[0065] According to an application of a fifth 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.

[0066] 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

[0067] Figure 1The above is the 1H NMR spectrum of the ligand obtained in Example 1 of this invention;

[0068] Figure 2 The above is the 1H NMR spectrum of the metal osmium complex obtained in Example 2 of this invention;

[0069] Figure 3 The UV-Vis absorption spectra of the metal osmium complex obtained in Example 2 of this invention in different solvents are shown.

[0070] Figure 4 The fluorescence excitation spectrum and fluorescence emission spectrum of the metal osmium complex obtained in Example 2 of this invention are shown.

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

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

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

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

[0075] Figure 9 This is a graph showing the test results of the photocatalytic oxidation of NADH and NADPH by the metal osmium complex prepared in Example 2 of this invention;

[0076] Figure 10 The figure shows the cytotoxicity test results of the photo-metallic osmium complex prepared in Example 2 of this invention against the mouse breast cancer cell line (4T1) under light and dark conditions. Detailed Implementation

[0077] 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.

[0078] 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.

[0079] 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.

[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)ethyl-1-one. Under an argon atmosphere (or other inert gases or nitrogen), 1-(pyrimidin-2-yl)ethyl-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 and stirred at 25 °C for 18 h. After the reaction was complete at room temperature, a 60 mL mixture of ammonium acetate (2500 mg, 32 mmol) in ethanol / acetic acid (2:1) was added. The reaction mixture was heated to reflux at 100 °C for 5 h. After cooling to room temperature, the mixture was poured onto ice and allowed to stand for 3 h. A solid precipitate was obtained by filtration. The crude product was purified by alumina column chromatography to obtain the ligand (277 mg, 29%).

[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 1 As shown. Specifically, the measured 1H NMR data are as follows:

[0087] 1¹H NMR (500MHz, Chloroform-d) δ 8.99 (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 indicates that the above operations yielded the target compound with the correct structure.

[0088] Example 2

[0089] This example demonstrates the preparation of a metal osmium complex with high efficiency in generating various reactive oxygen species. The structural formula of this complex is as follows:

[0090]

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

[0092] The chemical reaction equations for the above reactions are shown below:

[0093]

[0094] The 1H NMR spectrum of the product is as follows Figure 2 As shown. Specifically, the measured 1H NMR data are as follows:

[0095] 1 ¹H NMR (500MHz, DMSO-d⁶) δ 9.11 (s, 4H), 8.91 (dd, J = 4.7, 2.0Hz, 4H), 8.44 (d, J = 3.9Hz, 2H), 8.02 (dd, J = 5.9, 2.0Hz, 4H), 7.68 (d, J = 3.9Hz, 2H), 7.61 (dd, J = 5.1, 1.2Hz, 2H), 7.59 (d, J = 3.8Hz, 2H), 7.45–7.42 (m, 4H), 7.38–7.35 (m, 4H), 7.17 (dd, J = 5.1, 3.6Hz, 2H). This indicates that the above operations yielded the target compound with the correct structure.

[0096] Test case

[0097] To verify the application effect of the above-mentioned complex, its performance was tested, as follows:

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

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

[0100] The metal osmium complexes prepared in the above procedure were dissolved in water (H₂O), dichloromethane (DCM), and methanol (MeOH) to form 10 μM sample solutions, respectively. The UV-Vis absorption spectra of the metal osmium complexes in different solvents were then recorded using a double-beam UV-Vis spectrophotometer to characterize their absorbance. The results are as follows: Figure 3 As shown in the figure, the results indicate that the maximum absorption of the compound is around 520 nm, and the absorption wavelength in dichloromethane extends to 750 nm.

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

[0102] Using dichloromethane as a solvent, a 10 μM sample solution of the metal osmium complex prepared in the above operation was prepared. The fluorescence emission spectrum of the metal osmium complex was recorded using a fluorescence spectrophotometer at a fixed excitation wavelength of 550 nm. Similarly, the fluorescence excitation spectrum of the metal osmium complex in dichloromethane was recorded at a fixed emulation wavelength of 800 nm. The results are as follows: Figure 4 As shown in the figure, the optimal excitation wavelength in dichloromethane is 550 nm, and the optimal emission wavelength is 800 nm.

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

[0104] The metal osmium complexes prepared in the above procedure were prepared into 10 μM sample solutions using water (H₂O), dichloromethane (DCM), and methanol (MeOH), 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 550 nm to characterize their fluorescence intensity. The results are as follows: Figure 5 As shown in the figure, the compound exhibits strong fluorescence intensity in dichloromethane but weaker fluorescence in water.

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

[0106] To assess the photocatalytic ability of the synthesized osmium metal complex to generate singlet oxygen, the singlet oxygen probe 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) was used. When singlet oxygen is generated in the solution, ABDA immediately captures it, forming an internal peroxide. The singlet oxygen generation ability can be reflected by monitoring the changes in the UV absorption spectra of the test sample and the ABDA mixture under different illumination times using a UV spectrophotometer.

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

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

[0109] 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.

[0110] 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.

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

[0112] 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.

[0113] 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.

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

[0115] 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.

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

[0117] The resazurin assay was used to analyze the antiproliferative effect of osmium metalloproteinase complexes on a mouse breast cancer cell line (4T1 cells). Resazurin is a non-toxic, membrane-permeable blue dye with weak fluorescence. Live cells absorb resazurin dye into the cell via endocytosis. Inside the cell, resazurin is reduced to the red resorufin. The red fluorescence of resorufin can be detected by fluorescence microscopy at an excitation wavelength of 540 nm and an emission wavelength of 590 nm, or by measuring changes in absorbance using a photometer. This increase in fluorescence signal is related to cellular metabolic activity and proliferative capacity. By measuring the amount of resorufin inside the cell or the intensity of the fluorescence signal, cell viability and proliferation can be quantitatively assessed.

[0118] The experimental steps for the azure blade are as follows:

[0119] (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.

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

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

[0122] (4) After 4 hours of incubation, the dark group continued to be cultured under the same conditions, while the light group was illuminated for 30 minutes using a light source with a wavelength of 635 nm (33.12 J / cm²). 2 After the initial incubation, the culture medium was removed, and 80 μL of resazurin (1 mg / mL) was added to each well. The mixture was then incubated at 37°C for another 4 hours. EX was then detected using an ELISA reader. 540 EM 590 Calculate the cell proliferation inhibition rate and determine the IC50. 50 Value (drug concentration when the inhibition rate is equal to 50%).

[0123] The results are as follows Figure 10 As shown in the figure, the cytotoxic effects of different concentrations of osmium metal complexes on human and mouse breast cancer cell lines (4T1 cells) under light and dark conditions, as detected by the resazurite assay, are different. Under light conditions, the IC50 concentration of osmium metal complexes on mouse breast cancer cell lines is significantly lower than that under light conditions. 50 The concentration was 0.43 μM, which is superior to cisplatin (IC). 50 (The concentration was 12.39 μM). This indicates that the complexes of the present invention have a strong inhibitory effect on tumor proliferation.

[0124] 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. A ligand, characterized in that: The ligand has the following structural formula: .

2. A method for preparing the ligand as described in claim 1, characterized in that: Includes the following steps: The product is obtained by nucleophilic substitution of 1-(pyrimidin-2-yl)ethyl-1-one with 2,2':5',2''-trithiophene-5-carboxaldehyde.

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

4. The osmium complex according to claim 3, characterized in that: X - Represents PF6 - At least one of the following: halide ions, tetrafluoroborate, tetraphenylborate, trifluoromethanesulfonate, or tetra(3,5-di(trifluoromethyl)phenyl)borate.

5. The osmium complex according to claim 4, characterized in that: The halide ion is a chloride ion.

6. A method for preparing an osmium complex as described in any one of claims 3 to 5, comprising the following steps: causing a coordination reaction between a ligand and an osmium source to obtain the osmium complex; in, The structural formula of the ligand is as follows: .

7. The use of the osmium complex as described in any one of claims 3 to 5 in the preparation of antitumor drugs.

8. An antitumor drug, characterized in that: The antitumor drug contains an osmium complex as described in any one of claims 3 to 5.

9. The antitumor drug according to claim 8, characterized in that: The tumor is breast cancer.

10. 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 3 to 5.

11. A reagent or kit for biomedical imaging, characterized in that: It includes the osmium complex as described in any one of claims 3 to 5.

12. A chemical sensor, characterized in that: It includes the osmium complex as described in any one of claims 3 to 5.

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