Ruthenium (III) acid salt composition and application thereof

Abstract

The invention discloses a ruthenium (III) acid salt composition and application thereof. The composition comprises trans-[tetrachloro-bis (1H-indazole) ruthenium (III) acid salt] and at least one selected from the group consisting of 2-methylthiobenzeneboronic acid and / or a derivative thereof, and 2-aminophenol and / or a derivative thereof. When the trans-[tetrachloro-bis (1H-indazole) ruthenium (III) acid salt] compound is combined with at least one selected from 2-methylthiophenylboronic acid and / or a derivative thereof, and 2-aminophenol and / or a derivative thereof, the trans-[tetrachloro-bis (1H-indazole) ruthenium (III) acid salt] compound can well inhibit the growth of tumors. The ruthenium (III) acid salt composition provided by the invention has good stability and is not easily oxidized by air.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a ruthenium (III) salt composition and application thereof. Background Art

[0002] BOLD-100 (CAS No.: 197723-00-5) is an intravenously injected ruthenium-based anti-tumor clinical candidate drug that has passed Phase I clinical trials (Clinical Trial No.: NCT01415297). In the completed Phase II clinical trials, BOLD-100 was combined with 5-fluorouracil, calcium folinate, and oxaliplatin to treat solid tumors such as colorectal cancer, pancreatic cancer, and bile duct cancer (Clinical Trial No.: NCT04421820). Studies have shown that BOLD-100 is prone to hydrolysis under physiological conditions and irreversibly binds to proteins, causing its own structure to be destroyed, making it difficult to enter cells and reducing its anti-tumor activity. In several solid tumor models (colon cancer, breast cancer, and non-small cell lung cancer), BOLD-100 has a general effect in inhibiting tumor growth. In recent years, there have been a few studies on the anti-cancer activity of BOLD-100 in combination with other compounds, but the effect is not obvious.

[0003] In the in vivo experiments in mice, the anti-tumor effect of using BOLD-100 alone or a simple drug combination strategy was not ideal. Because BOLD-100 is easily hydrolyzed under physiological conditions and easily binds irreversibly to proteins, its bioavailability is reduced and its pharmacological activity is greatly weakened. Summary of the invention

[0004] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the object of the present invention is to provide a composition of ruthenate (III) and its application.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The first aspect of the present invention provides a composition of a ruthenium (III) salt, comprising trans-[tetrachlorobis(1H-indazole)ruthenium (III) salt] and at least one selected from 2-methylthiophenylboronic acid and / or its derivatives, 2-aminophenol and / or its derivatives.

[0007] In the present invention, the longer the incubation time of trans-[tetrachlorobis(1H-indazole)ruthenate(III) salt] in the culture medium, the lower the activity of the compound itself. However, when the compound is used in combination with at least one selected from 2-methylthiophenylboronic acid and / or its derivatives, 2-aminophenol and / or its derivatives, it shows a very strong tumor inhibitory effect.

[0008] In some embodiments of the present invention, the molar ratio of 2-methylthiophenylboronic acid and / or its derivatives to trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is ≥0.1, such as ≥0.5, ≥1.0, ≥2.0, ≥3.0, ≥4.0, 0.1 to 50. Excessive use of 2-methylthiophenylboronic acid and / or its derivatives may cause cytotoxicity.

[0009] In some embodiments of the present invention, the molar ratio of 2-aminophenol and / or its derivatives to trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is ≥0.1, such as ≥0.5, ≥1.0, ≥2.0, ≥3.0, ≥4.0, 0.1-50.

[0010] In some embodiments of the present invention, the structural formula of the trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is as shown in Formula I: Wherein, R is selected from Na + , Trans-[tetrachlorobis(1H-indazole)ruthenate(III)] will produce precipitation in PBS solution over time, and the solution will turn blue under full culture conditions. Newly prepared trans-[tetrachlorobis(1H-indazole)ruthenate(III)] will quickly non-covalently bind to human serum albumin (HSA) after entering the human body, but will produce irreversible covalent binding after a period of time, which will reduce the cellular uptake of the ruthenium complex and fail to exert an effective tumor inhibition effect. The present invention reveals the special "S-type" hydrolysis mechanism of trans-[tetrachlorobis(1H-indazole)ruthenate(III)] through UV-VIS and kinetic analysis (i.e., it starts very slowly, then increases exponentially, and finally becomes gentle), revealing the fundamental reason why the instability of the ruthenium complex leads to its reduced efficacy.

[0011] In some embodiments of the present invention, the trans-[tetrachlorobis(1H-indazole)ruthenate(III) salt] comprises

[0012]

[0013] In some embodiments of the present invention, the structural formula of the 2-methylthiophenylboronic acid and / or its derivatives is as shown in Formula II or a pharmaceutically acceptable salt of Formula II: Wherein, R1 and R2 are independently selected from H, C1-C6 alkyl, C1-C6 hydroxyalkyl, or R1, R2 and the atoms connected thereto form a 5- to 8-membered heterocycloalkyl; R3 is selected from C1-C6 alkyl, 3- to 10-membered cycloalkyl, 6- to 10-membered aryl, or S and the benzene ring connected thereto and R3 are connected to form a ring, such as a 6- to 10-membered arylbenzothiophene or a 6- to 10-membered arylbenzothiopyran; the alkyl, cycloalkyl and aryl groups are optionally unsubstituted or substituted by halogen; R4 is selected from H, halogen, amino, pyrazoleamino , C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, 6-10-membered aryl, heteroaryl with 5-10 rings (preferably 5, 6 or 9 rings); the alkyl, alkoxy, alkylthio, aryl, heteroaryl are optionally unsubstituted or substituted by halogen; when R4 is selected from H, halogen, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, m is selected from a natural number of 1-4; when R4 is selected from a 6-10-membered aryl, heteroaryl with 5-10 rings, m is 1. Preferably, the heteroatoms in the 5- to 8-membered heterocycloalkyl include O and N. The derivative also includes its salt form, such as potassium hydroxyl salt; the heteroaryl with 5-10 rings has 6, 10 or 14 shared π electrons in the cyclic arrangement; and has 1-5 heteroatoms in addition to carbon atoms. The term "heteroatom" refers to nitrogen, oxygen or sulfur and includes any oxidized form of nitrogen or sulfur and any quaternized form of basic nitrogen. Heteroaryl includes but is not limited to thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidyl, pyrazinyl, triazinyl, indolizinyl, purinyl, naphthyridinyl and pteridinyl. The term "heteroaryl" used in the present application also includes a group in which a heteroaromatic ring is fused to one or more aryl, cyclic aliphatic groups or heterocyclic rings. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, phenylquinazolinyl, phenylquinazolinyl-carbazolyl, biphenyl-benzoquinazolinyl-carbazolyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, phenylcarbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolyl, tetrahydroisoquinolyl and pyrido[2,3-b]1,4-oxazin-3(4H)-one, diphenyl-triazinyl. The heteroaryl group may be monocyclic or bicyclic. In the present invention, 2-methylthiophenylboronic acid and / or its derivatives can inhibit the self-hydrolysis of trans-[tetrachlorobis(1H-indazole)ruthenate(III) salt] and also inhibit its covalent binding to blood proteins, thereby enhancing the cellular uptake of trans-[tetrachlorobis(1H-indazole)ruthenate(III) salt] and further enhancing its anticancer activity in vivo.

[0014] In some embodiments of the present invention, the 2-methylthiophenylboronic acid and / or its derivatives are selected from

[0015]

[0016] In some embodiments of the present invention, the structural formula of the 2-aminophenol and / or its derivatives is as shown in Formula III:

[0017]

[0018] Formula III, wherein R5 is selected from H, halogen, C1-C6 alkyl. In the present invention, 2-methylthiophenylboronic acid and / or its derivatives can react with the hydrolyzed ruthenium complex to form a new complex, which has a good combined effect in cell experiments.

[0019] In some embodiments of the present invention, the 2-aminophenol and / or its derivatives are selected from

[0020] In some embodiments of the present invention, the ruthenate (III) salt composition further comprises a pharmaceutically acceptable excipient.

[0021] The composition of ruthenate (III) salt of the present invention is suitable for a variety of administration routes, and thus can be prepared into any pharmaceutically acceptable dosage form. For example, the composition of ruthenate (III) salt can be administered to patients or subjects who need such treatment by oral, parenteral, rectal or transpulmonary administration. When used for oral administration, the pharmaceutical composition can be prepared into oral preparations, for example, conventional oral solid preparations, such as tablets, capsules, pills, granules, etc.; it can also be prepared into oral liquid preparations, such as oral solutions, oral suspensions, syrups, etc. When preparing oral preparations, suitable fillers, adhesives, disintegrants, lubricants, etc. can be added. When used for parenteral administration, the pharmaceutical composition can also be prepared into injections, including injections, sterile powders for injection and concentrated solutions for injection. When preparing injections, conventional methods in the existing pharmaceutical field can be used for production. When preparing injections, additives can be omitted, or suitable additives can be added according to the properties of the drug. When used for rectal administration, the pharmaceutical composition can be prepared into suppositories, etc. When used for administration through the lungs, the pharmaceutical composition can be prepared into an inhalation preparation, an aerosol, a powder spray or a spray.

[0022] Pharmaceutically acceptable adjuvants refer to substances that are non-toxic, compatible with active ingredients, and otherwise biologically suitable for organisms. The selection of specific adjuvants will depend on the mode of administration or the type and state of disease used to treat a specific patient. Examples of pharmaceutically acceptable adjuvants include, but are not limited to, conventional solvents, diluents, dispersants, suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, adhesives, lubricants, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, ion exchangers, release agents, coating agents, flavoring agents, and antioxidants, etc. in the pharmaceutical composition. If necessary, flavoring agents, preservatives, and sweeteners, etc., may also be added to the pharmaceutical composition.

[0023] Tablets may contain an inert binder such as calcium carbonate, calcium phosphate, sodium phosphate or lactose; a granulating and distributing agent such as corn starch or alginate; a binder such as starch, gelatin or arabinose; and a lubricant such as aluminum or magnesium stearate, talc or silicone oil. They may also have a coating that is prepared to cause delayed release and reabsorption of the drug formulation in the gastrointestinal tract, thereby, for example, achieving improved compatibility, assimilation or retardation. Gelatin capsules may contain a drug substance with a solid such as calcium carbonate or kaolin or an oily substance such as olive oil, peanut oil or paraffin oil diluent.

[0024] Aqueous suspensions may contain suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth or gum arabic; dispersants or wetting agents, for example, polyoxyethylene stearate, heptadecaethylene-oxyglycerol, polyoxyethylene sorbitan monooleate or lecithin; preservatives, for example, methyl hydroxybenzoate or propyl hydroxybenzoate; flavor regulators; sweeteners, for example, sucrose, lactose, sodium cyclamate, glucose, invert sugar syrup.

[0025] Oily suspensions may be, for example, peanut oil, olive oil, sesame oil, coconut oil or paraffin oil and a thickener, such as beeswax, a high melting point wax or cetyl alcohol; as well as sweeteners, taste modifiers and antioxidants.

[0026] Powders and granules dispersible in water may contain a mixture of the composition of the invention with dispersing agents, wetting agents and suspending agents, such as those mentioned above, and sweetening, flavoring and coloring agents.

[0027] Emulsions may, for example, contain olive oil, peanut oil or paraffin oil together with emulsifiers such as arabinose, tragacanth, phospholipids, sorbitan monooleate, polyoxyethylene sorbitan monooleate and sweeteners and flavorings.

[0028] The aqueous solutions may contain preservatives such as methyl or propyl hydroxybenzoate; thickeners; flavor enhancers; sweeteners such as sucrose, lactose, sodium cyclamate, glucose, invert sugar syrup and flavoring and coloring agents.

[0029] A lyophilized preparation for injection generally comprises the composition of the present invention, a pH buffer and a cryoprotectant. The general method of providing the preparation comprises the following steps: preparing an aqueous buffer solution, preparing an aqueous cryoprotectant solution, dissolving the composition of the ruthenate (III) acid salt in a buffer solution, adding a cryoprotectant solution, sterilizing and filtering (e.g., aseptic filtration), filling a vial under aseptic conditions, and freeze-drying under aseptic conditions. Suitable buffers include, but are not limited to, citrate, TRIS, acetate, EDTA, HEPES, tricine (N-tris (hydroxymethyl) methylglycine) and imidazole. It is possible to use a phosphate buffer, but it is not preferred. A preferred aspect of the present invention is to use a citric acid / sodium citrate buffer. Suitable cryoprotectants include, but are not limited to, sugars, monosaccharides, disaccharides, polyols, mannitol, sorbitol, sucrose, trehalose, dextran and dextrose.

[0030] The second aspect of the present invention provides a use of the ruthenium (III) salt composition in the preparation of anti-tumor drugs.

[0031] The term "tumor" should be understood to refer to all forms of neoplastic cell growth, including tumors of the lung, liver, blood cells, skin, pancreas, stomach, colon, prostate, uterus, breast, lymph glands, bone and bladder.

[0032] In some embodiments of the present invention, the tumor comprises at least one of colon cancer, liver cancer, lung cancer, brain cancer, cervical cancer, breast cancer, melanoma, pancreatic cancer, multiple myeloma (MM) or blood cancer.

[0033] In some embodiments of the present invention, the therapeutically effective amount of the composition of ruthenium (III) salt is calculated based on the therapeutically effective amount of trans-[tetrachlorobis(1H-indazole)ruthenium (III) salt]. For example, the therapeutically effective amount of trans-[tetrachlorobis(1H-indazole)ruthenium (III) salt] is 320 mg / m 2 Up to 625 mg / m 2 The amount of body surface area (BSA) of the patient can be calculated using Modified Dubois, i.e., BSA (m 2 )=0.007184xHeight(cm) 0.725 x Weight (kg) 0.425 .

[0034] In some embodiments of the present invention, based on the combined therapeutic effects of the components in the composition of ruthenate (III) salts described in the present invention, the therapeutically effective amount of trans-[tetrachlorobis(1H-indazole)ruthenate (III) salt] is reduced to 0.05 to 0.95 times, such as 0.09 to 0.9 times, 0.09 to 0.8 times, or 0.09 to 0.7 times, of the single dosage.

[0035] "Therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome, at the dosage and duration necessary. The therapeutically effective amount of a formulation can vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit the desired response in the individual. The dosage regimen can be adjusted to provide the optimal therapeutic response. A therapeutically effective amount can also be an amount in which the therapeutic beneficial effects outweigh any toxic or adverse effects of the formulation or active compound.

[0036] The minimum and maximum carbon atom content in a hydrocarbon group is indicated by a prefix, for example, the prefix C a～b Alkyl refers to any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, "C 1～4 The term "alkyl" refers to an alkyl group containing 1 to 4 carbon atoms.

[0037] The "alkyl" described in the present invention refers to a saturated hydrocarbon group having a specified number of carbon atoms. For example, C1-6 alkyl refers to an alkyl group having 1 to 6 carbon atoms, for example, preferably 1 to 4 carbon atoms. The alkyl group may be straight chain or branched. Representative branched alkyl groups have one, two or three branches. The alkyl group may also be optionally substituted with one or more substituents as defined herein. Specific examples of alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, etc.

[0038] The "halogen" mentioned in the present invention is fluorine, chlorine, bromine or iodine.

[0039] The beneficial effects of the present invention are:

[0040] (1) When combined with at least one selected from 2-methylthiophenylboronic acid and / or its derivatives, 2-aminophenol and / or its derivatives, the trans-[tetrachlorobis(1H-indazole)ruthenate(III)ate] compound can well inhibit tumor growth, indicating that the trans-[tetrachlorobis(1H-indazole)ruthenate(III)ate] compound and at least one selected from 2-methylthiophenylboronic acid and / or its derivatives, 2-aminophenol and / or its derivatives can be used as an anticancer drug combination. In addition, the type of at least one selected from 2-methylthiophenylboronic acid and / or its derivatives, 2-aminophenol and / or its derivatives of the present invention can be adjusted as needed, thereby adjusting the absorption of the trans-[tetrachlorobis(1H-indazole)ruthenate(III)ate] compound in tumor tissue, thereby having good selectivity in anticancer in vivo.

[0041] (2) The present invention solves the problem that trans-[tetrachlorobis(1H-indazole)ruthenate(III)] has unstable structure, easy hydrolysis and irreversible binding with proteins under normal physiological conditions, which leads to its inability to effectively inhibit the proliferation of cancer cells by using trans-[tetrachlorobis(1H-indazole)ruthenate(III)] in combination with 2-methylthiophenylboronic acid and its derivatives or 2-aminophenol and its derivatives. The results of cell experiments show that the effect of 2-methylthiophenylboronic acid and its derivatives or 2-aminophenol and its derivatives in combination with the ruthenium complex is much stronger than the effect of the ruthenium complex alone.

[0042] (3) The trans-[tetrachlorobis(1H-indazole)ruthenate(III)] and 2-methylthiophenylboronic acid and its derivatives or 2-aminophenol and its derivatives of the present invention can be used as a combination of anticancer drugs. The combined use can greatly reduce the dosage of the ruthenium complex and reduce the potential toxic side effects of large doses of ruthenium. The composition has good stability, is not easily oxidized by air, and preferably exerts its anti-tumor effect by intraperitoneal injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a graph showing the effect of BOLD-100 combined with 2-methylthiophenylboronic acid on tumor volume in a mouse tumor model;

[0044] Figure 2 This is a graph showing the effect of BOLD-100 combined with 2-methylthiophenylboronic acid on the body weight of mice in a mouse tumor model;

[0045] Figure 3 This is a diagram showing the actual tumor inhibition effect (tumor image) of BOLD-100 combined with 2-methylthiophenylboronic acid in a mouse tumor model;

[0046] Figure 4These are H&E stained sections of the heart, liver, spleen, lung, and kidney of mice in each drug administration group after dissection;

[0047] Figure 5 This is the UV-visible absorption spectrum of BOLD-100 (400 μM) in PBS from 0 to 480 min;

[0048] Figure 6 It is the UV-visible absorption spectrum of BOLD-100 (400 μM) of the present invention mixed with 2-methylthiophenylboronic acid (40 μM) in PBS from 0 to 480 min (800 nm leveling);

[0049] Figure 7 It is a UV-visible 580nm absorption kinetic analysis diagram of BOLD-100 (400 μM) of the present invention and BOLD-100 (400 μM) and 2-methylthiophenylboronic acid (40 μM) under PBS conditions;

[0050] Figure 8 This is a UV-visible absorption spectrum of BOLD-100 (400 μM) and 2-aminophenol (200 μM) in PBS from 0 to 480 min. DETAILED DESCRIPTION

[0051] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0052] The cells are HCT116 (colon cancer cells), Huh-7 (liver cancer cells), A549 (lung cancer cells), U87 (human astrocytic glioma blast cells), Hela (cervical cancer cells), MCF-7 (breast cancer cells), A375 (melanoma cells), PANC-1 (pancreatic cancer cells) or Jurkat (blood cancer cells). Among them, A549, MCF-7, A375, and PANC-1 cells are from the American Type Culture Collection, HCT116 cells are from Wuhan Pronocell Life Science Co., Ltd., U87 cells are from Feng Min's research group at the School of Pharmacy, Sun Yat-sen University, and Huh-7, Jurkat, and Hela cells are from Wan Guohui's research group at the School of Pharmacy, Sun Yat-sen University.

[0053] In the following embodiments or tests, the cancer cells are cultured as follows:

[0054] Take the culture flask containing the cancer cells required for the experiment, discard the original culture medium, wash three times with 2mL PBS, add 1mL trypsin to digest for 30s, and add 2mL culture medium to stop digestion. Transfer the cells to a centrifuge tube, centrifuge at 1000 rpm for 3min, discard the supernatant, add 2mL culture medium and blow 10 times to mix. Take 10μL of cell suspension to the cell counter and count; spread 96-well plates, plant 5000 cells per well, calculate the required cell volume and cell well volume for 3 replicates for each compound, calculate the required cell suspension volume by cell counting, dilute the required number of cells with culture medium, 5000 cells per well, spread 100μL culture medium, mix by pipetting, use a spray gun to plant cells, place in an incubator for culture, and mark them.

[0055] Example 1

[0056] This example studies the effect of BOLD-100 combined with 2-methylthiophenylboronic acid on the inhibitory activity of various cancer cells. The specific process is as follows:

[0057] MTT colorimetric assay for detecting cancer cell proliferation inhibition

[0058] 24 hours after cell plating, add a certain amount of 2-methylthiophenylboronic acid to each well of the 96-well plate to make the concentration in each well 200 μM, then add BOLD-100 and dilute it step by step to act on the cells. After 48 hours, add 20 μL of MTT to each well of the 96-well plate with a dispenser (MTT concentration is 5 mg / mL in PBS); incubate at 37°C for 4 hours. After the incubation time, aspirate the MTT mixture and add DMSO at 130 μL / well with a dispenser; shake on a horizontal shaker for 10 minutes until formazan is fully dissolved, detect the absorbance at a wavelength of 490 nm with an enzyme marker, calculate the cell survival rate at each drug concentration as required, and make a scatter plot.

[0059] The specific composition of the composition of 2-methylthiophenylboronic acid compound and ruthenate (III) salt is shown in Table 1. The molar ratio in the table represents the molar ratio of the corresponding 2-methylthiophenylboronic acid compound to trans-[tetrachlorobis(1H-indazole)ruthenate (III) salt]:

[0060] Table 1

[0061]

[0062] The specific composition of the composition of 2-aminophenol compound and ruthenate (III) salt is shown in Table 2. The molar ratio in the table represents the molar ratio of the corresponding 2-aminophenol compound to trans-[tetrachlorobis(1H-indazole)ruthenate (III) salt]:

[0063] Table 2

[0064]

[0065] The survival rates of HCT116 colon cancer cells using BOLD-100, the ligand described in Examples 1-16, and the combination of the two are shown in Table 3.

[0066] Table 3

[0067]

[0068]

[0069] Example 2

[0070] This example studies the effect of BOLD-100 combined with 2-methylthiophenylboronic acid on the inhibitory activity of various cancer cells. The specific process is as follows:

[0071] 24 hours after cell plating, a certain amount of 2-methylthiophenylboronic acid was added to each well of the 96-well plate to make the concentration in each well 200 μM, and then BOLD-100 was added and diluted step by step to act on the cells. After 48 hours, 20 μL of MTT was added to each well of the 96-well plate using a spray gun (MTT concentration was 5 mg / mL in PBS); incubated at 37°C for 4 hours. After the incubation time, the MTT mixture was aspirated, and DMSO was added at 130 μL / well using a spray gun; the cells were shaken on a horizontal shaker for 10 minutes until formazan was fully dissolved, and the absorbance was detected at a wavelength of 490 nm using an enzyme marker. The cell survival rate at each concentration of the drug was calculated as required, and a scatter plot was made. The results of the effects of BOLD-100, 2-methylthiophenylboronic acid and the combination of the two compounds on cancer cell proliferation are shown in Table 4.

[0072] Table 4

[0073]

[0074]

[0075] From the results in Table 4, it can be seen that under normal physiological conditions (adding FBS to the empty culture medium can simulate the protein environment under physiological conditions), BOLD-100 has no obvious inhibitory effect on human colon cancer cells, human liver cancer cells, human lung cancer cells, human cervical cancer cells, human breast cancer cells, human brain glioblastoma cells, human melanoma cells, blood cancer and pancreatic cancer; however, when a non-toxic dose of 2-methylthiophenylboronic acid is added, BOLD-100 shows excellent inhibitory effects on the above 9 types of cancer cells, and 2-methylthiophenylboronic acid derivatives and 2-aminophenol and its derivatives also have similar effects.

[0076] Example 3

[0077] This example studies the ratio range of BOLD-100 and 2-methylthiophenylboronic acid, and the specific process is as follows:

[0078] The BOLD-100 concentration was fixed at 100 μM, and the 2-methylthiophenylboronic acid concentration was changed according to the ratio in Table 5. According to the experimental method of Example 2, the IC of BOLD-100 combined with 2-methylthiophenylboronic acid at different molar concentrations on the colon cancer cell line (HCT116) was tested. 50 .

[0079] Table 5

[0080]

[0081] From the results in Table 5, it can be seen that 2-methylthiophenylboronic acid has a combined effect with BOLD-100. When the combined concentration (ratio) of 2-methylthiophenylboronic acid increases, its combined effect with BOLD-100 is more obvious. When the ratio of 2-methylthiophenylboronic acid to BOLD-100 is greater than or equal to 2, the combined use has the strongest synergistic effect. 2-methylthiophenylboronic acid derivatives and 2-aminophenol and its derivatives also have similar effects.

[0082] Example 4

[0083] This example studies the effect of BOLD-100 combined with 2-methylthiophenylboronic acid on BOLD-100 cell absorption, and the detection steps are as follows:

[0084] HCT116 cells were used as experimental subjects.

[0085] The cells were cultured at 2 × 10 5 Each well was inoculated in a 6-well plate and incubated for 24 hours.

[0086] After the incubation, the cell culture medium was replaced with 30% FBS to simulate the high-concentration protein environment under normal physiological conditions. No reagent was added as the control group, and 10μM BOLD-100 and 200μM 2-methylthiophenylboronic acid were added to the experimental group for 6h (the final concentration of DMF in the system was ≤0.2%). The incubated cells were immediately washed 3 times with PBS, and 500μL ultrapure water was added to each well to break the cells. After 15min, the cell lysate was collected.

[0087] Aqua regia was added to the collected cell lysate to dissolve it, and then diluted to an appropriate ratio with ultrapure water. The ruthenium content was detected by inductively coupled plasma mass spectrometry (ICP-MS). The absorption rate of cells without BOLD-100 was used as a control, and the results are shown in Table 6.

[0088] Table 6 Effect of 2-methylthiophenylboronic acid on BOLD-100 cell absorption rate

[0089]

[0090] The above-mentioned absorption amount is the ruthenium content (ng) per gram of protein in the cell.

[0091] As shown in Table 6, in the medium with high FBS concentration, the absorption of ruthenium is reduced; when 2-methylthiophenylboronic acid is added, the absorption of BOLD-100 is greatly improved.

[0092] Example 5

[0093] This example studies the actual inhibitory effect of BOLD-100 combined with 2-methylthiophenylboronic acid in a mouse tumor model. The detection steps are as follows:

[0094] Establishment of mouse tumor model: Two million colon cancer cells HCT116 suspended in PBS were subcutaneously injected into the dorsal flank of 5-7 week old female BALB / c-nu / nu (nude mice) to establish a xenograft model. When the tumor volume reached about 50 mm 3 At 3-4 days after tumor inoculation, the mice were randomly divided into a control group (using castor oil) and a treatment group (BOLD-100 group, 2-methylthiophenylboronic acid group, and combined administration group). The combined administration group was treated with BOLD-100 (10 mg / kg mouse body weight / day) combined with 2-methylthiophenylboronic acid (40 mg / kg mouse body weight / day) by intraperitoneal injection for 5 consecutive days a week. When the tumor volume grew to 500 mm 3 Afterwards, the mice were anesthetized and then killed by cervical dislocation.

[0095] The results are as follows Figures 1 to 4 shown.

[0096] It can be seen that the tumor growth was not inhibited in the control group, the group receiving BOLD-100 alone, or the group receiving 2-methylthiophenylboronic acid alone; when BOLD-100 and 2-methylthiophenylboronic acid were used in combination, tumor growth was significantly inhibited.

[0097] Example 6

[0098] This example studies the UV-VIS spectra and absorption kinetic curves of BOLD-100 itself and after mixing with 2-methylthiophenylboronic acid or 2-aminophenol in PBS. The detection steps are as follows:

[0099] Dissolve BOLD-100 in PBS to prepare a 400μM stock solution. Then take three solutions, 2ml each. The first one is used as a self-control, the second one is further configured to contain 40μM 2-methylthiophenylboronic acid solution, and the third one is further configured to contain 200μM 2-aminophenol solution. Measure the UV-visible absorption spectra of these three solutions for 0-8h respectively. And select the 580nm wavelength of each of the first two solutions to make a kinetic absorption change curve.

[0100] The results are as follows Figures 5 to 8 As shown, UV-VIS shows that 2-methylthiophenylboronic acid, 2-aminophenol and their derivatives can effectively inhibit the formation of multi-nuclear ruthenium aggregates (absorption peak near 600nm) and enhance the anticancer activity.

[0101] It can be seen that the addition of 40 μM 2-methylthiophenylboronic acid can significantly inhibit the hydrolysis of BOLD-100 itself, and the addition of 200 μM 2-aminophenol will quickly react with BOLD-100, and the formation of new absorption peaks indicates that other compounds are generated.

[0102] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A composition of a ruthenate (III) salt, characterized in that: The invention comprises trans-[tetrachlorobis(1H-indazole)ruthenate(III) salt] and at least one selected from 2-methylthiophenylboronic acid and / or its derivatives, 2-aminophenol and / or its derivatives.

2. The composition of ruthenate (III) according to claim 1, characterized in that: The molar ratio of the 2-methylthiophenylboronic acid and / or its derivatives to trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is ≥0.

1.

3. The composition of ruthenate (III) according to claim 1, characterized in that: The molar ratio of the 2-aminophenol and / or its derivatives to trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is ≥0.

1.

4. The composition of ruthenate (III) according to claim 1, characterized in that: The structural formula of the trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is shown in Formula I: Wherein, R is selected from Na + , 5. The composition of ruthenate (III) according to claim 1, characterized in that: The structural formula of the 2-methylthiophenylboronic acid and / or its derivatives is as shown in Formula II or a pharmaceutically acceptable salt of Formula II: wherein R1 and R2 are independently selected from H, C1-C6 alkyl, C1-C6 hydroxyalkyl, or R1, R2 and the atoms to which they are connected form a 5- to 8-membered heterocycloalkyl; R3 is selected from C1-C6 alkyl, 3-10-membered cycloalkyl, 6-10-membered aryl, or S and the benzene ring connected thereto and R3 are connected to form a ring; the alkyl, cycloalkyl, aryl are optionally unsubstituted or substituted by halogen; R4 is selected from H, halogen, amino, pyrazole amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, 6-10 membered aryl, heteroaryl having 5-10 rings; the alkyl, alkoxy, alkylthio, aryl, heteroaryl are optionally unsubstituted or substituted by halogen; When R4 is selected from H, halogen, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, m is selected from a natural number of 1 to 4; when R4 is selected from a 6-10 membered aryl group, a heteroaryl group having 5 to 10 rings, m is 1.

6. The composition of ruthenate according to claim 1, characterized in that: The 2-methylthiophenylboronic acid and / or its derivatives are selected from 7. The composition of ruthenate (III) according to claim 1, characterized in that: The structural formula of the 2-aminophenol and / or its derivatives is shown in Formula III: Wherein, R5 is selected from H, halogen, C1-C6 alkyl.

8. The composition of ruthenate (III) according to claim 1, characterized in that: The ruthenate (III) salt composition further comprises a pharmaceutically acceptable auxiliary material.

9. Use of the ruthenium (III) salt composition in the preparation of anti-tumor drugs.

10. The use according to claim 8, characterized in that: The tumor includes at least one of colon cancer, liver cancer, lung cancer, brain cancer, cervical cancer, breast cancer, melanoma, pancreatic cancer, multiple myeloma or blood cancer.

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