A composition of a ruthenium (III) salt and its use

Abstract

This invention discloses a ruthenium(III) salt composition and its applications. The composition comprises 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. 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)ruthenium(III) salt] compound can effectively inhibit tumor growth. The ruthenium(III) salt composition of this invention exhibits good stability and is not easily oxidized by air.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a composition of ruthenium(III) salt and its applications. Background Technology

[0002] BOLD-100 (CAS No.: 197723-00-5) is a ruthenium-based antitumor drug candidate for intravenous injection, which has passed Phase I clinical trials (Clinical Trial No.: NCT01415297). In a completed Phase II clinical trial, BOLD-100 was used in combination with 5-fluorouracil, leucovorin, and oxaliplatin to treat solid tumors such as colorectal cancer, pancreatic cancer, and cholangiocarcinoma (Clinical Trial No.: NCT04421820). Studies have shown that BOLD-100 is easily hydrolyzed under physiological conditions, irreversibly binding to proteins, leading to structural damage, difficulty in entering cells, and reduced antitumor activity. In several solid tumor models (colon cancer, breast cancer, and non-small cell lung cancer), the effect of BOLD-100 in inhibiting tumor growth was moderate. In recent years, there have been a few studies on the anticancer activity of BOLD-100 in combination with other compounds; however, the effects have not been significant.

[0003] In in vivo experiments in mice, the antitumor effects of using BOLD-100 alone or in simple drug combination strategies were not ideal. This is because BOLD-100 is easily hydrolyzed under physiological conditions and readily binds irreversibly to proteins, leading to reduced bioavailability and significantly weakened pharmacological activity. Summary of the Invention

[0004] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide a composition of ruthenium(III) salt and its application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a composition of ruthenium(III) salts 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 this invention, the longer the trans-[tetrachlorobis(1H-indazole)ruthenium(III)ate] is incubated in the culture medium, the lower the activity of the compound itself becomes. However, when this 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 exhibits a very strong tumor-suppressing 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)ruthenium(III)ate] is ≥0.1, such as ≥0.5, ≥1.0, ≥2.0, ≥3.0, ≥4.0, or 0.1–50. Excessive amounts of 2-methylthiophenylboronic acid and / or its derivatives may lead to cytotoxicity.

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

[0010] In some embodiments of the present invention, the structural formula of the trans-[tetrachlorobis(1H-indazole)ruthenium(III)ate] is shown in Formula I: Wherein, R is selected from Na + , Trans-[tetrachlorobis(1H-indazole)ruthenium(III)ate] precipitates over time in PBS solution, turning the solution blue under full culture conditions. Freshly prepared trans-[tetrachlorobis(1H-indazole)ruthenium(III)ate] rapidly binds nonvalently to human serum albumin (HSA) upon entering the human body, but irreversibly covalently binds after a period of time, reducing cellular uptake of the ruthenium complex and preventing it from exerting an effective tumor-suppressive effect. This invention, through UV-VIS and kinetic analysis, reveals the unique "S-shaped" hydrolysis mechanism of trans-[tetrachlorobis(1H-indazole)ruthenium(III)ate] (i.e., initially slow, then exponentially increasing, and finally leveling off), revealing the fundamental reason for the reduced efficacy of this ruthenium complex due to its instability.

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

[0012]

[0013] In some embodiments of the present invention, the 2-methylthiophenylboronic acid and / or its derivatives have the structural formula shown in Formula II or pharmaceutically acceptable salts of Formula II: Wherein, R1 and R2 are independently selected from H, C1-C6 alkyl, and C1-C6 hydroxyalkyl, or R1 and R2 form a 5- to 8-membered heterocyclic alkyl group with the atoms attached to them; R3 is selected from C1-C6 alkyl, 3- to 10-membered cycloalkyl, and 6- to 10-membered aryl, or S is connected to the benzene ring attached to it and R3 to form a ring, such as 6- to 10-membered arylbenzothiophene and 6- to 10-membered arylbenzothiaran; the alkyl, cycloalkyl, and aryl groups are optionally unsubstituted or substituted with halogens; R4 is selected from H, halogens, amino groups, and pyrazole amino groups. The derivative comprises C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, 6-10 aryl, and heteroaryl groups having 5-10 rings (preferably 5, 6, or 9 rings); wherein the alkyl, alkoxy, alkylthio, aryl, and heteroaryl groups are optionally unsubstituted or substituted with halogens; when R4 is selected from H, halogens, amino, C1-C6 alkyl, C1-C6 alkoxy, or C1-C6 alkylthio, m is selected from a natural number from 1 to 4; when R4 is selected from 6-10 aryl groups or heteroaryl groups having 5-10 rings, m is 1. Preferably, the heteroatoms in the 5- to 8-membered heterocyclic alkyl groups include O and N. The derivative also includes its salt form, such as potassium hydroxyl salt; the heteroaryl groups having 5- to 10 rings have 6, 10, or 14 shared π electrons in the cyclic arrangement; and have 1-5 heteroatoms in addition to carbon atoms. The term "heteroatom" refers to nitrogen, oxygen, or sulfur, including any oxidized form of nitrogen or sulfur and any quaternized form of basic nitrogen. Heteroaryl groups include, but are not limited to, thiophene, furanyl, pyrrole, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridinyl, pyrazinyl, triazinyl, indazinyl, purine, diazonaphthyl, and pteridinyl. As used in this application, the term "heteroaryl" also includes groups in which a heteroaryl ring is fused with one or more aryl, cyclic aliphatic, or heterocyclic rings. Non-limiting examples include indolyl, isoindolyl, benzothiophenyl, benzofuranyl, dibenzofuranyl, indazoleyl, benzimidazolyl, benzothiazolyl, quinolinyl, isoquinolinyl, cyclolinyl, phthalazinyl, quinazolinyl, phenylquinazolinyl, phenylquinazolinyl-carbazoleyl, biphenyl-benzoquinazolinyl-carbazoleyl, quinoxalinyl, 4H-quinazinyl, carbazoleyl, phenylcarbazoleyl, acridineyl, phenazinyl, phenthiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]1,4-oxazin-3(4H)-one, diphenyl-triazinyl. The heteroaryl group can be monocyclic or bicyclic. In this invention, 2-methylthiophenylboronic acid and / or its derivatives can inhibit the self-hydrolysis of trans-[tetrachlorobis(1H-indazole)ruthenium(III)ate] and also inhibit its covalent binding to blood proteins, thereby enhancing the cellular uptake of trans-[tetrachlorobis(1H-indazole)ruthenium(III)ate] and thus enhancing its in vivo anticancer activity.

[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 shown in Formula III:

[0017]

[0018] Formula III, wherein R5 is selected from H, halogens, and C1-C6 alkyl groups. In this invention, 2-methylthiophenylboronic acid and / or its derivatives can react with hydrolyzed ruthenium complexes to form new complexes, which have excellent combined effects 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 invention, the ruthenium(III) salt composition further includes pharmaceutically acceptable excipients.

[0021] The ruthenium(III) salt compositions of the present invention are suitable for a variety of routes of administration and can therefore be formulated into any pharmaceutically acceptable dosage form. For example, the ruthenium(III) salt compositions described above can be administered orally, parenterally, rectally, or via the lungs to patients or subjects requiring such treatment. When used for oral administration, the pharmaceutical compositions can be formulated into oral preparations, such as conventional oral solid preparations, like tablets, capsules, pills, granules, etc.; or into oral liquid preparations, such as oral solutions, oral suspensions, syrups, etc. When formulating oral preparations, suitable fillers, binders, disintegrants, lubricants, etc., can be added. When used for parenterial administration, the pharmaceutical compositions described above can also be formulated into injections, including injection solutions, sterile powders for injection, and concentrated solutions for injection. When formulating injections, conventional methods in the existing pharmaceutical field can be used; when preparing injections, excipients may not be added, or suitable excipients may be added depending on the properties of the drug. When used for rectal administration, the pharmaceutical compositions can be formulated into suppositories, etc. When used for pulmonary administration, the pharmaceutical composition may be formulated as an inhalation formulation, aerosol, powder, or spray.

[0022] Pharmaceutically acceptable excipients are substances that are non-toxic, compatible with the active ingredient, and otherwise biologically suitable for use in organisms. The selection of specific excipients will depend on the route of administration or the type and state of disease in the treatment of a particular patient. Examples of pharmaceutically acceptable excipients include, but are not limited to, solvents, diluents, dispersants, suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, binders, lubricants, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, ion exchangers, release agents, coating agents, flavoring agents, and antioxidants commonly found in the pharmaceutical field. Flavoring agents, preservatives, and sweeteners may also be added to the pharmaceutical composition where necessary.

[0023] Tablets may contain inert binders such as calcium carbonate, calcium phosphate, sodium phosphate, or lactose; granulating and distributing agents such as corn starch or alginate; binding agents such as starch, gelatin, or arabinose; and lubricants such as aluminum stearate or magnesium stearate, talc, or silicone oil. They may also have a coating prepared to cause delayed release and reabsorption of the pharmaceutical formulation in the gastrointestinal tract, thereby achieving, for example, improved compatibility, assimilation, or inhibition. Gelatin capsules may contain a solid pharmaceutical substance 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 such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl cellulose, sodium alginate, polyvinylpyrrolidone, astragalus gum, or gum arabic; dispersants or wetting agents such as polyoxyethylene stearate, heptadecanoglycerate, polyoxyethylene sorbitan monooleate, or lecithin; preservatives such as methylparaben or propylparaben; flavor modifiers; and sweeteners such as sucrose, lactose, cyclamate, glucose, or invert sugar syrup.

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

[0026] Powders and granules dispersible in water may comprise mixtures of the compositions of the present invention with dispersants, wetting agents and suspending agents, such as those mentioned above, as well as sweeteners, flavoring agents and coloring agents.

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

[0028] Aqueous solutions may contain preservatives, such as methylparaben or propylparaben; thickeners; flavor modifiers; sweeteners, such as sucrose, lactose, cyclamate, glucose, invert sugar syrup, as well as flavoring and coloring agents.

[0029] Lyophilized formulations for injection typically comprise the composition described in this invention, a pH buffer, and a cryoprotectant. A general method for providing the formulation includes the following steps: preparing an aqueous buffer solution, preparing an aqueous cryoprotectant solution, dissolving the ruthenium(III) salt composition in the buffer solution, adding the cryoprotectant solution, sterilizing and filtering (e.g., aseptic filtration), filling vials under aseptic conditions, and lyophilizing under aseptic conditions. Suitable buffers include, but are not limited to, citrate, TRIS, acetate, EDTA, HEPES, tricine (N-tris(hydroxymethyl)methylglycine), and imidazole. The use of phosphate buffers is possible but not preferred. A preferred aspect of the invention is the use of a citrate / sodium citrate buffer. Suitable cryoprotectants include, but are not limited to, sugars, monosaccharides, disaccharides, polyols, mannitol, sorbitol, sucrose, trehalose, dextran, and dextrose.

[0030] A second aspect of the present invention provides the use of the aforementioned ruthenium(III) salt composition in the preparation of an antitumor drug.

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

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

[0033] In some embodiments of the present invention, the therapeutically effective amount of the ruthenium(III) salt composition 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 / m³. 2 Up to 625mg / m 2 The amount (based on the patient's body surface area (BSA)) can be calculated using Modified Dubois, i.e., BSA(m 2 = 0.007184 x height (cm) 0.725 x weight (kg) 0.425 .

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

[0035] A "therapeutic effective amount" refers to the amount that, at the necessary dose and duration, is effective in achieving the desired therapeutic outcome. The therapeutic effective amount of a formulation can vary depending on factors such as disease state, individual age, sex, and weight, and the compound's ability to elicit the desired response in the individual. Dosing regimens can be adjusted to provide the optimal therapeutic response. A therapeutic effective amount can also be the amount in which the beneficial therapeutic effect outweighs any toxicity or adverse effects of the formulation or active compound.

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

[0037] In this invention, "alkyl" 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, preferably specifically 1 to 4 carbon atoms. Alkyl groups can be straight-chain or branched. Representative branched alkyl groups have one, two, or three branches. Alkyl groups may also optionally be substituted by one or more substituents as defined herein. Specific examples of alkyl groups 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 this invention is fluorine, chlorine, bromine or iodine.

[0039] The beneficial effects of this 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)ruthenium(III)ate] compound can effectively inhibit tumor growth, indicating that the trans-[tetrachlorobis(1H-indazole)ruthenium(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. Furthermore, the type of at least one selected from 2-methylthiophenylboronic acid and / or its derivatives, 2-aminophenol and / or its derivatives can be adjusted as needed, thereby regulating the absorption of the trans-[tetrachlorobis(1H-indazole)ruthenium(III)ate] compound in tumor tissues, thus exhibiting good selectivity in in vivo anticancer activity.

[0041] (2) This invention addresses the shortcomings of trans-[tetrachlorobis(1H-indazole)ruthenium(III)ate] by combining it with 2-methylthiophenylboronic acid and its derivatives or 2-aminophenol and its derivatives. Under normal physiological conditions, trans-[tetrachlorobis(1H-indazole)ruthenium(III)ate] is structurally unstable, easily hydrolyzed, and irreversibly binds to proteins, thus failing to effectively inhibit cancer cell proliferation. Cell experiments show that the combined effect of 2-methylthiophenylboronic acid and its derivatives or 2-aminophenol and its derivatives with this ruthenium complex is far stronger than the effect of the ruthenium complex alone.

[0042] (3) The trans-[tetrachlorobis(1H-indazole)ruthenium(III)ate] 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. Combined use can significantly reduce the dosage of this ruthenium complex and reduce the potential toxic side effects of high-dose ruthenium. The composition has good stability and is not easily oxidized by air. It is preferably administered via intraperitoneal injection to exert its antitumor effect. Attached Figure Description

[0043] Figure 1 This is a graph showing the effect of the combination of BOLD-100 and 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 mouse body weight in a mouse tumor model.

[0045] Figure 3 This image shows the actual tumor-inhibiting effect of BOLD-100 combined with 2-methylthiophenylboronic acid in a mouse tumor model (tumor image).

[0046] Figure 4Images of H&E stained sections of the heart, liver, spleen, lungs, and kidneys of mice in each drug administration group after dissection according to the present invention;

[0047] Figure 5 The UV-Vis absorption spectrum of BOLD-100 (400 μM) in PBS from 0 to 480 min is shown below.

[0048] Figure 6 The UV-Vis absorption spectrum of BOLD-100 (400 μM) and 2-methylthiophenylboronic acid (40 μM) in PBS from 0 to 480 min (leveled at 800 nm) is shown below.

[0049] Figure 7 The diagram shows the UV-Vis absorption kinetics of BOLD-100 (400 μM) and BOLD-100 (400 μM) and 2-methylthiophenylboronic acid (40 μM) under PBS conditions at 580 nm.

[0050] Figure 8 The UV-Vis absorption spectrum of BOLD-100 (400 μM) and 2-aminophenol (200 μM) in PBS from 0 to 480 min is shown. Detailed Implementation

[0051] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0052] The cell lines used were HCT116 (colon cancer cells), Huh-7 (liver cancer cells), A549 (lung cancer cells), U87 (human astrocytoma-blastoma cells), HeLa (cervical cancer cells), MCF-7 (breast cancer cells), A375 (melanoma cells), PANC-1 (pancreatic cancer cells), or Jurkat (leukemia cells). A549, MCF-7, A375, and PANC-1 cells were sourced from the American Culture Collection Center; HCT116 cells were sourced from Wuhan Pronosei Biotechnology Co., Ltd.; U87 cells were sourced from Professor Feng Min's research group at the School of Pharmaceutical Sciences, Sun Yat-sen University; and Huh-7, Jurkat, and HeLa cells were sourced from Professor Wan Guohui's research group at the School of Pharmaceutical Sciences, Sun Yat-sen University.

[0053] In the following examples or tests, the cancer cells are cultured in the following manner:

[0054] Take the culture flask containing the desired cancer cells, discard the original culture medium, wash three times with 2 mL PBS, add 1 mL trypsin for 30 seconds, and add 2 mL culture medium to stop the digestion. Transfer the cells to a centrifuge tube, centrifuge at 1000 rpm for 3 minutes, discard the supernatant, add 2 mL culture medium, and mix by pipetting 10 times. Take 10 μL of the cell suspension into a cell counter and count the cells; seed 5000 cells per well, with 3 replicates per compound, and calculate the required cell volume and well volume. Calculate the required cell suspension volume using cell counting, dilute the required number of cells with culture medium to 5000 cells per well, and seed with 100 μL of culture medium. Mix by pipetting, seed the cells using a pipette, and incubate in an incubator, labeling as desired.

[0055] Example 1

[0056] This embodiment 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 the inhibitory effect on cancer cell proliferation

[0058] After 24 hours of cell seeding, a certain amount of 2-methylthiophenylboronic acid was added to each well of a 96-well plate to achieve a concentration of 200 μM per well. Then, BOLD-100 was added and serially diluted to treat the cells. After 48 hours, 20 μL of MTT (5 mg / mL in PBS) was added to each well of the 96-well plate using a multipipe. The plates were incubated at 37°C for 4 hours. After the incubation period, the MTT mixture was aspirated, and DMSO was added at 130 μL per well using a multipipe. The plates were shaken horizontally for 10 minutes to allow the formazan to dissolve completely. The absorbance was measured at 490 nm using a microplate reader. The cell viability at each drug concentration was calculated as required, and a scatter plot was plotted.

[0059] The specific compositions of the combinations of 2-methylthiophenylboronic acid compounds and ruthenium(III) salts are shown in Table 1. The molar ratios in the table represent the molar ratios of the corresponding 2-methylthiophenylboronic acid compounds to trans-[tetrachlorobis(1H-indazole)ruthenium(III) salt]:

[0060] Table 1

[0061]

[0062] The specific compositions of the combinations of 2-aminophenol compounds and ruthenium(III) salts are shown in Table 2. The molar ratios in the table represent the molar ratios of the corresponding 2-aminophenol compounds to trans-[tetrachlorobis(1H-indazole)ruthenium(III) salt]:

[0063] Table 2

[0064]

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

[0066] Table 3

[0067]

[0068]

[0069] Example 2

[0070] This embodiment 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] After 24 hours of cell seeding, a certain amount of 2-methylthiophenylboronic acid was added to each well of a 96-well plate to achieve a concentration of 200 μM per well. Then, BOLD-100 was added and serially diluted to treat the cells. After 48 hours, 20 μL of MTT (5 mg / mL in PBS) was added to each well of the 96-well plate using a multipipe. The plates were incubated at 37°C for 4 hours. After the incubation period, the MTT mixture was removed, and DMSO was added at 130 μL per well using a multipipe. The plates were shaken horizontally for 10 minutes to allow the formazan to dissolve completely. The absorbance was measured at 490 nm using a microplate reader. The cell viability at each drug concentration was calculated as required, and a scatter plot was plotted. 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] As shown in Table 4, under normal physiological conditions (adding FBS to the empty culture medium can simulate the protein environment under physiological conditions), BOLD-100 showed no significant inhibitory effect on human colon cancer cells, human liver cancer cells, human lung cancer cells, human cervical cancer cells, human breast cancer cells, human astrocytoma cells, human melanoma cells, leukemia, and pancreatic cancer. However, when a non-toxic dose of 2-methylthiophenylboronic acid was added, BOLD-100 showed excellent inhibitory effects on all nine types of cancer cells. 2-methylthiophenylboronic acid derivatives and 2-aminophenol and its derivatives also showed similar effects.

[0076] Example 3

[0077] This embodiment studies the mixing ratio range of BOLD-100 and 2-methylthiophenylboronic acid in combination. The specific process is as follows:

[0078] With the BOLD-100 concentration fixed at 100 μM, the concentration of 2-methylthiophenylboronic acid was varied according to the ratios in Table 5. Following the experimental method of Example 2, the IC50 of the combined use of BOLD-100 and different molar concentrations of 2-methylthiophenylboronic acid on the colon cancer cell line (HCT116) was tested. 50 .

[0079] Table 5

[0080]

[0081] The results in Table 5 show that 2-methylthiophenylboronic acid has a synergistic effect with BOLD-100. The synergistic effect is more pronounced when the concentration (ratio) of 2-methylthiophenylboronic acid increases. The strongest synergistic effect is achieved when the ratio of 2-methylthiophenylboronic acid to BOLD-100 is greater than or equal to 2. 2-methylthiophenylboronic acid derivatives, as well as 2-aminophenol and its derivatives, also exhibit similar effects.

[0082] Example 4

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

[0084] HCT116 cells were used as the experimental subjects.

[0085] Cells were loaded at 2×10 5 Inoculate one cell per well in a 6-well plate and incubate for 24 hours.

[0086] After incubation, the cell culture medium was replaced with 30% FBS to simulate a high-concentration protein environment under normal physiological conditions. A control group was used without any reagents, while the experimental group was incubated with 10 μM BOLD-100 and 200 μM 2-methylthiophenylboronic acid for 6 h (final DMF concentration ≤0.2%). Immediately after incubation, the cells were washed three times with PBS, and 500 μL of ultrapure water was added to each well to lyse the cells. Cell lysis was collected after 15 min.

[0087] The collected cell lysates were dissolved in aqua regia and then diluted with ultrapure water to an appropriate ratio. Ruthenium content was detected using inductively coupled plasma mass spectrometry (ICP-MS). Cell uptake rates without BOLD-100 were used as a control. The results are shown in Table 6.

[0088] Table 6. Effect of 2-methylthiophenylboronic acid on the uptake rate of BOLD-100 cells.

[0089]

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

[0091] As shown in Table 6, ruthenium absorption decreases in culture media with high FBS concentrations; however, the absorption of BOLD-100 is greatly improved when 2-methylthiophenylboronic acid is added.

[0092] Example 5

[0093] This embodiment 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] Establishing a mouse tumor model: Two million HCT116 colon cancer cells suspended in PBS were subcutaneously injected into the dorsal side of 5-7 week old female BALB / c-nu / nu (nude mice) to establish a xenograft model. When the tumor volume reached approximately 50 mm², the xenograft model was established. 3 (3-4 days after tumor inoculation) Mice were randomly divided into a control group (using castor oil) and a treatment group (BOLD-100 group, 2-methylthiophenylboronic acid group, and combined treatment group). The combined treatment 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) via intraperitoneal injection for 5 consecutive days in one week. When the tumor volume reached 500 mm, the treatment was initiated. 3 Afterwards, the mice were anesthetized and then euthanized by dislocating their cervical vertebrae.

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

[0096] It can be seen that tumor growth was not inhibited in the control group, the BOLD-100 monotherapy group, or the 2-methylthiophenylboronic acid monotherapy group; however, 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 kinetics curves of BOLD-100 itself and when mixed with 2-methylthiophenylboronic acid or 2-aminophenol in PBS. The detection steps are as follows:

[0099] BOLD-100 was dissolved in PBS to prepare a 400 μM stock solution. Three aliquots (2 ml each) were then prepared. The first aliquot served as a self-control; the second aliquot was further prepared to contain 40 μM 2-methylthiophenylboronic acid; and the third aliquot was further prepared to contain 200 μM 2-aminophenol. The UV-Vis absorption spectra of these three solutions were measured from 0 to 8 h. Kinetic absorption curves were plotted at 580 nm for the first two solutions.

[0100] The results are as follows Figures 5-8 As shown, UV-VIS revealed that 2-methylthiophenylboronic acid, 2-aminophenol, and their derivatives can effectively inhibit the formation of polynuclear ruthenium aggregates (with absorption peaks around 600 nm) and enhance 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 react quickly with BOLD-100, and the new absorption peaks formed indicate the formation of other compounds.

[0102] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A composition of ruthenium(III) salt, characterized in that: Including trans-[tetrachlorobis(1H-indazole)ruthenium(III)ate] and at least one selected from 2-methylthiophenylboronic acid and / or its derivatives, 2-aminophenol and / or its derivatives; The structural formula of the trans-[tetrachlorobis(1H-indazole)ruthenium(III)ate] is shown in Formula I: Wherein, R is selected from Na + , ; The structural formula of the 2-methylthiophenylboronic acid and / or its derivatives is shown in Formula II or a pharmaceutically acceptable salt of Formula II: ， R1 and R2 are independently selected from H, C1~C6 alkyl, and C1~C6 hydroxyl, respectively, or R1 and R2 form a 5- to 8-membered heterocyclic alkyl group with the atoms attached to them; R3 is selected from C1-C6 alkyl groups, or S and the benzene ring attached thereto, R3 are connected to form 6-10 arylbenzothiophene or 6-10 arylbenzothioran; the alkyl group is optionally unsubstituted or substituted with halogen; R4 is selected from H, halogen, amino, C1-C6 alkyl; the alkyl group is optionally unsubstituted or substituted with halogen; m is selected from natural numbers from 1 to 4; The structural formula of the 2-aminophenol and / or its derivatives is shown in Formula III: R5 is selected from H, halogens, and C1-C6 alkyl groups.

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

1.

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

1.

4. The composition of ruthenium(III) salt according to claim 1, characterized in that: The 2-methylthiophenylboronic acid and / or its derivatives are selected from... , , , , , , , , , , , , , , , , , , .

5. The composition of ruthenium(III) salt according to claim 1, characterized in that: The composition of the ruthenium(III) salt also includes pharmaceutically acceptable excipients.

6. The use of the ruthenium(III) salt composition described above in the preparation of an antitumor drug; wherein 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 leukemia.

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