A cyclic metal monocarbene platinum complex, its preparation method and application

Abstract

This invention discloses a cyclic metal monocarbene platinum complex, its preparation method, and its application. The cyclic metal monocarbene platinum complex is a compound of formula I or a pharmaceutically acceptable salt thereof. The cyclic metal monocarbene platinum complex of this invention can be rapidly taken up by tumor cells, and the complex has enhanced cytotoxicity to tumor cells, can better damage tumor cell DNA and induce ferroptosis in tumor cells, and can cause tumor cells to release a large number of extracellular vesicles during the process of inducing ferroptosis.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, and in particular to a cyclic metal monocarbene platinum complex, its preparation method, and its applications. Background Technology

[0002] Extracellular vesicles are membranous structures produced by cells through exocytosis, budding, and other processes. Their surface and interior often contain specific proteins, nucleic acids, lipids, and other biomolecules, serving as important intercellular communication mediators. Compared to normal cells, tumor cells exhibit a more vigorous extracellular vesicle secretion capacity, and tumor-derived extracellular vesicles play a crucial role in tumorigenesis and development. On one hand, tumor-derived extracellular vesicles possess antigen-presenting and information-transferring functions, making them potential new drug targets for intercellular communication intervention. On the other hand, the structural characteristics of extracellular vesicles make them well-suited as novel drug carriers for targeted drug delivery research. Therefore, specifically inducing the production of tumor-derived extracellular vesicles is of great significance for both novel drug development and drug carrier preparation.

[0003] Platinum-based drugs, represented by cisplatin, are widely used metallochemical chemotherapeutic agents in clinical practice. DNA is an important molecular target, leading to the development of various novel platinum complexes with good antitumor activity. When platinum complexes act on DNA, the ligands linked to the platinum center undergo hydrolysis. The unsaturated platinum then cross-links with guanine bases on the DNA, resulting in DNA damage. Studies have found that when tumor cells experience DNA damage stress, it promotes the production of tumor-derived extracellular vesicles, and the composition of vesicle contents changes depending on the damage pathway. Currently, no platinum complexes have been found that can induce the secretion of tumor-derived extracellular vesicles; furthermore, different complexes exhibit significant differences in their antitumor mechanisms and ability to induce the release of extracellular vesicles in tumor cells. 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 cyclic metal monocarbene platinum complex, its preparation method, and its applications.

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

[0006] A first aspect of the invention provides a compound of formula I or a pharmaceutically acceptable salt thereof:

[0007] ;

[0008] Wherein, X is selected from halogens;

[0009] R1 and R2 are independently selected from halogens and C1-C6 alkyl groups, respectively;

[0010] R3 is selected from C2-C10 alkyl groups.

[0011] In this invention, the compound of formula I, as a cyclic metalloid monocarbene platinum complex, can induce tumor ferroptosis, wherein the modification of the R3 alkyl chain can regulate the formation and excretion of cellular vesicles. Specifically, after tumor DNA damage, the number of intracellular micronuclei increases, and after gradual fusion with endosomes, multivesicles containing double-stranded DNA fragments are generated and released extracellularly to transmit tumor-specific molecular signals to homologous cells or different cell types; or the micronuclei encapsulating double-stranded DNA rupture in the cytoplasm, causing activation of the DNA sensor cGAS-STING pathway, promoting the production and release of extracellular vesicles. If double-stranded DNA is delivered to dendritic cells (DCs) via extracellular vesicles, it can also induce DC activation and the release of type I interferon (IFN I); if delivered to homologous cells, it can cause a death cascade reaction, which can not only effectively kill the core cells in the tumor parenchyma that are difficult for drugs to directly infiltrate, but also expand the inflammation and immune response through a "domino effect," promoting chemoimmunotherapy.

[0012] In some embodiments of the present invention, X is selected from F, Cl, Br, and I.

[0013] In some embodiments of the present invention, R1 and R2 are independently selected from F, Cl, Br, I, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, and hexyl, respectively.

[0014] In some embodiments of the present invention, R3 is selected from C2-C10 straight-chain alkyl groups.

[0015] In some embodiments of the present invention, R3 is selected from ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl.

[0016] In some embodiments of the present invention, the compound is selected from the following compounds:

[0017] , , , , , .

[0018] A second aspect of the present invention provides a method for preparing the aforementioned compound, comprising the following steps:

[0019] S1: A platinum precursor is prepared by coordinating a tetrahaloplatinate with a compound of formula II.

[0020] S2: The platinum precursor is reacted with the compound of formula III to obtain the compound described above;

[0021] , ;

[0022] The definitions of R1, R2, and R3 are as described above.

[0023] In some embodiments of the present invention, the tetrahaloplatinate includes at least one of potassium tetrafluoroplatinate, potassium tetrachloroplatinate, sodium tetrachloroplatinate, ammonium tetrachloroplatinate, potassium tetrabromoplatinate, potassium tetraiodoplatinate, magnesium tetrachloroplatinate, and tetrachloroplatinate tetrabutylammonium.

[0024] In some embodiments of the present invention, in S1, the molar ratio of the tetrahaloplatinate to the compound of formula II is 1:(1.5-3); such as 1:(1.7-2.5), 1:2.1, etc.

[0025] In some embodiments of the present invention, in S2, the molar ratio of the platinum precursor to the compound of formula III is 1:(1.5-3); such as 1:(1.7-2.5), 1:2.1, etc.

[0026] In some embodiments of the present invention, in S2, the reaction is carried out under an inert atmosphere and a strong base; the molar ratio of the platinum precursor to the strong base is 1:(3-10), such as 1:(4-6); the strong base includes at least one of sodium hydroxide, potassium hydroxide, and triethylamine; the inert atmosphere includes a nitrogen atmosphere or an argon atmosphere.

[0027] In some embodiments of the present invention, in S1, the reaction temperature of the coordination reaction is 90-130°C, such as 100-120°C; the reaction time of the coordination reaction is 12-48h, such as 20-30h or 22-26h; the reaction solvent of the coordination reaction includes water and / or ethylene glycol ethyl ether; such as a mixed solvent of ethylene glycol ethyl ether and water; or a mixed solvent of ethylene glycol ethyl ether and water in a volume ratio of (2-4):1 (such as 3:1).

[0028] In some embodiments of the present invention, in S2, the reaction temperature is 70-100℃, such as 80-100℃, 90℃, etc.; the reaction time is 12-48h, such as 20-30h, 22-26h.

[0029] In some embodiments of the present invention, in S2, the reaction solvent of the reaction includes dichloromethane and / or methanol.

[0030] In some embodiments of the present invention, the preparation method of the compound of formula III includes the following steps: reacting the compound of formula IV with the compound of formula V to obtain the compound of formula III;

[0031] , Wherein, X and R3 are defined as described above; the reaction temperature is 140-180℃, such as 150-170℃; the reaction time is 24-60h, such as 35-55h.

[0032] A third aspect of the invention provides a pharmaceutical composition comprising the said compound; and optionally, a pharmaceutically acceptable carrier, diluent, and / or adjuvant.

[0033] Pharmaceutically acceptable carriers can be liquids or solids and can be selected based on the planned route of administration to provide the desired volume, consistency, and other relevant transport and chemical properties when combined with one or more therapeutic compounds. Typical pharmaceutically acceptable carriers include, but are not limited to: water, saline solutions, binders (e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose), fillers (e.g., lactose and other sugars, gelatin, or calcium sulfate), lubricants (e.g., starch, polyethylene glycol, or sodium acetate), disintegrants (e.g., starch or sodium glycolate), and wetting agents (e.g., sodium dodecyl sulfate). Pharmaceutically acceptable carriers also include aqueous pH buffer solutions or liposomes (small vesicles composed of various types of lipids, phospholipids, and / or surfactants that can be used to deliver drugs to mammals). Other examples of pharmaceutically acceptable carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™ polyethylene glycol (PEG) and PLURONICS™.

[0034] Pharmaceutical compositions include, but are not limited to, solutions, emulsions, aqueous suspensions, and liposome-containing formulations. These compositions can be generated from a variety of components, including, for example, pre-formulated liquids, self-emulsifying solids, and self-emulsifying semi-solids. Emulsions are typically biphase systems, consisting of two immiscible liquid phases tightly mixed and dispersed between each other; generally, emulsions are of the water-in-oil (w / o) or oil-in-water (o / w) type. Emulsion formulations are widely used for the oral delivery of therapeutic agents due to their ease of formulation and solubilization, absorption, and bioavailability.

[0035] In some embodiments of the present invention, the pharmaceutical composition comprises the compound and liposomes encapsulating the compound.

[0036] The pharmaceutical composition also comprises a chemotherapeutic agent. In some embodiments of the invention, the chemotherapeutic agent is selected from one or more of alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, mTor inhibitors, or other chemotherapeutic agents, or pharmaceutically acceptable salts thereof.

[0037] In some embodiments of the present invention, the antitumor antibiotic is selected from doxorubicin, mitoxantrone, bleomycin, daunorubicin, cytomegalocin, epirubicin, idarubicin, procainoxicin, mitomycin, pentostatin, and pentorubicin, or pharmaceutically acceptable salts thereof.

[0038] In some embodiments of the present invention, the antimetabolite is selected from gemcitabine, 5-fluorouracil, capecitabine, hydroxyurea, mercaptopurine, pemetrexed, fludarabine, nerabine, cladribine, clofarabine, cytarabine, decitabine, pralatrexate, fluorouridine, methotrexate, and thioguanine, or pharmaceutically acceptable salts thereof.

[0039] In some embodiments of the present invention, the alkylating agent is selected from carboplatin, cisplatin, cyclophosphamide, chlorambucil, melphalan, carmustine, busulfan, lomustine, dacarbazine, oxaliplatin, ifosfamide, nitrogen mustard, temozolomide, thiotepa, bendamustine, strepzoline, or pharmaceutically acceptable salts, hydrates, solvates, or polymorphs thereof.

[0040] In some embodiments of the present invention, the mitotic inhibitor is selected from one or more of irinotecan, topotecan, rubitecan, cabazitaxel, docetaxel, paclitaxel, etoposide, vincristine, ixaprilone, vinorelbine, vinblastine, teniposide, or pharmaceutically acceptable salts thereof.

[0041] In some embodiments of the present invention, the mTor inhibitor is selected from one or more of everolimus, sirolimus, tesimolimus, or pharmaceutically acceptable salts thereof.

[0042] A fourth aspect of the invention provides the use of the said compound, or the said pharmaceutical composition, in the preparation of at least one molecular probe, tumor imaging agent, or antitumor drug.

[0043] In some embodiments of the present invention, the tumor includes at least one of breast cancer, cervical cancer, or lung cancer.

[0044] The beneficial effects of this invention are:

[0045] The cyclic metal monocarbene platinum complex of the present invention can be rapidly taken up by tumor cells, and the complex has enhanced cytotoxicity to tumor cells, can better damage tumor cell DNA and induce tumor cell ferroptosis, and can cause tumor cells to release a large number of extracellular vesicles during the process of inducing ferroptosis.

[0046] The preparation method of the cyclic metal monocarbene platinum complex of the present invention is simple, easy to implement, and low in cost; therefore, the complex prepared by the present invention has good application prospects and broad development space in the preparation and acquisition of antitumor drugs and extracellular vesicles. Attached Figure Description

[0047] Figure 1 It is the ultraviolet spectrum of the response of the complex Pt1 to proteins, lipids, dsDNA, and G4 DNA in buffer solution.

[0048] Figure 2 It shows the uptake and localization of the complex Pt1 by tumor cells.

[0049] Figure 3 This is a confocal image of DNA damage in tumor cells caused by the complex Pt1; where "γ-H2A.X" represents the excitation of the fluorescent secondary antibody labeled with γ-H2A.X; "BF" represents bright field excitation; "Merge" represents merging; in the figure, a is the bright field excitation channel image, b is the fluorescent secondary antibody excitation channel image, and c is the image after overlaying a and b.

[0050] Figure 4 This is a Western blot image and its quantitative results after the complex Pt1 was applied to tumor cells.

[0051] Figure 5 This is a confocal image of tumor cells generating micronuclei induced by the complex Pt1; where "Lamin A / C" represents the excitation of the fluorescent secondary antibody labeled with nuclear membrane protein Lamin A / C, "DAPI" represents the excitation of DAPI; "Overlay" represents superposition; in the figure, a is a photo of the excitation channel of the secondary antibody, b is a photo of the excitation channel of DAPI, and c is a photo of the superposition of a and b.

[0052] Figure 6 This is a transmission electron microscope image of multivesicular bodies induced by the complex Pt1 in tumor cells. In the figure, a is a transmission electron microscope image at a lower magnification (scale bar is 2.0 μm), and b is a magnified image of the area within the red box in figure a, where the multivesicular bodies are marked by red arrows.

[0053] Figure 7 These are images of multivesicular bodies within HeLa cells obtained by transmission electron microscopy and extracellular vesicles obtained by differential centrifugation. Detailed Implementation

[0054] 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.

[0055] The term "pharmaceutically acceptable salt" includes, but is not limited to, inorganic or organic acid salts with basic groups such as amines; and alkali metal or organic salts with acidic groups such as carboxylic acids. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of parent compounds, for example, formed from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, aminosulfonic acid, phosphoric acid, and nitric acid; and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, dihydroxynaphthyl acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, and hydroxyethanesulfonic acid, etc. The pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing basic or acidic moieties using conventional chemical methods. Typically, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture thereof; typically, a non-aqueous medium, such as diethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile, is preferred.

[0056] Example 1

[0057] This embodiment prepares a cyclic metal monocarbene platinum complex, the specific process of which is as follows:

[0058]

[0059] Butylimidazole (0.6 g, 5 mmol) and 2-chloropyridine (1.0 g, 5 mmol) were mixed in a high-temperature pressure-resistant flask and heated to 160 °C for 48 h. After the reaction was completed, the system was cooled to room temperature, and the product was washed several times with a chloroform / ether system and then dried to obtain a black viscous liquid product, thus preparing the ligand 2-(imidazolium-1-butylene)-pyridine.

[0060] ESI-MS: Theoretical value: m / z = 202.28 [M-Cl] + Experimental value: m / z = 202.33 [M-Cl] +

[0061] S1: Potassium tetrachloroplatinate and 2-(2,4-difluorophenyl)-pyridine were mixed at a molar ratio of 1:2.1 and dissolved in a mixed solvent of ethylene glycol ethyl ether and ultrapure water (volume ratio of 3:1). The mixture was subjected to a coordination reaction at 110 °C for 24 h under reflux and stirring. After cooling to room temperature and filtration, the resulting solid product was washed three times with ultrapure water and ether, respectively, and dried under vacuum to obtain the platinum precursor.

[0062] S2: The platinum precursor (380 mg, 0.5 mmol) obtained in step S1 was mixed with 2-(imidazolium-1-butylene)-pyridine (248 mg, 1.05 mmol), dissolved in dichloromethane, and triethylamine (252 mg, 2.5 mmol) was added under N2 protection. The reaction was carried out at 90 °C for 25 h. After the reaction was completed, the reaction solvent was removed by rotary evaporation, and the solid was precipitated. The solid was filtered and washed three times with ultrapure water and diethyl ether, respectively. The solid was further purified by column chromatography (eluent: dichloromethane:methanol = 10:1, volume ratio), and dried under vacuum to obtain the cyclic metal monocarbene platinum complex Pt1.

[0063] In step S2, the molar ratio of platinum precursor, 2-(2,4-difluorophenyl)pyridine to triethylamine is 1:2.1:5.

[0064] The structural formula of the complex Pt1 is: The characterization data are as follows:

[0065] 1H NMR (400 MHz, DMSO-d6) δ 9.54 – 9.48 (m, 1H), 9.03 (d, J = 8.2 Hz,1H), 8.52 (dd, J = 4.9, 2.0 Hz, 1H), 8.19 – 8.11 (m, 1H), 8.06 (dd, J = 5.3,3.1 Hz, 2H), 7.93 (td, J = 7.8, 1.9 Hz, 1H), 7.79 (d, J = 2.2 Hz, 1H), 7.59(t, J = 6.2 Hz, 1H), 7.39 (dd, J = 7.5, 5.0 Hz, 1H), 6.85 (ddd, J = 12.2,9.4, 2.4 Hz, 1H), 5.71 (dd, J = 9.0, 2.4 Hz, 1H), 4.48 (dt, J = 14.1, 7.2 Hz,1H), 4.32 (dt, J = 13.5, 7.1 Hz, 1H), 2.02 – 1.79 (m, 2H), 1.26 (qd, J =21.3, 20.8, 9.3 Hz, 2H), 0.81 (t, J = 7.4 Hz, 3H).

[0066] ESI-MS: Theoretical value: m / z = 586.53 [M-Cl] + Experimental value: m / z = 586.66 [M-Cl] + .

[0067] Example 2

[0068] This embodiment prepares a cyclic metal monocarbene platinum complex, the specific process of which is as follows:

[0069] The ligand 2-(imidazol-1-octyl)-pyridine was prepared according to the preparation method in Example 1.

[0070] S1: Potassium tetrachloroplatinate and 2-(2,4-difluorophenyl)-pyridine were mixed at a molar ratio of 1:2.1 and dissolved in a mixed solvent of ethylene glycol ethyl ether and ultrapure water (volume ratio of 3:1). The mixture was subjected to a coordination reaction at 110 °C for 24 h under reflux and stirring. After cooling to room temperature and filtration, the resulting solid product was washed three times with ultrapure water and ether, respectively, and dried under vacuum to obtain the platinum precursor.

[0071] S2: The platinum precursor (380 mg, 0.5 mmol) obtained in step S1 was mixed with 2-(imidazol-1-octyl)-pyridine (307 mg, 1.05 mmol), dissolved in dichloromethane, and triethylamine (252 mg, 2.5 mmol) was added under N2 protection. The reaction was carried out at 90 °C for 25 h. After the reaction was completed, the reaction solvent was removed by rotary evaporation, and the solid was precipitated. The solid was filtered and washed three times with ultrapure water and diethyl ether, respectively. The solid was further purified by column chromatography (eluent: dichloromethane:methanol = 10:1, volume ratio), and dried under vacuum to obtain the cyclic metal monocarbene platinum complex Pt2.

[0072] In step S2, the molar ratio of the platinum precursor, 2-(2,4-difluorophenyl)pyridine, and triethylamine is 1:2.1:5.

[0073] The structural formula of the complex Pt2 is: The characterization data are as follows:

[0074] 1 H NMR (400 MHz, DMSO-d6) δ 9.50 (d, J = 5.8 Hz, 1H), 9.01 (d, J =8.2 Hz, 1H), 8.55 – 8.49 (m, 1H), 8.15 (t, J = 7.8 Hz, 1H), 8.06 (d, J = 7.4Hz, 1H), 7.79 (d, J = 2.2 Hz, 1H), 7.59 (t, J = 6.6 Hz, 1H), 7.48 (d, J = 7.8Hz, 1H), 7.39 (dd, J = 7.5, 4.9 Hz, 1H), 7.27 (t, J = 6.9 Hz, 1H), 6.85 (ddd,J = 12.4, 9.4, 2.4 Hz, 1H), 5.94 – 5.38 (m, 1H), 4.22 (p, J = 6.8 Hz, 2H), 2.10 – 1.72 (m, 2H), 1.34 – 0.88 (m, 10H), 0.75 (t, J = 7.0 Hz, 3H).

[0075] ESI-MS: Theoretical value: m / z = 642.64 [M-Cl] + Experimental value: m / z = 642.78 [M-Cl] + .

[0076] Example 3

[0077] In this embodiment, the cyclic metal monocarbene platinum complex Pt1 is used to bind to DNA. The specific process is as follows:

[0078] Using the cyclic metal monocarbene platinum complex Pt1 from Example 1, a 1 μM solution of the cyclic metal monocarbene platinum complex Pt1 was prepared at room temperature using 1M Tris-HCl buffer at pH = 7.4. Protein (BSA), lipid (cholesterol), and DNA (double-stranded DNA and G4 DNA) were added to the platinum complex solution, and the UV-Vis absorption spectra of the platinum complex Pt1 at different concentrations of titrant were measured and plotted.

[0079] The binding behavior of complex Pt1 to various biomolecules, including proteins, lipids, and DNA, is as follows: Figure 1 As shown, although the absorption peaks of DNA before 300 nm increased significantly with the addition of DNA, the absorption peaks of the complex in the 325-400 nm range showed a significant decreasing trend, especially after the addition of G4 DNA.

[0080] The results show that the cyclic metal monocarbene platinum complex prepared in this invention has good binding ability to a variety of biomacromolecules, including G4 DNA.

[0081] Example 4

[0082] This embodiment uses the cyclic metal monocarbene platinum complex Pt1 for antitumor activity. The specific process is as follows:

[0083] Using the cyclic metal monocarbene platinum complex Pt1 from Example 1 as the experimental group and cisplatin as the control group, its cytotoxicity against tested tumor cells (MDA-MB-231 (human breast cancer cell line) and HeLa (human cervical cancer cell line) was determined. The specific determination methods are as follows:

[0084] The MTT assay was used to determine the concentration of tumor cells. The tested tumor cells were digested with trypsin into single-cell suspensions, and cell counts were performed using a hemocytometer. The cell concentration was adjusted to 5 × 10⁻⁶ cells / mL. 4 Cells were seeded at 160 μL / well in 96-well plates and cultured for 24 h. Then, different concentrations of the drug (cyclic monocarbene platinum complex or cisplatin) were added, and the plates were incubated at 37 °C for 48 h under normal oxygen conditions (cells were cultured in an incubator containing 5% CO2). Four h before the end of incubation, 20 μL of MTT was added per well. After 4 h, the supernatant was discarded, and 150 μL of DMSO was added per well. After shaking for 5 minutes, the OD value was measured using a microplate reader at a wavelength of 595 nm.

[0085] The cell viability rate is calculated using the following formula: (average optical density of treated cells / average optical density of control cells) × 100%. The viability rate of the tested tumor cells is then plotted and the IC50 is determined. 50 The antitumor activity of cyclic platinum monocarbene complexes was evaluated by using the value of [value missing].

[0086] Example 1: The IC50 of cyclic metal monocarbene platinum complex Pt1 on tumor cells 50 As shown in Table 1, it can be seen that the complex Pt1 has good antitumor activity and its toxicity to tumor cells is slightly higher than that of cisplatin.

[0087] The results show that the cyclic metal monocarbene platinum complex Pt1 prepared in this invention has good antitumor activity.

[0088] Table 1. IC50 of the cyclic metal monocarbene platinum complex Pt1 against tumor cells in Example 1. 50 value

[0089]

[0090] Example 5

[0091] This embodiment tests the uptake and localization distribution of the cyclic metal monocarbene platinum complex Pt1 by tumor cells. The specific process is as follows:

[0092] Prepare a clean, sterile 15 mm Corning culture dish. Digest HeLa (human cervical cancer cell line) in good growth condition with trypsin and passage it in the dish. Incubate at 37 °C under normoxic conditions (cells are cultured in an incubator containing 5% CO2). When the HeLa cell density reaches 70%, add the cyclic metalloid monocarbene platinum complex Pt1 prepared in this invention to a final concentration of 10 μM. Continue culturing for 12 h, then remove the culture medium, wash twice with PBS, count the number of drug-treated cells using a cell counter, and extract organelles such as nuclei, mitochondria, endoplasmic reticulum, and Golgi apparatus. Nitrify the cells overnight with 0.5 mL of concentrated nitric acid, then dilute with ultrapure water to 10 mL, add 1% indium internal standard, and immediately analyze by ICP-MS. A standard working curve was prepared using standard solutions with platinum contents of 0, 1 ppb, 2 ppb, 5 ppb, 10 ppb, 20 ppb, 50 ppb, and 100 ppb. The platinum content in the sample was obtained by comparing the solution with the standard working curve.

[0093] Tumor cell uptake of the cyclic metal monocarbene platinum complex Pt1 is as follows: Figure 2As shown, the complex Pt1 is mainly enriched in the nucleus of tumor cells, indicating that the complex can be well taken up by tumor cells and is significantly enriched in the nucleus.

[0094] Results show that the cyclic metal monocarbene platinum complex prepared in this invention can be taken up by tumor cells and enriched in the cell nucleus.

[0095] Example 6

[0096] This embodiment uses the cyclic metal monocarbene platinum complex Pt1 for antitumor activity. The specific process is as follows:

[0097] This embodiment explains the mechanism of the antitumor effect of the cyclic metal monocarbene platinum complex Pt1 prepared in this invention from multiple aspects. Immunofluorescence and Western blot experiments were used to determine the complex's ability to induce DNA damage and micronucleus generation in HeLa cells, and Western blot experiments were used to determine the complex's ability to induce ferroptosis in HeLa cells. Specific experimental methods and results are as follows:

[0098] 1. Ability to induce DNA damage in HeLa cells

[0099] (1) Experimental methods

[0100] HeLa cells in good growth condition were digested with trypsin, diluted into a single-cell suspension in DMEM medium, and seeded in confocal dishes. After cell attachment, 10 μM of complex Pt1 was added and incubated for 12 h, with cells not treated with the complex serving as a control group. The medium was removed, and cells were fixed with 4% paraformaldehyde, then permeabilized with 0.2% Trinton X-100 for 15 min, followed by washing with PBS; blocking buffer was applied for 30 min; the blocking buffer was removed, and cells were washed with washing buffer, then incubated overnight at 4 °C with primary antibody (mouse anti-γ-H2A.X, Mouse IgG1, 80312, CST). After removing the primary antibody, cells were washed three times with washing buffer, and then incubated with FITC-labeled secondary antibody (goat anti-mouse IgG H&L, Fluorescein(FITC)-conjugated Goat Anti-Mouse IgG(H+L), SA00003-1, Proteintech) at room temperature in the dark for 1 h. Rinse with PBS and then observe under a laser confocal microscope.

[0101] (2) Experimental results

[0102] like Figure 3As shown in the confocal image, compared with the blank control group without drug treatment, the fluorescence intensity of γ-H2A.X in HeLa cells treated with the cyclic metal monocarbene platinum complex Pt1 was significantly enhanced, indicating that DNA damage in HeLa cells increased, suggesting that the complex has the ability to damage the DNA of HeLa cells.

[0103] Results show that the cyclic metal monocarbene platinum complex prepared in this invention causes DNA damage to tumor cells.

[0104] 2. Ability to induce ferroptosis in HeLa cells

[0105] (1) Experimental methods

[0106] HeLa cells were seeded in Corning 60 mm culture dishes and cultured to approximately 70% confluence. The cells were then treated with a cyclic monocarbene platinum complex (Pt1) for 12 h. Cells were collected using a cell scraper, centrifuged, washed twice with pre-chilled PBS, and lysed on ice for 30 min using RIPA cell lysis buffer. The supernatant was collected by centrifugation. The total protein concentration was determined using a BCA assay kit. All proteins were then homogenized using lysis buffer, heated at 100 °C for 10 min to fully denature the proteins, and then subjected to SDS-PAGE gel electrophoresis. A rabbit anti-β-Actin antibody (45 kDa) was used as the reference control.

[0107] (2) Experimental results

[0108] like Figure 4 As shown in the Western blot, compared with the untreated control group, the band corresponding to γ-H2A.X was upregulated and GPX4 was downregulated after treatment with the platinum complex Pt1, indicating that the platinum complex induced DNA damage and ferroptosis.

[0109] The results show that the cyclic metal monocarbene platinum complex prepared in this invention not only induced DNA damage in tumor cells, but also caused ferroptosis in tumor cells.

[0110] 3. Generation of micronuclei induced by cyclic metal monocarbene platinum complexes

[0111] (1) Experimental methods

[0112] HeLa cells in good growth condition were digested with trypsin, diluted into a single-cell suspension in DMEM medium, and seeded in confocal dishes. After cell attachment, 10 μM of complex Pt1 was added and incubated for 12 h, with cells not treated with the complex serving as a control group. The medium was removed, and cells were fixed with 4% paraformaldehyde, then permeabilized with 0.2% Trinton X-100 for 15 min, followed by washing with PBS; blocking buffer was applied for 30 min; the blocking buffer was removed, and cells were washed with washing buffer, then incubated overnight at 4 °C with primary antibody (rabbit anti-Lamin A / C, Rabbit / IgG, 10298-1-AP, Proteintech). After removing the primary antibody, cells were washed three times with washing buffer, and Cy3-conjugated secondary antibody (goat anti-rabbit IgG H&L, Cy3-conjugated Goat Anti-Rabbit IgG(H+L), SA00009-2, Proteintech) was added and incubated at room temperature in the dark for 1 h. The cells were washed with PBS, stained with DAPI to label the nuclei, and then observed under a laser confocal microscope.

[0113] (2) Experimental results

[0114] like Figure 5 As shown in the confocal image, compared with the blank control group without drug treatment, the cytoplasm of HeLa cells treated with the cyclic metal monocarbene platinum complex Pt1 showed fluorescence of the nuclear membrane protein Lamin A / C, indicating that micronuclei were formed in HeLa cells under the induction of the platinum complex.

[0115] Results show that the cyclic metal monocarbene platinum complex prepared in this invention induces the generation of micronuclei in tumor cells.

[0116] Example 7

[0117] In this embodiment, the cyclic metal monocarbene platinum complex Pt1 is used to induce tumor cells to release extracellular vesicles. The specific process is as follows:

[0118] Prepare a clean, sterile 15 mm Corning culture dish. Digest healthy HeLa (human cervical cancer cell line) cells with trypsin and passage them into the dish. Culture at 37 °C under ambient oxygen conditions (cells cultured in an incubator containing 5% CO2). When the HeLa cell density reaches 70%, add the cyclic metalloid monocarbene platinum complex Pt1 from Example 1 to a final concentration of 10 μM. Continue culturing for 12 h. Collect the cells after trypsin digestion of the lower layer, carefully add 2.5% electron microscopy fixative, fix overnight at 4 °C, prepare samples, and image under a transmission electron microscope. Centrifuge the upper culture medium at 1000, 2500, 10000, and 100000 rcf. Wash the resulting precipitate twice with PBS and analyze the precipitate using NTA and TEM, respectively.

[0119] The cells were observed to have a distinct classical multivesicular structure in the cytoplasm under transmission electron microscopy. Figure 6 As shown in Table 2, the NTA results of extracellular vesicles obtained after centrifugation of the supernatant are shown in Table 2. Compared with the control group without drug treatment, the release of extracellular vesicles after treatment with the cyclic metal monocarbene platinum complex Pt1 was significantly increased. The results of transmission electron microscopy (TEM) are shown in Table 2. Figure 7 As shown, the obtained precipitate particles contain a membrane structure and are identified as exosome vesicles.

[0120] Table 2

[0121]

[0122] Results show that the cyclic metal monocarbene platinum complex prepared in this invention induces tumor cells to release a large number of extracellular vesicles.

[0123] 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 compound of formula I or a pharmaceutically acceptable salt thereof: ； in, X is selected from halogens; R1 and R2 are independently selected from halogens and C1-C6 alkyl groups, respectively; R3 is selected from C2-C10 alkyl groups.

2. The compound according to claim 1, characterized in that: X is selected from F, Cl, Br, and I.

3. The compound according to claim 1, characterized in that: R1 and R2 are independently selected from F, Cl, Br, I, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, and hexyl, respectively.

4. The compound according to claim 1, characterized in that: R3 is selected from ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl.

5. The compound according to claim 1, characterized in that: The compound is selected from the following compounds: 、 、 、 、 、 。 6. A method for preparing the compound according to any one of claims 1-5, characterized in that: Includes the following steps: S1: A platinum precursor is prepared by coordinating a tetrahaloplatinate with a compound of formula II. S2: The platinum precursor is reacted with the compound of formula III to obtain the compound described above; 、 ； The definitions of R1, R2, R3, and X are as described in any one of claims 1-5.

7. A pharmaceutical composition, characterized in that: Includes the compounds according to any one of claims 1-5; and optionally, pharmaceutically acceptable carriers, diluents, and / or adjuvants.

8. The pharmaceutical composition according to claim 7, characterized in that: The pharmaceutical composition further includes a chemotherapeutic agent; the chemotherapeutic agent is selected from one or more of alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, mTor inhibitors or other chemotherapeutic agents, or pharmaceutically acceptable salts thereof.

9. The use of a compound according to claims 1-5, or a pharmaceutical composition according to claim 7 or 8, in the preparation of at least one molecular probe, tumor imaging agent, or antitumor drug; wherein the tumor is selected from at least one of breast cancer, cervical cancer, or lung cancer.

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