Metal iridium complex as well as preparation method and application thereof

Abstract

The invention discloses a metal iridium complex as well as a preparation method and application thereof. The structural formula of the metal iridium complex is as shown in a formula I compound # imgabs0 #. After being wrapped by lipidosome, the liposome can be taken in by tumor cells, and has a phosphorescent tracing characteristic; the cytotoxicity of the complex to tumor cells is at least four times that of cis-platinum, tumor cell ATP and HMGB-1 exosome up-regulation can be better induced, and finally ferroptosis and necroptosis of the tumor cells are induced, so that the complex has excellent anti-tumor activity and has a good application prospect and a wide development space in preparation of anti-tumor drugs. The preparation method of the compound shown in the formula I is simple and easy to implement and low in cost.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a metal iridium complex and a preparation method and application thereof. Background Art

[0002] Immunogenic Cell Death (ICD) is a regulatory cell death (RCD) mode that can stimulate adaptive immune responses in immune-competent hosts. ICD is a special form of cell death that not only causes cell death but also stimulates the body's specific immune response to antigens released by dying cells. This mode of death is driven by stress and involves activating the immune system against dead cells in an immunocompetent host. The ICD concept has now been widely used in tumor immunity. Various common treatments - including specific chemotherapy drugs, radiotherapy, and some targeted anticancer drugs - can lead to positively activated ICD, thereby generating an immune response against the tumor to support the therapeutic effect. Therefore, the development of new metal anti-tumor drugs with different anti-tumor mechanisms is of great significance for improving the therapeutic effect of tumors.

[0003] Ferroptosis is an iron-dependent form of cell death that is different from traditional death modes such as apoptosis, necrosis, and autophagy. This form of cell death is characterized by iron-dependent lipid peroxidation, which is usually accompanied by the accumulation of lipid peroxides and the destruction of cell membrane integrity. Ferroptosis is the depletion of glutathione, the decrease in the activity of glutathione peroxidase (GPX4), and the inability of lipid oxides to be metabolized by the glutathione reductase reaction catalyzed by GPX4. Then, divalent iron ions oxidize lipids to produce reactive oxygen species, thereby promoting the occurrence of ferroptosis.

[0004] Necroptosis is a form of programmed cell death similar to necrosis. When cells fail to undergo apoptosis normally after inflammatory, oxidative or ischemic stress, necroptosis is used as a "substitute" for apoptosis. Necroptosis eliminates damaged cells without the activation of caspases. When apoptosis is blocked, it is a process of cell self-destruction activated by extracellular signals (death receptor-ligand binding) or intracellular signals (exogenous microbial nucleic acids). During necroptosis, swelling of organelles, rupture of cell membranes, and decomposition of cytoplasm and nucleus can be observed. Summary of the invention

[0005] 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 metal iridium complex and a preparation method and application thereof.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is: The first aspect of the present invention provides a compound of formula I, or a tautomer thereof, or a pharmaceutically acceptable salt thereof: .

[0007] The second aspect of the present invention provides a method for preparing the compound of formula I, comprising the following steps: S1: Coordination reaction of iridium trichloride and 2-phenylpyridine to obtain an iridium precursor; S2: reacting (2S,3R,E)-2-aminooctadec-4-ene-1,3-diol with [2,2'-bipyridine]-4-carboxylic acid to obtain sphingosine; S3: reacting an iridium precursor with sphingosine, and then adding an ammonium hexafluorophosphate solution for precipitation to obtain the compound of formula I.

[0008] In some embodiments of the present invention, the molar ratio of iridium trichloride to 2-phenylpyridine is 1:2-3, for example, 1:2-2.4, 1:2.1.

[0009] In some embodiments of the present invention, the molar ratio of (2S, 3R, E)-2-aminooctadec-4-ene-1,3-diol to [2,2'-bipyridine]-4-carboxylic acid is 1:1-2, for example, 1:1-1.6, 1:1.1.

[0010] In some embodiments of the present invention, the molar ratio of the iridium precursor to sphingosine is 1:2-3, for example, 1:2-2.4, 1:2.2.

[0011] In some embodiments of the present invention, the ammonium hexafluorophosphate solution is a supersaturated ammonium hexafluorophosphate aqueous solution.

[0012] In some embodiments of the present invention, the temperature of the coordination reaction is 100°C to 130°C, for example, 110°C; the time of the coordination reaction is 20 to 48 h, for example, 22 to 36 h, 24 h; the solvent of the coordination reaction includes at least one of dichloromethane, methanol, ethylene glycol ethyl ether, and ultrapure water, for example, a mixed solvent of dichloromethane and methanol, and a mixed solvent of ethylene glycol ethyl ether and ultrapure water; the volume ratio of dichloromethane to methanol is 2 to 3:1, for example, 2 to 2.4:1, 2:1; the volume ratio of ethylene glycol ethyl ether to ultrapure water is 2 to 4:1, for example, 3 to 4:1, 3:1.

[0013] In some embodiments of the present invention, the temperature of the reaction in S2 is 18°C ​​to 30°C, for example, 20°C; the reaction time is 20 to 48 h, for example, 22 to 36 h, 24 h; the solvent of the reaction includes DMF; the catalyst used in the reaction includes at least one of HATU; the reaction is carried out under a strong base, and the strong base includes at least one of triethylamine.

[0014] In some embodiments of the present invention, the reaction temperature in S3 is 60°C to 75°C, for example, 60°C to 70°C, 68°C; the reaction time is 12 to 36 hours, for example, 18 to 24 hours, 20 hours; the reaction is carried out under an inert atmosphere such as a nitrogen atmosphere.

[0015] The third aspect of the present invention provides a pharmaceutical composition comprising the compound of formula I, or its isomer, a pharmaceutically acceptable salt thereof, and optionally, a pharmaceutically acceptable carrier, diluent and / or adjuvant.

[0016] Pharmaceutically acceptable carriers can be liquid or solid and can be selected based on the planned mode of administration so as to provide the desired volume, consistency and other relevant transport and chemical properties when combined with one or more therapeutic compounds or any other ingredients of a given pharmaceutical composition. Typical pharmaceutically acceptable carriers include, for example, but are not limited to: water, saline solutions, binders (e.g., polyvinyl pyrrolidone 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 starch glycolate), and wetting agents (e.g., sodium lauryl sulfate). Pharmaceutically acceptable carriers also include aqueous pH buffered 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 phosphate, citrate and other organic acids; antioxidants such as ascorbic acid, low molecular weight (less than about ten residues) polypeptides; 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 dextrins, 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™.

[0017] Pharmaceutical compositions include, but are not limited to, solutions, emulsions, aqueous suspensions, and liposome-containing preparations. These compositions can be produced from a variety of components, including, for example, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. Emulsions are typically biphasic systems, consisting of two immiscible liquid phases that are closely mixed and mutually dispersed; typically, emulsions are water-in-oil (w / o) or oil-in-water (o / w) types. Emulsion formulations are widely used for oral delivery of therapeutic agents because of their ease of preparation and solubilization, absorption, and bioavailability.

[0018] In some embodiments of the present invention, the pharmaceutical composition includes the compound of formula I, or its isomer, or its pharmaceutically acceptable salt, and liposomes encapsulating the compound of formula I, or its isomer, or its pharmaceutically acceptable salt.

[0019] The pharmaceutical composition further comprises a chemotherapeutic agent. In some embodiments of the present invention, the chemotherapeutic agent is selected from one or more of an alkylating agent, an antimetabolite, an antitumor antibiotic agent, a mitotic inhibitor, an mTor inhibitor or other chemotherapeutic agent, or a pharmaceutically acceptable salt thereof.

[0020] In some embodiments of the present invention, the antitumor antibiotic is selected from doxorubicin, mitoxantrone, bleomycin, daunorubicin, dactinomycin, epirubicin, idarubicin, plicamycin, mitomycin, pentostatin and valrubicin, or a pharmaceutically acceptable salt thereof.

[0021] In some embodiments of the invention, the antimetabolite is selected from gemcitabine, 5-fluorouracil, capecitabine, hydroxyurea, mercaptopurine, pemetrexed, fludarabine, nelarabine, cladribine, clofarabine, cytarabine, decitabine, pralatrexate, floxuridine, methotrexate and thioguanine or a pharmaceutically acceptable salt thereof.

[0022] 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, streptozocin, or a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof.

[0023] 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, ixabepilone, vinorelbine, vinblastine, teniposide, or pharmaceutically acceptable salts thereof.

[0024] In some embodiments of the present invention, the mTor inhibitor is selected from one or more of everolimus, sirolimus, temsirolimus or pharmaceutically acceptable salts thereof.

[0025] The fourth aspect of the present invention provides a use of the compound of formula I, or its isomer, its pharmaceutically acceptable salt, or the pharmaceutical composition in the preparation of at least one of a molecular probe, a tumor imaging agent, and an anti-tumor drug.

[0026] In some embodiments of the invention, the tumor comprises at least one of breast cancer, cervical cancer or lung cancer.

[0027] Throughout the specification and the appended claims, a given chemical formula or name shall encompass all stereoisomers and optical isomers and racemates thereof (where such isomers exist). Unless otherwise indicated, all chiral (enantiomers and diastereomers) and racemic forms are within the scope of the present invention. Many geometric isomers of C=C double bonds, C=N double bonds, ring systems, etc. may also be present in the complex, and all such stable isomers are encompassed in the present invention. The cis and trans (or E- and Z-) geometric isomers of the compounds of the present invention are described and can be separated into mixtures of isomers or separated isomeric forms. The compounds of the present invention can be separated in optically active or racemic form. Optically active forms can be prepared by resolution of racemic forms or by synthesis from optically active starting materials. All methods for preparing the compounds of the present invention and intermediates prepared therein are considered to be part of the present invention. When preparing enantiomeric or diastereomeric products, they can be separated by conventional methods, such as by chromatography, fractional crystallization or by using chiral reagents. Depending on the process conditions, the final product of the present invention can be obtained in the form of free (neutral) or salt. The free forms and salts of these final products are within the scope of the present invention. If necessary, one form of the compound can be converted into another form. A free base or acid can be converted into a salt; a salt can be converted into a free compound or another salt; a mixture of isomeric compounds of the present invention can be separated into individual isomers. The compounds of the present invention, free forms and salts thereof can exist in a variety of tautomeric forms, in which hydrogen atoms are translocated to other parts of the molecule, and the chemical bonds between the atoms of the molecule are thus rearranged. It should be understood that all tautomeric forms, as long as they may exist, are included in the present invention. In addition, a given chemical formula or name should cover all its conformers, rotational isomers or conformational isomers, where such isomers exist. Different conformations can have different energies, can usually be mutually converted, and are rarely separable. Some molecules can be separated in multiple conformations. For example, atropisomers are isomers produced by hindered rotation around a single bond, in which the steric strain barrier of rotation is high enough to allow the separation of conformational isomers. It is to be understood that all conformers, rotational isomers or conformational isomeric forms, insofar as they may exist, are included within the scope of the present invention.

[0028] The term "pharmaceutically acceptable salt" includes, but is not limited to, inorganic or organic acid salts of basic groups such as amines; alkali metal or organic salts of acidic groups such as carboxylic acids. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound, 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, pamoic 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 isethionic acid, etc. The pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing basic or acidic moieties by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media such as diethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.

[0029] The beneficial effects of the present invention are: The compound of formula I of the present invention is a cyclometallated iridium sphingosine complex, which can be taken up by tumor cells after being encapsulated by liposomes and has phosphorescent tracing characteristics; the cytotoxicity of the complex to tumor cells is at least 6 times that of cisplatin, and it can better induce up-regulation of ATP and HMGB-1 exocytosis of tumor cells, and finally induce ferroptosis and necroptosis of tumor cells, indicating that the complex has excellent anti-tumor activity and has good application prospects and broad development space in the preparation of anti-tumor drugs.

[0030] The preparation method of the compound of formula I of the present invention is simple, easy and has low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is the uptake of the liposome-encapsulated cyclometallated iridium sphingosine complex by tumor cells of the present invention; wherein, "Brightfield" represents bright field, "LTDR" represents lysosome deep red probe, and "Overlay" represents overlay; wherein, from left to right are respectively the bright field photo, the photo of the complex 405nm excitation channel, the photo of the lysosome deep red probe, and the overlay photo.

[0032] Figure 2This is the situation of HMGB-1 exocytosis induced by the liposome-encapsulated complex Ir1 of the present invention; wherein "Brightfield" represents bright field, "DAPI" represents cell nucleus, "HMGB-1" represents HMGB-1 protein, and "Overlay" represents overlay; wherein, from left to right, they are respectively bright field photo, cell nucleus photo, HMGB-1 protein photo, and overlay photo.

[0033] Figure 3 This is a diagram showing the ATP excretion of tumor cells by the complex Ir1 encapsulated by the liposome of the present invention.

[0034] Figure 4 The morphological diagram of tumor cells observed by transmission electron microscopy of the complex Ir1 encapsulated by the liposome of the present invention; wherein, Figure a is a photo of the blank control group, Figure b is a photo of the morphological characteristics of necroptosis, and Figure c is a photo of the morphological characteristics of ferroptosis.

[0035] Figure 5 This is a graph showing the effect of pre-incubation of liposome-encapsulated complex Ir1 with different death mode inhibitors on the survival rate of tumor cells. DETAILED DESCRIPTION

[0036] 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.

[0037] Example 1: This example prepares a cyclometallated iridium sphingosine complex Ir1 , the specific process is: ;in, The structural formula is ;

[0038] S1: Iridium trichloride hydrate and 2-phenylpyridine were mixed at a molar ratio of 1:2.1, dissolved in a mixed solvent of ethylene glycol ethyl ether and ultrapure water (volume ratio of 3:1), and subjected to coordination reaction at 110°C for 24 hours under reflux stirring, cooled to room temperature and filtered, and the obtained solid product was washed three times with ultrapure water and ether respectively, and dried in vacuo to obtain an iridium precursor; S2: (2S, 3R, E)-2-aminooctadec-4-ene-1,3-diol and [2,2'-bipyridine]-4-carboxylic acid were mixed and dissolved in DMF at a molar ratio of 1:1.1, stirred for 10 minutes, HATU was added at a molar ratio of 1:1.5 to (2S, 3R, E)-2-aminooctadec-4-ene-1,3-diol, and stirred for 10 minutes, and then triethylamine was added at a molar ratio of 1:3 to (2S, 3R, E)-2-aminooctadec-4-ene-1,3-diol at 20°C and stirred for 24 hours. After rotary evaporation, ultrapure water was added and filtered, and the obtained solid product was washed 3 times with ultrapure water and vacuum dried to obtain sphingosine; S3: The iridium precursor (107.2 mg, 0.1 mmol) obtained in step S1 was mixed with sphingosine (96.4 mg, 0.2 mmol), dissolved in a dichloromethane / methanol mixed solvent (volume ratio of 2:1), and heated under N 2 The reaction was carried out at 68 °C for 20 h under protection. After the reaction was completed, the reaction solvent was evaporated by rotary evaporation, 0.5 mL of DMF was added to dissolve, and saturated NH 4 PF 6 The solid was filtered and washed three times with ultrapure water and ether respectively to obtain a relatively pure solid, which was further purified by column chromatography (eluent: dichloromethane: methanol = 100: 1, volume ratio) and dried in vacuo to obtain the complex Ir1.

[0039] 1 H NMR (400 MHz, DMSO- d 6 ) δ 9.14 (d, J = 5.5 Hz, 1H), 8.95 (t, J = 8.0Hz, 1H), 8.76 (s, 1H), 8.59 (d, J = 9.0 Hz, 1H), 8.41 (s, 1H), 8.35 (d, J =8.0 Hz, 1H), 8.27 (s, 1H), 7.94 (s, 4H), 7.79 (d, J = 5.0 Hz, 1H), 7.75 (s,1H), 7.66 (d, J = 5.4 Hz, 1H), 7.58 (s, 1H), 7.48 (d, J = 6.6 Hz, 1H), 7.15(t, J = 6.7 Hz, 2H), 7.03 (t, J= 7.6 Hz, 2H), 6.91 (d, J = 7.0 Hz, 2H), 6.19(d, J = 7.1 Hz, 2H), 5.59 – 5.55 (m, 1H), 5.43 (s, 1H), 4.98 (d, J = 11.1 Hz,1H), 4.64 – 4.59 (m, 1H), 4.04 (s, 1H), 3.95 (s, 1H), 3.66 (s, 2H), 1.93 –1.90 (m, 2H), 1.24 (s, 22H), 0.85 (s, 3H). ESI-MS: Calculated value: m / z = 982.42 [M-PF 6 ] + ; Exp. value: m / z = 982.84 [M-PF 6 ] + .

[0040] Example 2: This example uses liposomes to encapsulate the cyclometallated iridium sphingosine complex Ir1. The specific process is as follows: 100 mg of lecithin, 30 mg of cholesterol and 10 mg of mPEG-DSPE, MW 2000 98% MW 2000 reagent were mixed with 5 mg of Ir1 and dissolved in 20 mL of ultra-dry dichloromethane. The mixture was subjected to rotary evaporation to form a film. The liposome-encapsulated Ir1 was obtained after being dissolved in ultrapure water and extruded through a liposome extruder.

[0041] Application Example 1 Determination of the Antitumor Activity of Cyclometallated Iridium Sphingosine Complexes 1. Experimental methods The liposome-encapsulated Ir1 prepared in Example 2 was used as the experimental group, and cisplatin was used as the control group. The cytotoxicity of the liposome-encapsulated Ir1 to the tested tumor cells (MDA-MB-231 (human breast cancer cells), HLF (human lung fibroblast-like cells), HeLa (human cervical cancer cell line), A549 (human lung cancer cell line), LLC (mouse lung cancer cells), 4T1 (mouse breast cancer cells)) was determined respectively. The specific determination method is as follows: The MTT colorimetric method was used to determine the number of cells in the test tumor. The test tumor cells were digested with trypsin into single cell suspensions and counted using a hemocytometer. The cell concentration was adjusted to 5 × 10 4 / mL, inoculated in 96-well plates, 160 μL per well, cultured for 24 h, then added different concentrations of drugs (cyclometallated iridium sphingosine complex Ir1 or cisplatin), and placed in normoxia (cells in a 5% CO 2Under the condition of incubation in a 5% CO incubator, the cells were incubated at 37°C for 48 hours. 20 μL / well of MTT was added 4 hours before the end of the incubation. After 4 hours, the supernatant was discarded and 150 μL / well of DMSO was added. After shaking for 5 minutes, the OD value was measured with an enzyme-labeled instrument at a wavelength of 595 nm.

[0042] According to the following formula: The formula for calculating cell survival rate is: (average optical density of treated cells / average optical density of control cells) × 100%, calculate the survival rate of the tested tumor cells, and draw a graph and calculate the IC 50 The antitumor activity of liposome-encapsulated cyclometallated iridium sphingosine complexes was evaluated.

[0043] 2. Experimental results The IC of the cyclometallated iridium sphingosine complex prepared by the present invention to tumor cells under normoxic conditions is 50 The values ​​are shown in Table 1. It can be seen that the complex Ir1 has good anti-tumor activity and the toxicity of the complex Ir1 to tumor cells is significantly higher than that of cisplatin.

[0044] Table 1

[0045] The results show that the cyclometallated iridium sphingosine complex Ir1 prepared by the present invention has good anti-tumor activity.

[0046] Application Example 2 Uptake of Cyclometallated Iridium Sphingosine Complexes by Tumor Cells 1. Experimental methods Prepare three clean, sterilized Corning laser / confocal culture dishes with a diameter of 35 mm, digest the well-growing human breast cancer cells (MDA-MB-231 cells) with trypsin, subculture them in confocal culture dishes, and culture them in normoxia (cells are heated in a humidified atmosphere containing 5% CO). 2 The cells were cultured in an incubator at 37°C. When the density of MDA-MB-231 cells reached 70%, the liposome-encapsulated cyclometallated iridium sphingosine complex Ir1 prepared by the present invention was added to a final concentration of 5 μM, and the culture was continued for 24 hours. The culture medium was then removed, and the cells were washed twice with PBS. The cells were then incubated with a lysosomal probe for 15 minutes, and the culture medium was removed, and the cells were washed twice with PBS. The cells were immediately observed using a laser confocal microscope, and the fluorescence intensity of the complex Ir1 at 620±20 nm was measured using 405 nm excitation. The probe was excited at 633 nm, and the fluorescence intensity of 650±20 nm was measured.

[0047] 2. Experimental results Uptake of liposome-encapsulated cyclometallated iridium sphingosine complexes by tumor cells Figure 1As shown, it can be seen that the complex Ir1 is mainly enriched in the lysosomes of tumor cells, indicating that the liposome-encapsulated complex Ir1 can be well taken up by tumor cells and enriched in lysosomes, and has phosphorescence tracing characteristics.

[0048] The above results show that the cyclometallated iridium sphingosine complex Ir1 prepared by the present invention can be well taken up by tumor cells and aggregated in lysosomes after being encapsulated by liposomes.

[0049] Application Example 3 Study on the Antitumor Effect of Cyclometallated Iridium Sphingosine Complexes This application example explains the anti-tumor mechanism of the cyclometallated iridium sphingosine complex prepared by the present invention from multiple aspects, and determines the necroptosis and ferroptosis of MDA-MB-231 cells induced by the liposome-encapsulated complex Ir1 through inhibitor experiments, and the ability of the liposome-encapsulated complex Ir1 to induce immunogenic death through immunofluorescence experiments. The specific experimental methods and results are as follows: 1. Immunofluorescence staining of intracellular HMGB-1 (1) Experimental methods Antibodies were purchased from Abcam and operated according to the instructions. MDA-MB-231 cells were seeded into 6-well plates and incubated in the dark with liposome-encapsulated Ir1 (5.0 μM) for 24 h. For HMGB-1 detection, cells were fixed with 4% PFA for 15 min, washed three times with PBS, treated with triton X-100 (0.1%) for 15 min, and washed three times with PBS. After blocking with immunostaining blocking buffer for 30 min, cells were incubated with Alexa Fluor® 488 rabbit anti-HMGB-1 monoclonal antibody at 4 °C overnight. After removing the primary antibody, the cells were washed three times with washing buffer, and Alexa Fluor® 488 rabbit secondary antibody was added and incubated at room temperature in the dark for 1 h. After rinsing with PBS, Hoechst was added to stain the cell nuclei. Then the cells were observed under a laser confocal microscope.

[0050] (2) Experimental results The complex Ir1 causes HMGB-1 excretion. Figure 2 As shown, it can be seen that after treatment with the complex Ir1, the fluorescence of the cell nucleus weakened and fluorescence appeared in the cytoplasm, indicating that the complex Ir1 can cause the excretion of HMGB-1.

[0051] 2. Determination of extracellular ATP concentration (1) Experimental methods MDA-MB-231 cells were seeded in 6-well plates and cultured in an atmosphere containing 5% CO. 2The cells were cultured in complete DMEM medium at 37°C for 24 h in a cell culture incubator. Subsequently, the cells were incubated with untreated or liposome-encapsulated Ir1 (2.5 μM, 5 μM, 10 μM) for 24 h at 37°C in the dark. After incubation, the extracellular ATP concentration was determined by luciferase luminescence using an enhanced ATP detection kit (Biyuntian #S0027).

[0052] (2) Experimental results The results of the effect of complex Ir1 on ATP excretion are as follows Figure 3 As shown, it can be seen that ATP excretion increased after treatment with complex Ir1, and the degree of ATP excretion increased with the increase of the concentration of complex Ir1, indicating that complex Ir1 can induce immunogenic death.

[0053] 3. Transmission electron microscopy to observe cell morphology (1) Experimental methods MDA-MB-231 cells were inoculated in a Corning 100 mm culture dish and cultured for 24 hours. Ir1 (2 μM) was added and incubated in the dark in an incubator for 12 hours. Then the adherent and suspended cells were scraped directly without washing. The cells were collected by centrifugation. Sesame-sized cell clusters, about the size of mung beans, were visible to the naked eye. 1 mL of electron microscopy fixative was gently added and slowly moved to a 4°C refrigerator for temporary storage and transported with 4°C ice packs. During storage and transportation, do not freeze the fixative and send it for inspection later.

[0054] (2) Experimental results The experimental results are as follows Figure 4 The control group ( Figure 4 The mitochondria and endoplasmic reticulum of a) were normal; the drug group treated with liposome-encapsulated Ir1 ( Figure 4 b in Figure 4 (c) The cells are swollen and the membranes are ruptured, showing morphological characteristics of necroptosis, and the mitochondria are broken and condensed, showing morphological characteristics of ferroptosis.

[0055] 4. Study on the mode of cell death (1) Experimental methods To investigate the cell death pattern of MDA-MB-231 cells induced by liposome-encapsulated Ir1, the cells were incubated with inhibitors (Z-VAD-fmk: 50 μM; 3MA: 500 μM; fer-1: 10 μM; Lip-1: 200 nM; rapamycin: 100 nM; NSA: 10 μM) for 1 h and then incubated with Ir1 (2 μM, 24 h). The cell viability was detected by MTT assay.

[0056] (2) Experimental results The results of the model of MDA-MB-231 cell death induced by complex Ir1 are as follows Figure 5 As shown, it can be seen that the survival rate of tumor cells pre-incubated with ferroptosis inhibitors and necroptosis inhibitors increased after treatment with the inhibitor and liposome-encapsulated complex Ir1, indicating that the complex Ir1 can induce ferroptosis and necroptosis in MDA-MB-231 cells.

[0057] The above results show that the complex Ir1 prepared by the present invention can significantly induce necroptosis and ferroptosis of tumor cells, and has a significant ability to induce necroptosis and ferroptosis of MDA-MB-231 cells.

[0058] The cyclometallated iridium sphingosine complex Ir1 prepared by the invention has significant anti-tumor activity and immunogenicity.

[0059] 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 compound of formula I, or a tautomer thereof, or a pharmaceutically acceptable salt thereof: .

2. A method for preparing the compound of formula I according to claim 1, characterized in that: The following steps are involved: S1: Coordination reaction of iridium trichloride and 2-phenylpyridine to obtain an iridium precursor; S2: reacting (2S,3R,E)-2-aminooctadec-4-ene-1,3-diol with [2,2'-bipyridine]-4-carboxylic acid to obtain sphingosine; S3: reacting an iridium precursor with sphingosine, and then adding an ammonium hexafluorophosphate solution for precipitation to obtain the compound of formula I.

3. The method for preparing the compound of formula (I) according to claim 2, characterized in that: At least one of the following conditions must be met: (I) the molar ratio of iridium trichloride to 2-phenylpyridine is 1:2-3; (II) the molar ratio of (2S, 3R, E)-2-aminooctadec-4-ene-1,3-diol to [2,2'-bipyridine]-4-carboxylic acid is 1:1-2; (III) The molar ratio of the iridium precursor to sphingosine is 1:2-3.

4. The method for preparing the compound of formula (I) according to claim 2, characterized in that: At least one of the following conditions must be met: (I) The temperature of the coordination reaction is 100°C to 130°C; (II) the temperature of the reaction in S2 is 18°C ​​to 30°C; (III) The temperature of the reaction in S3 is 60°C to 75°C.

5. A pharmaceutical composition, characterized in that: The invention comprises the compound of formula I as claimed in claim 1, or its isomer, a pharmaceutically acceptable salt thereof, and optionally, a pharmaceutically acceptable carrier, diluent and / or adjuvant.

6. The pharmaceutical composition according to claim 5, characterized in that: The pharmaceutical composition includes a solution, an emulsion, an aqueous suspension or a liposome-containing formulation.

7. The pharmaceutical composition according to claim 5, characterized in that: The pharmaceutical composition also includes liposomes encapsulating the compound of formula I, or its isomers, or pharmaceutically acceptable salts thereof.

8. The pharmaceutical composition according to claim 5, characterized in that: The pharmaceutical composition also comprises a chemotherapeutic agent.

9. Use of the compound of formula I according to claim 1, or its isomer, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to any one of claims 5 to 8 in the preparation of at least one of a molecular probe, a tumor imaging agent, and an anti-tumor drug.

10. The use according to claim 9, characterized in that: The tumor includes at least one of breast cancer, cervical cancer or lung cancer.

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