A metal iridium complex and its preparation method and application

Abstract

The present invention discloses a metal iridium complex, its preparation method, and application. The structural formula of the metal iridium complex is shown as compound #imgabs0# of Formula I. After being encapsulated in liposomes, the complex can be taken up by tumor cells and has phosphorescence tracing properties. The complex has at least four times the cytotoxicity of cisplatin for tumor cells, can better induce upregulation of ATP and HMGB-1 excretion in tumor cells, and ultimately induces ferroptosis and necroptosis in 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. The preparation method of the compound of Formula I of the present invention is simple, easy, and low-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 form of regulated cell death (RCD) that can stimulate an adaptive immune response in an immunocompetent host. ICD is a specialized form of cell death that not only results in cell death but also stimulates a specific immune response to antigens released by dying cells. This mode of death is stress-driven and involves the activation of the immune system against dying cells in an immunocompetent host. The concept of ICD has now been widely applied to tumor immunotherapy. Common treatments—including specific chemotherapies, radiotherapy, and some targeted anticancer drugs—can positively activate ICD, thereby generating an immune response against the tumor to support therapeutic efficacy. Therefore, the development of novel metallo-antitumor drugs with distinct anti-tumor mechanisms is of great significance for improving the efficacy of tumor treatment.

[0003] Ferroptosis is an iron-dependent form of cell death that differs from traditional cell death pathways such as apoptosis, necrosis, and autophagy. This cell death is characterized by iron-dependent lipid peroxidation, often accompanied by the accumulation of lipid peroxides and the destruction of cell membrane integrity. Ferroptosis is caused by glutathione depletion, which leads to decreased glutathione peroxidase (GPX4) activity. This prevents lipid peroxides from being metabolized by the glutathione reductase reaction catalyzed by GPX4. Subsequently, divalent iron ions oxidize lipids to produce reactive oxygen species, thereby promoting ferroptosis.

[0004] Necroptosis is a form of programmed cell death similar to necrosis. It is a "backup" to apoptosis when cells fail to undergo normal apoptosis following inflammatory, oxidative, or ischemic stress. Necroptosis eliminates damaged cells without the activation of caspases. When apoptosis is blocked, it triggers a process of cellular self-destruction activated by extracellular signals (death receptor-ligand binding) or intracellular signals (foreign microbial nucleic acids). During necroptosis, organelle swelling, cell membrane rupture, and decomposition of the 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 present invention aims to provide a metal iridium complex and its preparation method and application.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides a compound of formula I, or a tautomer thereof, or a pharmaceutically acceptable salt thereof: .

[0008] The second aspect of the present invention provides a method for preparing the compound of formula I, comprising the following steps:

[0009] S1: iridium trichloride is subjected to coordination reaction with 2-phenylpyridine to prepare an iridium precursor;

[0010] S2: (2S,3R,E)-2-aminooctadec-4-ene-1,3-diol reacts with [2,2'-bipyridine]-4-carboxylic acid to produce sphingosine;

[0011] S3: reacting an iridium precursor with sphingosine, and then adding an ammonium hexafluorophosphate solution for precipitation to obtain the compound of formula I.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] In some embodiments of the present invention, the ammonium hexafluorophosphate solution is a supersaturated ammonium hexafluorophosphate aqueous solution.

[0016] 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, or 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.

[0017] 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 hours, for example, 22 to 36 hours, 24 hours; 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.

[0018] 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; and the reaction is carried out under an inert atmosphere such as a nitrogen atmosphere.

[0019] The third aspect of the present invention provides a pharmaceutical composition comprising the compound of formula I, or its isomers, pharmaceutically acceptable salts thereof, and optionally, a pharmaceutically acceptable carrier, diluent and / or adjuvant.

[0020] Pharmaceutically acceptable carriers can be liquid or solid and can be selected based on the intended 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™.

[0021] Pharmaceutical compositions include, but are not limited to, solutions, emulsions, aqueous suspensions, and liposome-containing formulations. 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 intimately mixed and dispersed with one another; 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.

[0022] In some embodiments of the present invention, the pharmaceutical composition includes the compound of formula I, or its isomers, or pharmaceutically acceptable salts thereof, and liposomes encapsulating the compound of formula I, or its isomers, or pharmaceutically acceptable salts thereof.

[0023] 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, a mitotic inhibitor, an mTor inhibitor or other chemotherapeutic agent, or a pharmaceutically acceptable salt thereof.

[0024] 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.

[0025] In some embodiments of the present 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] The fourth aspect of the present invention provides a use of the compound of formula I, or its isomers, pharmaceutically acceptable salts, or the pharmaceutical composition in the preparation of at least one of a molecular probe, a tumor imaging agent, and an anti-tumor drug.

[0030] In some embodiments of the present invention, the tumor comprises at least one of breast cancer, cervical cancer, or lung cancer.

[0031] Throughout the specification and the appended claims, a given chemical formula or name should encompass all stereoisomers and optical isomers and their racemates (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. 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 enantiomers or diastereomeric products, they can be separated by conventional methods, for example, 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 a free (neutral) or salt. Both the free form and the salt 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 multiple 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 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 encompass all its conformers, rotational isomers or conformers, where such isomers exist. Different conformations can have different energies, can usually be converted into each other, 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 for rotation is high enough to allow separation of conformers. It is to be understood that all conformers, rotamers or conformational isomeric forms, so far as they may exist, are included within the scope of the present invention.

[0032] 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, sulfamic 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, among others. Pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing a basic or acidic moiety 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.

[0033] The beneficial effects of the present invention are:

[0034] 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 in liposomes and has phosphorescence tracing properties. The complex has a cytotoxicity to tumor cells that is at least 6 times that of cisplatin, can better induce upregulation of ATP and HMGB-1 excretion in tumor cells, and ultimately induces ferroptosis and necroptosis in 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.

[0035] 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

[0036] Figure 1 This 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 lysosomal deep red probe, and "Overlay" represents overlay; wherein, from left to right are respectively a bright field photograph, a photograph of the complex in the 405nm excitation channel, a photograph of the lysosomal deep red probe, and an overlay.

[0037] 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 a bright field photo, a cell nucleus photo, a HMGB-1 protein photo, and an overlay photo.

[0038] Figure 3 This is a diagram showing the ATP excretion of tumor cells by the liposome-encapsulated complex Ir1 of the present invention.

[0039] Figure 4 The morphological diagram of tumor cells observed by transmission electron microscopy using the liposome-encapsulated complex Ir1 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.

[0040] Figure 5 This figure shows the effect of pre-incubation of liposome-encapsulated complex Ir1 with different death mode inhibitors on the survival rate of tumor cells. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below using specific examples. Unless otherwise specified, the raw materials, reagents, and apparatus used in the examples and comparative examples are available from conventional commercial sources or by conventional methods. Unless otherwise specified, all experimental or testing methods are conventional in the art.

[0042] Example 1: This example prepared a cyclometalated iridium sphingosine complex Ir1 The specific process is: ;in, The structural formula is ;

[0043] 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 ether and ultrapure water (volume ratio of 3:1), and subjected to coordination reaction at 110°C under reflux and stirring for 24 hours. The reaction was cooled to room temperature and filtered. The obtained solid product was washed three times with ultrapure water and ether, respectively, and dried in vacuo to obtain an iridium precursor.

[0044] S2: (2S,3R,E)-2-aminooctadeca-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, and stirred for 10 min. HATU was added at a molar ratio of 1:1.5 to (2S,3R,E)-2-aminooctadeca-4-ene-1,3-diol, and stirred for 10 min. Triethylamine was then added at a molar ratio of 1:3 to (2S,3R,E)-2-aminooctadeca-4-ene-1,3-diol, and the mixture was stirred at 20°C for 24 h. After rotary evaporation, ultrapure water was added and filtered. The obtained solid product was washed three times with ultrapure water and dried in vacuo to obtain sphingosine.

[0045] 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 reacted at 68°C for 20 hours under N2 protection. After the reaction was completed, the reaction solvent was rotary evaporated, 0.5 mL of DMF was added to dissolve it, and then a saturated NH4PF6 aqueous solution was added to precipitate the solid. The solid was filtered and washed three times with ultrapure water and ether respectively to obtain a relatively pure solid. The solid was further purified by column chromatography (eluent: dichloromethane: methanol = 100:1, volume ratio) and vacuum dried to obtain the complex Ir1.

[0046] 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).

[0047] ESI-MS: Calculated: m / z = 982.42 [M-PF6] + ; Exp. value: m / z = 982.84 [M-PF6] + .

[0048] Example 2: This example uses liposomes to encapsulate the cyclic metal iridium sphingosine complex Ir1. The specific process is as follows:

[0049] 5 mg of Ir1 was mixed with 100 mg of lecithin, 30 mg of cholesterol, and 10 mg of mPEG-DSPE, MW 2000 98% MW 2000 reagent and dissolved in 20 mL of ultra-dry dichloromethane. The mixture was rotary evaporated to form a film, which was dissolved in ultrapure water and extruded through a liposome extruder to obtain liposome-encapsulated Ir1.

[0050] Application Example 1 Determination of the Antitumor Activity of Cyclometallated Iridium Sphingosine Complexes

[0051] 1. Experimental methods

[0052] 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 liposomes against 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), and 4T1 (mouse breast cancer cells)) was determined. The specific determination method is as follows:

[0053] The MTT colorimetric assay was used to determine the concentration of cells. The tumor cells were digested with trypsin to form single cell suspensions, and counted using a hemocytometer. The cell concentration was adjusted to 5 × 10 4 / mL, seeded in 96-well plates, 160 μL per well. After 24 hours of incubation, different concentrations of drugs (cyclometallated iridium sphingosine complex Ir1 or cisplatin) were added. The cells were incubated at 37°C under normoxia (cells were cultured in an incubator containing 5% CO2) for 48 hours. Four hours before the end of incubation, 20 μL of MTT was added per well. After 4 hours, 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.

[0054] The cell survival rate was calculated as follows: (average optical density of treated cells / average optical density of control cells) × 100%, and the survival rate of the tested tumor cells was calculated. The IC 50 The antitumor activity of liposome-encapsulated cyclometallated iridium sphingosine complexes was evaluated.

[0055] 2. Experimental results

[0056] 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.

[0057] Table 1

[0058]

[0059] The results show that the cyclometallated iridium sphingosine complex Ir1 prepared by the present invention has good anti-tumor activity.

[0060] Application Example 2: Uptake of Cyclometallated Iridium Sphingosine Complexes by Tumor Cells

[0061] 1. Experimental methods

[0062] Three clean, sterilized Corning laser / confocal culture dishes with a diameter of 35 mm were prepared. Healthy human breast cancer cells (MDA-MB-231 cells) were trypsinized and passaged into the confocal culture dishes. The cells were cultured at 37°C under normoxia (cells were cultured in an incubator containing 5% CO2). When the MDA-MB-231 cell density reached 70%, the liposome-encapsulated cyclometallated iridium sphingosine complex Ir1 prepared according to the present invention was added to a final concentration of 5 μM. The cells were cultured for 24 hours, after which the culture medium was removed and the cells were washed twice with PBS. The cells were then incubated with a lysosomal probe for 15 minutes. The culture medium was then removed and the cells were washed twice with PBS. The cells were immediately observed using a laser confocal microscope. The fluorescence intensity of the complex Ir1 was measured at 620±20 nm using 405 nm excitation, while the fluorescence intensity of the probe was measured at 650±20 nm using 633 nm excitation.

[0063] 2. Experimental results

[0064] Uptake of liposome-encapsulated cyclometallated iridium sphingosine complexes by tumor cells Figure 1 As 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.

[0065] The above results indicate that the cyclometallated iridium sphingosine complex Ir1 prepared by the present invention can be well taken up by tumor cells and accumulated in lysosomes after being encapsulated by liposomes.

[0066] Application Example 3: Study on the Antitumor Effect of Cyclometallated Iridium Sphingosine Complexes

[0067] This application example explains the anti-tumor mechanism of the cyclometallated iridium sphingosine complex prepared by the present invention from multiple perspectives. Inhibitor experiments were performed to determine the ability of the liposome-encapsulated complex Ir1 to induce necroptosis and ferroptosis in MDA-MB-231 cells. Immunofluorescence experiments were also performed to determine the ability of the liposome-encapsulated complex Ir1 to induce immunogenic cell death. The specific experimental methods and results are as follows:

[0068] 1. Immunofluorescence staining of intracellular HMGB-1

[0069] (1) Experimental methods

[0070] Antibodies were purchased from Abcam and used according to the manufacturer's instructions. MDA-MB-231 cells were seeded into 6-well plates and incubated with liposome-encapsulated Ir1 (5.0 μM) for 24 hours in the dark. For HMGB-1 detection, cells were fixed with 4% PFA for 15 minutes, washed three times with PBS, treated with 0.1% Triton X-100 for 15 minutes, and washed three times with PBS. After blocking with immunostaining blocking buffer for 30 minutes, cells were incubated with Alexa Fluor® 488 rabbit anti-HMGB-1 monoclonal antibody overnight at 4°C. 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 for 1 hour at room temperature in the dark. After rinsing with PBS, Hoechst was added to stain the nuclei. Cells were then observed under a laser confocal microscope.

[0071] (2) Experimental results

[0072] 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.

[0073] 2. Determination of extracellular ATP concentration

[0074] (1) Experimental methods

[0075] MDA-MB-231 cells were seeded in 6-well plates and cultured in complete DMEM medium at 37°C in a cell culture incubator with 5% CO₂ for 24 hours. Subsequently, the cells were incubated with untreated or liposome-encapsulated Ir1 (2.5 μM, 5 μM, and 10 μM) for 24 hours at 37°C in the dark. Following incubation, extracellular ATP concentration was measured using the enhanced ATP assay kit (Beyotime #S0027) using a luciferase-based luminescence assay.

[0076] (2) Experimental results

[0077] 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.

[0078] 3. Observation of Cell Morphology by Transmission Electron Microscopy

[0079] (1) Experimental methods

[0080] MDA-MB-231 cells were seeded in Corning 100 mm culture dishes and cultured for 24 hours. Ir1 (2 μM) was then added and incubated in the dark for 12 hours. Adherent and suspended cells were then scraped directly from the dish without rinsing. Cells were harvested by centrifugation. Sesame-sized and mung bean-sized cell clusters were visible to the naked eye. 1 mL of electron microscopy fixative was gently added and the dish was slowly transferred to a 4°C refrigerator for temporary storage. Transported on ice at 4°C, the fixative was carefully protected from freezing during storage and transport. The dish was then sent for examination.

[0081] (2) Experimental results

[0082] The experimental results are as follows Figure 4 As shown. Figure 4 The mitochondria and endoplasmic reticulum in a) were normal; the drug group treated with liposome-encapsulated Ir1 ( Figure 4 b in Figure 4 In (c), the cells swelled and the membrane ruptured, showing morphological characteristics of necroptosis, and the mitochondria had broken cristae and condensed, showing morphological characteristics of ferroptosis.

[0083] 4. Study on cell death mode

[0084] (1) Experimental methods

[0085] 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.

[0086] (2) Experimental results

[0087] 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.

[0088] 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.

[0089] The cyclometallated iridium sphingosine complex Ir1 prepared by the invention has significant anti-tumor activity and immunogenicity.

[0090] 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 considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A compound of formula I, 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: iridium trichloride and 2-phenylpyridine are subjected to a coordination reaction at a molar ratio of 1:2 to 3 at 100° C. to 130° C. to prepare an iridium precursor; S2: (2S,3R,E)-2-aminooctadec-4-ene-1,3-diol and [2,2'-bipyridine]-4-carboxylic acid are reacted at a molar ratio of 1:1-2 at 18°C ​​to 30°C to prepare N-[(4E)-1,3-dihydroxyoctadec-4-ene-2-yl]-[2,2'-bipyridine]-4-carboxamide; S3: reacting an iridium precursor with N-[(4E)-1,3-dihydroxyoctadec-4-en-2-yl]-[2,2'-bipyridine]-4-carboxamide at a molar ratio of 1:2-3 at 60°C to 75°C, and then adding ammonium hexafluorophosphate solution for precipitation to obtain the compound of formula I.

3. A pharmaceutical composition, characterized in that: The invention comprises the compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, and optionally, a pharmaceutically acceptable carrier, diluent and / or adjuvant.

4. The pharmaceutical composition according to claim 3, wherein: The pharmaceutical composition includes a solution, an emulsion, an aqueous suspension or a liposome-containing formulation.

5. The pharmaceutical composition according to claim 3, characterized in that: The pharmaceutical composition further comprises liposomes encapsulating the compound of formula I or a pharmaceutically acceptable salt thereof.

6. The pharmaceutical composition according to claim 3, characterized in that: The pharmaceutical composition also comprises a chemotherapeutic agent.

7. Use of the compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to any one of claims 3 to 6 in the preparation of at least one of a molecular probe, a tumor imaging agent, and an anti-tumor drug; wherein the tumor comprises at least one of breast cancer, cervical cancer, or lung cancer.

Images