A metal ion affinity cyclometalated iridium complex, its preparation method and application

Abstract

The present invention discloses a metal ion-affinity cyclometalated iridium complex, its preparation method and application. The cyclometalated iridium complex has the structure of a compound of Formula I:. The cyclometalated iridium complex of the present invention can be well taken up by tumor cells and has good anti-tumor activity. This complex can redistribute endogenous Zn<supgt;2+< / supgt; from the cytoplasm to the mitochondria, resulting in the accumulation of excessive Zn<supgt;2+< / supgt> in the mitochondria of cancer cells, regulating mitochondrial zinc homeostasis without causing a significant change in the overall Zn<supgt;2+< / supgt> level of the whole cell; this complex has excellent cytotoxicity, about 10 times that of cisplatin. The in vivo anti-tumor results show that the inhibition rate of this complex on the primary tumor reaches 50.5%, higher than that of cisplatin; this complex can also target mitochondria, increase the intracellular ROS level, induce mitochondrial dysfunction and mitochondrial DNA damage.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal chemistry, and particularly relates to a metal ion-affinity cyclometalated iridium complex and its preparation method and application. Background Art

[0002] Zinc is the second most abundant transition metal element in the human body, and the precise regulation of its intracellular homeostasis and distribution plays a key role in physiological and pathological processes. Approximately 30% - 40% of cellular zinc is distributed in the nucleus, about 50% is present in the cytoplasm and other subcellular organelles, and the rest is distributed in the cell membrane. Most zinc ions (Zn²⁺) bind to proteins and enzymes to maintain their structure and stability, resulting in a relatively low concentration of free Zn²⁺ in the cytoplasm or sequestered in organelles and vesicles. However, free Zn²⁺ is also indispensable for many key cellular processes such as signal transduction, cell metabolism, cytoskeleton assembly, and immune regulation. Studies have found that Zn²⁺ overload can generate reactive oxygen species (ROS) through electron leakage during mitochondrial aerobic respiration, leading to mitochondrial DNA (mtDNA) damage and the production of high levels of interferon and inflammatory cytokines. At the same time, Zn²⁺ also participates in the formation of zinc-dependent transcription factors and proteins related to DNA repair, significantly affecting the process of DNA damage-mediated cell death. Therefore, regulating intracellular zinc homeostasis and distribution may become a potential strategy for anti-tumor therapy.

[0003] However, most current studies mainly interfere with the zinc homeostasis of cancer cells by a large amount of exogenous zinc ion input. For example, pH-sensitive zeolitic imidazolate framework (ZIF-8) can release Zn²⁺ in lysosomes, resulting in a sudden increase in Zn²⁺ concentration in cancer cells, and ultimately highly activating anti-tumor immunity by simultaneously inducing pyroptosis, necrosis, and ICD. In contrast, metal drugs can regulate zinc homeostasis by interfering with intracellular zinc distribution (rather than changing the overall zinc level), thus avoiding the side effects caused by exogenous zinc input. However, how to regulate the level of zinc in mitochondria by metal drugs to synergistically treat tumors is still an under-explored area. Summary of the Invention

[0004] The present invention aims to at least solve one of the above technical problems existing in the prior art. To this end, the object of the present invention is to provide a metal ion-affinity cyclometalated iridium complex and its preparation method and application.

[0005] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0006] In the first aspect of the present invention, there is provided a compound of formula I, or its isomers, its pharmaceutically acceptable salts: .

[0007] The second aspect of the present invention provides a method for preparing the compound of formula I, comprising the following steps:

[0008] S1: Performing a coordination reaction between iridium(III) chloride and 2-phenylpyridine to obtain an iridium precursor;

[0009] S2: Performing a Mannich reaction between 1,10-phenanthroline-5,6-dione and 2-thiophenecarboxaldehyde, and then performing a methylation reaction to obtain a ligand;

[0010] S3: Reacting the iridium precursor with the ligand to obtain the compound of formula I.

[0011] In some embodiments of the present invention, the molar ratio of iridium(III) chloride to 2-phenylpyridine is 1:2 to 3, such as 1:2 to 2.4, 1:2.2.

[0012] In some embodiments of the present invention, the iridium(III) chloride can be a hydrate of iridium(III) chloride.

[0013] In some embodiments of the present invention, the molar ratio of 1,10-phenanthroline-5,6-dione to 2-thiophenecarboxaldehyde is 1:0.9 to 2, such as 1:1 to 1.5, 1:1.2.

[0014] In some embodiments of the present invention, the catalyst for the Mannich reaction includes any one of ammonium acetate, ammonium carbonate, and ammonium chloride.

[0015] In some embodiments of the present invention, the molar ratio of 1,10-phenanthroline-5,6-dione to the catalyst is 1:5 to 10, such as 1:6 to 9; 1:8 to 9, etc.

[0016] In some embodiments of the present invention, in S3, the molar ratio of the iridium precursor to the ligand is 1:2 to 3, such as 1:2 to 2.4, 1:2.2.

[0017] In some embodiments of the present invention, in S1, the temperature of the coordination reaction is 120°C to 150°C, such as 130°C to 140°C; the time of the coordination reaction is 12 to 48 h, such as 18 to 36 h; the solvent for the coordination reaction includes at least one of dichloromethane, methanol, ethylene glycol monoethyl ether, and ultrapure water, such as a mixed solvent of dichloromethane and methanol, a mixed solvent of ethylene glycol monoethyl ether and ultrapure water; the volume ratio of dichloromethane to methanol is 2 to 3:1, such as 2 to 2.4:1, 2:1; the volume ratio of ethylene glycol monoethyl ether to ultrapure water is 2 to 4:1, such as 3 to 4:1, 3:1.

[0018] In some embodiments of the present invention, in S2, the reaction temperature of the Mannich reaction is 100°C to 130°C, such as 110 to 120°C; the reaction time of the Mannich reaction is 3 to 9 h, such as 4 to 8 h, 5 to 7 h, etc.; the reaction solvent of the Mannich reaction includes at least one of glacial acetic acid, formic acid, ethanol, methanol, dimethyl sulfoxide (DMSO), N,N -dimethylformamide.

[0019] In some embodiments of the present invention, in S2, the methylation reagent used in the methylation reaction includes haloalkane, such as any one of chloromethane, bromomethane, and iodomethane; the reaction temperature of the methylation reaction is 35°C to 55°C, such as 40 to 50°C; the reaction time of the methylation reaction is 1 to 5 h, such as 2 to 4 h; the methylation reaction is carried out under alkaline conditions; the solvent of the methylation reaction includes at least one of dimethyl sulfoxide, N,N -dimethylformamide, N-methylpyrrolidone, and acetonitrile.

[0020] In some embodiments of the present invention, in S3, the reaction is carried out in an inert environment; the reaction temperature is 40°C to 60°C, such as 45°C to 55°C; the reaction time is 4 to 8 h, such as 5 to 7 h; the reaction solvent includes at least one of dichloromethane, methanol, ethylene glycol monoethyl ether, and ultrapure water, for example, a mixed solvent of dichloromethane and methanol, a mixed solvent of ethylene glycol monoethyl ether and ultrapure water; the volume ratio of dichloromethane to methanol is 2 to 3:1, such as 2 to 2.4:1, 2:1; the volume ratio of ethylene glycol monoethyl ether to ultrapure water is 2 to 4:1, such as 3 to 4:1, 3:1.

[0021] The third aspect of the present invention provides a pharmaceutical composition, comprising the compound of formula I, or its isomers, its pharmaceutically acceptable salts, and optionally, a pharmaceutically acceptable carrier, diluent, and / or adjuvant.

[0022] The pharmaceutical composition of the present invention is applicable to various administration routes and can thus be formulated into any pharmaceutically acceptable dosage form. For example, the above-mentioned pharmaceutical composition can be administered to patients or subjects in need of such treatment by oral, parenteral, rectal, or pulmonary administration, etc. For oral administration, the pharmaceutical composition can be formulated into oral preparations, such as conventional oral solid preparations, such as tablets, capsules, pills, granules, etc.; it can also be formulated into oral liquid preparations, such as oral solutions, oral suspensions, syrups, etc. When formulated into oral preparations, suitable fillers, binders, disintegrants, lubricants, etc. can be added. For parenteral administration, the above-mentioned pharmaceutical composition can also be formulated into injections, including injection solutions, sterile powders for injection, and concentrated solutions for injection. When formulating into injections, conventional methods in the existing pharmaceutical field can be used. When preparing injections, no additives can be added, or suitable additives can be added according to the nature of the drug. For rectal administration, the pharmaceutical composition can be formulated into suppositories, etc. For pulmonary administration, the pharmaceutical composition can be formulated into inhalation preparations, aerosols, powder aerosols, or sprays, etc.

[0023] A pharmaceutically acceptable carrier can be liquid or solid and can be selected according to the intended mode of administration so as to provide, in combination with one or more therapeutic compounds, the desired volume, consistency, and other relevant transport and chemical properties for any other components of a given pharmaceutical composition. Typical pharmaceutically acceptable carriers include, for example but not limited to: water, saline solutions, DMSO, binders (e.g., polyvinylpyrrolidone or hydroxypropylmethylcellulose), 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 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) 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 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™.

[0024] The 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 semi-solids. Emulsions are generally biphasic systems, consisting of two immiscible liquid phases intimately mixed and dispersed in each other; typically, 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 efficacy.

[0025] In some embodiments of the present invention, the pharmaceutical composition includes the compound of formula I, or an isomer thereof, a pharmaceutically acceptable salt thereof, and a liposome encapsulating the compound of formula I, or an isomer thereof, a pharmaceutically acceptable salt thereof.

[0026] The pharmaceutical composition further contains a chemotherapeutic agent. In some embodiments of the present invention, the chemotherapeutic agent is selected from one or more of alkylating agents, antimetabolites, antitumor antibiotic agents, mitotic inhibitors, mTor inhibitors, or other chemotherapeutic agents, or pharmaceutically acceptable salts thereof.

[0027] 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 pharmaceutically acceptable salts thereof.

[0028] 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 pharmaceutically acceptable salts thereof.

[0029] 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 pharmaceutically acceptable salts, hydrates, solvates, or polymorphs thereof.

[0030] 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, and teniposide, or pharmaceutically acceptable salts thereof.

[0031] 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.

[0032] The fourth aspect of the present invention provides an application of the compound of formula I, or its isomers, its 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.

[0033] In some embodiments of the present invention, the tumor includes at least one of breast cancer, cervical cancer, or lung cancer.

[0034] In some embodiments of the present invention, the anti-tumor effect of the anti-tumor drug includes at least one of inhibiting tumor growth, inducing DNA (such as mtDNA) damage in tumor cells, inducing mitochondrial dysfunction in tumor cells, and inducing an increase in the ROS level in tumor cells.

[0035] Throughout the specification and the appended claims, a given chemical formula or name shall cover 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. can also exist in the complexes, and all such stable isomers are covered 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 as a mixture of isomers or in the form of isolated isomers. The compounds of the present invention can be separated in optically active or racemic forms. The optically active forms can be prepared by resolution of the racemic form or by synthesis from optically active starting materials. All methods for preparing the compounds of the present invention and the 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 products of the present invention can be obtained in free (neutral) or salt form. Both the free form and the salts of these final products are within the scope of the present invention. If desired, one form of the compound can be converted into another form. The free base or acid can be converted into a salt; the salt can be converted into the free compound or another salt; a mixture of isomeric compounds of the present invention can be separated into the individual isomers. The compounds of the present invention, their free forms and their salts can exist in a variety of tautomeric forms, in which a hydrogen atom is transposed to another part of the molecule and the chemical bonds between the atoms of the molecule are thus rearranged. It should be understood that all tautomeric forms, insofar as they may exist, are included in the present invention. In addition, a given chemical formula or name shall cover all its conformational isomers, rotational isomers or conformers, where such isomers exist. Different conformations can have different energies, can generally interconvert, and are rarely separable. Some molecules can be separated in multiple conformations. For example, atropisomers are isomers generated by hindered rotation around a single bond, where the steric strain barrier to rotation is high enough to allow the separation of conformers. It should be understood that all conformational isomers, rotational isomers or conformer forms, insofar as they may exist, are included in the present invention.

[0036] The term "pharmaceutically acceptable salts" includes, but is not limited to, inorganic or organic acid salts of basic groups such as amines; alkali metal salts 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, etc. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent complex containing basic or acidic moieties by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or in a mixture of both; generally, a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile is preferred.

[0037] The beneficial effects of the present invention are:

[0038] The compound of formula I of the present invention can be well taken up by tumor cells and has good anti-tumor activity. This complex can redistribute endogenous Zn 2+ from the cytoplasm to the mitochondria, resulting in an excess of Zn in the mitochondria of cancer cells 2+ accumulation, regulating mitochondrial zinc homeostasis and not causing a significant change in the overall Zn 2+ level of the whole cell; this complex has excellent cytotoxicity, about 10 times that of cisplatin. The in vivo anti-tumor results show that the inhibition rate of this complex on the primary tumor reaches 50.5%, higher than that of cisplatin; this complex can also target mitochondria, increase the intracellular ROS level, induce mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage.

[0039] The compound of formula I of the present invention, as a cyclometalated iridium complex, has good anti-tumor activity, and its preparation method is simple, feasible and low-cost; therefore, the complex prepared by the present invention provides new clues for the development of new metal anti-cancer complexes. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is the electrospray mass spectrum of the ligand prepared in the example of the present invention.

[0041] Figure 2 is the 1H nuclear magnetic resonance spectrum of the ligand prepared in the example of the present invention.

[0042] Figure 3It is the electrospray mass spectrum of the cyclometalated iridium complex Th-Ir prepared in the embodiments of the present invention.

[0043] Figure 4 It is the 1H NMR spectrum of the cyclometalated iridium complex Th-Ir prepared in the embodiments of the present invention.

[0044] Figure 5 It is the 13C NMR spectrum of the cyclometalated iridium complex Th-Ir prepared in the embodiments of the present invention.

[0045] Figure 6 It is the ultraviolet-visible absorption (B) and emission spectrum (A) of the cyclometalated iridium complex Th-Ir of the present invention in different solvents.

[0046] Figure 7 It is the change diagram of the 600 MHz 1H NMR spectrum of the cyclometalated iridium complex Th-Ir of the present invention in DMSO-d6 before and after adding Zn 2+ before and after. 1 It is the change diagram of the 1H NMR spectrum.

[0047] Figure 8 It is the cellular localization diagram of the cyclometalated iridium complex Th-Ir of the present invention in MAD-MB-231 cells; wherein, "LTDR" and "MTDR" respectively represent lysosome deep red probe and mitochondrion deep red probe; "Brightfield" represents bright field; "Overlay" represents overlay; "Probe" represents probe.

[0048] Figure 9 It is the zinc ion distribution diagram of the cyclometalated iridium complex Th-Ir of the present invention in cells.

[0049] Figure 10 It is the functional effect diagram of the cyclometalated iridium complex Th-Ir of the present invention in destroying the mitochondria of MAD-MB-231 cells.

[0050] Figure 11 It is the result diagram of the influence of the cyclometalated iridium complex Th-Ir of the present invention on the ROS level in MAD-MB-231 cells.

[0051] Figure 12 It is the effect diagram of the mitochondrial DNA damage of the cyclometalated iridium complex Th-Ir of the present invention on MAD-MB-231 cells.

[0052] Figure 13 It is the diagram of the change of the primary tumor volume (left) and body weight (right) of the mice of the present invention during in vivo drug treatment. Detailed implementation manners

[0053] The content of the present invention will be further described in detail through specific embodiments below. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial channels or can be obtained by existing technical methods. Unless otherwise specified, the test or testing methods are conventional methods in the art.

[0054] Example 1, in this example, a cyclometalated iridium complex Th-Ir (i.e., the compound of formula I) was prepared. The specific process was as follows:

[0055]

[0056] Among them, The structural formula of is

[0057] S1. Iridium(III) chloride hydrate and the ligand were mixed at a molar ratio of 1:2.2 and dissolved in a mixed solvent of ethylene glycol monoethyl ether and ultrapure water (volume ratio 3:1). The coordination reaction was carried out at 135 °C under reflux stirring for 24 h, cooled to room temperature and filtered by suction. The obtained solid product was washed 3 times with ultrapure water and diethyl ether respectively, and dried under vacuum to obtain the iridium precursor;

[0058] S2. 1,10-Phenanthroline-5,6-dione (0.50 g, 2.3 mmol), 2-thiophenecarboxaldehyde (0.26 mL, 2.76 mmol), and ammonium acetate (1.50 g, 19.5 mmol) were added to 15 mL of glacial acetic acid. Under nitrogen protection, the mixture was heated to 115 °C and refluxed, and continuously stirred for 4 h. After the reaction was completed, the mixture was cooled to room temperature, and the precipitated yellow solid product was collected by suction filtration. The solid was washed with a small amount of cold ethanol to remove impurities. Finally, the product was dried in a vacuum drying oven to obtain a yellow powder; the obtained yellow powder (0.6 g, 1.987 mmol) and NaOH (0.088 g, 2.2 mmol) were dissolved in anhydrous DMSO (3 mL), and stirred at room temperature for 2 h. Methyl iodide (0.338 g, 2.384 mmol) was added to the reaction system, and stirred at 40 °C for 3 h. The mixture was added to 200 mL of water to precipitate a brown solid, and filtered to obtain the ligand;

[0059] S3. Mix the iridium precursor (1 eq.) obtained in step S1 with the ligand (2.2 eq.) obtained in step S2, dissolve the mixture in a dichloromethane / methanol mixed solvent (V / V = 2 / 1), and stir and reflux the solution for 6 h under a nitrogen atmosphere at 50 °C. Cool the solution to room temperature, and add a saturated NH4PF6 (6.0 eq.) solution. Use a dichloromethane / methanol mixed solvent (V / V = 20 / 1) as the eluent, and separate and purify the reaction product by silica gel column chromatography, and then dry it under vacuum to obtain the cyclometalated iridium complex Th-Ir;

[0060] Among them, the ligand prepared in S2 is 2-(thiophen-2-yl)-1-methylimidazo[4,5-f][1,10]phenanthroline, and its electrospray mass spectrum and 1H nuclear magnetic resonance spectrum are as shown in Figure 1 and Figure 2 shown. Specifically:

[0061] 1 1H NMR (400 MHz, chloroform-d) δ 9.17 (dt, J = 4.4, 1.5 Hz, 2H), 9.05(dd, J = 8.1, 1.8 Hz, 1H), 8.76 (dd, J = 8.4, 1.6 Hz, 1H), 7.70 (ddd, J =15.8, 8.2, 4.3 Hz, 2H), 7.62 – 7.52 (m, 2H), 7.26 – 7.24 (m, 1H), 4.41 (s,3H).

[0062] ESI-MS: Theoretical value: m / z = 316.08; Experimental value: m / z = 317.43.

[0063] The electrospray mass spectrum, 1H nuclear magnetic resonance spectrum and 13C nuclear magnetic resonance spectrum of the compound of formula I are as shown in Figure 3 、 Figure 4 and Figure 5 shown. Specifically:

[0064] 11H NMR (600 MHz, DMSO-d6) δ 9.38 (d, J = 8.6 Hz, 1H), 9.21 – 9.15 (m,1H), 8.28 (t, J = 7.5 Hz, 2H), 8.21 (d, J = 4.5 Hz, 2H), 8.09 (ddd, J = 10.4,8.4, 5.1 Hz, 2H), 8.01 – 7.86 (m, 6H), 7.50 (dd, J = 16.5, 5.9 Hz, 2H), 7.42– 7.36 (m, 1H), 7.08 (td, J = 7.8, 2.4 Hz, 2H), 6.99 (dq, J = 21.3, 7.2 Hz,4H), 6.35 – 6.28 (m, 2H), 4.52 (s, 3H).

[0065] 13 13C NMR (151 MHz, DMSO-d6) δ 166.82, 166.80, 150.56, 150.12, 149.28,148.99, 148.91, 148.07, 144.26, 144.00, 143.90, 143.87, 138.61, 136.06,132.22, 131.95, 131.13, 130.55, 130.20, 130.17, 129.74, 128.43, 127.46,127.39, 126.59, 125.67, 125.00, 123.75, 123.67, 122.39, 122.28, 119.94,119.88, 35.78.

[0066] ESI-MS: Calculated: m / z = 817.17 [M-PF6] + ; Found: m / z = 817.42 [M-PF6] + 。

[0067] Application Example 1. In this application example, the ultraviolet-visible absorption and emission spectra of the cyclometalated iridium complex were tested. The specific process is as follows:

[0068] The cyclometalated iridium complex Th-Ir prepared in Example 1 was used as the experimental group. Samples were prepared into solutions with a concentration of 20 μM using PBS (phosphate buffered saline), CH3CN (acetonitrile solution), and CH2Cl2 (dichloromethane solution) as solvents respectively. Then, a two-photon ultraviolet-visible spectrophotometer was used to record the ultraviolet-visible absorption spectrum of the cyclometalated iridium complex Th-Ir and the ultraviolet emission spectrum at an excitation wavelength of 405 nm.

[0069] The ultraviolet-visible absorption spectrum of the cyclometalated iridium complex Th-Ir prepared in Example 1 ( Figure 6 B in Figure 6 ) and the ultraviolet emission spectrum at an excitation wavelength of 405 nm ( Figure 6 A in

[0070] are shown in 2+ . It can be seen that in PBS, CH3CN, and CH2Cl2, the ultraviolet-visible spectra of Th-Ir show similar absorption peaks, which are attributed to the π-π* transition (250 - 350 nm) of the inner ligand and the metal-ligand charge transfer (350 - 470 nm) respectively. When excited at 405 nm, Th-Ir exhibits a strong emission at 605 nm.

[0071] 1 1H-NMR titration: The 1H-NMR spectrum of Th-Ir dissolved in deuterated DMSO was recorded; then, a solution of ZnSO4 (Zn 1 ) dissolved in deuterated water was added to the above sample, and the recording was done again after 24 h. 2+

[0072] As shown in Figure 7 , 1 the 1H NMR spectrum shows that after Zn 2+ titration, the signals of Th-Ir at positions q, t, s, v, r, and y shift to higher fields.

[0073] The above results indicate that the binding of Th-Ir to Zn 2+ is mainly achieved through the N atom in the imidazole ring and the S atom in the thiophene ring, and the methyl substitution on the imidazole ring will weaken but not eliminate the binding ability of Zn 2+ .

[0074] Application Example 3. In this application example, the uptake and distribution of the cyclometalated iridium complex in tumor cells and the zinc ion regulation effect were measured. The specific process is as follows:

[0075] MDA-MB-231 (human breast cancer cell line) cells were seeded in confocal dishes overnight and co-incubated with Th-Ir (10 μM, 1 h) and organelle-specific probes: MTDR (150 nM, 30 min) and LTDR (50 nM, 30 min) at 37 °C, then washed three times with sterile PBS and imaged by confocal microscopy. For Ir(III) complex: λex = 405 nm, λem = 605±20 nm; for LTDR / MTDR: λex = 633 nm, λem = 665±20 nm. The ability of Th-Ir to regulate intracellular Zn 2+ distribution was also studied in MAD-MB-231 cells using ICP-MS.

[0076] As Figure 8 shown, Th-Ir was found to effectively penetrate into MAD-MB-231 cells within 1 h, and Th-Ir mainly accumulated in mitochondria, with good co-localization with the lysosome-specific stain (LTDR) and the mitochondria-specific stain (MTDR), and the Pearson correlation coefficients were 0.88 and 0.86, respectively.

[0077] The zinc ion distribution of the cyclometalated iridium complex Th-Ir in cells was as Figure 9 shown. After treatment with Th-Ir (5 μM, 8 h) in MAD-MB-231 cells, the total intracellular zinc ion content did not change significantly; however, compared with control cells, the zinc content in the cytoplasm decreased, while the zinc content in mitochondria increased significantly.

[0078] The above results indicate that the complex Th-Ir can target mitochondria well and redistribute endogenous zinc from the cytoplasm and vesicles to mitochondria, resulting in excessive Zn 2+ accumulation in cancer cell mitochondria to regulate mitochondrial zinc homeostasis.

[0079] Application Example 4. In this application example, the damage of the cyclometalated iridium complex to subcellular organelle functions was determined, that is, the damage of the subcellular organelle functions of the cyclometalated iridium complex prepared in Example 1 was explained from multiple aspects. The destruction of mitochondrial function by Th-Ir, the effect on the ROS level in cells, and the damage to mitochondrial DNA were observed by confocal microscopy. The specific process is as follows:

[0080] 1. Destruction of mitochondrial function by the cyclometalated iridium complex

[0081] (1) Experimental method

[0082] MDA-MB-231 cells were seeded in 35 mm confocal dishes and incubated overnight in an atmosphere of 37 °C and 5% CO2. After treating the cells with Th-Ir (5 μM) for 8 h, the cells were washed three times with sterile PBS and stained with JC-1 (5 μg / mL) for 20 min in the dark at 37 °C. The cells were washed again with sterile PBS and detected using a confocal microscope. The excitation wavelength λex = 488 nm; the monomer fluorescence emission wavelength λem = 530 ± 20 nm; the aggregate fluorescence emission wavelength λem = 585 ± 20 nm.

[0083] (2)Experimental results

[0084] As Figure 10 shown, according to the JC-1 staining assay, Th-Ir induced a significant loss of mitochondrial membrane potential (MMP), which was reflected by the red-to-green color change of 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1).

[0085] The above results indicate that the cyclometalated iridium complex Th-Ir prepared in the present invention can significantly cause mitochondrial dysfunction.

[0086] 2. Effect of cyclometalated iridium complex on the level of ROS in cells

[0087] (1)Experimental method

[0088] MDA-MB-231 cells were seeded in 35 mm confocal dishes and cultured overnight. After treating the cells with Th-Ir (5 μM) for 8 h, the cells were incubated with 10 μM 2',7'-dichlorofluorescein (H2DCFDA) for 20 min at 37 °C in the dark. Then, the cells were rinsed twice with serum-free DMEM. Observation was performed using a laser confocal scanning microscope (λex = 488 nm; λem = 530±20 nm).

[0089] (2)Experimental results

[0090] As Figure 11 shown, compared with untreated cells, Th-Ir could increase the fluorescence intensity of 2',7'-dichlorofluorescein (DCF), indicating that more reactive oxygen species (ROS) were generated.

[0091] The above results indicate that the cyclometalated iridium complex Th-Ir prepared in the present invention can significantly increase the level of intracellular ROS.

[0092] 3. Damage to mitochondrial DNA by cyclometalated iridium complex

[0093] (1)Experimental method

[0094] The MDA-MB-231 cells were incubated with Th-Ir (5 μM) for 8 h, then stained with Pico-Green for 30 min, rinsed with sterile PBS, and immediately imaged with a confocal microscope. λex = 488 nm; λem = 520±20 nm.

[0095] (2)Experimental results

[0096] As Figure 12 shown, compared with the control cells, the fluorescence of Pico-Green in Th-Ir-treated cells was significantly reduced, indicating that Th-Ir can induce mitochondrial DNA damage.

[0097] The above results indicate that: the cyclometalated iridium complex Th-Ir prepared in the present invention can significantly induce mitochondrial DNA damage.

[0098] Application Example 5. This application example studies the anti-tumor effect of the cyclometalated iridium complex, that is, explains the mechanism of the anti-tumor effect of the cyclometalated iridium complex prepared in the present invention from multiple aspects. The cytotoxicity of the complex Th-Ir against different tumor cells was determined by the tetrazolium salt (MTT) colorimetric method, and the in vivo anti-tumor effect of the complex Th-Ir was also determined. The specific process is as follows:

[0099] 1. Determination of the cytotoxicity of the complex Th-Ir against different tumor cells

[0100] (1)Experimental method

[0101] Using the cyclometalated iridium complex Th-Ir prepared in Example 1 as the experimental group and cisplatin as the control group, their cytotoxicity against the test tumor cells (MDA-MB-231, A549 (human lung cancer cell line), HeLa (human cervical cancer cell line), 4T1 (mouse breast cancer cell line), and MCF-10A (non-tumorigenic mammary epithelial cell line)) was determined respectively. The specific determination method is as follows:

[0102] Determined by the tetrazolium salt (MTT) colorimetric method. The test tumor cells were digested into single-cell suspensions with trypsin respectively, counted using a hemocytometer, and the cell concentration was adjusted to 5×10 4 / mL, inoculated into 96-well plates, 160 μL per well. After culturing for 24 h, different concentrations of drugs (cyclometalated iridium complex Th-Ir and cisplatin) were added, and the cells were placed under normoxic conditions (cultured in an incubator containing 5% CO2) or hypoxic conditions (cultured under hypoxic conditions of 1% O2, 5% CO2, and 94% N2), and incubated at 37 °C for 48 h. 20 μL of MTT was added per well 4 h before the end of incubation. After 4 h, the supernatant was discarded, 150 μL of DMSO was added per well, and after vibrating for 5 minutes, the OD value was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the wavelength was set at 595 nm.

[0103] Calculate the survival rate of the tested tumor cells, and plot the graph and calculate the IC 50 value to evaluate the anti-tumor activity of the cyclometalated iridium complex.

[0104] (2)Experimental results

[0105] The IC 50 value of the cyclometalated iridium complex prepared in Example 1 against tumor cells under normoxic conditions is shown in Table 1. It can be seen that the IC 50 value of Th-Ir is in the range of 2.0 - 3.2 μM, about 10 times that of cisplatin.

[0106] Table 1 IC 50 value of the cyclometalated iridium complex prepared in the present invention against tumor cells

[0107]

[0108] The results show that the cyclometalated iridium complex Th-Ir prepared in the present invention has good anti-tumor activity.

[0109] 2. Determination of in vivo anti-tumor efficacy of Th-Ir

[0110] (1)Experimental method

[0111] After 7 days of adaptive feeding of experimental mice, 4T1 tumor cells (containing 2 × 10 6Cells (100 μL PBS) were subcutaneously inoculated on the right side of female BALB / c mice, and then subcutaneously inoculated on the left side 7 days later. The tumor-bearing mice were randomly divided into three groups: (a) control group, (b) cisplatin group, and (c) Th-Ir group. Before each use, the compound was freshly dissolved in 2% DMSO, 4% PEG400, 4% Tween80 and 90% PBS, and cisplatin was dissolved in physiological saline. The mice in the experimental groups were injected intratumorally with Th-Ir and cisplatin (50 μL, 3 mg / kg) on days 0 / 4 / 8, and the mice in the control group were injected with physiological saline (50 μL) synchronously. The body weight and tumor volume of the mice were recorded every 2 days. The tumor volume (V) was calculated as V = W 2 × L / 2, (where W is the tumor width and L is the tumor length). The mice were sacrificed on day 14 for further determination.

[0112] (2) Experimental results

[0113] Figure 13 The body weight (right figure) and tumor volume (left figure) of the mice during drug administration were shown. During the treatment process, the body weights of the mice in each group remained stable. At the same time, after calculation, the inhibition rate of the primary tumor volume in the Th-Ir treatment group was 50.5%, and the tumor growth inhibition rate of cisplatin was 36.9%.

[0114] The results showed that the cyclometalated iridium complex Th-Ir prepared in Example 1 had excellent in vivo antitumor efficacy.

[0115] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope 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, Comprising the following steps: S1: Performing a coordination reaction between iridium trichloride and 2-phenylpyridine to obtain an iridium precursor; S2: Performing a Mannich reaction between 1,10-phenanthroline-5,6-dione and 2-thiophenecarboxaldehyde, and then performing a methylation reaction to obtain a ligand; S3: Reacting the iridium precursor with the ligand to obtain the compound of formula I; In S3, the molar ratio of the iridium precursor to the ligand is 1:2 to 3.

3. The method for preparing the compound of formula I according to claim 2, characterized in that: The molar ratio of the 1,10-phenanthroline-5,6-dione to the 2-thiophenecarboxaldehyde is 1:0.9 to 2; and / or, the catalyst for the Mannich reaction is selected from any one of ammonium acetate, ammonium carbonate, and ammonium chloride.

4. The method for preparing the compound of formula I according to claim 2, characterized in that: In S2, the reaction temperature of the Mannich reaction is 100°C to 130°C; and / or, the reaction time of the Mannich reaction is 3 to 9 h.

5. The preparation method of the compound of formula I according to claim 2, wherein: In S3, the reaction temperature is 40°C to 60°C; and / or, the reaction time is 4 to 8 h.

6. A pharmaceutical composition, characterized in that: Comprising the compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof, and optionally, a pharmaceutically acceptable carrier, diluent, and / or adjuvant.

7. Use of the compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as claimed in claim 6 in the preparation of at least one of a molecular probe, a tumor imaging agent, and an anti-tumor drug; the tumor is selected from at least one of breast cancer, cervical cancer, and lung cancer.

8. The application according to claim 7, wherein: The anti-tumor effect of the anti-tumor drug is selected from at least one of inhibiting tumor growth, inducing DNA damage in tumor cells, inducing mitochondrial dysfunction in tumor cells, and increasing the intracellular ROS level in tumor cells.

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