Metal ion affinity cyclometalated iridium complex as well as preparation method and application thereof

Abstract

The invention discloses a metal ion affinity cyclometalated iridium complex as well as a preparation method and application thereof. The cyclometalated iridium complex has a structure of a compound as shown in a formula I: # imgabs0 #. The cyclometalated iridium complex can be well taken by tumor cells, has good anti-tumor activity, and can redistribute endogenous Zn < 2 + > from cytoplasm to mitochondria, so that excessive Zn < 2 + > in the mitochondria of cancer cells is accumulated, the zinc homeostasis of the mitochondria is adjusted, and the obvious change of the Zn < 2 + > level of the whole cell is not caused; the complex has excellent cytotoxicity which is about 10 times that of cis-platinum, and an in-vivo anti-tumor result shows that the primary tumor inhibition rate of the complex reaches 50.5% and is higher than that of the cis-platinum; the complex can also target mitochondria, improve the ROS level in cells and induce mitochondrial dysfunction and mitochondrial DNA damage.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical chemistry, and in particular to a metal ion affinity cyclometallated iridium complex and a preparation method and application thereof. Background Art

[0002] Zinc is the second most abundant transition metal in the human body, and the precise regulation of its intracellular homeostasis and distribution plays a key role in physiological and pathological processes. About 30% to 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⁺) are bound to proteins and enzymes to maintain their structure and stability, resulting in low concentrations of free Zn²⁺ in the cytoplasm or isolated in organelles and vesicles. However, free Zn²⁺ is also indispensable for many key cellular processes (such as signal transduction, cell metabolism, cytoskeleton assembly, immune regulation, etc.). Studies have found that Zn²⁺ overload can produce reactive oxygen species (ROS) through electron leakage during mitochondrial aerobic respiration, leading to mitochondrial DNA (mtDNA) damage and high levels of interferon and inflammatory cytokine production. At the same time, Zn²⁺ is also involved in the formation of zinc-dependent transcription factors and proteins related to DNA repair, significantly affecting the process of cell death mediated by DNA damage. 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 inputting large amounts of exogenous zinc ions. For example, the pH-sensitive zeolite imidazolate framework (ZIF-8) can release Zn²⁺ in lysosomes, resulting in a sudden increase in the concentration of Zn²⁺ in cancer cells, and ultimately efficiently activates 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), thereby avoiding the side effects caused by exogenous zinc input. However, how to regulate zinc levels in mitochondria through metal drugs to coordinate anti-tumor therapy remains an underexplored area. Summary of the invention

[0004] The present invention aims to solve at least 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 a preparation method and application thereof.

[0005] 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 an isomer thereof, or a pharmaceutically acceptable salt thereof: .

[0006] 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: 1,10-phenanthroline-5,6-dione and 2-thiophenecarboxaldehyde are subjected to a Mannich reaction and then subjected to a methylation reaction to obtain a ligand; S3: reacting the iridium precursor with the ligand to obtain the compound of formula I.

[0007] 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, 1:2.2.

[0008] In some embodiments of the present invention, the iridium trichloride may be a hydrate of iridium trichloride.

[0009] In some embodiments of the present invention, the molar ratio of 1,10-phenanthroline-5,6-dione to 2-thiophenecarboxaldehyde is 1:0.9-2, such as 1:1-1.5, 1:1.2.

[0010] In some embodiments of the present invention, the catalyst for the Mannich reaction includes any one of ammonium acetate, ammonium carbonate, and ammonium chloride.

[0011] In some embodiments of the present invention, the molar ratio of the 1,10-phenanthroline-5,6-dione to the catalyst is 1:5-10, such as 1:6-9; 1:8-9, etc.

[0012] In some embodiments of the present invention, in S3, the molar ratio of the iridium precursor to the ligand is 1:2-3, for example, 1:2-2.4, 1:2.2.

[0013] In some embodiments of the present invention, the temperature of the coordination reaction in S1 is 120°C~150°C, such as 130°C~140°C; the time of the coordination reaction is 12~48h, such as 18~36h; the solvent of the coordination reaction includes at least one of dichloromethane, methanol, ethylene glycol ethyl ether, and ultrapure water, such as a mixed solvent of dichloromethane and methanol, and a mixed solvent of ethylene glycol ethyl ether and ultrapure water; the volume ratio of dichloromethane and methanol is 2~3:1, such as 2~2.4:1, 2:1; the volume ratio of ethylene glycol ethyl ether and ultrapure water is 2~4:1, such as 3~4:1, 3:1.

[0014] 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 hours, such as 4 to 8 hours, 5 to 7 hours, etc.; the reaction solvent of the Mannich reaction includes glacial acetic acid, formic acid, ethanol, methanol, dimethyl sulfoxide (DMSO),N,N - at least one dimethylformamide.

[0015] In some embodiments of the present invention, in S2, the methylation reagent used in the methylation reaction includes methyl halide, such as any one of methyl chloride, methyl bromide, and methyl iodide; the reaction temperature of the methylation reaction is 35°C to 55°C, such as 40°C to 50°C; the methylation reaction time is 1 to 5 hours, such as 2 to 4 hours; the methylation reaction is carried out under alkaline conditions; the solvent of the methylation reaction includes dimethyl sulfoxide, N,N - At least one of dimethylformamide, N-methylpyrrolidone and acetonitrile.

[0016] In some embodiments of the present invention, in S3, the reaction is carried out under an inert environment; the reaction temperature is 40°C~60°C, such as 45°C~55°C; the reaction time is 4~8h, such as 5~7h; the reaction solvent includes at least one of dichloromethane, methanol, ethylene glycol ethyl ether, and ultrapure water, such as a mixed solvent of dichloromethane and methanol, and a mixed solvent of ethylene glycol ethyl ether and ultrapure water; the volume ratio of dichloromethane and methanol is 2~3:1, such as 2~2.4:1, 2:1; the volume ratio of ethylene glycol ethyl ether and ultrapure water is 2~4:1, such as 3~4:1, 3:1.

[0017] 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.

[0018] The pharmaceutical composition of the present invention is suitable for a variety of administration routes, and thus can be prepared into any pharmaceutically acceptable dosage form. For example, the pharmaceutical composition can be administered to patients or subjects who need such treatment by oral, parenteral, rectal or transpulmonary administration. When used for oral administration, the pharmaceutical composition can be prepared into oral preparations, for example, conventional oral solid preparations, such as tablets, capsules, pills, granules, etc.; it can also be prepared into oral liquid preparations, such as oral solutions, oral suspensions, syrups, etc. When preparing oral preparations, suitable fillers, binders, disintegrants, lubricants, etc. can be added. When used for parenteral administration, the pharmaceutical composition can also be prepared into injections, including injections, sterile powders for injection, and concentrated solutions for injection. When preparing injections, conventional methods in the existing pharmaceutical field can be used for production. When preparing injections, additives can be omitted or appropriate additives can be added according to the properties of the drug. When used for rectal administration, the pharmaceutical composition can be prepared into suppositories, etc. When used for transpulmonary administration, the pharmaceutical composition can be prepared into inhalation preparations, aerosols, powder sprays or sprays, etc.

[0019] 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 not limited to: water, saline solution, DMSO, 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™.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] In some embodiments of the invention, the tumor comprises at least one of breast cancer, cervical cancer or lung cancer.

[0030] 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 tumor cell DNA (such as mtDNA) damage, inducing tumor cell mitochondrial dysfunction, and inducing increased ROS levels in tumor cells.

[0031] 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.

[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, 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.

[0033] The beneficial effects of the present invention are: The compound of formula I of the present invention can be well taken up by tumor cells and has good anti-tumor activity. The complex can convert endogenous Zn 2+ Redistribution from the cytoplasm to the mitochondria, resulting in excess Zn in cancer cell mitochondria 2+ accumulation, regulating mitochondrial zinc homeostasis without causing overall cellular Zn 2+ The complex has excellent cytotoxicity, which is about 10 times that of cisplatin. The in vivo anti-tumor results show that the complex has an inhibition rate of 50.5% on primary tumors, which is higher than cisplatin. The complex can also target mitochondria, increase intracellular ROS levels, and induce mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage.

[0034] The compound of formula I of the present invention has good anti-tumor activity as a cyclometallated iridium complex, and its preparation method is simple and easy with low cost; therefore, the complex prepared by the present invention provides new clues for the development of new metal anticancer complexes. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is the electrospray mass spectrum of the ligand prepared in the embodiment of the present invention.

[0036] Figure 2 It is the nuclear magnetic resonance hydrogen spectrum of the ligand prepared in the embodiment of the present invention.

[0037] Figure 3It is the electrospray mass spectrum of the cyclometalated iridium complex Th-Ir prepared in the embodiment of the present invention.

[0038] Figure 4 It is the hydrogen nuclear magnetic resonance spectrum of the cyclometalated iridium complex Th-Ir prepared in the embodiment of the present invention.

[0039] Figure 5 It is the carbon nuclear magnetic resonance spectrum of the cyclometalated iridium complex Th-Ir prepared in the embodiment of the present invention.

[0040] Figure 6 The UV-visible absorption (B) and emission spectra (A) of the cyclometalated iridium complex Th-Ir of the present invention in different solvents.

[0041] Figure 7 The cyclometalated iridium complex Th-Ir of the present invention is prepared by adding Zn to DMSO-d6. 2+ 600 MHz before and after 1 HNMR spectrum changes.

[0042] Figure 8 It is a cellular localization map of the cyclometallated iridium complex Th-Ir of the present invention in MAD-MB-231 cells; wherein, "LTDR" and "MTDR" represent lysosomal deep red probe and mitochondrial deep red probe, respectively; "Brightfield" represents bright field; "Overlay" represents overlay; and "Probe" represents probe.

[0043] Fig. 9 The figure is the zinc ion distribution diagram of the cyclometalated iridium complex Th-Ir of the present invention in cells.

[0044] Fig.10 This is a functional effect diagram of the cyclometalated iridium complex Th-Ir of the present invention in destroying the mitochondria of MAD-MB-231 cells.

[0045] Fig.11 This is a graph showing the effect of the cyclometalated iridium complex Th-Ir of the present invention on the ROS level in MAD-MB-231 cells.

[0046] Fig.12 This is a diagram showing the damage effect of the cyclometalated iridium complex Th-Ir of the present invention on mitochondrial DNA of MAD-MB-231 cells.

[0047] Fig.13 Figure 2 is a graph showing changes in primary tumor volume (left) and body weight (right) of the mice of the present invention during in vivo drug treatment. DETAILED DESCRIPTION

[0048] 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.

[0049] Example 1: This example prepares a cyclometalated iridium complex Th-Ir (i.e., a compound of formula I), and the specific process is as follows:

[0050] in, The structural formula is .

[0051] S1. After mixing iridium trichloride hydrate and the ligand at a molar ratio of 1:2.2, the mixture was dissolved in a mixed solvent of ethylene glycol ethyl ether and ultrapure water (volume ratio of 3:1), and the coordination reaction was carried out at 135 °C under reflux stirring for 24 h. The mixture was cooled to room temperature and filtered. The obtained solid product was washed three times with ultrapure water and ether respectively, and dried in vacuum to obtain an iridium precursor. S2. 1,10-phenanthroline-5,6-dione (0.50 g, 2.3 mmol), 2-thiophenecarboxaldehyde (0.26 mL, 2.76mmol), 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. The reaction was stirred for 4 hours. After the reaction, the mixture was cooled to room temperature. 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 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 hours. Methyl iodide (0.338 g, 2.384 mmol) was added to the reaction system and stirred at 40°C for 3 hours. The mixture was added with 200 mL of water to precipitate a brown solid, which was filtered to obtain the ligand; S3. The iridium precursor (1 eq.) obtained in step S1 and the ligand (2.2 eq.) obtained in step S2 are mixed and dissolved in a dichloromethane / methanol mixed solvent (V / V = 2 / 1), and stirred and refluxed at 50 °C under a nitrogen atmosphere for 6 h. The solution is cooled to room temperature, and a saturated NH4PF6 (6.0 eq.) solution is added. The reaction product is separated and purified by silica gel column chromatography using a dichloromethane / methanol mixed solvent (V / V = 20 / 1) as an eluent, and then vacuum dried to obtain the cyclometalated iridium complex Th-Ir; Among them, the ligand prepared by S2 is 2-(thiophene-2-yl)-1-methylimidazo[4,5-f][1,10]phenanthroline, and its electrospray ionization mass spectrum and nuclear magnetic resonance hydrogen spectrum are as follows: Figure 1 and Figure 2 As shown, specifically: 1 H 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). ESI-MS: Calculated: m / z = 316.08; Found: m / z = 317.43.

[0052] The electrospray mass spectrometry, hydrogen nuclear magnetic resonance spectrum and carbon nuclear magnetic resonance spectrum of the compound of formula I are as follows: Figure 3 , Figure 4 and Figure 5 As shown, specifically: 1H 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). 13 C 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. ESI-MS: Calculated: m / z = 817.17 [M-PF6] + ; Exp. value: m / z = 817.42 [M-PF6] + .

[0053] Application Example 1: This application example tests the UV-visible absorption and emission spectra of a cyclometallated iridium complex. The specific process is as follows: The cyclometalated iridium complex Th-Ir prepared in Example 1 was used as the experimental group, and PBS (phosphate buffered saline solution), CH3CN (acetonitrile solution) and CH2Cl2 (dichloromethane solution) were used as solvents to prepare sample solutions with a concentration of 20 μM, and then a two-photon UV-visible spectrophotometer was used to record the UV-visible absorption spectrum of the cyclometalated iridium complex Th-Ir and the UV emission spectrum under an excitation wavelength of 405 nm.

[0054] The ultraviolet-visible absorption spectrum of the cyclometallated iridium complex Th-Ir prepared in Example 1 is ( Figure 6 B) and UV emission spectra using an excitation wavelength of 405 nm ( Figure 6 A) Figure 6 As shown, it can be seen that in PBS, CH3CN and CH2Cl2, the UV-visible spectra of Th-Ir show similar absorption peaks, which are attributed to the π-π* transition of the internal ligand (250~350 nm) and the metal-ligand charge transfer (350~470 nm), respectively. Under 405 nm excitation, Th-Ir exhibits a strong emission at 605 nm.

[0055] Application Example 2: This example determines the effect of cyclometalated iridium complexes on Zn 2+ The specific process is: 1 H-NMR titration: recording the Th-Ir dissolved in deuterated DMSO 1 H-NMR spectrum; ZnSO4 (Zn 2+ ) solution was added to the above sample and recorded again after 24h.

[0056] like Figure 7 As shown, 1 H NMR spectrum shows that Zn 2+ After titration, the signals of Th-Ir at the q, t, s, v, r, and y positions all shifted to the high field.

[0057] The above results show that: Th-Ir and Zn 2+ The binding is mainly achieved through the N atom in the imidazole ring and the S atom in the thiophene ring, while the methyl substitution on the imidazole ring will weaken but not eliminate the Zn 2+ The combining ability.

[0058] Application Example 3: This application example determines the uptake and distribution of cyclometallated iridium complexes in tumor cells and the regulatory effect of zinc ions. The specific process is as follows: MDA-MB-231 (human breast cancer cell line) cells were seeded in confocal culture dishes overnight, 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. Ir (III) complex: λex = 405 nm, λem = 605±20 nm; LTDR / MTDR: λex = 633 nm, λem = 665±20 nm. At the same time, ICP-MS was used to study the regulation of intracellular Zn by Th-Ir in MAD-MB-231 cells. 2+ Distributed capabilities.

[0059] like Figure 8 As shown in the results, Th-Ir was observed to effectively penetrate into MAD-MB-231 cells within 1 h and was mainly accumulated in mitochondria. It co-localized well with lysosome-specific stain (LTDR) and mitochondria-specific stain (MTDR), with Pearson correlation coefficients of 0.88 and 0.86, respectively.

[0060] The distribution of zinc ions in cells of the cyclometallated iridium complex Th-Ir is as follows Fig. 9 As shown in the results, after treatment with Th-Ir (5 μM, 8 h) in MAD-MB-231 cells, the total intracellular zinc content did not change significantly; however, the zinc content in the cytoplasm was decreased, while the zinc content in the mitochondria was significantly increased compared with the control cells.

[0061] The above results show that the complex Th-Ir can target mitochondria well and redistribute endogenous zinc from the cytoplasm and vesicles to mitochondria, thereby leading to excessive Zn in cancer cell mitochondria. 2+ accumulation to regulate mitochondrial zinc homeostasis.

[0062] Application Example 4: This application example measures the damage of cyclometallated iridium complexes to subcellular organelle functions, that is, explains the damage of subcellular organelle functions of cyclometallated iridium complexes prepared in Example 1 from multiple aspects, and observes the damage of Th-Ir to mitochondrial function, the influence of ROS levels in cells and the damage to mitochondrial DNA by confocal microscopy. The specific process is as follows: 1. Damage to mitochondrial function by cyclometallated iridium complexes (1) Experimental methods MDA-MB-231 cells were seeded in 35 mm confocal culture dishes and incubated overnight at 37°C in an atmosphere of 5% CO2. After treatment 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 examined with a confocal microscope. Excitation wavelength λex = 488 nm; monomer fluorescence emission wavelength λem = 530 ± 20 nm; aggregate fluorescence emission wavelength λem = 585 ± 20 nm.

[0063] (2) Experimental results like Fig.10 As shown, Th-Ir triggered a significant loss of mitochondrial membrane potential (MMP) as detected by JC-1 staining, which was reflected by the red to green color change of 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarboxycyanine iodide (JC-1).

[0064] The above results indicate that the cyclometallated iridium complex Th-Ir prepared by the present invention can significantly cause mitochondrial dysfunction.

[0065] 2. Effects of cyclometallated iridium complexes on ROS levels in cells (1) Experimental methods MDA-MB-231 cells were seeded in 35 mm confocal dishes and cultured overnight. After being treated with Th-Ir (5 μM) for 8 h, the cells were incubated with 10 μM 2',7'-dichlorofluorescein (H2DCFDA) at 37 °C in the dark for 20 min. Then, the cells were washed twice with serum-free DMEM. The images were observed using a laser confocal scanning microscope (λex = 488 nm; λem = 530±20 nm).

[0066] (2) Experimental results like Fig.11 As shown, Th-Ir increased the fluorescence intensity of 2',7'-dichlorofluorescein (DCF) compared with untreated cells, indicating that more reactive oxygen species (ROS) were generated.

[0067] The above results show that the cyclometallated iridium complex Th-Ir prepared by the present invention can significantly increase the level of intracellular ROS.

[0068] 3. Damage of cyclometallated iridium complexes to mitochondrial DNA (1) Experimental methods 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 by confocal microscopy. λex = 488 nm; λem = 520 ± 20 nm.

[0069] (2) Experimental results like Fig.12 As shown, the fluorescence of Pico-Green was significantly attenuated in Th-Ir-treated cells compared with control cells, indicating that Th-Ir can induce mitochondrial DNA damage.

[0070] The above results show that the cyclometallated iridium complex Th-Ir prepared by the present invention can significantly induce mitochondrial DNA damage.

[0071] Application Example 5: This application example studies the anti-tumor effect of cyclometallated iridium complexes, that is, explains the anti-tumor mechanism of the cyclometallated iridium complex prepared by the present invention from multiple aspects, and determines the cytotoxicity of the complex Th-Ir to different tumor cells by using tetrazolium salt (MTT) colorimetry, and determines the in vivo anti-tumor effect of the complex Th-Ir. The specific process is as follows: 1. Determination of cytotoxicity of Th-Ir complex on different tumor cells (1) Experimental methods The cyclometalated iridium complex Th-Ir prepared in Example 1 was used as the experimental group, and cisplatin was used as the control group to measure the cytotoxicity of the tested 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 breast epithelial cell line)). The specific measurement 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 (cyclometalated iridium complex Th-Ir and cisplatin), placed in normoxia (cells cultured in an incubator containing 5% CO2) or hypoxia (cells cultured under hypoxic conditions of 1% O2, 5% CO2, 94% N2), incubated at 37 °C for 48 h, and added MTT 20 μL / well 4 h before the end of incubation. After 4 h, the supernatant was discarded, DMSO 150 μL / well was added, and the OD value was measured with a microplate reader after shaking for 5 minutes, with the wavelength set to 595 nm.

[0072] Calculate the survival rate of the tested tumor cells, plot and calculate the IC50 The antitumor activity of cyclometallated iridium complexes was evaluated.

[0073] (2) Experimental results Example 1 The IC of the cyclometallated iridium complex prepared in tumor cells under normoxic conditions 50 The values ​​are shown in Table 1. It can be seen that the IC of Th-Ir 50 The values ​​were in the range of 2.0~3.2 μM, which was approximately 10 times that of cisplatin.

[0074] Table 1 IC values ​​of the cyclometallated iridium complexes prepared by the present invention on tumor cells 50 value

[0075] The results show that the cyclometallated iridium complex Th-Ir prepared by the present invention has good anti-tumor activity.

[0076] 2. Determination of the anti-tumor efficacy of Th-Ir in vivo (1) Experimental methods After the experimental mice were adaptively fed for 7 days, 4T1 tumor cells (containing 2 × 10 6 cells, 100 μL PBS) were subcutaneously inoculated into the right side of BALB / c female mice, and then subcutaneously inoculated into the left side 7 days later. The tumor-bearing mice were randomly divided into 3 groups: (a) control group, (b) cisplatin group, and (c) Th-Ir group. Before each use, the compounds were freshly dissolved in 2% DMSO, 4% PEG400, 4% Tween80 and 90% PBS, and cisplatin was dissolved in normal saline. The mice in the experimental group were injected with Th-Ir and cisplatin (50 μL, 3 mg / kg) intratumorally on days 0 / 4 / 8, and the mice in the control group were injected with normal saline (50 μL) at the same time. The weight and tumor volume of the mice were recorded every 2 days. The tumor volume (V) was calculated as V = W 2 × L / 2, (W is tumor width, L is tumor length). Mice were sacrificed on day 14 for further determination.

[0077] (2) Experimental results Fig.13 The mouse weight (right) and tumor volume (left) during the drug administration period are shown. During the treatment, the weight of mice in each group remained stable. At the same time, it was calculated that the primary tumor volume inhibition rate in the Th-Ir treatment group was 50.5%, and the cisplatin tumor growth inhibition rate was 36.9%.

[0078] The results show that the cyclometallated iridium complex Th-Ir prepared in Example 1 has excellent in vivo anti-tumor efficacy.

[0079] 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 made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. A compound of formula I, or an isomer 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: 1,10-phenanthroline-5,6-dione and 2-thiophenecarboxaldehyde are subjected to a Mannich reaction and then to a methylation reaction to obtain a ligand; S3: reacting the iridium precursor with the ligand to obtain the compound of formula I.

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 2-thiophenecarboxaldehyde is 1:0.9-2; and / or the catalyst of the Mannich reaction includes 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 S3, the molar ratio of the iridium precursor to the ligand is 1:2-3.

5. 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 hours.

6. The method for preparing the compound of formula (I) according to claim 2, characterized in that: In S3, the reaction temperature is 40°C to 60°C; and / or the reaction time is 4 to 8 hours.

7. 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.

8. 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 claim 7 in the preparation of at least one of a molecular probe, a tumor imaging agent, and an anti-tumor drug.

9. The use according to claim 8, characterized in that: The tumor includes at least one of breast cancer, cervical cancer or lung cancer.

10. The use according to claim 8, characterized in that: The anti-tumor effect of the anti-tumor drug includes at least one of inhibiting tumor growth, inducing DNA damage in tumor cells, inducing mitochondrial dysfunction in tumor cells, and inducing an increase in the level of ROS in tumor cells.

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