Ring metal iridium (III) complex as well as preparation method and application thereof

Abstract

The invention relates to the technical field of antitumor drugs, in particular to a cyclometalated iridium (III) complex as well as a preparation method and application thereof. The cyclometalated iridium (III) complex has a structure as shown in a formula I which is described in the specification. Experimental results show that the cyclometalated iridium complex provided by the invention has excellent anti-tumor activity, can enter tumor cells in an energy-dependent manner, and gathers on endoplasmic reticulum and lysosome. Caspase-1 can be activated to induce cells to generate a large amount of ROS, the cells are further induced to generate pyroptosis by activating the Caspase-1, and a large amount of inflammatory factors are released. In in-vitro experiments, migration of tumor cells can be effectively inhibited. In further in-vivo experiments, the cyclometalated iridium complex can influence normal development of zebrafish embryo blood vessels to different degrees. The cyclometalated iridium complex provided by the invention can induce tumor cell death by inhibiting angiogenesis.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor drugs, and in particular to a cyclometallated iridium (III) complex and a preparation method and application thereof. Background Art

[0002] Tumor metastasis is an important cause of death in cancer patients. Although the development of anticancer drugs continues to advance, residual tumor cells in the body often increase the risk of tumor metastasis. The survival rate of patients with tumor metastasis is very low, and the 5-year survival rate of patients with solid tumors ranges from only 5% to 30%. Tumor metastasis is a multi-program process, including the invasion stage, circulation stage, and colonization stage. During tumor metastasis, cancer cells leave the original lesion and spread to different parts of the body through the blood or lymphatic system. Tumor cells will settle at the distal end, attach and grow to form secondary sites, and eventually lead to serious complications. At present, only late-stage secondary tumors can be detected clinically, but at this time, metastasis to multiple sites has often occurred, greatly increasing the difficulty of treatment. At present, the main treatment options for cancer metastasis include blocking epithelial-mesenchymal transition, inhibiting tumor angiogenesis, and targeting metastasis inhibitors. In clinical studies, Bevacizumab has been approved for the treatment of lung cancer, colon cancer, and brain cancer, and can inhibit the formation of tumor angiogenesis. Denosumab has been approved for the prevention of multiple myeloma and bone metastasis patients, which can inhibit the activation of osteoclasts and thus inhibit the bone metastasis of cancer cells. However, during use, due to the high toxicity, strong side effects and the emergence of drug resistance, the patient's quality of life has not changed significantly.

[0003] Therefore, the development of drugs that can inhibit the formation of tumor angiogenesis is of great significance in the field of anti-cancer. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a cyclometallated iridium (III) complex and a preparation method and application thereof. The prepared cyclometallated iridium (III) complex can induce tumor cell death by inhibiting angiogenesis.

[0005] To achieve the above object, the present invention provides a cyclometallated iridium (III) complex having a structure shown in Formula I:

[0006]

[0007] in, Select from any of the following structures:

[0008]

[0009] Indicates the connection location.

[0010] Preferably, the anion of the cyclometalated iridium (III) complex is PF 6 - .

[0011] The general chemical formula of the cyclometallated iridium (III) complex is [Ir(CN) 2 L](PF 6 ), wherein the ligand L is 1-ethyl-7-methyl-4-oxo-N-(1,10-phenanthroline-5-yl)-1,4-dihydro-1,8-naphthyridine-3-carboxamide, CN=2-(p-tolyl)pyridine (tpy), denoted as Ir1; CN=2-(2,4-difluorophenyloxazole)pyridine (dfppy), denoted as Ir2; CN=1-phenylisoquinoline (piq), denoted as Ir3.

[0012] Specifically, the cyclometallated iridium (III) complex has any of the following structures:

[0013]

[0014] The present invention provides a method for preparing the above-mentioned cyclometallated iridium (III) complex, comprising the following steps:

[0015] A) reacting the nalidixic acid of formula Ⅰ-a with the 1,10-phenanthroline-5-amino group of formula Ⅰ-b to obtain the ligand of formula Ⅰ-c;

[0016] B) reacting the ligand represented by formula Ⅰ-c with the metal iridium complex represented by formula Ⅰ-d, formula Ⅰ-e or formula Ⅰ-f to obtain the cyclometalated iridium complex represented by formula Ⅰ;

[0017]

[0018] X is halogen.

[0019] The X is F, Cl or Br, preferably Cl.

[0020] The present invention uses nalidixic acid (shown in formula I-a) and 5-amino-1,10-phenanthroline (shown in formula I-b) to react to prepare a ligand (shown in formula I-c).

[0021] The present invention has no particular limitation on the sources of the nalidixic acid and 5-amino-1,10-phenanthroline, which can be commercially available or prepared according to methods well known to those skilled in the art.

[0022] The reaction in step A) is preferably carried out under the catalytic action of a catalyst.

[0023] Specifically, nalidixic acid and 5-amino-1,10-phenanthroline are dissolved in organic solvents respectively, and reacted under the catalytic action of a catalyst.

[0024] The organic solvent is preferably dichloromethane.

[0025] The catalyst is preferably one or more of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 4-dimethylaminopyridine (DMAP).

[0026] The reaction temperature is preferably 30-50°C, more preferably 45°C; the reaction time is preferably 20-24h, more preferably 24h.

[0027] After the reaction is completed, post-treatment is performed. Preferably, the reaction solution is cooled, filtered, and the precipitate is washed with anhydrous ethanol and dried to obtain the ligand represented by formula Ⅰ-c.

[0028] Then the ligand is reacted with a metal iridium precursor compound shown in formula I-d, formula I-e or formula I-f to obtain a cyclometalated iridium complex shown in formula I.

[0029] Preferably, the reaction is carried out under reflux in N,N-dimethylformamide.

[0030] Preferably, the reaction is carried out under light-proof conditions.

[0031] The reaction is preferably carried out in an inert atmosphere.

[0032] The present invention has no particular limitation on the inert atmosphere, and it can be an inert atmosphere well known to those skilled in the art, preferably a nitrogen or argon atmosphere.

[0033] After the reaction is completed, preferably, an excess of saturated NH 4 PF 6 solution, and collect the precipitated solid, which is the cyclometalated iridium complex.

[0034] Preferably, the crude cyclometalated iridium complex is purified by column chromatography.

[0035] The eluent for the column chromatography is preferably a mixed solvent of dichloromethane and methanol.

[0036] The reaction scheme of the above reaction is as follows:

[0037]

[0038] The present invention has detected the anti-tumor activity of the above-mentioned cyclometal iridium complex, and the results show that the above-mentioned cyclometal iridium complex provided by the present invention has a high inhibitory effect on tumor cells (MCF-7 cells, Hela cells), mainly enters cells in an energy-dependent manner, and is located in the endoplasmic reticulum and lysosomes. The cyclometal iridium complexes Ir1-Ir3 prepared in the specific embodiments of the present invention can induce cells to produce a large amount of reactive oxygen, and finally induce cell pyroptosis by activating Caspase-1. The results of in vitro experiments show that the cyclometal iridium complexes Ir1-Ir3 can inhibit cell migration. In further in vivo experiments, Ir1-Ir3 can inhibit the normal development of blood vessels in zebrafish embryos to varying degrees. This shows that the cyclometal iridium complex provided by the present invention can induce tumor cell death by inhibiting angiogenesis.

[0039] Based on this, the present invention provides the use of the above-mentioned cyclometallated iridium (III) complex or the cyclometallated iridium (III) complex prepared by the above-mentioned preparation method in the preparation of anti-tumor drugs.

[0040] Preferably, the tumor is a solid tumor.

[0041] More preferably, the tumor is one or more of breast cancer and cervical cancer.

[0042] Compared with the prior art, the present invention provides a cyclometallated iridium (III) complex, characterized in that it has a structure shown in Formula I. The cyclometallated iridium (III) complex contains metal ions in its structure and carries a charge. Compared with traditional organic small molecules, it enhances the in vivo penetration and retention effect. Moreover, the metal complex has a multi-coordinated configuration and can be modified with different ligands to achieve its excellent biological activity.

[0043] The experimental results show that the cyclometal iridium complex provided by the present invention has excellent anti-tumor activity. It can enter tumor cells in an energy-dependent manner and accumulate in the endoplasmic reticulum and lysosomes. It can induce cells to produce a large amount of ROS, activate Caspase-1 and induce cell pyroptosis, and release a large amount of inflammatory factors. In in vitro experiments, it can effectively inhibit the migration of tumor cells. In further in vivo experiments, the cyclometal iridium complex can affect the normal development of zebrafish embryonic blood vessels to varying degrees. This shows that the cyclometal iridium complex provided by the present invention can induce tumor cell death by inhibiting angiogenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a diagram showing the effect of the complex Ir1 in Example 2 on the vascular development in zebrafish embryos;

[0045] Figure 2This is a diagram showing the effect of the complex Ir2 in Example 2 on the vascular development in zebrafish embryos;

[0046] Figure 3 This is a diagram showing the effect of the complex Ir3 in Example 2 on the vascular development in zebrafish embryos. DETAILED DESCRIPTION

[0047] In order to further illustrate the present invention, the following is a detailed description in conjunction with the embodiments. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the invention.

[0048] All raw materials of the present invention are not particularly limited in their sources and can be purchased from the market or prepared according to conventional methods well known to those skilled in the art.

[0049] Example 1

[0050] (1) Preparation of ligand L:

[0051] 5-Amino-1,10-phenanthroline (0.40 g, 2 mmol), nalidixic acid (0.46 g, 2 mmol), EDC (0.76 g, 4 mmol) and DMAP (0.48 g, 4 mmol) were dissolved in dichloromethane (75 mL) and refluxed at 45°C for 24 h. After the reaction, the solution was cooled to room temperature, filtered, and the precipitate was washed with anhydrous ethanol and dried to obtain 0.18 g of light yellow powder with a yield of 21.9%.

[0052] Elemental Analysis C 24 H 19 N 5 O 2 (Molecular weight is 409.15), theoretical value: C, 70.40%; H, 4.68%; N, 17.10%; experimental value: C, 70.35%; H, 4.61%; N, 17.15%.

[0053] ESI-MS: [(M+H)] + Theoretical value: m / z=410.16, experimental value: m / z=409.8.

[0054] (2) Preparation of complex Ir1

[0055] Weigh [Ir(tpy) 2 Cl] 2 ·2H 2 O (0.22 g, 0.2 mmol) and ligand L (0.16 g, 0.4 mmol) were dissolved in N,N-dimethylformamide (25 mL), and argon was introduced for protection. The solution was refluxed at 130 °C in the dark for 5.5 h, and turned red. After the reaction was completed, the solution was cooled to room temperature and NH4 PF 6 The crude product was dissolved in a small amount of dichloromethane and separated by silica gel column chromatography using dichloromethane:methanol (85:1, v / v) as the eluent to obtain 0.12 g of bright orange powder with a yield of 27.9%.

[0056] Elemental Analysis C 48 H 39 F 6 Ir 7 O 2 P (molecular weight is 1083.24), theoretical value: C, 53.23%; H, 3.63%; N, 9.05%; experimental value: C, 53.19%; H, 3.59%; N, 9.11%.

[0057] ESI-MS: [M-PF 6 - ] + Theoretical value: m / z=938.28, experimental value: m / z=938.9.

[0058] (3) Preparation of complex Ir2

[0059] Weigh [Ir(dfppy) 2 Cl] 2 ·2H 2 O (0.23 g, 0.2 mmol) and ligand L (0.16 g, 0.4 mmol) were dissolved in N,N-dimethylformamide (25 mL), and argon was introduced for protection. The mixture was refluxed at 130 °C in the dark for 5.5 h. After the reaction was completed, the mixture was cooled to room temperature and NH 4 PF 6 The precipitate was stirred and filtered to obtain a crude product. The crude product was dissolved in a small amount of dichloromethane and separated by silica gel column chromatography using dichloromethane:methanol (110:1, v / v) as an eluent to obtain 0.13 g of a bright yellow powder with a yield of 28.9%.

[0060] Elemental Analysis C 46 H 31 F 10 Ir 7 O 2 P (molecular weight is 1127.17), theoretical value: C, 49.03%; H, 2.77%; N, 8.70%; experimental value: C, 48.97%; H, 2.72%; N, 8.78%.

[0061] ESI-MS: [M-PF 6 - ] +Theoretical value: m / z=982.21, experimental value: m / z=980.2.

[0062] (4) Preparation of complex Ir3

[0063] Weigh [Ir(piq) 2 Cl] 2 ·2H 2 O (0.24 g, 0.2 mmol) and ligand L (0.16 g, 0.4 mmol) were dissolved in N,N-dimethylformamide (25 mL), and argon was introduced for protection. The mixture was refluxed at 130 °C in the dark for 5.5 h. After the reaction was completed, the mixture was cooled to room temperature and NH 4 PF 6 The precipitate was precipitated by stirring, and the precipitate obtained by filtering was the crude product. The crude product was dissolved in dichloromethane and separated by silica gel column chromatography using dichloromethane:methanol (80:1, v / v) as eluent to obtain 0.16 g of bright red powder with a yield of 34.8%.

[0064] Elemental Analysis C 54 H 39 F 6 Ir 7 O 2 P (molecular weight is 1155.24), theoretical value: C, 56.15%; H, 3.40%; N, 8.49%; experimental value: C, 56.10%; H, 3.33%; N, 8.52%.

[0065] ESI-MS: [M-PF 6 - ] + Theoretical value: m / z=1010.28, experimental value: m / z=1010.2.

[0066] Example 2 In vitro antitumor activity experiment

[0067] (1) Anti-tumor activity test (MTT method): The anti-tumor ability of iridium complexes is mainly determined by the MTT method: The MTT method is a classic method for determining the anti-tumor activity of drugs. The operating steps are as follows:

[0068] When the cells grow to the logarithmic phase, they are digested with 0.25% trypsin to form a single cell suspension, the live cells are counted, and 5,000 cells (160 μL) are seeded in each well of a 96-well plate. The empty wells at the edge can be filled with PBS. 5% CO 2After 24 hours of incubation in the incubator, 40 μL of drugs of different concentrations diluted with culture medium were added respectively. After 44 hours of incubation in the incubator, 20 μL of MTT (dissolved in PBS) was added to each well. After 4 hours, the culture medium was removed and 100 μL of DMSO was added to each well. The wells were protected from light and shaken on a shaker for about 10 minutes. The OD value at 595 nm was immediately measured with an enzyme marker. The cell survival rate was calculated according to the formula, and the half lethal concentration (IC 50 This analysis method provides a reliable screening method based on the MTT classical experiment, which can be used to directly determine whether a compound has the ability to have anti-tumor activity.

[0069] Survival rate % = average OD value of drug-treated wells / average OD value of control wells × 100%

[0070] The test results are shown in Table 1 below:

[0071] Table 1

[0072]

[0073] a IC 50 The concentration of the complex corresponding to 50% inhibition of cell proliferation. The experimental data are the average values ​​obtained after three parallel experiments.

[0074] It can be seen from the experimental results that the three complexes have excellent anti-tumor activity and have a high inhibitory effect on the selected tumor cell lines.

[0075] (2) Experiment on inhibiting angiogenesis in zebrafish embryos:

[0076] Inhibiting angiogenesis is an important therapeutic goal for inhibiting tumor metastasis. The vascular structure of zebrafish is highly similar to that of other vertebrates, and vascular development can be studied by using the early embryonic stage of zebrafish. Since the vascular endothelial cells of Tg (Fli1: ​​EGFP) vascular green fluorescent transgenic zebrafish can express enhanced green fluorescent protein, its vascular development can be observed by fluorescence microscopy, and it is often used for the in vivo evaluation of angiogenesis inhibitors. When observing whether the complex affects angiogenesis, the development of the dorsal long axis vessels (DLAV) and intersegmental vessels (ISVs) of zebrafish embryos is mainly observed between 22h-48h after embryo fertilization. The main operating steps are as follows:

[0077] Tg (Fli1: ​​EGFP) vascular green fluorescent transgenic zebrafish were selected. After feeding brine shrimp feed for 4 hours at 16:00 the day before, active zebrafish were selected for mating. One male and one female fish were placed in the breeding tank, separated by a partition. The partition was removed at 9:00 the next day, and the zebrafish were waited for to mate on their own. One hour later, the zebrafish embryos were collected in a culture dish, and the residual feces were picked out under a microscope. After standing for 2 hours, milky white unfertilized eggs or dead fetuses were picked out under a microscope. Qualified zebrafish fertilized eggs were selected and washed several times, placed in a constant temperature zebrafish breeding system at 28.5℃, and incubated for 22 hours under the condition of alternating light for 14 hours and dark for 10 hours. Under a microscope, forceps were used to peel off the embryonic membrane of the zebrafish embryo, and the peeled embryos were washed several times with culture water, and zebrafish embryos with normal development and successful demembraning were selected under a microscope. The embryos were divided into a control group and three drug groups with different concentrations, with 20 embryos in each group. Drug intervention was carried out for 26 hours 22 hours after embryo fertilization. After the zebrafish embryos in each group developed for 48 hours, the embryos in each group were immediately anesthetized, and the development of the intersegmental vessels (ISVs) and dorsal long axis vessels (DLAV) of each group of embryos was observed under a fluorescence microscope.

[0078] The experimental results are as follows Figure 1-Figure 3 As shown in the figure, when the drug was used within the safe concentration range, the development of DLAV and ISVs of each group of embryos was observed. Compared with the control group, the green fluorescence in the zebrafish embryos in the Ir1 (10 μM) group was weakened. When the concentration reached 30 μM, the green fluorescence was significantly weakened, and the development of ISVs was completely impaired. In the Ir2 (15 μM) group, the green fluorescence of the blood vessels in the zebrafish embryos was significantly weakened, and the DLAV and ISVs were incompletely developed. The inhibitory effect of Ir3 on angiogenesis is slightly weaker than that of Ir1 and Ir2. In general, Ir1-Ir3 can inhibit angiogenesis in zebrafish embryos to varying degrees.

[0079] This proves that the cyclometallated iridium complex prepared by the present invention can be used to prepare anti-tumor drugs.

[0080] The above embodiments are only used to help understand the method and core idea of ​​the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A cyclometallated iridium (III) complex, characterized in that It has the structure shown in formula I: in, Select from any of the following structures: Indicates the connection location.

2. The cyclometallated iridium (III) complex according to claim 1, characterized in that The anion of the cyclometalated iridium (III) complex is PF6 - .

3. The method for preparing the cyclometallated iridium (III) complex according to any one of claims 1 to 2, characterized in that: The following steps are involved: A) reacting the nalidixic acid of formula Ⅰ-a with the 1,10-phenanthroline-5-amino group of formula Ⅰ-b to obtain the ligand of formula Ⅰ-c; B) reacting the ligand represented by formula Ⅰ-c with the metal iridium complex represented by formula Ⅰ-d, formula Ⅰ-e or formula Ⅰ-f to obtain the cyclometalated iridium complex represented by formula Ⅰ; X is halogen.

4. The preparation method according to claim 3, characterized in that: In the step A), the reaction temperature is 30-50°C and the reaction time is 20-24h; The reaction in step A) is carried out under the action of a catalyst; The catalyst is one or more of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine.

5. The preparation method according to claim 3, characterized in that: The reaction solvent in step B) is N,N-dimethylformamide.

6. The preparation method according to claim 3, characterized in that: The step B) further comprises: After the reaction was completed, an excess of saturated NH4PF6 solution was added to the system and the precipitated solid was collected.

7. Use of the cyclometallated iridium (III) complex according to any one of claims 1 to 2 or the cyclometallated iridium (III) complex prepared by the preparation method according to any one of claims 3 to 6 in the preparation of antitumor drugs.

8. The use according to claim 7, characterized in that: The tumor is a solid tumor.

9. The use according to claim 8, characterized in that: The tumor is one or more of breast cancer and cervical cancer.

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