Cu (II) complex of mixed ligand as well as preparation method and application of Cu (II) complex

Abstract

The invention discloses a Cu (II) complex of a mixed ligand as well as a preparation method and application of the Cu (II) complex. The chemical formula of the target Cu (II) complex is [CuII (L) (Ind)]. NO3. CH3OH, HL is (E)-N '-((2-hydroxynaphthalene-1-yl) methylene) butyryl hydrazine, and Ind is an indazole ligand. The Cu (II) complex is simple to prepare, high in yield and easy to purify. The Cu (II) complex can effectively inhibit proliferation and migration of airway smooth muscle cells. Flow cytometry reveals that the Cu (II) complex can induce apoptosis and cycle arrest of airway smooth muscle cells. A Western blot experiment shows that the Cu (II) complex can induce autophagy of airway smooth muscle cells by changing expression of LC3 II protein. The proliferation of airway smooth muscle cells promotes airway remodeling in asthma, so that the Cu (II) complex disclosed by the invention can be used for treating asthma and other diseases caused by the proliferation of airway smooth muscle cells.

Description

Technical Field

[0001] The invention relates to a copper complex, in particular to a Cu(II) complex of a mixed ligand of (E)-N'-((2-hydroxynaphthalene-1-yl)methylene)butyric acid hydrazide and indazole, and a preparation method and application thereof. Background Art

[0003] Airway smooth muscle is a regulator of airway responsiveness. A notable feature of asthma is the loss of homeostatic control of airway smooth muscle. Any factor that reduces airway pressure, including thickening of the lamina or connective tissue surrounding the bronchi, shallow breathing, failure to breathe deeply, and inflammation, will reduce the beneficial effects of muscle load fluctuations and promote airway closure. These features are prominent in asthma. Hyperplasia of airway smooth muscle cells contributes to airway remodeling in asthma. Increased airway smooth muscle mass enhances bronchoconstriction in response to stimulation, exacerbates airway hyperresponsiveness, and thickens the airway wall, leading to basal airway resistance and irreversible airflow obstruction, ultimately leading to increased morbidity and even mortality. Hyperplasia of airway smooth muscle cells is mainly caused by changes in cell behavior, excessive growth, insufficient apoptosis, enhanced migration and secretion, and changes in autophagy. Exploring drugs that can modulate the behavior of airway smooth muscle cells may be promising in the treatment of asthma. In summary, airway smooth muscle cells play a central role in the pathogenesis of asthma, are involved in airway hyperresponsiveness and the inflammatory and remodeling processes observed in asthmatic patients, and are an important target for treatment.

[0004] Currently, the most recommended treatment for asthma is the use of corticosteroids. However, long-term oral corticosteroids may cause a variety of side effects, including hypertension, gastrointestinal bleeding ulcers, depression, and sepsis. Although some progress has been made in the understanding of asthma, the development of alternative treatments has been relatively slow, and new effective therapies are still needed to control severe asthma. In recent years, the study of the application of metal-based complexes in the treatment of various diseases has become increasingly attractive. Metal-based complexes offer the potential for unique drug mechanisms of action by selecting metals, their oxidation states, the type and amount of ligands, and coordination geometry. Whether metal-based complexes can modulate the behavior of airway smooth muscle cells and thus participate in airway remodeling in asthma remains to be elucidated. Summary of the invention

[0005] The invention provides a Cu(II) complex of a (E)-N'-((2-hydroxynaphthalene-1-yl)methylene)butyric acid hydrazide and an indazole mixed ligand. The complex can effectively inhibit the proliferation and migration of airway smooth muscle cells, induce apoptosis, autophagy and cycle arrest of airway smooth muscle cells, provide a potential method for the treatment of asthma, and expand the research field of copper complexes.

[0006] The technical solution for achieving the purpose of the present invention is as follows:

[0007] A mixed ligand Cu(II) complex having the chemical formula [Cu II (L)(Ind)]·NO3·CH3OH, wherein HL is (E)-N'-((2-hydroxynaphthalen-1-yl)methylene)butyric acid hydrazide and Ind is an indazole ligand;

[0008] The Cu(II) metal center is coordinated with the ligand HL, the hydroxyl hydrogen on HL will be removed, and the ligand will become an anion, so it is represented by L in the chemical formula.

[0009] The structural formula of the complex is as follows:

[0010]

[0011] X-ray single crystal diffraction shows that the Cu(II) complex belongs to the monoclinic system with space group P21 / c;

[0012] The unit cell parameters are: α( o )90,β( o )97.603(5),γ( o )90;

[0013] The Cu(II) ion in the complex forms a square planar coordination geometry with the tridentate keto mode HL and the monodentate indazole.

[0014] The preparation method of the mixed ligand Cu(II) complex shown in the above formula is as follows:

[0015] (1) dissolving butyrylhydrazide, 2-hydroxy-1-naphthaldehyde and indazole in an organic solution in a molar ratio of 1:1:0.8 and stirring at room temperature for 30-60 minutes;

[0016] (2) adding Cu(NO3)2·3H2O organic solution to the mixed solution in step (1), heating and stirring for 2-3 hours; then, crystallizing at room temperature to obtain a mixed ligand Cu(II) complex;

[0017] The molar ratio of the Cu(NO3)2·3H2O compound to the indazole compound is 1:1.

[0018] During preparation, raw materials can be added in multiples of the above ratio.

[0019] In the steps of the above preparation method, the organic solvent is one or a combination of two or more of methanol, acetonitrile, chloroform and ethanol.

[0020] In the steps of the above preparation method, the ratio of the organic solvent to butyrylhydrazide, 2-hydroxy-1-naphthaldehyde, indazole and Cu(NO3)2·3H2O is 5-50mL:1mmol.

[0021] When the organic solvent is methanol, its amount is preferably such that it can dissolve the raw materials participating in the reaction. Usually, based on 1 mmol of Cu(NO3)2·3H2O compound, all the raw materials participating in the reaction are usually dissolved in 5 to 50 mL of methanol.

[0022] In step (2) of the above preparation method, the reaction is preferably carried out under heating conditions, usually refluxed at the boiling point of methanol, 64-65° C. According to experiments, when the reaction is carried out at the boiling point of methanol, the reaction time is more appropriately controlled at 120 min to 180 min.

[0023] Another object of the present invention is to provide the use of the mixed ligand Cu(II) complex in the preparation of drugs for inhibiting the proliferation and migration of airway smooth muscle cells.

[0024] Another object of the present invention is to provide the use of the mixed ligand Cu(II) complex in the preparation of anti-asthma drugs.

[0025] Advantages of the present invention:

[0026] (1) The mixed ligand Cu (II) complex of the present invention has easy synthesis conditions, high synthesis yield and purity, and is easy to separate.

[0027] (2) The mixed ligand Cu(II) complex of the present invention can inhibit the proliferation and migration of airway smooth muscle cells.

[0028] (3) The mixed ligand Cu(II) complex of the present invention can treat the proliferation of airway smooth muscle cells through various mechanisms, such as apoptosis and autophagy, and can be used for the preparation of anti-asthma drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the crystal structure of the mixed ligand Cu (II) complex of the present invention. For simplicity, H atoms are deleted.

[0030] Figure 2 The UV-vis stability diagram of the mixed ligand Cu(II) complex in aqueous solution is shown in FIG.

[0031] Figure 3 The figure is a graph showing the cell viability of rat airway smooth muscle cells treated with mixed ligand Cu(II) complexes of different concentrations for 48 hours.

[0032] Figure 4A is an example of an airway smooth muscle cell treated with mixed ligand Cu(II) complexes at different concentrations for 48 hours, and then an EdU staining experiment was performed. Then, a fluorescence microscope was used to observe the proliferating cells. Figure 4 B is the quantification of the proportion of proliferating cells in A, **p<0.005.

[0033] Figure 5 A is a graph showing the anti-migration effect of the mixed ligand Cu (II) complex on airway smooth muscle cells in the example. Figure 5 B is quantification of cell migration in A, **p<0.005.

[0034] Figure 6 A is a diagram showing the apoptosis of airway smooth muscle cells treated with the mixed ligand Cu (II) complex of the embodiment using flow cytometry. Figure 6 B is quantification of apoptosis in A, **p<0.005.

[0035] Figure 7 A is a diagram showing the cycle status of airway smooth muscle cells treated with the mixed ligand Cu (II) complex of the embodiment using flow cytometry. Figure 7 B is the quantification of apoptosis in A, *p<0.05, **p<0.005.

[0036] Figure 8 A is an example of a Western blot experiment to detect the expression of autophagy-related protein LC3. Figure 8 B is the immunoblot density quantification of LC3-II and LC3-I. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described in detail below in conjunction with the embodiments and drawings, but the present invention is not limited thereto.

[0038] Example

[0039] Methanol, butyric acid hydrazide, 2-hydroxy-1-naphthaldehyde, indazole, and Cu(NO3)2·3H2O were purchased from Sigma-Aldrich. All other reagents were from commercial sources and used without further purification.

[0040] (1) Preparation and structural characterization of mixed ligand Cu(II) complexes:

[0041] 1 mmol of butyrylhydrazide, 1 mmol of 2-hydroxy-1-naphthaldehyde and 0.8 mmol of indazole were added to 30 mL of methanol, and the mixed liquid was stirred at room temperature for 30-60 minutes;

[0042] Then, 10 mL of a methanol solution containing 0.8 mmol of Cu(NO3)2·3H2O was added, and the mixture was refluxed at 64 degrees for 2 hours. The mixed solution was then placed at room temperature, and the filtrate solvent was slowly evaporated to obtain a dark blue block-like Cu(II) complex with a yield of 71%.

[0043] Calculated value C 23 H 25 CuN5O6(531.02):C,52.02;H,4.75;N,13.18.

[0044] Measured values: C, 50.53; H, 5.13; N, 12.01.

[0045] The crystallographic data of the mixed-ligand Cu(II) complex were obtained on a Bruker SMART Apex IICCD diffractometer using Mo-Kα (λ=0.71069 Å) radiation at room temperature.

[0046] Cambridge Crystallographic Data Centre (CCDC) storage number: 2382973.

[0047] The crystal parameters and bonding parameters of the selected mixed-ligand Cu(II) complexes are listed in Tables 1 and 2.

[0048] Table 1. Crystallographic data of mixed-ligand Cu(II) complexes.

[0049]

[0050] Table 2. Bond lengths of mixed ligand Cu(II) complexes and bond angles (°).

[0051]

[0052] X-ray single crystal diffraction shows that the mixed ligand Cu(II) complex belongs to the monoclinic system and the space group P21 / c. The Cu(II) ion in the complex forms a planar square coordination geometry with the 3-dentate keto mode HL and the monodentate indazole.

[0053] (2) UV-vis studies of mixed ligand Cu(II) complexes:

[0054] The stability of the Cu(II) complexes in deionized water was evaluated using a UV spectrophotometer.

[0055] Cu(II) complexes at a concentration of 30 μM were introduced into aqueous solution or complete culture medium, and subsequently, the absorption spectra were recorded at specified time intervals (0 min, 5 min, 10 min, 30 min, 60 min, 120 min) in the wavelength range of 280 to 800 nm.

[0056] like Figure 2 As shown, the mixed ligand Cu(II) complexes mainly exhibit spin-allowed ligand center n→π* and / or π→π* transitions in the 300-500 nm band. Importantly, at room temperature, the time-dependent absorption spectra of the mixed ligand Cu(II) complexes in aqueous solution did not detect significant changes within 2 hours, indicating that these Cu(II) complexes can maintain relatively stable structures in these two media.

[0057] (3) Study on the inhibition of airway smooth muscle cell proliferation by mixed ligand Cu(II) complexes:

[0058] Airway smooth muscle cells were derived from the following method (Eur. Respir. J., 1996, 9: 808). Male SD rats aged 6-8 weeks were purchased from the Experimental Animal Center of Guilin Medical College. After anesthesia with sodium pentobarbital, the trachea was removed and the connective tissue was removed using aseptic operation. The epithelial cells on the surface of the tracheal lumen were gently scraped with a blade. The airway smooth muscle strips were carefully dissected out from the surrounding parenchymal tissue, while ensuring that all connective tissue was removed. Then, the airway smooth muscle strips were washed three times in Hanks balanced salt solution (HBSS) containing 50U / ml penicillin and 20μg / ml streptomycin. Next, the muscle tissue was minced and digested with 1mg / ml type I collagenase for one hour at 37°C and 5% CO2. After digestion, twice the volume of DMEM containing 10% fetal bovine serum was added to terminate digestion, followed by centrifugation at 3000rpm for 5 minutes. The pellet obtained after centrifugation was resuspended and cultured in DMEM containing 20% ​​fetal bovine serum. The cells were passaged until confluence, usually within two weeks. The identity of the smooth muscle cells was confirmed by positive staining for α-smooth muscle actin. The experiments used airway smooth muscle cells at passages 4-6 and were passaged using 0.25% trypsin and 0.01% EDTA solution when the cells were confluent.

[0059] After AMS cells were obtained, we evaluated the ability of mixed-ligand Cu(II) complexes to inhibit AMS cell proliferation using CCK8 assay and EdU staining.

[0060] The CCK8 analysis method is as follows: After trypsin digestion, each group of cells was diluted to 5×10 4 Then, 100 μl of cell suspension was added to each well of a 96-well plate, with each well containing 5×10 3Cells were cultured for 24 hours, and triplicate wells were set up for each group. After 24 hours of cell culture, each group was given the designated treatment, and then a mixture of CCK8 reagent and DMEM medium containing 10% FBS in a ratio of 1:10 was added to the wells (100 μl per well). Subsequently, the cells were incubated at 37°C and 5% CO2 for 1-4 hours. The absorbance was measured at 450nm using a spectrophotometer, and a cell growth curve was drawn based on the absorbance values ​​obtained.

[0061] For EdU staining, cells were plated at 1 × 10 4 Cells were seeded on sterile coverslips in 24-well plates at a density of 100 μM. Cells were then treated with different concentrations of mixed ligand Cu(II) complexes (0, 0.8, 1.6 μM) for 24 h. After the incubation period, EdU detection was performed using the BeyoClicks detection kit (C0071S, Beyotime Biotech, Shanghai, China) according to the manufacturer's instructions. Briefly, cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature and then permeabilized with 0.3% Triton-X100 for 15 min. Click reaction solution was then added and incubated for 30 min at room temperature in the dark. Cell nuclei were then stained with Hoechst 33342 in the dark for 10 min. EdU-positive cells were observed and counted using a fluorescence microscope (Olympus BX53, Tokyo, Japan).

[0062] like Figure 3 As shown, the mixed ligand Cu(II) complex inhibited the proliferation of airway smooth muscle cells in a dose-dependent manner (*P<0.05, **P<0.01). After 24 hours of treatment with 1.2 μM compound, the inhibition rate exceeded 40%.

[0063] To further confirm the inhibitory effect of mixed ligand Cu(II) complex on airway smooth muscle cell proliferation, EdU staining was used to detect proliferating cells. Figure 4 As shown in A, after 24 h of treatment at 0.8 and 1.6 μM doses, the number of proliferating cells decreased by 35.8% and 23.8%, respectively (*P<0.05) ( Figure 4 B). These results indicate that the mixed-ligand Cu(II) complex can effectively inhibit the proliferation of airway smooth muscle cells.

[0064] (4) Study on the inhibition of airway smooth muscle cell migration by mixed ligand Cu(II) complexes:

[0065] Inhibition of airway smooth muscle cell migration was investigated by wound healing assay. Airway smooth muscle cells were cultured to confluence in 6-well plates for 24 hours, and then scratches were made on the cell monolayer using a sterile 200 mL pipette tip in a predetermined grid pattern. After scratching, airway smooth muscle cells were washed three times with serum-free medium and then treated with serum-free medium from different dose groups. Wounds were photographed at baseline, 6 hours, 12 hours, and 24 hours. The number of cells that migrated into the wound area was quantified.

[0066] like Figure 5 As shown in A, after 6-24 hours of treatment with mixed ligand Cu(II) complexes, the migration of airway smooth muscle cells was inhibited. Further quantification revealed that after 24 hours of treatment with mixed ligand Cu(II) complexes, the migration rates of airway smooth muscle cells were 41.93%, 11.57% and 6.66% respectively ( Figure 5 B). These results indicate that the mixed-ligand Cu(II) complex inhibits the migration of airway smooth muscle cells in a dose-dependent manner.

[0067] (5) Study on the apoptosis of airway smooth muscle cells induced by mixed ligand Cu(II) complexes:

[0068] Apoptosis analysis was performed using the FITC Annexin V Apoptosis Detection Kit I from BD Biosciences. Airway smooth muscle cells were plated in 6-well plates at 3 × 10 5 The cells were cultured at a density of 100 cells / well for 24 hours at 37°C and 5% CO2. The cells were then treated with mixed ligand Cu(II) complexes at different concentrations for 24 hours. After the prescribed cell treatment, the cells were co-stained with FITC Annexin V and PI according to the manufacturer's instructions. The stained cells were then analyzed by flow cytometry (FACSCalibur, BD Biosciences). The acquired data were further analyzed using FlowJo V10 software.

[0069] To evaluate whether the mixed-ligand Cu(II) complexes affect the apoptosis of airway smooth muscle cells, apoptosis was detected by flow cytometry after FITCAnnexin V / PI double staining. Figure 6 As shown in A, mixed ligand Cu(II) complexes induced apoptosis of airway smooth muscle cells in a dose-dependent manner. After treatment with 0, 0.8 and 1.6 μM doses of mixed ligand Cu(II) complexes, the apoptosis rates were 3.63%, 6.29% and 39.5%, respectively. When treated with 1.6 μM concentration of mixed ligand Cu(II) complexes, the apoptosis rate increased significantly, and the difference was statistically significant (**P<0.01, Figure 6 B).

[0070] (6) Study on the blockade of airway smooth muscle cell cycle by mixed ligand Cu(II) complexes:

[0071] Airway smooth muscle cells were cultured in 6-well plates at 3 × 10 5 The cells were cultured at a density of 100 cells / well for 24 hours and incubated at 37°C and 5% CO2. The cells were then treated with mixed ligand Cu(II) complexes at different concentrations for 24 hours. Afterwards, the cells were collected and washed with ice-cold PBS, resuspended in PBS, and 70% pre-cooled ethanol was quickly added, mixed by pipetting and left overnight at 4°C. The cells were stained with PI staining solution containing RNase A at 37°C for 15 minutes. The cell cycle of airway smooth muscle cells was detected by flow cytometry (FACS Calibur, BD Biosciences). The data were analyzed using FlowJo V10 software.

[0072] The cell cycle status was determined by PI staining and flow cytometry. Figure 7 As shown in A, after 24 hours of treatment with 0, 0.8 and 1.6 μM mixed ligand Cu (II) complex, the proportion of cells in the S phase was 27.72%, 22.68% and 19.89%, respectively. The proportion of cells in the G2 phase was 8.04%, 12.01% and 19.89%, respectively. That is, after 24 hours of treatment with 1.6 μM mixed ligand Cu (II) complex, the proportion of cells in the G2 phase increased from 8.04% to 19.89% (**P<0.01, Figure 7 B), however, there were no significant changes in G1 and S phases. These results indicate that the mixed-ligand Cu(II) complex caused cell cycle arrest in G2 / M phase in a concentration-dependent manner.

[0073] (7) Study on the induction of autophagy in airway smooth muscle cells by mixed ligand Cu(II) complexes:

[0074] Autophagy is a cellular process involved in cell survival and apoptosis. Autophagy is involved in many cellular physiological and pathophysiological processes and plays an important role in maintaining cellular homeostasis. Autophagy has a dual role in apoptosis. Therefore, we finally tested the effect of mixed ligand Cu(II) complexes on autophagy in airway smooth muscle cells. The ratio of lipidated LC3B-II to normal LC3B-I increased, which is a reliable marker of autophagy. Therefore, we examined the expression of lipidated LC3B-II and LC3B-I proteins by Western blot experiments. Western blot showed ( Figure 8 ), the ratio of LC3-II / LC3-I increased significantly after 24 h of treatment with 1.6 μM mixed ligand Cu(II) complex, indicating that mixed ligand Cu(II) complex activated autophagy in airway smooth muscle cells.

[0075] In summary, these results indicate that the Cu(II) complex of the mixed ligand of (E)-N'-((2-hydroxynaphthalen-1-yl)methylene)butyric acid hydrazide and indazole of the present invention can inhibit the proliferation of airway smooth muscle cells by regulating multiple pathways (such as apoptosis, cycle arrest and autophagy), confirming that the Cu(II) complex can be used as a promising drug for the treatment of asthma.

Claims

1. A mixed ligand Cu(II) complex, characterized in that: The chemical formula of the complex is [Cu II (L)(Ind)]·NO3·CH3OH, wherein HL is (E)-N'-((2-hydroxynaphthalen-1-yl)methylene)butyric acid hydrazide and Ind is an indazole ligand; The structural formula of the complex is as follows: X-ray single crystal diffraction shows that the Cu(II) complex belongs to the monoclinic system with space group P21 / c; The unit cell parameters are: α( o )90,β( o )97.603(5),γ( o )90; The Cu(II) ion in the complex forms a square planar coordination geometry with the tridentate keto mode HL and the monodentate indazole.

2. The method for preparing a mixed ligand Cu(II) complex according to claim 1, characterized in that: The following steps are involved: (1) dissolving butyrylhydrazide, 2-hydroxy-1-naphthaldehyde and indazole in an organic solution in a molar ratio of 1:1:0.8 and stirring at room temperature for 30-60 minutes; (2) adding Cu(NO3)2·3H2O organic solution to the mixed solution in step (1) and heating and stirring for 2-3 hours; then, crystallizing at room temperature to obtain a mixed ligand Cu(II) complex; The molar ratio of the Cu(NO3)2·3H2O compound to the indazole compound is 1:

1.

3. The method for preparing a mixed ligand Cu(II) complex according to claim 2, characterized in that: The organic solvent is one or a combination of two or more of methanol, acetonitrile, chloroform and ethanol.

4. The method for preparing a mixed ligand Cu(II) complex according to claim 2, characterized in that: The organic solvent, butyrylhydrazide, 2-hydroxy-1-naphthaldehyde, indazole and Cu(NO3)2·3H2O are mixed in a ratio of 5 to 50 mL:1 mmol.

5. The use of the mixed ligand Cu(II) complex according to claim 1, characterized in that: The Cu(II) complex is used in preparing drugs for inhibiting the proliferation and migration of airway smooth muscle cells.

6. The use of the mixed ligand Cu(II) complex according to claim 1, characterized in that: The Cu(II) complex is used in preparing medicine for treating asthma.

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