A dithioformate Zn(II) complex, a preparation method and application thereof

Abstract

The application discloses a dithioformate Zn(II) complex and a preparation method and application thereof, and the chemical formula of the Zn(II) complex is [Zn II (L)2], wherein HL is 2-(bis(pyridin-2-yl)methylene)dithiohydrazine methyl ester. The Zn(II) complex is simple to prepare and easy to purify. The complex can be obviously fluorescent in a visible spectrum region of 520-600 nm under excitation of 470 nm wavelength light, and can be imaged in a single-layer cell model and a 3D cell ball model. The complex can not only kill lung cancer cells through photodynamic therapy, but also kill lung cancer cells through chemotherapy. Further mechanism research reveals that the chemotherapy mechanism depends on multiple pathways; the mechanism of the photodynamic effect is to kill cancer cells by generating superoxide anion free radicals. The complex can not only be used for cell imaging, but also realize a remarkable anti-lung cancer effect through multi-mode photodynamic therapy / chemotherapy, and has good application value.

Description

TECHNICAL FIELD

[0001] The present application relates to a zinc complex, in particular a dithioformate Zn(II) complex, a preparation method and application thereof. BACKGROUND

[0002] Lung cancer is a highly prevalent cancer and an important obstacle to improving life expectancy worldwide. According to the Global Cancer Network (GLOBOCAN) estimates in 2020, lung cancer is the leading cause of cancer death worldwide (18.0%), and the second most commonly diagnosed cancer (11.4%), with nearly 1.8 million deaths and 2.2 million new cancer cases each year. In addition, in most countries, the 5-year relative survival rate of lung cancer patients after diagnosis is only 10%-20%. Currently, various lung cancer therapies have been developed, including surgery, radiotherapy, chemotherapy, photodynamic therapy and immunotherapy. However, due to the high heterogeneity of lung cancer, the efficacy of single therapy is often limited. Therefore, multi-modal therapy combining different treatment methods has attracted great interest; these therapies take advantage of different treatment mechanisms, which can exert synergistic effects and thus improve treatment efficiency.

[0003] Photodynamic therapy is a minimally invasive treatment with few side effects. In photodynamic therapy, photosensitizers accumulate in tumors and produce reactive oxygen species (ROS) under light irradiation, thereby inducing tumor regression. After photodynamic therapy, a series of therapeutic effects occur, such as tumor vascular damage or permeability, tumor cell apoptosis, tumor cell necrosis and activation of the immune system, thereby inducing tumor regression. However, as a single therapy, photodynamic therapy often results in incomplete tumor killing due to limited penetration of light in tissues, survival of cells after photodynamic therapy leading to disease recurrence, tumor hypoxia and tumor migration, etc. Multiple studies have shown that photodynamic therapy combined with chemotherapy has multiple advantages: (1) photodynamic therapy can be used for local treatment of tumor tissue with minimal damage to adjacent tissues, while chemotherapy can eliminate surviving tumor cells, especially in deep or large tumors where laser penetration is difficult; (2) photodynamic therapy can reduce tumor intrinsic pressure and reduce the extravascular barrier, thereby reducing tumor vascular permeability, which can enhance the accumulation of chemotherapeutic drugs in tumors and improve efficacy; (3) ROS generated in photodynamic therapy can directly damage lysosomes and endosomes, promoting the transport of intracellular drugs to mitochondria, nuclei and microtubules, etc.; (4) DNA damage caused by the combined action of photodynamic therapy and chemotherapy is not easily repaired by drug-resistant DNA repair enzymes. To date, many studies have attempted to achieve synergistic effects of chemical photodynamic therapy through chemical linkage or nano-loading of photosensitizers and anticancer drugs to achieve higher therapeutic effects. Nano-loading often requires complex operations, and due to the accumulation of nanomaterials in the body, it often causes toxicity problems. Therefore, designing and developing molecules with both chemotherapy and photodynamic effects is a promising strategy for tumor treatment. SUMMARY

[0004] The present application provides a dithioformate Zn(II) complex, which can simultaneously exert photodynamic and chemotherapy effects, provides a simple method for multi-mode treatment of cancer, and expands the field of anticancer research of zinc complexes.

[0005] The technical solution for achieving the object of the present application is as follows:

[0006] A dithioformate Zn(II) complex, which has a chemical formula of [Zn II (L)2], wherein HL is methyl 2-(bis(pyridin-2-yl)methylene)dithiohydrazinate; the Zn(II) metal is coordinated with the ligand HL, and the hydrogen on the ligand HL is removed, so that the ligand becomes an anion, and therefore L is used in the chemical formula.

[0007] The dithioformate Zn(II) complex has the following structural formula:

[0008]

[0009] X-ray single crystal diffraction shows that the dithioformate Zn(II) complex belongs to a monoclinic system, and has a space group P21 / n; the cell parameters are as follows: a 10.9912(3),b 16.5190(4),c 15.7993(4),α(°)90,β(°)95.001(3),γ(°)90; the Zn(II) ion in the complex forms a six-coordinated structure with two thiol modes of HL.

[0010] The preparation method of the dithioformate Zn(II) complex of the present application is as follows:

[0011] ZnCl2 is added to a methanol solution containing methyl 2-(bis(pyridin-2-yl)methylene)dithiohydrazinate, and stirred at room temperature for 2-3 h; then, crystallization is carried out at room temperature, to obtain the dithioformate Zn(II) complex.

[0012] The molar ratio of ZnCl2 to methyl 2-(bis(pyridin-2-yl)methylene)dithiohydrazinate is 1:2.

[0013] The ratio of methyl 2-(bis(pyridin-2-yl)methylene)dithiohydrazinate to the methanol solution is 1 mmol:20 mmol.

[0014] During preparation, the raw materials can be added according to multiples of the above ratio.

[0015] Another object of the present application is to provide the application of the dithioformate Zn(II) complex in lung cancer cell imaging.

[0016] Another object of the present application is to provide the application of the dithioformate Zn(II) complex in the preparation of anti-lung cancer drugs for photodynamic therapy.

[0017] Another object of the present application is to provide the application of the dithioformate Zn(II) complex in the preparation of anti-lung cancer drugs for chemotherapy.

[0018] Another object of the present application is to provide the application of the dithioformate Zn(II) complex in the preparation of anti-lung cancer drugs for photodynamic therapy-chemotherapy combination.

[0019] Advantages of the present application:

[0020] (1) The dithioformate Zn(II) complex can be synthesized in high yield and high purity through simple experimental procedures and experimental conditions.

[0021] (2) The Zn(II) complex can be used for single-layer cell and 3D cell ball model imaging.

[0022] (3) The Zn(II) complex can effectively kill lung cancer cells through photodynamic therapy and chemotherapy at the same time.

[0023] The present application proposes a dithioformate Zn(II) complex as a potential photodynamic therapy and chemotherapy drug for lung cancer treatment. Porphyrin, phthalocyanine and chloride constitute the basic structure of many photodynamic therapy agents, which are not only difficult to synthesize and purify, but also have limitations such as aggregation and poor stability in biological liquids. In this work, the synthesized Zn(II) complex shows good water stability and low hemolysis, as well as good chemotherapy and photodynamic activity. The present application uses various methods to study the photodynamic and chemotherapy mechanisms of the Zn(II) complex. The photodynamic mechanism can be mainly divided into two types: type I and type II. Recent studies have shown that type I photosensitizers can produce strong cytotoxicity under hypoxic conditions, which is beneficial to overcome tumor hypoxia. The Zn(II) complex can cause cell damage using type I process. In addition, the study of the chemotherapy mechanism of the Zn(II) complex shows that it can inhibit cancer cells by triggering various mechanisms. The research results of the present application provide new insights for the development and utilization of Zn(II) complex and the treatment of lung cancer. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Schematic diagram of the crystal structure of the dithioformate Zn(II) complex of the present application, in which H atoms are omitted for simplicity.

[0025] Figure 2 Graph showing the photophysical property changes of the dithioformate Zn(II) complex of Example 2, in which the dithioformate Zn(II) complex is represented as Zn in the graph for simplicity;

[0026] Figure 2 In (A), the absorption spectrum of the dithioformate Zn(II) complex (30 μM) in methanol is shown;

[0027] (B) is a graph showing the photostability changes of the dithioformate Zn(II) complex (30 μM) under continuous irradiation with a 470 nm wavelength LED;

[0028] (C) is a fluorescence spectrum of the dithioformate Zn(II) complex (10 μM) in methanol after excitation at 470 nm.

[0029] Figure 3 Schematic diagram of the determination of ROS production in A549 cells using a fluorescence microscope in Example 2, in which the dithioformate Zn(II) complex is represented as Zn in the graph for simplicity.

[0030] Figure 4 Fluorescence image of live A549 cells incubated with the dithioformate Zn(II) complex (10 μM) in real time (15, 30, 60, and 120 minutes) in Example 2; λex= 488 nm, λem= 520-570 nm.

[0031] Figure 5 Image of three-dimensional A549 tumor spheroids after incubation with the dithioformate Zn(II) complex (10 μM) for 12 hours in Example 2.

[0032] Figure 6 Representative image of the detection of reactive oxygen species (ROS) in A549 cells exposed to light (470 nm, 20 mW / cm2) in Example 2, in which the dithioformate Zn(II) complex is represented as Zn in the graph for simplicity.

[0033] Figure 7 Graph showing the pathway analysis of differentially expressed proteins based on the KEGG database in Example 2.

[0034] Figure 8 Schematic diagram of the effect of the dithioformate Zn(II) complex on A549 cell apoptosis and cell cycle in Example 2;

[0035] Figure 8Figure (A) is a graph showing the apoptotic effect of dithioformate Zn(II) complex on A549 cells;

[0036] Figure (B) is a graph showing the protein expression level of cleaved caspase-3, PARP-1, cleaved PARP-1, ITGA5, MMP-9, CDKN1A, HIF1A, LTBR and GAPDH in A549 cells analyzed by Western blot;

[0037] Figure (C) is a histogram showing the cell cycle distribution of A549 cells treated with dithioformate Zn(II) complex.

[0038] For brevity, dithioformate Zn(II) complex is denoted as Zn in the figures.

[0039] Figure 9 Figure (D) is a schematic diagram showing the detection of anti- graft effect of A549 cells by wound healing assay in the examples. For brevity, dithioformate Zn(II) complex is denoted as Zn in the figure. DETAILED DESCRIPTION

[0040] The present application will be further described with reference to the following examples and figures, but is not limited to the examples.

[0041] In the examples, ZnCl2, methyl hydrazine dithioformate and bis(pyridin-2-yl)methanone were purchased from Sigma-Aldrich. All other reagents and solvents were obtained from commercial sources and used without further purification.

[0042] EXAMPLE

[0043] (1) Synthesis and characterization of methyl 2-(bis(pyridin-2-yl)methylene)dithiohydrazinecarboxylate ligand (HL):

[0044] Methyl hydrazine dithioformate (2 mmol) and bis(pyridin-2-yl)methanone (2 mmol) were refluxed in MeOH for 1 h to give a yellow solution containing methyl 2-(bis(pyridin-2-yl)methylene)dithiohydrazinecarboxylate. The yellow product of HL was obtained by slow evaporation of the solvent at 4 °C with a yield of 0.41 g (71 %). Calculated for C 13 H 12 N4S2(288.39): C, 54.14; H, 4.19; N, 19.43. Found: C, 54.06; H, 4.33; N, 19.12. m / z: 287.04 [M-H] + .

[0045] (2) Synthesis and structural characterization of dithioformate Zn(II) complex:

[0046] ZnCl2(0.5 mmol) was added to a methanolic (20 mL) solution containing 1 mmol of HL ligand and stirred at room temperature for 2-3 h; then the mixture solution was left at room temperature and the filtrate solvent was obtained by slow evaporation to get brown block-like dithiomethanate Zn(II) complex, yield: 86%. Calc. for C 26 H 22 N8S4Zn (640.13): C, 48.78; H, 3.46; N, 17.50. Found: C, 47.53; H, 3.57; and N, 17.36.

[0047] Crystallographic data of dithiomethanate Zn(II) complex was obtained on a Bruker SMART Apex II CCD diffractometer with Mo-Ka (l = 0.71069) radiation at 296 K. Cambridge Crystallographic Data Centre (CCDC) deposition number: 2265561. Selected dithiomethanate Zn(II) complex [Zn II (L)2] crystal parameters and bonding parameters are listed in Table 1 and Table 2.

[0048] Table 1. Crystal data of dithiomethanate Zn(II) complex.

[0049]

[0050]

[0051] Table 2. Bond lengths and bond angles (°) of dithiomethanate Zn(II) complex.

[0052]

[0053] X-ray single crystal diffraction shows that the dithiomethanate Zn(II) complex belongs to monoclinic system, space group P21 / n. The Zn(II) ion in the complex forms a six-coordinated structure with two HL thiol mode.

[0054] (3) The photophysical properties of dithiomethanate Zn(II) complex were studied, as shown in Figure 2

[0055] The UV-Vis absorption spectrum of dithiomethanate Zn(II) complex in methanol is shown in Figure 2 A, where the 300-500 nm wavelength band is mainly spin-allowed ligand center n→π* and / or π→π* transition. The photostability of dithiomethanate Zn(II) complex was tested by monitoring the UV-Vis absorption spectrum of their methanol solution under continuous LED light irradiation at 470 nm wavelength for 30 minutes. During the whole experiment, the absorption characteristics of all complexes did not change significantly, as​​Figure 2 B, which proves their photostability.

[0056] Upon excitation at 470 nm, the dithioformate Zn(II) complexes emit green fluorescence, as shown in Figure Figure 2 C, the emission lifetime of the dithioformate Zn(II) complexes is between 2-5 nanoseconds, which indicates the fluorescent nature of the emission. These complexes show fluorescence in the green region of the visible spectrum, indicating that they can be used for cell fluorescence imaging.

[0057] (4) Study of the in vitro anti-lung cancer activity of dithioformate Zn(II) complexes:

[0058] Cells were cultured in McCoy's A5 (for human lung cancer cell line Calu-1) and RPMI 1640 (for human lung cancer cell lines A549 and A549cisR) culture medium, supplemented with 1% antibiotic-antimycotic solution and 10% fetal bovine serum, in an environment containing 5% CO2, at a culture temperature of 37°C.

[0059] The cytotoxicity of dithioformate Zn(II) complexes in the dark and under light was determined separately. For dark cytotoxicity, cells were incubated with different concentrations (0-32 μΜ) of the test complexes for 48 hours; for light cytotoxicity, different concentrations (0-10 μΜ) of dithioformate Zn(II) complexes were added to the cells, which were incubated for 2 hours, washed with PBS, and then irradiated with blue LEDs (470 nm, 20 mW / cm 2 ) for 15 minutes. After irradiation, the cells were incubated for another 46 hours in culture medium without complexes. Then, MTT (10 μL, 5 mg / mL) in PBS was added to each well of the 96-well plate, which was then incubated at 37°C in 5% CO2for 4 hours. Subsequently, the supernatant in each well of the 96-well plate was replaced with 100 μL of dimethyl sulfoxide to dissolve the MTT-formhydrazine crystals. The optical density of each well was measured at a wavelength of 570 nm using an Infinite M200 Pro microplate reader.

[0060] Table 3. IC50values (48 h, μΜ) of dithioformate Zn(II) complexes for inhibiting the growth of human lung adenocarcinoma cell lines. 50

[0061]

[0062] To investigate the efficacy of dithioformate Zn(II) complexes as anticancer agents, the inventors investigated their efficacy in the dark and under 470 nm wavelength (15 minutes, 20 mW / cm 2 ​Cytotoxicity of human lung cancer cells (A549, A549 / DDP, and Calu-1) under irradiation (photocytotoxicity) was studied. The dithiomethyl ester Zn(II) complex caused cell death in all studied lung cancer cell lines, with an IC50 value of [missing value] under dark conditions. 50 The values ​​ranged from 4.55 to 8.63 μM. Under light irradiation, the dithiomethyl ester Zn(II) complex exhibited higher anticancer activity (see Table 3), indicating that the complex has strong phototoxicity.

[0063] To confirm the close relationship between ROS and phototoxicity, the inventors investigated the generation of ROS in A549 cells after irradiation. Intracellular ROS generated by the studied dithiomethyl ester Zn(II) complex after irradiation was detected using a red fluorescent cell ROS detection kit (abcam, ab186027). A549 cells were seeded in 6-well plates and incubated overnight at 37°C under 5% CO2. Cells were then incubated with a ROS probe (λ) in an incubator. ex =520nm, λ em Incubate at 590-625 nm for 1 hour, then treat in the dark with 5 μM of the studied dithiomethyl ester Zn(II) complex for 2 hours. After irradiation with blue light (or incubation in the dark) for 15 minutes, wash the sample with PBS and examine it under a fluorescence microscope. Figure 3 As shown, in the dark, cells treated with methyl thioester Zn(II) complexes and ROS probes exhibited almost no fluorescence; conversely, upon light irradiation after treatment with methyl thioester Zn(II) complexes, ROS generation significantly increased (strong red fluorescence). This confirms that methyl thioester Zn(II) complexes are promising photodynamic therapy compounds, as ROS generation is a major mechanism by which photodynamic therapy induces cell death. Tumor recurrence in surviving cells after photodynamic therapy is a significant problem, partly due to the limited diffusion distance and short half-life of ROS within cells. Chemotherapy can eliminate surviving tumor cells after photodynamic therapy, thereby reducing the likelihood of cancer recurrence. Therefore, formulations combining photodynamic and chemotherapeutic properties could more effectively ablate cancer cells.

[0064] (5) Imaging studies of dithiomethyl ester Zn(II) complexes in monolayer cells and 3D tumor spheroids:

[0065] The fluorescence imaging properties of dithioformate Zn(II) complexes in monolayer living lung cancer A549 cells were investigated using a confocal microscope. Monolayer cell imaging of dithioformate Zn(II) complexes was performed using a Nikon (AX+N-STORM) confocal microscope. Briefly, A549 cells were seeded in glass-bottom dishes and cultured in RPMI 1640 containing 10% fetal bovine serum; after 24 hours of cell seeding, the cells were incubated with Zn1-Zn4 complexes (5 μM) at 37°C for 30-120 minutes, then the cells were washed three times with phosphate-buffered saline (PBS) to remove excess dithioformate Zn(II) complexes, and the cells were preserved in fresh RPMI 1640 for fluorescence imaging. The Zn(II) complexes were excited using a 488-nanometer laser and their emission was detected in the 520- to 570-nanometer wavelength range, as shown in Figure 4 Figure 6. The dithioformate Zn(II) complexes were effectively absorbed by the cells and could be used for cell imaging, as the fluorescence signal could be clearly observed in living cells. Inventors' results showed that the dithioformate Zn(II) complexes were mainly accumulated in the cytoplasm within the first 60 minutes and partially migrated to the nucleus during further incubation (120 minutes).

[0066] After evaluating the cytotoxicity of monolayer cells, the inventors investigated the imaging ability of dithioformate Zn(II) complexes with good chemotherapy and photodynamic therapy ability in three-dimensional multicellular tumor spheroids. First, 1,500 A549 cells were seeded per well in an RPMI 1640 ultra-low attachment 96-well plate (Corning) to prepare tumor spheroids. The culture medium was changed daily. Within 2-4 days, cell suspensions formed tumor spheroids. For imaging, the tumor spheroids were incubated with dithioformate Zn(II) complexes (5 μM) in normal culture medium for 24 hours. Then they were rinsed twice with PBS and observed using a Nikon (AX+N-STORM) confocal microscope. In Z-stack mode, fluorescence images along the Z-axis were captured using excitation (λ ex = 488 nm, λ em = 520-570 nm).

[0067] It is noteworthy that three-dimensional tumor spheroids more closely resemble tumor models used in clinical treatment than monolayer cell models, and are therefore a widely used tissue model for evaluating drug delivery efficacy. Three-dimensional tumor spheroids can also replicate certain pathological elements of solid tumors, such as spatial structure and tumor-center hypoxia. Therefore, three-dimensional A549 multicellular tumor spheroids were co-cultured with a methyl dithioester Zn(II) complex. Many studied anticancer drugs fail during the conversion from cancer monolayer cells to in vivo models, partly due to impaired drug delivery caused by the permeability across the extracellular barrier. The permeability of the complex was first analyzed using Z-stack imaging microscopy. After 12 hours of culture, fluorescence signals were observed at each section depth, such as… Figure 5 As shown, the dithiomethyl ester Zn(II) complex completely penetrates the three-dimensional tumor sphere and can be used for imaging.

[0068] (6) Study on the photodynamic mechanism of dithiomethyl ester Zn(II) complex:

[0069] To determine the type of ROS produced in A549 cells, the cells were pretreated with a selective ROS scavenger and incubated at 37°C in 5% CO2 for 1 hour, followed by treatment with a methyl dithioester Zn(II) complex (1.0 μM). Hydroxyl radicals were scavenged using 50 mM Mannitol. - Hydrogen peroxide (H₂O₂) was removed using sodium pyruvate at a final concentration of 10 mM, and singlet oxygen was removed using sodium azide at 5 mM. 1 O2), using 50 μM selenium to remove overnitrite anions (ONOO). - Using 5 mMtiron to scavenge superoxide anion free radicals ( · O2 - Antioxidants were present throughout the experiment. Cells were pretreated with a selective ROS scavenger and then treated with a Zn(II) dithiomethyl ester complex for 2 hours. Cells were then washed with PBS and subjected to blue light (470 nm, 20 mW / cm²). 2 Irradiate for 15 minutes. After irradiation, image the cells using a microscope (OLYMPUS Co., Japan).

[0070] Upon photoexcitation, cells treated with the dimethyl thioester Zn(II) complex in the presence of NaN3 / D-mannitol / sodium pyruvate / ebuselenium exhibited similar cell morphology to cells treated with the dimethyl thioester Zn(II) complex alone, which ruled out the possibility of... 1 O2, · OH, H2O2, and ONOO - The possibility of participating in the photodynamic effect, such as Figure 6The production of intracellular ROS was significantly inhibited in the presence of tiron, indicating that intracellular ROS was produced after light irradiation in the presence of the dithioformate Zn(II) complex · O2 - Since photodynamic therapy requires molecular oxygen to initiate cell death, low concentrations of oxygen in solid tumors remain a barrier to the use of classic photosensitizers. In contrast to type II photosensitizers, type I photosensitizers can still produce strong cytotoxicity under hypoxic conditions. Therefore, the dithioformate Zn(II) complex is expected to be used for photodynamic therapy of tumors because it utilizes the type I pathway, which is beneficial for overcoming the problem of tumor hypoxia.

[0071] (6) Chemotherapy mechanism of the dithioformate Zn(II) complex:

[0072] Given the good chemotherapy ability (in the dark) of the dithioformate Zn(II) complex, its chemotherapy mechanism was also carefully studied. Four-dimensional data-independent acquisition (4D-DIA) proteomics analysis is a fast, efficient, and sensitive method that helps to elucidate the potential anticancer mechanism of drugs at the protein level. Therefore, the inventors performed 4D-DIA proteomics analysis in the diaPASEF mode to understand the details of the chemotherapy mechanism of the dithioformate Zn(II) complex.

[0073] The total number of identified peptides was 86,379, and the total number of corresponding quantified proteins was 7,897. Among these quantified proteins, 1065 proteins were found to have significant differential expression, with the following criteria: FC>1.5 or FC<0.6667, P<0.05: FC>1.5 or FC<0.6667, P<0.05. Among these differentially expressed proteins (DEPs), 520 were up-regulated, and 545 were down-regulated.

[0074] Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis helps to systematically understand cellular biological processes, disease mechanisms or characteristics, and drug action mechanisms. Therefore, the inventors performed KEGG pathway enrichment analysis based on DEPs to elucidate the action pathways significantly affected by the dithioformate Zn(II) complex. As shown in Table 2, the top 20 pathways significantly affected by the dithioformate Zn(II) complex were identified. Figure 7The most important 20 pathways shown cover complement and coagulation cascades and protein digestion and absorption, environmental information processing (cytokine-cytokine receptor interaction, ECM-receptor interaction, PI3K-Akt signaling pathway, and HIF-1 signaling pathway), genetic information processing (mismatch repair, fanconi anemia pathway, base excision repair, DNA replication, and Homologous recombination), human diseases (microRNAs in cancer, small cell lung cancer, malaria, pathways in cancer, and toxoplasmosis), cellular processes (p53 signaling pathway and mitophagy-animal), and metabolism (glycosaminoglycan biosynthesis and sphingolipid metabolism). Among these KEGG pathways, the number of differentially expressed proteins related to cancer pathways is the most (43), of which 20 are up-regulated and 23 are down-regulated. In addition, among these KEGG pathways, the pathways closely related to cell proliferation, death and migration, including p53 signaling pathway, ECM-receptor interaction, cytokine-cytokine receptor interaction, PI3K-Akt signaling pathway and HIF-1 signaling pathway, are jointly involved in the composition of pathways in cancer. Therefore, the inventors further verified these pathways by Western blotting and flow cytometry.

[0075] Disregulation of p53 signaling pathway is detected in almost all tumors, so developing therapeutic drugs targeting p53 signaling pathway has attracted widespread attention. Activation of p53 signaling pathway mainly leads to cell cycle arrest or apoptosis. Therefore, the inventors studied the effects of dithioformate Zn(II) complex on apoptosis and cell cycle of A549 cells, as shown in Figure 8 Figure 8 ​As shown in Figure A, the dithioformate Zn(II) complex induced dose-dependent apoptosis in A549 cells. To further confirm that the dithioformate Zn(II) complex induces apoptosis, the inventors performed Western blot analysis to monitor the expression level of cleaved caspase-3 and cleaved PARP-1. As shown in Figure Figure 8 B, the levels of cleaved caspase-3 and cleaved PARP-1 were upregulated, which further indicated that the apoptosis program had been executed. In addition, the effect of the dithioformate Zn(II) complex on the cell cycle was also monitored by flow cytometry. Flow cytometry analysis showed that the dithioformate Zn(II) complex arrested the cell cycle at the G1 phase, as shown in Figure Figure 8 C.

[0076] The ECM–receptor interaction pathway can directly or indirectly control cell activities such as adhesion, migration, and proliferation. KEGG studies showed that the upregulation of ITGA5, COL4A1, COL4A2, ITGA2, and LAMB3 and the downregulation of AGRN, LAMA5, LAMB1, LAMC1, SDC1, HSPG2, DAG1, and LAMA3 interfered with the ECM–receptor interaction pathway. ITGA5, as an important protein in the ECM–receptor interaction pathway, plays a key role in different tumor cell subpopulations and cell-to-cell interactions. Western blotting showed that treatment of A549 cells resulted in a dose-dependent upregulation of ITGA5 levels (Figure Figure 8 B), which was consistent with the results of proteomics. In addition, given that the ECM–receptor interaction pathway plays a key role in cell migration, the inventors investigated the effect of the dithioformate Zn(II) complex on A549 cell migration by wound healing assay and Western blotting, as shown in Figure Figure 9 The control cells migrated rapidly, and the gap was significantly reduced. In contrast, after the addition of the dithioformate Zn(II) complex, fewer cells migrated, and the gap was larger. The inventors also detected the expression of MMP-9, an extracellular matrix protein that promotes tumor cell migration, by Western blotting. The dithioformate Zn(II) complex inhibited the expression of MMP-9 (Figure Figure 8 B), which was consistent with the results of the wound healing assay.

[0077] In addition, the inventors also selected LTBR protein in cytokine-cytokine receptor interaction pathway, CDKN1A protein in PI3K-Akt signaling pathway and LTBR, CDKN1A and HIF1A proteins in HIF-1 signaling pathway to further verify their expression by Western blot. Western blot showed that the expression of CDKN1A and HIF1A increased in a dose-dependent manner and the expression of LTBR decreased in a dose-dependent manner after treating A549 cells with the dithioformate Zn(II) complex Figure 8 B). The Western blot data were consistent with the proteomics results, and the dithioformate Zn(II) complex could activate the following pathways: cytokine-cytokine receptor interaction, PI3K-Akt signaling pathway, and HIF-1 signaling pathway.

[0078] In summary, these results showed that the dithioformate Zn(II) complex exerted a chemotherapeutic effect by regulating multiple pathways such as pathways in cancer, p53 signaling pathway, and ECM-receptor interactions.

Claims

1. A dithiomethyl ester Zn(II) complex, characterized in that, The chemical formula of this complex is [Zn]. II [(L)2], where HL is methyl 2-(bis(pyridin-2-yl)methylene)dithiohydrazine, Zn(II) metal coordinates with ligand HL, the hydrogen on HL will be removed, and the ligand becomes an anion, so L is used in the chemical formula; The structural formula of the complex is as follows: X-ray single-crystal diffraction showed that the dithiomethyl ester Zn(II) complex belongs to the monoclinic crystal system, space group P21 / n; the unit cell parameters are: a 10.9912(3),b 16.5190(4),c 15.7993(4),α( o )90,β( o )95.001(3),γ( o )90; The Zn(II) ions in this complex form a six-coordinate structure with the two HL thiol configurations.

2. The method for preparing the dithiomethyl ester Zn(II) complex as described in claim 1, characterized in that: Add ZnCl2 to a methanol solution containing methyl 2-(bis(pyridin-2-yl)methylene)dithiohydrazine at a molar ratio of 1:2, stir at room temperature for 2-3 hours, and then crystallize at room temperature to obtain the dithiomethyl ester Zn(II) complex.

3. The method for preparing the dithiomethyl ester Zn(II) complex as described in claim 2, characterized in that: The ratio of methyl 2-(bis(pyridin-2-yl)methylene)dithiohydrazide to methanol solution is 1 mmol: 20 mmol.

4. The application of the dithiomethyl ester Zn(II) complex as described in claim 1, characterized in that: The application of the dithiomethyl ester Zn(II) complex in the preparation of fluorescent probes for cell imaging.

5. The application of the dithiomethyl ester Zn(II) complex as described in claim 1, characterized in that: The application of the dithiomethyl ester Zn(II) complex in the preparation of anti-lung cancer drugs for photodynamic therapy.

6. The application of the dithiomethyl ester Zn(II) complex as described in claim 1, characterized in that: The application of the dithiomethyl ester Zn(II) complex in the preparation of chemotherapeutic drugs for lung cancer.

7. The application of the dithiomethyl ester Zn(II) complex as described in claim 1, characterized in that: The application of the dithiomethyl ester Zn(II) complex in the preparation of a combined photodynamic therapy-chemotherapy anti-lung cancer drug.

Images