Preparation method and application of TrxR-targeted arsenic inhibitor and drunkenness-quitting sulfur coupling medicine

Abstract

The invention relates to a preparation method and application of a coupled drug molecule for synergistic anti-tumor treatment, and belongs to the technical field of medical chemistry and biological medicine. The invention designs and synthesizes a combined prodrug molecule (pAs-DTC) which is formed by connecting an arsenic inhibitor of targeted thioredoxin reductase (TrxR) with dithiocarbamate (DTC) through a pathological switch disulfide bond. The prodrug can be selectively broken under the action of reducing species in tumor cells, the two active components are released, and the aim of synergistically killing the tumor cells is achieved through the combination of targeted inhibition of TrxR activity by the arsenic inhibitor and chelating of DTC with Cu. The invention provides a novel anticancer combined treatment strategy, provides a theoretical basis and a practical basis for developing high-efficiency and low-toxicity antitumor drug combinations, and has a very good clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biochemistry and medicine, and more particularly, to a method for preparing a conjugate drug molecule for synergistic anti-tumor therapy and its application. Background Art

[0002] Arsenic compounds have significant anti-cancer activity and have been successfully applied to the clinical treatment of acute promyelocytic leukemia. Organic arsenic exerts its anti-tumor effect by targeting thioredoxin reductase (TrxR). TrxR is a key enzyme that maintains cellular redox homeostasis and is widely present in various cell types. Aberrant expression of TrxR is closely related to tumors, inflammation, and neurodegenerative diseases. Selective inhibition of TrxR can effectively disrupt the redox balance of tumor cells and induce cell death. Dithiocarbamate (DTC) is a class of bioactive organosulfur compounds that are widely used in anti-cancer research. DTC can form a complex Cu(DTC)2 with copper ions and exhibits significant killing activity in a variety of cancer cells. Cu(DTC)2 can induce oxidative stress, mitochondrial dysfunction, and apoptosis, and has good selective toxicity. Combination therapy strategies can enhance anti-tumor efficacy and reduce the risk of drug resistance. The combination of a TrxR inhibitor and the DTC-Cu system can synergistically disrupt the redox homeostasis within tumor cells. High concentrations of biological thiols in the pathological microenvironment can cleave disulfide bonds to achieve specific activation and drug efficacy release of prodrugs. Based on this, an arsenic inhibitor and a DTC molecule linked by a pathological disulfide bond are designed to construct a controllable-release anti-cancer prodrug. This prodrug responds to the biological thiol signal in the tumor microenvironment, enabling highly selective release of active molecules, targeting TrxR, and synergistically inducing cellular oxidative stress. This strategy is expected to improve the therapeutic effect, reduce systemic toxicity, and expand the development path of combination therapy drugs. Summary of the Invention

[0003] Based on the current status of the above-mentioned prior art research, the present invention designed and synthesized a conjugate prodrug molecule ( P As-DTC) formed by linking an arsenic inhibitor targeting thioredoxin reductase (TrxR) and DTC through a pathological switch disulfide bond. This prodrug can be selectively cleaved under the action of reducing species within tumor cells, releasing two active components, and achieving the purpose of synergistically killing tumor cells by targeting and inhibiting TrxR activity with the arsenic inhibitor and chelating Cu 2+ with DTC.

[0004] Specifically, the present invention provides a prodrug for inhibitor combination therapy, the structure of which is shown in the following formula (I):

[0005]

[0006] The drug molecules involved in the present invention have excellent targeting, synergy, and low toxicity and side effects, so they can be successfully applied to the combined treatment targeting TrxR in cancer treatment. The synergistic effect of arsenic inhibitors and the DTC-Cu system is realized to precisely treat abnormal glutathione content in tumor cells. The present invention provides a new strategy for the development of multi-mechanism combined anti-cancer drugs, opens up new ideas for optimizing anti-cancer treatment regimens, and reveals the potential of prodrug systems activated by pathological microenvironment response in targeted therapy. The specific preparation method of the inhibitor combined treatment prodrug shown in the present invention is as follows:

[0007] Synthesis of compound SS-DTC: Dissolve disulfiram (2.97 g, 10 mmol, 1.0 eq) in 50 mL of absolute ethanol, add 2-mercaptoethanol (780 mg, 10 mmol, 1.0 eq), and reflux the reaction for 4 h. After the reaction is completed, concentrate the reaction solution and purify the product by silica gel column chromatography (petroleum ether / ethyl acetate = 4:1) to obtain the black oily compound SS-DTC.

[0008] Compound p As-DTC and o Synthesis of As-DTC: Dissolve the arsenic compound (1 mmol, 1 eq) in 50 mL of DCM, add TEA (3 mmol, 3 eq) at room temperature, then slowly add triphosgene (0.5 mmol, 0.5 eq), and after reacting at room temperature for 1 h, slowly dropwise add a 10 mL DCM solution containing SS-DTC (1 mmol, 1 eq) to the reaction system, and then add TEA (2 mmol, 2 eq) and react overnight. After the reaction is completed, directly evaporate the reaction solution to dryness and mix the sample, and obtain the target compound by column chromatography with DCM. Description of the Drawings

[0009] Figure 1 It is a schematic diagram of the cytotoxicity test of the As-DTC combined treatment drug.

[0010] Figure 2 It is a schematic diagram of the cytotoxicity test of the As-DTC combined treatment drug on different transfected HeLa cells.

[0011] Figure 3 It is a schematic diagram of measuring the activity of TrxR in living HL-60 cells with the fluorescent probe TRFS-Green.

[0012] Figure 4 Compound p It is a schematic diagram of the production of ROS in HL-60 cells treated with As-DTC.

[0013] Figure 5 Compoundp Schematic diagram of verification related to apoptosis of HL-60 cells under As-DTC treatment.

[0014] Experimental Example 1: Cytotoxicity test experiment of As-DTC combined with therapeutic drugs

[0015] Experimental method: Different cells in the logarithmic growth phase were inoculated in a sterile 96-well plate in the form of a single-cell suspension (1×10 4 cells per well, final volume 100 μL). After adhering for 24 h in an incubator at 37 °C and 5% CO2, gradient concentrations of the combined therapeutic prodrug molecules or the control solution containing Cu 2+ (5 μM) were added respectively (maintaining a total volume of 100 μL / well), and a blank control group containing only complete medium was set. After 24 h of drug treatment, 10 μL of CCK-8 reagent was added to each well, and incubation was continued in the dark for 2 h. The absorbance value of each well was measured at a wavelength of 450 nm using a Thermo Scientific multi-functional microplate reader, and the IC-50 value was further obtained through calculation.

[0016] Experimental results: The toxicity of para-substituted p As-DTC in cells was generally stronger than that of o As-DTC. Further research found that in the case of introducing Cu 2+ , the toxic effect of this molecule on HL-60 cells was significantly higher than that on other cells, which might be because As compounds have a unique toxicological effect on the HL-60 cell line.

[0017] Experimental Example 2: Cytotoxicity test experiment of As-DTC combined with therapeutic drugs on different transfected HeLa cells

[0018] Experimental method: Different cells in the logarithmic growth phase were inoculated in a sterile 96-well plate in the form of a single-cell suspension (1×10 4 cells per well, final volume 100 μL). After adhering for 24 h in an incubator at 37 °C and 5% CO2, gradient concentrations of the combined therapeutic prodrug molecules or the control solution containing Cu 2+ (5 μM) were added respectively (maintaining a total volume of 100 μL / well), and a blank control group containing only complete medium was set. After 24 h of drug treatment, 10 μL of CCK-8 reagent was added to each well, and incubation was continued in the dark for 2 h. The absorbance value of each well was measured at a wavelength of 450 nm using a Thermo Scientific multi-functional microplate reader, and the IC-50 value was further obtained through calculation.

[0019] Experimental results: As Figure 2As shown, two transfected cell lines, namely HeLa-shNT cells and HeLa-shTrxR1 cells, were generated by transfecting non-targeting (NT) control plasmids and plasmids specifically targeting the TrxR1 gene. Compared with HeLa-shNT cells, HeLa-shTrxR1 cells were more sensitive to the treatment with two compounds, and the same p As-DTC exhibited stronger cytotoxicity than o As-DTC..

[0020] Test Example 3: Experiment on measuring the activity of TrxR in living HL-60 cells using the fluorescent probe TRFS-Green

[0021] Experimental method: HL-60 cells were seeded into imaging dishes containing 2 mL of medium at a density of 1×10 5 cells per dish, and then incubated with p As-DTC (10 μM) at 37 °C for 12 h, and then incubated with TRFS-Green (10 μM) for 4 h. After completion, the cells in the dish were washed three times with PBS buffer, and then fluorescence images were collected using a laser confocal microscope (FV-3000).

[0022] Experimental results: It was observed that the control cells showed strong fluorescence emission, while the drug treatment led to dose-dependent inhibition of cellular TrxR activity. The introduction of Cu 2+ had no obvious effect on the fluorescence intensity. The imaging results of TRFS-Green staining supported the demonstration that the compound p As-DTC could effectively inhibit the activity of intracellular TrxR, which was consistent with the detection results of the insulin reduction method shown in the intracellular enzyme activity experiment.

[0023] Test Example 4: Experiment on the production of ROS in HL-60 cells treated with the compound p As-DTC

[0024] Experimental method: HL-60 cells were seeded into imaging dishes containing 2 mL of medium at a density of 1×10 5 cells per dish, and then incubated with As-DTC (10 μM) at 37 °C for 12 h, and then incubated with DCFH-DA (10 μM) or DHE (10 μM) for 30 min. After completion, the cells in the dish were washed three times with PBS buffer, and then fluorescence images were collected using a laser confocal microscope (FV-3000).

[0025] Experimental results: The results showed that the control group of HL-60 cells only showed weak fluorescence, while the fluorescence inside the cells increased significantly after adding p As-DTC. At the same time, the introduction of Cu 2+The fluorescence of the parallel experimental group is slightly stronger than that of the group without Cu. 2+ This is because p As-DTC will be opened by intracellular GSH to release DCT, and the DTC-Cu chelate will also cause cellular oxidative stress. The fluorescence intensity of each group shows an increasing trend with the increase in the compound concentration. The experimental results consistently show that with the increase in the compound concentration, the red fluorescence intensity in HL-60 cells significantly increases, further supporting the dose-dependent increase in the intracellular ROS level. In summary, combining the experimental results of the two probes, it can be clearly confirmed that this compound can effectively induce the generation of ROS in HL-60 cells.

[0026] Test Example 5: Study on the apoptosis mechanism of compound p As-DTC in HL-60 cells

[0027] HL-60 cells were seeded into imaging dishes containing 2 mL of medium at a density of 1×10 5 cells per dish and cultured with the compound p As-DTC at a specified concentration for 24 h, and then Hoechst 33342 dye with a final concentration of 5 μg / mL was added and cultured for another 20 min. After that, the cells in the dish were washed three times with PBS buffer, and then fluorescence images were collected using a laser confocal microscope (FV-3000).

[0028] Experimental results: Weak and uniform blue fluorescence was observed in the control group cells, while after adding the compound p As-DTC, high-fluorescence apoptosis appeared in the cell nuclei, indicating that apoptosis occurred in the cells.

Claims

1. The present invention discloses a preparation method and application of a conjugate drug molecule for collaborative anti-tumor treatment, and its structure is shown as the following formula (I). The inhibitor conjugate prodrug shown in the present invention can be used to specifically inhibit the activity of TrxR enzyme by targeting the thioredoxin reductase system, and simultaneously release DTC with anti-tumor activity to achieve efficient killing of tumor cells:

2. A combined therapeutic drug of an As inhibitor targeting thioredoxin reductase and a DTC-Cu system, characterized in that Design of a conjugate therapeutic drug molecule with a disulfide bond structure, and its structure is shown as the following formula (I):

3. A new use of an inhibitor combination prodrug as described in claim 1, characterized in that Design of a conjugate therapeutic drug molecule with a disulfide bond structure can be used to specifically inhibit the activity of TrxR enzyme by targeting the thioredoxin reductase system, and simultaneously release DTC with anti-tumor activity to achieve efficient killing of tumor cells.

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