A prodrug compound of metal dithiocarbamate complex and its preparation method and application

Abstract

The application discloses a prodrug compound of metal dithiocarbamate complex, a preparation method and application thereof. The prodrug compound can simultaneously carry dithiocarbamic acid and metal ions in a single small molecule, and keeps the two in an inert state; under specific conditions, the dithiocarbamic acid prototype is triggered to be released, the metal chelation function is restored, the intramolecular metal coordination reaction with the metal ions carried in the molecule is generated, and the biologically active metal dithiocarbamate complex is formed. The prodrug compound can realize selective in-situ activation of metal complexes, has a good application prospect in tumor prevention and treatment and diseases related to abnormal metabolism of metal ions, and provides a feasible idea and method for the prodrug design of metal coordination type drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a prodrug compound of dithiocarbamate metal complex, its preparation method and application. Background Technology

[0002] prodrug

[0003] Prodrugs are a classic strategy in drug development. Typically, key groups of an active drug are linked through chemical modification to obtain inactive or low-activity prodrugs, which can be converted into the original active drug under specific in vivo conditions to exert their effects. In recent years, prodrugs that can be selectively activated at disease sites have attracted increasing attention from researchers. For example, in antitumor drug design, researchers utilize the differences between tumor tissue and normal tissue in terms of microenvironment, specific enzymes, and specific surface antigens to design prodrugs, thereby improving drug targeting, reducing adverse reactions, expanding the therapeutic window, or improving the drug's physicochemical properties and bioavailability.

[0004] In-situ synthesized prodrugs

[0005] With the development of bioorthogonal reactions, the concept of novel in-situ synthetic prodrugs has been proposed. In-situ synthetic prodrugs refer to prodrugs that can be synthesized in situ within a living organism through a chemical reaction. This differs from traditional prodrug design. The advantage of this prodrug model lies in the significant structural differences between the prodrug and the active drug; typically, the prodrug structure does not contain the pharmacophore of the active drug, resulting in a substantial difference in activity between the in-situ synthesized active drug and the prodrug. Based on this, this type of prodrug exhibits better performance in improving drug targeting and expanding the therapeutic window. However, activation of prodrugs based on bioorthogonal reactions usually requires the simultaneous presence of the prodrug and another compound (i.e., a bioorthogonal reaction pair), and the reaction occurs upon contact. Therefore, these two compounds need to be administered separately, which greatly limits the efficacy and practical application of this type of prodrug.

[0006] Metal coordination compounds

[0007] Metal coordination compounds hold immense potential in the biomedical field, particularly in oncology and metal metabolism-related diseases. However, because they are typically composed of metal ions and organic ligands linked by coordinate bonds, they possess properties distinct from ordinary small organic molecules, making most traditional prodrug design methods unsuitable for them. Current techniques often utilize the differences in bioactivity between different metal oxidation states to design prodrugs, such as the common tetravalent platinum drugs. This limits the prodrug response to specific stimuli or disease conditions. Therefore, developing broader prodrug design strategies for metal coordination compounds is of significant practical importance. Summary of the Invention

[0008] To overcome the shortcomings of existing technologies, this invention provides a prodrug compound of dithiocarbamate metal complex. The prodrug compound disclosed in this invention can simultaneously carry dithiocarbamate and metal ions in a single small molecule while maintaining both in an inert state. Under specific conditions, it triggers the release of the original dithiocarbamate, restoring its metal chelating function, and undergoes an intramolecular metal coordination reaction with the metal ions carried in the molecule to form a biologically active dithiocarbamate metal complex.

[0009] Purpose of the invention

[0010] The main objective of this invention is to provide a prodrug compound of dithiocarbamate metal complex, its preparation method and application.

[0011] Technical solution

[0012] The objective of this invention can be achieved through the following design scheme:

[0013] A prodrug compound of a dithiocarbamate metal complex, as shown in general formula I:

[0014] General Formula I:

[0015] in, Represents the dithiocarbamate group.

[0016] R1 and R2 can be independent of each other, C1-C1-C2. 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne, C1-C6 alkyl groups optionally substituted with one or more hydroxyl, amino, halogen, cyano, nitro, sulfonyl, aromatic ring, aromatic heterocyclic, and fused-ring aryl groups, or those that can be combined into C3-C7 cycloalkyl, C3-C7 heterocyclic alkyl, C6-C 10 Aryl, 5-10 membered heteroaryl, polycyclic aryl, C6-C 10 Heterocyclic group.

[0017] In some specific embodiments of the present invention, R1 and R2 are two independent diethyl groups, or are combined into a C5 cycloalkyl group.

[0018] L represents a sensitively cleavable group. In this invention, ROS-sensitive bonds such as phenylboronic acid or phenylboronic ester bonds, thioclase bonds or oxalate bonds are preferred; GSH-sensitive bonds such as disulfide bonds are preferred; enzymatically cleavable chemical bonds such as cathepsins, podocyte proteases, matrix metalloproteinases, FAP-α enzymes are preferred; light-sensitive chemical bonds such as o-nitrobenzyl alcohol / sulfur / nitrogen are preferred; more preferably phenylboronic acid or phenylboronic ester bonds, disulfide bonds, and chemical bonds cleavable by FAP-α enzymes are preferred.

[0019] The ROS-sensitive phenylboronic acid bond is a group containing a phenylboronic acid structure. In some specific embodiments of the present invention, the ROS-sensitive phenylboronic acid bond preferably has the following structure:

[0020] ,

[0021] The ROS-sensitive phenylboronic acid bond is a group containing a phenylboronic ester structure. In some specific embodiments of the present invention, the ROS-sensitive phenylboronic ester bond preferably has the following structure:

[0022] ,

[0023] The structure of the GSH-sensitive disulfide bond is -SS-, where one S comes from a dithiocarbamate group. In some specific embodiments of the present invention, the following structure is preferred:

[0024]

[0025] The chemical bonds involved in FAP-α cleavage are groups containing proline and glycine dipeptides that can be specifically recognized and cleaved by the FAP-α enzyme. In other specific embodiments of the present invention, the following structure is preferred:

[0026]

[0027] Y is an organic ligand that can coordinate with metal ions, and its structural characteristics are represented by the following general formula I-a:

[0028] General formula I-a:

[0029] Where X represents C and N.

[0030] R3 and R4 can be two identical or different coordinating groups, or one can be a polydentate coordinating group and the other can be hydrogen. The structural characteristics of the coordinating groups are: containing one or more coordinating groups such as carboxyl groups, O and O electron donors, N and O electron donors, or N, O, and S electron donors, including but not limited to the following groups:

[0031] , , , , ,

[0032] More preferably, Y has the following structure:

[0033] , , , , , , , ,

[0034] In some embodiments of the present invention, Y is:

[0035] , ,

[0036] --- represents a coordinate bond; M is a metal ion, preferably Cu. 2+ Cu + Fe 3+ Fe 2+ Zn 2+ Ca 2+ , Mn 2+ ;

[0037] More preferably Cu 2+ Zn 2+ .

[0038] In some specific embodiments of the present invention, M is Cu 2+ .

[0039] Preferably, the prodrug compounds of the dithiocarbamate metal complex of the present invention are of the following general formulas II-a and II-b:

[0040] General formula II-a:

[0041] General Formula II-b:

[0042] Wherein, L is preferably:

[0043] , , , , ,

[0044] Y is preferably:

[0045] , , , , , , , ,

[0046] --- represents a coordinate bond; M is a metal ion, preferably Cu. 2+ .

[0047] In some specific embodiments of the present invention, a prodrug compound of a ROS-sensitive copper diethyldithiocarbamate coordination compound, abbreviated as DPBD-Cu, is obtained, wherein,

[0048] R1 and R2 are both ethyl groups, and L is... Its carbonyl end is connected to Y, where Y is M is Cu 2+ .

[0049] In some specific embodiments of the present invention, a prodrug compound of a ROS-sensitive copper diethyldithiocarbamate coordination compound, abbreviated as DPPBD-Cu, is obtained, wherein,

[0050] R1 and R2 are both ethyl groups, and L is... Y is M is Cu 2+ .

[0051] The aforementioned prodrug compound of the ROS-sensitive copper diethyldithiocarbamate coordination compound can oxidize phenylboronic acid / phenylboronic ester to phenol under the action of ROS, releasing diethyldithiocarbamate (DTC) through a self-elimination reaction. This compound can react with Cu in the molecule. 2+ A metal coordination reaction occurs to form a biologically active copper diethyldithiocarbamate complex (Cu(DTC)2).

[0052] The ROS mentioned include: superoxide anion, hydrogen peroxide, hydroxyl radical, hypochlorite ion, singlet oxygen, etc.

[0053] This invention provides a method for preparing the prodrug compound of the above-mentioned ROS-sensitive copper diethyldithiocarbamate coordination compound, comprising the following steps:

[0054] First, synthesize the ROS-sensitive component. It is reacted with sodium diethyldithiocarbamate, and the resulting product is demethylated and protected before being reacted with... A condensation reaction yields the compound shown below, abbreviated as DPBD. Mixing DPBD with copper nitrate trihydrate yields the coordination compound DPBD-Cu, with a coordination molar ratio of 1:1. (The figure shows its possible coordination forms.)

[0055] DPBD:

[0056] DPBD-Cu:

[0057] Alternatively, synthesize the ROS-sensitive component. It reacts with sodium diethyldithiocarbamate, and then with... A condensation reaction yields the compound shown below, abbreviated as DPPBD. Mixing DPPBD with copper nitrate trihydrate yields the coordination compound DPPBD-Cu, with a coordination molar ratio of 1:1. (The figure shows its possible coordination forms.)

[0058] DPPBD:

[0059] DPPBD-Cu:

[0060] The beneficial effects of this invention are:

[0061] Compared with existing technologies, the present invention provides a prodrug compound of dithiocarbamate metal complex, its preparation method and application.

[0062] The prodrug compound of dithiocarbamate metal coordination provided by this invention is a single small molecule compound that can trigger the release of dithiocarbamate under specific conditions, restore its metal chelating function, and undergo intramolecular metal coordination reaction with metal ions carried in the molecule to form a biologically active dithiocarbamate metal complex.

[0063] Compared to traditional prodrug molecules or prodrug molecules designed based on bioorthogonal reactions, the dithiocarbamate metal complex prodrug compound provided by this invention is a single small molecule compound, avoiding separate administration and exhibiting better drug-like properties. Furthermore, its in-situ triggering of intramolecular metal coordination reactions results in a prodrug activation mode where the formed active metal complex exhibits enhanced activity differences compared to the prodrug compound, significantly broadening the therapeutic window of the metal complex. This demonstrates excellent clinical applicability.

[0064] The prodrug compounds disclosed in this invention can achieve selective in-situ activation of metal complexes, and have good application prospects in tumor prevention and treatment and diseases related to abnormal metal ion metabolism. At the same time, they provide feasible ideas and methods for the prodrug design of metal coordination drugs, overcoming the problem of the current single type of prodrug design for metal coordination drugs. Attached Figure Description

[0065] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings, wherein:

[0066] Figure 1 The image shows the 1H NMR spectrum of the DPBD in Example 1.

[0067] Figure 2 This is the mass spectrum of DPBD in Example 1.

[0068] Figure 3 This is the mass spectrum of DPBD-Cu in Example 1.

[0069] Figure 4 The image shows the hydrogen NMR spectrum of DPPBD in Example 2.

[0070] Figure 5 The image shows the hydrogen NMR spectrum of the DPD in Example 3.

[0071] Figure 6 This shows the ROS-sensitive cleavage of DPBD-Cu in Example 4. In the figure, A represents the change in the UV absorbance of DPBD-Cu at 420 nm under different hydrogen peroxide concentrations. B represents the change in the HPLC-MS / MS spectrum of DPBD-Cu under hydrogen peroxide incubation. This indicates that DPBD-Cu can undergo a series of reactions under hydrogen peroxide, ultimately forming Cu(DTC)₂.

[0072] Figure 7 The results show the cytotoxicity of each prodrug compound in Example 5.

[0073] Figure 8 This study investigates the mechanism of action of DPBD-Cu on 4T1 cells in Example 6. In the figures, A represents the result of DPBD-Cu inducing the accumulation of ubiquitinated proteins in cells, and BC represents the result of inducing ROS production in cells. This indicates that DPBD-Cu can induce the accumulation of ubiquitinated proteins and the production of ROS in 4T1 cells by forming Cu(DTC)2.

[0074] Figure 9 The figure shows the antitumor effect of DPBD-Cu in the mouse 4T1 tumor model in Example 7. It indicates that DPBD-Cu has a better antitumor effect, which is more significant than the combined effect of either drug alone.

[0075] Specific implementation methods

[0076] The preferred embodiments will be explained in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the invention.

[0077] Example 1: Preparation of DPBD-Cu, a prodrug compound of ROS-sensitive copper diethyldithiocarbamate complex.

[0078] (1) The preparation process of DPBD compound is as follows:

[0079]

[0080] 1) Preparation of intermediate compound 1: Mono-Boc-ethylenediamine (4.00 g, 24.97 mmol, 1 eq), 2-chloromethylpyridine hydrochloride (9.01 g, 54.93 mmol, 2.2 eq), and anhydrous sodium sulfate (13.23 g, 124.84 mmol, 5 eq) were dissolved in 200 mL of methanol and heated under reflux for 72 h under nitrogen protection. After the reaction was complete, the solvent was evaporated, and the mixture was redissolved in dichloromethane and filtered. The filtrate was concentrated and purified by silica gel column chromatography (dichloromethane / methanol = 20:1) to obtain the intermediate compound 1.

[0081] 2) Preparation of DPA-NH2: Intermediate compound 1 (6.00 g, 17.52 mmol, 1 eq) was dissolved in 150 mL of dichloromethane and placed in an ice bath. Trifluoroacetic acid (26 mL, 20 eq) was slowly added dropwise under ice bath conditions, and the reaction was stirred at room temperature for 2 h. After the reaction was complete, the pH of the reaction solution was adjusted to 12-14 with 4M NaOH, then extracted with dichloromethane and washed with saturated brine. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness to obtain the final product.

[0082] 3) Preparation of intermediate compound 2: 2-(4-bromophenyl)-2-hydroxyacetic acid (5.00 g, 21.64 mmol, 1 eq) was dissolved in 50 mL of methanol. H₂SO₄ (1.15 mL, 11.76 mmol, 1 eq) was dissolved in 10 mL of methanol and slowly added dropwise to the above solution. The mixture was heated under reflux for 2 h. After the reaction was complete, the solvent was evaporated, the mixture was reconstituted with ethyl acetate, and washed three times with saturated brine. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness to obtain the final product.

[0083] 4) Preparation of intermediate compound 3: Intermediate compound 2 (5.13 g, 20.93 mmol, 1 eq), bis(pinacolyl)diboron (7.97 g, 31.40 mmol, 1.5 eq), 1,1'-bis(diphenylphosphino)ferrocene palladium(II) dichloromethane complex (0.51 g, 0.63 mmol, 0.03 eq), and sodium acetate (6.16 g, 62.80 mmol, 3 eq) were dissolved in 150 mL of anhydrous 1,4-dioxane and reacted in an oil bath at 80 °C for 12 h under nitrogen protection. After the reaction was complete, the reaction solution was concentrated, diluted with ethyl acetate, and washed with saturated ammonium chloride, water, and saturated brine, respectively. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1).

[0084] 5) Preparation of intermediate compound 4: Intermediate compound 3 (2.69 g, 9.21 mmol, 1 eq) was dissolved in 50 mL of anhydrous dichloromethane and placed in an ice bath. Thionyl chloride (13.36 mL, 184.16 mmol, 20 eq) was dissolved in a small amount of dichloromethane and slowly added dropwise to the reaction solution under an ice bath. The mixture was heated under reflux overnight under nitrogen protection. After the reaction was complete, the reaction was quenched with 20 mL of water under an ice bath, followed by washing with water (50 mL × 3) and saturated brine (50 mL × 3) successively. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain the final product.

[0085] 6) Preparation of intermediate compound 5: Intermediate compound 4 (2.21 g, 7.12 mmol, 1 eq) was dissolved in 15 mL of anhydrous acetonitrile, and sodium diethyldithiocarbamate (1.28 g, 7.47 mmol, 1.05 eq) was dissolved in 15 mL of anhydrous acetonitrile. The dissolved acetonitrile was added dropwise to the above solutions, and the mixture was stirred at room temperature until a white precipitate formed. After the reaction was complete, the reaction solution was filtered, the filtrate was concentrated, and dissolved in 50 mL of dichloromethane. The solution was washed with water (50 mL × 3) and saturated brine (50 mL × 1), respectively. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 20:1).

[0086] 7) Preparation of intermediate compound 6: Intermediate compound 5 (2.12 g, 5.01 mmol, 1 eq) was dissolved in 50 mL of tetrahydrofuran. Sodium hydroxide (0.40 g, 10.01 mmol, 2 eq) was dissolved in 50 mL of water and added dropwise to the above solution. The reaction mixture was stirred at room temperature. After approximately 2 h, the reaction solution was extracted with ethyl acetate. The organic phase was collected and extracted three times with water, and the aqueous phase was collected. The pH of the aqueous phase was adjusted to approximately 5 with 1 M HCl, and the mixture was extracted with dichloromethane (100 mL × 3), then washed once with water (100 mL × 1), and all dichloromethane layers were collected. The mixture was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain the final product.

[0087] 8) Preparation of DPBD: Intermediate compound 6 (1.60 g, 3.91 mmol, 1 eq) and DPA-NH2 (1.14 g, 4.69 mmol, 1.2 eq) were dissolved in 100 mL of anhydrous dichloromethane. Under nitrogen protection, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDCI (1.50 g, 7.82 mmol, 2 eq), 4-dimethylaminopyridine DMAP (0.05 g, 0.39 mmol, 0.1 eq), and triethylamine (0.87 g, 8.60 mmol, 2.2 eq) were added to the above solution, and the mixture was stirred at room temperature for 24 h. After the reaction was complete, the reaction solution was immediately washed with water. Collect the organic phase, dry it with anhydrous sodium sulfate, filter it, concentrate the filtrate, and purify it by silica gel column chromatography (dichloromethane:methanol = 13:1).

[0088] (2) Preparation of DPBD-Cu: DPBD is soluble in DMSO, acetonitrile, or methanol to obtain a DPBD mother liquor of a certain concentration. Copper nitrate trihydrate or copper chloride dihydrate is dissolved in water. According to the concentration requirement, the two are mixed in a molar ratio of 1:1 to obtain DPBD-Cu.

[0089] Example 2: Preparation of DPPBD-Cu, a prodrug compound of ROS-sensitive copper diethyldithiocarbamate complex.

[0090] (1) The preparation process of DPPBD compound is shown below:

[0091]

[0092] 1) Preparation of intermediate compound 7: 2,6-bis(hydroxymethyl)-p-cresol (10 g, 59.46 mmol, 1 eq) and imidazole (8.91 g, 130.80 mmol, 2.2 eq) were dissolved in 40 mL of anhydrous DMF and placed in an ice bath. Tert-butyldimethylchlorosilane was dissolved in 40 mL of anhydrous DMF and added dropwise to the above reaction solution under ice bath conditions. The reaction mixture was stirred at room temperature for 2 h. The reaction solution was diluted with 200 mL of ethyl acetate and washed three times with water (100 mL × 3). The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 50:1) to obtain the final product.

[0093] 2) Preparation of intermediate compound 8: Intermediate compound 7 (5.11 g, 12.88 mmol, 1 eq) was dissolved in 25 mL of anhydrous DMF and placed in an ice bath. Potassium carbonate (2.14 g, 15.46 mmol, 1.2 eq) was added and the mixture was stirred for 10 minutes. Then, 4-bromomethylphenylboronic acid pinacol ester (3.83 g, 12.88 mmol, 1 eq) was added to the above reaction solution, and the mixture was stirred at room temperature. After the reaction was complete, the solution was diluted with 200 mL of ethyl acetate and washed with saturated ammonium chloride, water, and saturated brine, respectively. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 50:1) to obtain the final product.

[0094] 3) Preparation of intermediate compound 9: Intermediate compound 8 (5.80 g, 9.46 mmol, 1 eq) and p-toluenesulfonic acid (0.36 g, 1.89 mmol, 0.2 eq) were dissolved in 25 mL of methanol and stirred at room temperature for 2 h. After the reaction was complete, the reaction solution was concentrated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain the final product.

[0095] 4) Preparation of intermediate compound 10: Under nitrogen protection, intermediate compound 9 (2.46 g, 6.40 mmol, 1 eq) was dissolved in 10 mL of anhydrous tetrahydrofuran. Phosphorus tribromide (3.01 mL, 32.01 mmol, 5 eq) in anhydrous tetrahydrofuran was added in an ice bath, and the reaction was stirred in an ice bath. After the reaction was complete, the solution was concentrated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5:1).

[0096] 5) Preparation of DPPBD: Intermediate compound 10 (1.77 g, 3.47 mmol, 1 eq) was dissolved in 10 mL of anhydrous acetonitrile, sodium diethyldithiocarbamate (1.28 g, 7.47 mmol, 1.05 eq) was dissolved in 10 mL of anhydrous acetonitrile, and added dropwise to the above solutions. The reaction was stirred at room temperature, and the reaction was monitored by TLC (petroleum ether: ethyl acetate = 5:1). After the reaction was complete, the solvent was evaporated to dryness, and the product was directly used for the next reaction without purification. Under nitrogen protection, the above product (0.382 g, 0.66 mmol, 1 eq), dimethylpyridinium chloride (0.132 g, 0.66 mmol, 1 eq), and potassium carbonate (0.091 g, 0.66 mmol, 1 eq) were dissolved in 5 mL of anhydrous acetonitrile, and the reaction was stirred at room temperature. After the reaction is complete, the reaction solution is filtered, the filtrate is concentrated, and purified by silica gel column chromatography (petroleum ether: acetone = 8:1) to obtain the final product.

[0097] (2) Preparation of DPPBD-Cu: DPBD is soluble in DMSO, acetonitrile, or methanol to obtain a certain concentration of DPPBD mother liquor. Copper nitrate trihydrate or copper chloride dihydrate is dissolved in water. As needed, the two are mixed in a molar ratio of 1:1 to obtain DPPBD-Cu.

[0098] Example 3: Preparation of negative control DPD-Cu

[0099] (1) The preparation process of DPD compounds is as follows:

[0100]

[0101] 1) Preparation of intermediate compound 11: α-Bromophenylacetic acid (0.50 g, 2.33 mmol, 1 eq) was dissolved in 5 mL of anhydrous acetonitrile, and sodium diethyldithiocarbamate (0.42 g, 2.44 mmol, 1.05 eq) was dissolved in 5 mL of anhydrous acetonitrile. The α-bromophenylacetic acid was added dropwise to the above solution, and the mixture was stirred at room temperature until a white precipitate formed. After the reaction was complete, the reaction solution was filtered, concentrated, and redissolved in 25 mL of double-distilled water. The pH was adjusted to approximately 5 with 1M HCl, and the mixture was extracted with dichloromethane (50 mL × 3), collecting all the dichloromethane layers. The extract was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain the intermediate compound 11.

[0102] 2) Preparation of DPD: Intermediate compound 11 (0.425 g, 1.50 mmol, 1 eq) and DPA-NH2 (0.436 g, 1.80 mmol, 1.2 eq) were dissolved in 50 mL of anhydrous dichloromethane. Under nitrogen protection, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDCI (0.575 g, 3.00 mmol, 2 eq), 4-dimethylaminopyridine DMAP (0.018 g, 0.15 mmol, 0.1 eq), and triethylamine (0.334 g, 3.30 mmol, 2.2 eq) were added to the above solution, and the mixture was stirred at room temperature for 24 h. After the reaction was complete, the reaction solution was immediately washed with water (50 mL × 1). Collect the organic phase, dry it with anhydrous sodium sulfate, filter it, concentrate the filtrate, and purify it by silica gel column chromatography (dichloromethane:methanol = 15:1).

[0103] (2) Preparation of DPD-Cu: DPD can be dissolved in DMSO, acetonitrile, or methanol to obtain a DPD mother liquor of a certain concentration. Copper nitrate trihydrate or copper chloride dihydrate is dissolved in water. As needed, the two are mixed in a molar ratio of 1:1 to obtain DPD-Cu.

[0104] Example 4: ROS sensitivity study of DPBD-Cu

[0105] The ROS sensitivity of DPBD-Cu was investigated using UV spectrophotometry and HPLC-MS / MS, respectively. DPBD and copper nitrate trihydrate were dissolved in DMSO and pure water, respectively, and DPBD-Cu was mixed with them at a molar ratio of 1:1. DPBD-Cu (2 mM) was mixed with different concentrations of hydrogen peroxide (0, 5, 10, 20 mM), and the UV-Vis spectra were scanned at 5-minute intervals after mixing using a UV spectrophotometer to examine the change in absorbance at 420 nm. Furthermore, a mixed sample of DPBD-Cu (10 μM) and hydrogen peroxide (50 μM) was prepared (solvent ratio of methanol / water / DMSO = 90 / 9.9 / 0.1), and the changes in DPBD-Cu were examined in the HPLC-MS / MS system.

[0106] Example 5: Cytotoxicity investigation of DPD-Cu and DPBD-Cu

[0107] 4T1 cells were fed at a rate of 1×10 4 The cells were seeded at a density of 100% in each well of a 96-well plate and cultured overnight in a cell culture incubator. After cell attachment, Cu, DPD, DPD-Cu, DPD-Cu+H2O2, DPBD, DPBD-Cu, and DPBD-Cu+H2O2 were added to each well and incubated for 24 h. Subsequently, 20 μL of MTT (5 mg / mL in PBS) was added to each well, and the cells were incubated in the dark for 4 h. The culture medium was then discarded, and 100 μL of DMSO was added. The cells were shaken at 100 rpm for 10 min in the dark, and the absorbance at 490 nm was measured using a microplate reader. The control group (no drug added) and the blank group (no cells added) were used as the control group. The cell viability after drug administration was calculated using the following formula: (OD sample - OD blank / OD control - OD blank) × 100%.

[0108] Example 6: Investigation of the cellular mechanism of DPBD-Cu

[0109] Intracellular ROS levels were determined using the DCFH-DA method. 4T1 cells were loaded with 1×10⁻⁶ cells. 5 The cells were seeded at a density of 1:1 in each well of a 6-well plate and incubated overnight in a cell culture incubator. After cell attachment, DPBD-Cu was added and the cells were incubated for 12 h. Then, the culture medium was discarded, and serum-free culture medium of DCFH-DA (10 μM) was added to each well, and the cells were incubated for another 30 min. Subsequently, the culture medium was discarded, and the cells were washed three times with PBS. The cells were digested with trypsin and collected by centrifugation. The fluorescence of the collected cells was analyzed by flow cytometry.

[0110] The level of ubiquitinated proteins in cells was determined using Western blot. 4T1 cells were sputtered at a concentration of 1 × 10⁻⁶. 5The cells were seeded at a density of 1:1 in each well of a 6-well plate and incubated overnight in a cell culture incubator. After cell attachment, DPBD-Cu was added and the cells were incubated for 12 h. Then, the original culture medium was discarded, the cells were washed three times with PBS, lysis buffer was added, and the cells were lysed on ice for 30 min. All cells and liquid were then collected in 1.5 mL EP tubes and centrifuged at 12000 rpm for 20 min at 4 °C. The supernatant was collected. Protein concentration was quantified using a BCA protein quantification kit. Proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking in 5% skim milk at room temperature for 2 hours, the PVDF membrane was incubated overnight at 4 °C with ubiquitin protein and β-Actin primary antibody, respectively. Subsequently, the PVDF membrane was incubated with a secondary antibody bound to HRP at room temperature for 2 hours and finally developed by chemiluminescence.

[0111] Example 7: In vivo antitumor activity of DPBD-Cu

[0112] First, a mouse subcutaneous 4T1 tumor model was constructed. 4T1 cells were cultured in DMEM medium, digested with trypsin, and the cells were collected and dispersed in PBS solution. 4T1 cells (1×10⁻⁶) were then... 6 A 4T1 breast cancer ectopic subcutaneous transplantation model was established in female BALB / c mice by injecting a suspension of 1 cell / mouse into the subcutaneous segment of the right back of the mice after 5-6 weeks of gestation. Tumor cell inoculation sites were observed from the second day onwards, and tumor growth was recorded. The long axis (L) and short axis (W) of the subcutaneous tumor were measured using calipers, and the volume was calculated using the formula (V) = L*W. 2 / 2 Calculate tumor volume.

[0113] When the subcutaneous tumor of 4T1 breast cancer in BALB / c mice grows to 100 mm 3 Mice were randomly divided into three groups (n=7): saline group, DPBD+Cu group (both injected separately twice), and DPBD-Cu group. All were administered via tail vein injection, once every two days, for a total of five administrations. Starting from the day of administration, the tumor size of mice in each group was measured every other day, and changes in mouse weight were recorded. Tumor volume was calculated using the volume formula. Mice were euthanized by cervical dislocation on day 18.

Claims

1. A prodrug compound of a dithiocarbamate metal complex, characterized in that, The compound represented by general formula I: General Formula I: in, It is a dithiocarbamic acid group. R1 and R2 are either identical or different C1-C6 alkyl groups; L is a group that can sensitively respond to cleavage, selected from... , ; Y is an organic ligand that coordinates with a metal ion, selected from... , ; --- is the coordinate key; M is a metal ion selected from Cu. 2+ .

2. The prodrug compound of the dithiocarbamate metal complex according to claim 1, characterized in that, R1 and R2 are either the same or different independent methyl and ethyl groups, respectively.

3. The prodrug compound of the dithiocarbamate metal complex according to claim 2, characterized in that, R1 and R2 are the same independent ethyl groups.

4. The use of the prodrug compound of the dithiocarbamate metal complex according to any one of claims 1-3 in the preparation of medicaments for the prevention and / or treatment of tumor diseases.

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