Preparation method and application of glutathione-activated diagnosis and treatment prodrug FR-SS-DTC

Abstract

The invention belongs to the technical field of medicines, and discloses a diagnosis and treatment prodrug FR-SS-DTC with a disulfide bond unit as a glutathione response site. An anti-cancer drug dithiocarbamate molecule (DTC) is connected with a fluorescence labeling molecule through a disulfide chain for the first time, the disulfide chain is reduced and broken under the action of glutathione, and then DTC with high anti-cancer activity and signal molecules emitting red fluorescence are released. The diagnosis and treatment prodrug molecule disclosed by the invention can release fluorescent molecules and drug groups after specifically responding to glutathione in a living body, so that the purpose of integrating diagnosis and treatment is achieved, and the application prospect is very good.

Description

Technical Field

[0001] The present invention relates to the field of biochemical medicine, and in particular to a preparation method and application of a novel diagnostic and therapeutic prodrug molecule using a disulfide bond as a glutathione recognition site. Background Art

[0002] Diagnostic and therapeutic prodrugs are an important research direction in the field of precision medicine in recent years. The core is to integrate diagnostic and therapeutic functions into a single drug system, and to achieve precise disease management through intelligent responses at the molecular level. This type of drug is usually composed of an active drug molecule, a cleavable linker arm and a fluorescent group, and has the dual functions of lesion localization and imaging and controlled drug release. In terms of design strategy, diagnostic and therapeutic prodrugs mainly use pathological microenvironment-specific biomarkers (such as pH, enzymes, redox gradients) or exogenous stimuli (light, magnetism, ultrasound) as triggering mechanisms. DTC is a class of sulfur-containing organic compounds whose unique chemical structure gives it metal chelation, antioxidant and bioactivity regulation capabilities. In recent years, it has shown a wide range of application value in the field of chemical biology. DTC specifically binds to Cu through its strong metal chelation ability. 2+ , showing a unique role in cancer treatment. Copper, as a key factor in promoting cancer, is involved in the regulation of angiogenesis, metastasis and drug resistance. DTC chelates Cu 2+ Destroy its cancer-promoting pathway, while DTC carries Cu 2+ After that, it showed considerable anti-cancer activity, which makes it a promising new strategy for targeted therapy. The DTC prodrugs reported so far are all activated and released by a single molecule without changes in the fluorescence signal, which makes it impossible to monitor and capture the release site and release efficiency of the drug molecule in the body. In summary, the development of DTC diagnostic and therapeutic prodrugs based on fluorescence signals has very good application prospects. Summary of the invention

[0003] Based on the above situation, the present invention provides for the first time a method for preparing a glutathione-activated diagnostic and therapeutic prodrug based on a disulfide bond as a recognition unit and its use. The diagnostic and therapeutic prodrug shown in the present invention can be used for fluorescent labeling of cells and inhibition of tumor activity:

[0004] Specifically, the present invention provides a diagnostic and therapeutic prodrug, the structure of which is shown in the following formula (I):

[0005]

[0006] The present invention has excellent specificity for tumor cells, high sensitivity, and a good inhibitory effect on cell activity. Therefore, it can be successfully applied to the monitoring of the state and inhibition of the activity of cancer cells in biological systems. It realizes the specific recognition of glutathione and can efficiently kill cancer cells. The present invention provides a new tool for the diagnosis and treatment evaluation of diseases related to abnormal expression of glutathione, provides a new option for promoting the development of glutathione-activated diagnostic and therapeutic prodrugs, and reveals the potential of disulfide bond units as novel and specific glutathione receptor moieties.

[0007] The specific preparation method of the fluorescent molecular probe shown in the present invention is as follows:

[0008]

[0009] Reagents and Condition: (a) EtOH, reflux; (b) Methanesulfonic acid, HCIO4, 5h. (c) Triphosgene, TEA, DCM, 12h.

[0010] Synthesis of compound SS-DTC: Dissolve disulfiram (10 mmol, 1.0 eq) in 50 mL of anhydrous ethanol, add 2-mercaptoethanol (10 mmol, 1.0 eq), and heat under reflux 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 a black oily compound SS-DTC (yield: 42%).

[0011] Synthesis of compound 1: Dissolve 4-(diethylamino)salicylaldehyde (1 mmol, 1 eq) and 6-amino-1,2,3,4-tetrahydro-1-naphthone (1.1 mmol, 1.1 eq) in methanesulfonic acid, stir at 90 °C for 5 h, cool to room temperature, pour the reaction mixture into 200 mL of ice water, and then slowly pour perchloric acid into the system. Filter by suction and dry under reduced pressure to obtain a black solid product.

[0012] Synthesis of compound FR-SS-DTC: Dissolve compound 1 (0.5 mmol, 1 eq) in 50 mL of dry DCM, add DMAP (1 mmol, 2 eq), add triphosgene (0.25 mmol, 0.5 eq) at 0 °C, and then raise the temperature to room temperature and react for 2 h. Then add TEA (100 mg, 1 mmol, 2 eq), and finally dissolve SS-DTC (225 mg, 1 mmol, 2 eq) in 10 mL of dry DCM and slowly add it dropwise to the reaction solution at 0 °C. After the addition is complete, react at room temperature for 8 h. After the reaction is completed, concentrate the reaction solution and purify the reaction system by silica gel column chromatography (DCM:MeOH = 100:1) to obtain the target compound. Description of the Drawings

[0013] Figure 1 It is a schematic diagram of the UV-visible absorption spectrum of the reaction between FR-SS-DTC and GSH.

[0014] Figure 2 It is a schematic diagram of the time-dependent fluorescence spectrum of the reaction between FR-SS-DTC and GSH.

[0015] Figure 3 It is a schematic diagram of the concentration-dependent fluorescence spectrum of the reaction between FR-SS-DTC and GSH.

[0016] Figure 4 It is a schematic diagram of the fluorescence spectrum of FR-SS-DTC towards GSH at different pH values.

[0017] Figure 5 It is a schematic diagram of the selective fluorescence spectrum of FR-SS-DTC towards biothiols.

[0018] Figure 6 It is a schematic diagram of the high performance liquid chromatography of the reaction between FR-SS-DTC and GSH.

[0019] Figure 7 It is a schematic diagram of the cytotoxic activity results of FR-SS-DTC against HepG2 and HeLa cells.

[0020] Figure 8 It is a schematic diagram of the cellular imaging of FR-SS-DTC towards biothiols (A) and the cellular imaging after treatment with NEM (B).

[0021] Figure 9 It is a schematic diagram of the zebrafish imaging of FR-SS-DTC towards biothiols.

[0022] Experimental Example 1: UV-visible Absorption Spectrum Experiment of the Reaction between FR-SS-DTC and GSH

[0023] Experimental method: The absorbance of FR-SS-DTC was measured by incubating FR-SS-DTC (10 μM) with GSH in PBS (10 mM, pH = 7.4) at 37 °C for 70 minutes.

[0024] Experimental results: FR-SS-DTC showed an obvious absorption peak at 550 nm. After incubation with GSH, a red shift occurred at 585 nm, indicating the reactive release of the fluorophore towards GSH.

[0025] Experimental Example 2: Time-dependent Fluorescence Response Experiment of the Reaction between FR-SS-DTC and GSH

[0026] Experimental method: The time-dependent fluorescence response of FR-SS-DTC was measured by incubating FR-SS-DTC (10 μM) with GSH in PBS (10 mM, pH = 7.4) at 37 °C and measuring every five minutes for a total of 70 minutes.

[0027] Experimental results: As Figure 2 shown, an attenuated fluorescence signal centered at λ em = 625 nm was initially observed for FR-SS-DTC, and this signal gradually increased with time and reached a peak within about 70 minutes. These results confirmed the release of the unmasked fluorophore triggered by GSH, resulting in enhanced fluorescence.

[0028] Test Example 3: Concentration-dependent fluorescence response experiment of the reaction between FR-SS-DTC and GSH

[0029] Experimental method: The concentration-dependent fluorescence response of FR-SS-DTC was measured by incubating FR-SS-DTC with different concentrations of GSH in PBS (10 mM, pH 7.4) at 37 °C for 70 minutes.

[0030] Experimental results: The fluorescence response of FR-SS-DTC to different concentrations of GSH (0 mM to 2 mM) was observed. As Figure 3 shown, the fluorescence signal centered at λ em = 625 nm increased significantly with the increase in GSH concentration. These results further supported the excellent and quantitative response characteristics of FR-SS-DTC to biothiols.

[0031] Test Example 4: Fluorescence spectral response experiment of FR-SS-DTC to GSH at different pH values

[0032] Experimental method: FR-SS-DTC and GSH (1 mM) were added to buffer solutions with different pH values to prepare solutions with appropriate concentrations and incubated for a certain time, and then their fluorescence spectra were measured.

[0033] Experimental results: FR-SS-DTC showed weak fluorescence signals in the pH range of 6.0 - 9.0. When FR-SS-DTC was incubated with GSH (1 mM), the fluorescence intensity increased sharply in the pH range of 7.0 - 8.5. In the pH range of 7.0 - 9.0, the relative fluorescence intensity remained stable, indicating that this molecule is suitable for physiological conditions.

[0034] Test Example 5: Selective fluorescence spectral experiment of FR-SS-DTC for biothiols

[0035] Experimental method: FR-SS-DTC and various test substances were respectively added to PBS buffer solution to prepare solutions with appropriate concentrations and incubated for 70 minutes, and then their fluorescence spectra were measured.

[0036] Experimental results: Even at higher concentrations (100 eq.), the incubation of FR-SS-DTC (10 μM) with all these potential interfering species had a weak effect on the fluorescence signal of the designed prodrug.

[0037] Test Example 6: High-performance liquid chromatography experiment to clarify the reaction mechanism between FR-SS-DTC and GSH

[0038] Experimental method: FR-SS-DTC was added to PBS buffer solution to prepare a solution with an appropriate concentration, and then an appropriate amount of GSH was added, and then its high-performance liquid chromatography was measured.

[0039] Experimental results: When FR-SS-DTC with a retention time of 8.2 minutes was incubated with GSH (1 mM), a product FR-NH2 with a retention time of 6.7 minutes was produced. These results indicate that FR-SS-DTC reacts with GSH, resulting in the cleavage of the carbamate bond and the formation of FR-NH2.

[0040] Test Example 7: Cytotoxicity experiment of FR-SS-DTC on HepG2, HeLa, and 4T1 cells

[0041] Experimental method: In a 96-well plate, 1×10 4 HepG2, HeLa, and 4T1 cells were added to each well, and the compounds in FR-SS-DTC were used to act for 24 hours, and finally the cytotoxicity was measured by the CCK-8 method.

[0042] Experimental results: As Figure 7 shown, since the toxicity of DTC depends on the presence of Cu 2+ we also set up a group with only the prodrug molecule and a group containing the prodrug molecule and Cu 2+ The results showed that all five molecules exhibited very good cytotoxicity in the presence of Cu 2+ while having almost no toxic effect on various cells in the absence of Cu 2+ The results showed that all five molecules exhibited very good cytotoxicity in the presence of Cu, while having almost no toxic effect on various cells in the absence of Cu.

[0043] Test Example 8: Cellular imaging experiment of FR-SS-DTC on glutathione

[0044] Experimental method: FR-SS-DTC was applied to image glutathione in HepG2 cells, and fluorescence images were taken at 30-minute intervals for a total of 90 minutes. Next, HepG2 cells were incubated with N-ethylmaleimide (NEM) at 37 °C for 30 minutes to block cellular biothiols. Then, the NEM-incubated cells were incubated with FR-SS-DTC at 37 °C for 90 minutes.

[0045] Experimental results: Strong fluorescence signals appeared in HepG2 cells treated with FR-SS-DTC, as shown in Figure 8 (A), and the signal was observed to gradually increase over time. After incubation with NEM, a weakened fluorescence signal was observed in the test cells due to the blockage of cellular biothiols by NEM, as shown in Figure 8 (B). These results further verified the selective reaction of FR-SS-DTC with glutathione to release fluorescence and its ability to serve as a highly selective theranostic prodrug for monitoring and inhibiting cancer cell activity.

[0046] Test example 9: FR-SS-DTC imaging test of GSH in zebrafish Experimental method: FR-SS-DTC was applied to image zebrafish. Next, the zebrafish were incubated with NEM, then treated with FR-SS-DTC and imaged, and then treated with GSH and imaged.

[0047] Experimental results: As shown in Figure 9 , there was no fluorescence in the control group, while the zebrafish treated with FR-SS-DTC showed strong fluorescence signals. The zebrafish were pretreated with NEM and then treated with FR-SS-DTC, and there was almost no fluorescence, indicating that glutathione was blocked. After treatment with GSH, the fluorescence release was significantly enhanced. The above results verified that FR-SS-DTC could effectively detect and image GSH in vivo.

Claims

1. The present invention discloses a preparation method and use of a diagnostic and therapeutic prodrug FR-SS-DTC with a disulfide bond unit as a glutathione-responsive site, and its structure is shown in the following formula (I). The diagnostic and therapeutic prodrug shown in the present invention can be used for specific recognition of glutathione and achieve integrated diagnosis and treatment:

2. A dithiol bond structure derivative, characterized in that By connecting a red fluorescent molecule and an antitumor drug molecule through a disulfide bond structure, and its structure is shown in the following formula (I):

3. A new use of the pre-diagnosis and treatment drug as described in claim 1, characterized in that, It can be specifically cleaved by high-concentration glutathione in tumor cells, releasing antitumor drug molecules and red fluorescent molecules, and finally achieving the integration of tumor diagnosis and treatment.

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