A probe prodrug for ros activation of inflammation and methods of making and using the same

Abstract

The application relates to a ROS-activated probe prodrug for inflammation and a preparation method and application thereof, a structural formula of the probe prodrug is as follows, and a preparation method and application of the probe prodrug are disclosed. The probe prodrug can be activated by hypochlorous acid in an inflammation microenvironment to release methylene blue molecules with fluorescence and ciprofloxacin with anti-infection activity.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a ROS-activated probe prodrug for inflammation, its preparation method, and its application. Background Technology

[0002] Inflammation is the body's defensive mechanism against infection and injury. However, uncontrolled inflammation (such as severe inflammation and sepsis) can trigger a systemic cytokine storm, leading to multiple organ dysfunction syndrome and becoming one of the leading causes of death in critical clinical conditions. Currently, the treatment of inflammation mainly focuses on broad-spectrum anti-infective and anti-inflammatory symptomatic support. However, traditional anti-inflammatory / anti-infective drugs generally suffer from insufficient specificity in targeting inflammation, making it difficult to effectively distinguish between inflamed and normal tissues. This results in insufficient local drug concentrations, limited therapeutic effects, and a higher risk of side effects such as bacterial resistance, intestinal flora imbalance, and liver and kidney damage. To overcome this bottleneck, the "activated prodrug strategy," designed based on the physicochemical differences between the inflammatory microenvironment and normal tissues, has emerged. This strategy can improve the local therapeutic effect of inflammation while reducing systemic toxicity, and has become a research hotspot in the field of precision inflammation treatment. Currently, researchers have constructed a variety of controllable activated prodrug systems by utilizing specific stimuli such as excessive reactive oxygen species (ROS), neutrophil-specific proteases, and acidic pH generated in the inflammatory microenvironment. Among them, ROS-activated prodrugs, due to the close correlation between abnormally elevated ROS levels and the occurrence, development, and severity of inflammation, and their high sensitivity to changes in ROS levels, demonstrate unique advantages in the field of targeted inflammation diagnosis and treatment.

[0003] Hypochlorous acid (HClO), a core member of reactive oxygen species (ROS), exhibits a positive correlation between its concentration and the intensity of inflammation in inflamed tissues. Hypochlorous acid serves as a specific biomarker for inflammation / sepsis and is also an ideal activator for targeted prodrug activation. However, existing hypochlorous acid-related probes have significant limitations: most focus solely on in vitro detection, releasing signals via fluorophores and failing to effectively release therapeutic drug molecules; furthermore, they suffer from long response times and high activation thresholds, failing to meet the demands for rapid treatment and real-time monitoring of acute conditions such as sepsis. Therefore, developing multifunctional intelligent prodrugs with rapid response and high sensitivity to achieve integrated "detection-imaging-treatment" for related inflammation / sepsis has significant scientific value and application prospects for the precise treatment of inflammation / sepsis. Summary of the Invention

[0004] To address the shortcomings of existing methods, this invention provides a probe prodrug for ROS activation in inflammation, its preparation method, and its application.

[0005] The technical solution adopted by this invention to solve its technical problem is: a probe prodrug for ROS activation in inflammation, with the following structural formula. .

[0006] Preferably, the probe prodrug can release methylene blue molecules and ciprofloxacin in a hypochlorous acid environment.

[0007] A method for preparing a ROS-activated probe prodrug for inflammation, characterized in that the probe prodrug has the structural formula described above, and the preparation steps are as follows: S1, Compound 1 is subjected to reduction and chloroformylation in a protective atmosphere of inert gas or nitrogen to obtain Compound 2. S2, compound 2 was subjected to an acyl chloride nucleophilic substitution reaction with compound 3 to obtain compound 4; S3, compound 6 is obtained by reacting compound 4, N,N-diisopropylethylamine and compound 5; S4, reacting compound 6, N,N-diisopropylethylamine and compound 7 to obtain the probe prodrug; Wherein, compound 1 is methylene blue; the structural formula of compound 2 is , Compound 3 is p-aminobenzyl alcohol, and the structural formula of compound 4 is... , Compound 5 is phenyl p-nitrochloroformate, and the structural formula of compound 6 is... Compound 7 is ciprofloxacin.

[0008] Preferably, step S1 includes the following steps: S1a, Compound 1 is dispersed in a mixed solvent of water and dichloromethane, then sodium carbonate is added, and the mixture is stirred and heated to 40 °C under a nitrogen atmosphere to obtain solution 1; S1b, under a nitrogen protective atmosphere, an aqueous solution of sodium dithionite is added to solution 1 through a syringe and stirred thoroughly to obtain solution 2, with a reaction time of 20-40 min; S1c, cool the temperature of solution 2 to room temperature, and slowly add triphosgene dissolved in dichloromethane to the cooled solution 2 over 10 min. After the addition is complete, continue stirring the reaction for 1-3 h. After the reaction was complete (S1d), the compound was separated and purified by column chromatography to obtain compound 2.

[0009] Preferably, step S2 includes the following steps: S2a, compound 2 and triethylamine are dispersed in acetonitrile to obtain solution 3; S2b, add compound 3 to solution 3, stir at 50 °C for 12-20 h; After the reaction S2c was completed, the compound was separated and purified by column chromatography to obtain compound 4.

[0010] Preferably, step S3 includes the following steps: S3a, compound 4 and N,N-diisopropylethylamine were dispersed in dichloromethane to obtain solution 4; S3b, add compound 5 to solution 4, stir the mixture at room temperature, and react for 6-10 hours; After the reaction was completed (S3c), compound 6 was obtained by separation and column chromatography purification. The molar ratio of compound 4, N,N-diisopropylethylamine, and compound 5 is 1:3:1.2.

[0011] Preferably, step S4 includes the following steps: S4a, compound 6 and N,N-diisopropylethylamine were dispersed in N,N-dimethylformamide to obtain solution 5; S4b, add compound 7 to solution 5, stir the reaction at room temperature, and the reaction time is 12-20h; After the reaction S4c is completed, the probe prodrug is separated and purified by column chromatography. The molar ratio of compound 6, N,N-diisopropylethylamine, and compound 7 is 1:6:2.5.

[0012] Preferably, the column chromatography purification is performed using silica gel column chromatography with a solvent system consisting of n-hexane and ethyl acetate mixed in different proportions.

[0013] Preferably, the ratio of n-hexane to ethyl acetate is 1-6:1 by volume.

[0014] A drug comprising a ROS-activated probe prodrug as described above for inflammation or sepsis.

[0015] The beneficial effects of this invention are as follows: the probe prodrug of this invention can be rapidly activated by hypochlorous acid overexpressed in the inflammatory / septic microenvironment, and release methylene blue (MB) molecules with fluorescence imaging function and ciprofloxacin (CIP) drug molecules with strong antibacterial activity, which can realize the visual diagnosis and treatment of inflammation. Attached Figure Description

[0016] Figure 1 This is the 1H NMR spectrum of the probe prodrug of this invention; Figure 2 This is the carbon NMR spectrum of the probe prodrug of this invention; Figure 3 This is the mass spectrometry characterization of the probe prodrug of this invention; Figure 4The fluorescence spectra of the probe prodrug of the present invention before and after activation by hypochlorous acid in solution are shown (black indicates before activation, orange indicates after activation). Figure 5 This is a comparative diagram showing the specificity of the probe prodrug of this invention in response to hypochlorous acid; Figure 6 This is a toxicity test of different concentrations of the probe prodrug of the present invention against RAW264.7 cells; Figure 7 These are confocal fluorescence images of the probe prodrug of this invention at a fixed concentration in inflamed RAW264.7 cells at different incubation times; Figure 8 These are confocal fluorescence images of different concentrations of the probe prodrug of this invention in inflammatory RAW264.7 cells incubated for the same time; Figure 9 This is a graph showing the effect of the probe prodrug of the present invention on inflammatory cytokines; Figure 10 This is a real-time fluorescence image of the probe prodrug of the present invention tracing ROS in septic mice; Figure 11 This is a graph showing the effect of the probe prodrug of the present invention on inflammatory factors in the serum of septic mice. Detailed Implementation

[0017] To more clearly illustrate the objectives, technical solutions, and advantages of the embodiments of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and embodiments. It is clear and complete that the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the protection scope of the present invention.

[0018] An embodiment of the present invention provides a probe prodrug for ROS activation in inflammation, the structural formula of which is shown below. This is called MB-CIP. In a hypochlorous acid environment, this probe prodrug releases methylene blue molecules and ciprofloxacin. Specifically, methylene blue is linked to a rearrangeable linker via a urea bond and exhibits no fluorescence, while ciprofloxacin, linked to methylene blue via the linker, also shows no anti-infective activity; at this point, MB-CIP is in a "closed" state. When this probe prodrug enters an inflammatory or sepsis microenvironment, it is specifically recognized by the overexpressed hypochlorous acid in that microenvironment, triggering the breakage of the linker bond, resulting in molecular rearrangement and the release of methylene blue molecules with fluorescent imaging capabilities and ciprofloxacin drug molecules with anti-infective activity. The mechanism by which the probe prodrug responds to hypochlorous acid is as follows: .

[0019] A method for preparing a ROS-activated probe prodrug for inflammation, the probe prodrug having the structural formula described above, and the reaction route is as follows: ; The specific preparation steps are as follows: S1, under a protective atmosphere of inert gas or nitrogen, compound 1 (methylene blue) is successively reduced and chloroformylated to obtain compound 2; compound 1 is methylene blue, and the structural formula of compound 2 is... The specific details of this step are as follows: S1a, compound 1 (3.87 g, 12.1 mmol) was dispersed in a mixed solvent of water and dichloromethane (40 ml water + 40 ml dichloromethane), followed by the addition of sodium carbonate (5.13 g, 48.4 mmol). The reaction mixture was stirred and heated to 40 °C under a nitrogen atmosphere to obtain solution 1. S1b, dissolve sodium dithionite (8.43 g, 48.4 mmol) in 30 ml of water to prepare sodium dithionite aqueous solution. Under a nitrogen protective atmosphere, add the sodium dithionite aqueous solution to solution 1 through a syringe. Observe that the reaction mixture gradually turns yellow. Stir thoroughly for 20-40 min to obtain solution 2, preferably stirring thoroughly for 30 min. S1c, cool the temperature of solution 2 to room temperature, and slowly add triphosgene (2.16 g, 7.3 mmol) dissolved in dichloromethane (30 ml) to the cooled solution 2 through a dropping funnel over 10 min. After the addition is completed within 10 min, continue stirring for 1-3 h, preferably for 2 h. S1d, after the previous reaction step is completed, water (20 ml) and dichloromethane (100 ml) are added to the reaction solution. After thorough mixing, the dichloromethane phase is separated through a separatory funnel. The solvent is evaporated to dryness to obtain the crude product. The crude product is then purified by silica gel column chromatography (the solvent is a mixed solution of n-hexane and ethyl acetate, and their volume ratio in the mixed solution is n-hexane:ethyl acetate = 6:1). After purification, a white solid compound 2 (2.02 g, 48%) is obtained. S2, compound 2 is subjected to a nucleophilic substitution reaction involving acyl chloride hydrolysis of compound 3 to yield compound 4; compound 3 is p-aminobenzyl alcohol, and the structural formula of compound 4 is […]. The specific details of this step are as follows: S2a, compound 2 (1.85 g, 5.32 mmol) and triethylamine (2.22 mL, 15.96 mmol) were dispersed in acetonitrile (50 mL) to obtain solution 3; S2b, add compound 3 (0.79 g, 6.38 mmol) to solution 3, stir at 50 °C for 12-20 h, preferably at 50 °C for 16 h; After the reaction S2c was completed, the solvent was evaporated to obtain the crude product, which was then purified by silica gel column chromatography (the solvent system was n-hexane:ethyl acetate = 4:1 by volume). After purification, a white solid compound 4 (1.29 g, 56%) was obtained. S3, compound 6 is obtained by reacting compound 4, N,N-diisopropylethylamine, and compound 5; compound 5 is phenyl p-nitrochloroformate, and the structural formula of compound 6 is [insert structural formula here]. The molar ratio of compound 4, N,N-diisopropylethylamine, and compound 5 is 1:3:1.2; the specific details of this step are as follows: S3a, compound 4 (0.98 g, 2.25 mmol) and N,N-diisopropylethylamine (0.65 ml, 3.75 mmol) were dispersed in dichloromethane (50 ml) to obtain solution 4; S3b, add compound 5 (0.55 g, 2.70 mmol) to solution 4, and stir the reaction at room temperature for 6-10 h, preferably for 8 h at room temperature; After the reaction in S3c was completed, the solvent was evaporated to obtain the crude product. The crude product was purified by silica gel column chromatography (the solvent system was n-hexane:ethyl acetate = 6:1 by volume). After purification, a pale yellow solid compound 6 (1.12 g, 83%) was obtained. S4, reacting compound 6, N,N-diisopropylethylamine, and compound 7 to obtain the probe prodrug; compound 7 is ciprofloxacin, and the molar ratio of compound 6, N,N-diisopropylethylamine, and compound 7 is 1:6:2.5; the specific details of this step are as follows: S4a, compound 6 (0.30 g, 0.50 mmol) and N,N-diisopropylethylamine (0.38 ml, 1.50 mmol) were dispersed in N,N-dimethylformamide (10 ml) to obtain solution 5; S4b, add compound 7 (0.41 g, 1.25 mmol) to solution 5 and stir the reaction at room temperature for 12-20 h, preferably 15 h at room temperature; After the reaction in S4c was completed, water (20 ml) and ethyl acetate (100 ml) were added to the reaction solution. After thorough mixing, the ethyl acetate phase was separated by a separatory funnel. The solvent was evaporated to dryness to obtain the crude product. The crude product was purified by silica gel column chromatography (the solvent system was n-hexane:ethyl acetate = 1:1 by volume) to obtain the yellow solid compound MB-CIP (0.23 g, 58%).

[0020] The prodrug NMR spectrum of the probe is shown below. Figure 1 As shown: 1 H NMR (400 MHz, DMSO-d6) δ ppm: 15.17(s, 1H), 8.64 (s, 1H), 8.20 (s, 1H), 7.92 (d, J = 13.2 Hz, 1H), 7.56 (d, J =7.2 Hz, 1H), 7.47 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.8 Hz, 2H), 7.28 (d, J =8.4 Hz, 2H), 6.74 (d, J = 2.4 Hz, 2H), 6.68 (dd, J = 9.2, 2.8 Hz, 2H), 5.04 (s,2H), 3.77 (brs, 1H), 3.62 (s, 4H), 3.33 (s, 4H), 2.90 (s, 12H), 1.31 (d, J =6.4 Hz, 2H), 1.16 (s, 2H).

[0021] The carbon NMR spectrum of the probe prodrug is shown below. Figure 2 As shown: 13 C{ 1 H}NMR (100.6 MHz, DMSO-d6) δ ppm:176.84, 176.83, 166.38, 154.95, 154.68, 153.26, 152.20, 149.05, 148.51,145.51, 145.41, 140.00, 139.58, 133.57, 130.91, 128.82, 128.55, 127.41,120.42, 119.39, 119.32, 116.64, 111.58, 111.35, 110.86, 107.26, 107.19,86.86, 49.70, 43.58, 40.67, 36.34, 8.05.

[0022] Mass spectrometric characterization of the probe prodrug is shown in [reference needed]. Figure 3 As shown: HRMS (ESI): m / z calcd for C 42 H 43 N7O6FS [M+H] +,792.2974; found, 792.2950.

[0023] The following examples illustrate the determination of the performance of the probe prodrug: First, the probe prodrug was dissolved in DMSO to prepare a prodrug stock solution (concentration of 5 mM), and then the following tests were performed respectively; like Figure 4 The figure shows the fluorescence spectra (620 nm excitation) of the probe prodrug MB-CIP molecule (5 μM) before and after activation by hypochlorous acid (20 μM) in solution. The test steps are as follows: The prodrug stock solution was diluted to a concentration of 5 µM using a prepared mixed solution (DMSO:PBS = 1:9, v / v), and then its fluorescence spectrum (620 nm excitation) was tested, which is the fluorescence spectrum of the probe prodrug before activation by hypochlorous acid; The prodrug stock solution was diluted to a concentration of 5 µM using a prepared mixed solution (DMSO:PBS = 1:9, v / v), and a calculated hypochlorous acid solution was added to make the concentration of hypochlorous acid 20 µM. The reaction was carried out at room temperature in the dark for 2 hours. After the reaction was completed, the fluorescence spectrum (620 nm excitation) of the solution after the reaction was completed was tested under the same conditions, which is the fluorescence spectrum of the probe prodrug after activation by hypochlorous acid; As can be seen from the figure, the compound MB-CIP is non-fluorescent before activation by hypochlorous acid; after activation by hypochlorous acid, it emits strong fluorescence, with a maximum emission wavelength of 676 nm. Figure 5 This is a comparative diagram showing the specificity of MB-CIP's response to hypochlorous acid. The prodrug stock solution was diluted to multiple 5µM solutions using a prepared mixed solution (DMSO:PBS = 1:9, v / v). Then, one 5µM MB-CIP solution was incubated with a 0.5mM interfering substance in an aqueous solution. Each interfering substance corresponded to one MB-CIP solution. After incubation at room temperature for 2 hours, the fluorescence spectra of each solution were measured using 620 nm excitation. Figure 5 This is a comparison fluorescence image after testing. The red line is the fluorescence spectrum of the response to hypochlorous acid, and the straight line corresponding to the vertical axis 0 is the fluorescence spectrum of various interfering substances. The results in the image show that the compound MB-CIP can respond to hypochlorous acid and thus emit fluorescence, but it does not respond to other interfering substances and does not emit fluorescence. Figure 6 For the cytotoxicity test of MB-CIP: RAW264.7 cells were first seeded in 96-well plates and grown for 24 hours. Then, different concentrations of MB-CIP (0, 0.5 μM, 1 μM, 5 μM, 10 μM, 20 μM) were added and incubated in a cell culture incubator for 24 hours. Cell viability was then detected by the MTT assay. The results in the figure show that MB-CIP has very low cytotoxicity and excellent biocompatibility. Figure 7 To obtain confocal fluorescence images of RAW264.7 cells incubated with a fixed concentration of probe prodrug for different time periods, RAW264.7 cells were first induced with lipopolysaccharide (LPS) (0.5 μg / ml) for 24 hours to establish an inflammation / septic cell model. This involved co-incubating RAW264.7 macrophages with 0.5 μg / mL LPS for 24 hours to construct the inflammation cell model. The LPS-induced inflammation cells were then incubated with MB-CIP (10 µM) in a cell culture incubator for different time periods. Fluorescence images were captured using confocal fluorescence microscopy at each time point. The results show that the fluorescence intensity of RAW264.7 sepsis cells increased with prolonged incubation time, indicating that MB-CIP can serve as a probe for inflammation / septic cells. Figure 8 Confocal fluorescence images of RAW264.7 cells incubated with different concentrations of the probe prodrug for the same duration are shown. Similarly, RAW264.7 cells were first induced with LPS (0.5 μg / ml) for 24 h to establish an inflammation / septic cell model. Then, cells were incubated with different concentrations of MB-CIP in a cell culture incubator for 2 h, and fluorescence images were captured using a confocal fluorescence microscope. The results show that the fluorescence intensity of RAW264.7 cells in inflammation / septic cells increased with increasing MB-CIP concentration, further demonstrating that MB-CIP can serve as a probe for inflammation / septic cells. Figure 9 This study investigated the therapeutic effect of MB-CIP in an inflammation / septic cell model, specifically the effect of the probe prodrug on inflammatory cytokines. RAW264.7 cells were first induced with LPS (0.5 μg / ml) for 24 hours to establish an inflammation / septic cell model. Then, 10 μM MB-CIP was added and incubated in a cell culture incubator for different durations. Cell supernatant was extracted at each time point, and the levels of inflammatory cytokines (IL-1β, IL-6, TNF-α) in the culture medium were detected by ELISA. The results in the figure indicate that MB-CIP significantly reduced the levels of inflammatory factors in septic cells, demonstrating a good therapeutic effect. Figure 10This study investigated the real-time tracking effect of MB-CIP on ROS in a mouse model of sepsis, specifically by obtaining fluorescence images of the probe prodrug for real-time ROS tracking in sepsis-affected mice. First, 6-8 week old male Balb / c mice were intraperitoneally injected with LPS at a dose of 10 mg / kg. Twelve hours later, the LPS-injected mice were randomly divided into a PBS group and an MB-CIP group, with four mice in each group. The PBS group received an intravenous injection of sterile PBS equal to the amount of MB-CIP, while the MB-CIP group received an intravenous injection of MB-CIP at a dose of 10 mg / kg. Finally, fluorescence images were captured using a small animal in vivo imaging system at 0 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 12 h, and 24 h. These fluorescence images allowed for real-time tracking of ROS distribution in the mice. The results indicate that MB-CIP exhibits excellent fluorescence imaging performance in the mouse model of sepsis, enabling real-time tracking of ROS in sepsis-affected mice. Figure 11 This study investigated the therapeutic effect of MB-CIP in a sepsis animal model, specifically the effect of the probe prodrug on inflammatory factors in the serum of septic mice. First, 6-8 week old male Balb / c mice were intraperitoneally injected with LPS at a dose of 10 mg / kg. Twelve hours later, the LPS-injected mice were randomly divided into a PBS group and an MB-CIP group, with eight mice in each group. The PBS group received an equal volume of sterile PBS injected via the tail vein, while the MB-CIP group received MB-CIP at a dose of 10 mg / kg injected via the tail vein. Serum samples were collected at different time points, and the levels of cellular inflammatory factors (IL-1β, IL-6, TNF-α) in the serum were detected by ELISA. The results in the figure indicate that MB-CIP significantly reduced the levels of inflammatory factors (IL-1β, IL-6, TNF-α) in the serum of septic mice, demonstrating a good therapeutic effect.

[0024] A drug for inflammation or sepsis comprising a ROS-activated probe prodrug as described above, which is specifically recognized and triggers the breaking of linkage bonds in the inflammatory microenvironment using hypochlorous acid as an activator, releasing fluorescent methylene blue molecules and simultaneously releasing ciprofloxacin, a drug with anti-infective activity, thus enabling visualized diagnosis and treatment of inflammation; it can be directly added to cell culture medium for co-culture with cells, efficiently exerting anti-inflammatory effects on the inflammatory microenvironment and emitting fluorescence.

[0025] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A probe prodrug for ROS activation in inflammation, characterized in that, The structural formula is shown below. .

2. The ROS-activating probe prodrug for inflammation according to claim 1, characterized in that, The probe prodrug can release methylene blue molecules and ciprofloxacin in a hypochlorous acid environment.

3. A method for preparing a ROS-activated probe prodrug for inflammation, characterized in that, The probe prodrug has the structural formula as described in claim 1, and its preparation steps are as follows: S1, Compound 1 is subjected to reduction and chloroformylation in a protective atmosphere of inert gas or nitrogen to obtain Compound 2. S2, compound 2 was subjected to an acyl chloride nucleophilic substitution reaction with compound 3 to obtain compound 4; S3, compound 6 is obtained by reacting compound 4, N,N-diisopropylethylamine and compound 5; S4, reacting compound 6, N,N-diisopropylethylamine, and compound 7 to obtain the probe prodrug; wherein, compound 1 is methylene blue; the structural formula of compound 2 is [insert structural formula here]. , Compound 3 is p-aminobenzyl alcohol, and the structural formula of compound 4 is... , Compound 5 is phenyl p-nitrochloroformate, and the structural formula of compound 6 is... Compound 7 is ciprofloxacin.

4. The method for preparing the ROS-activated probe prodrug for inflammation according to claim 3, characterized in that, Step S1 includes the following steps: S1a, Compound 1 is dispersed in a mixed solvent of water and dichloromethane, then sodium carbonate is added, and the mixture is stirred and heated to 40 °C under a nitrogen atmosphere to obtain solution 1; S1b, under a nitrogen protective atmosphere, an aqueous solution of sodium dithionite is added to solution 1 through a syringe and stirred thoroughly to obtain solution 2, with a reaction time of 20-40 min; S1c, cool the temperature of solution 2 to room temperature, and slowly add triphosgene dissolved in dichloromethane to the cooled solution 2 over 10 min. After the addition is complete, continue stirring the reaction for 1-3 h. After the reaction was complete (S1d), the compound was separated and purified by column chromatography to obtain compound 2.

5. The method for preparing the ROS-activated probe prodrug for inflammation according to claim 3, characterized in that, Step S2 includes the following steps: S2a, compound 2 and triethylamine are dispersed in acetonitrile to obtain solution 3; S2b, add compound 3 to solution 3, stir at 50 °C for 12-20 h; After the reaction S2c was completed, the compound was separated and purified by column chromatography to obtain compound 4.

6. The method for preparing the ROS-activated probe prodrug for inflammation according to claim 3, characterized in that, Step S3 includes the following steps: S3a, compound 4 and N,N-diisopropylethylamine were dispersed in dichloromethane to obtain solution 4; S3b, add compound 5 to solution 4, stir the mixture at room temperature, and react for 6-10 hours; After the reaction was completed (S3c), compound 6 was obtained by separation and column chromatography purification. The molar ratio of compound 4, N,N-diisopropylethylamine, and compound 5 is 1:3:1.

2.

7. The method for preparing a ROS-activated probe prodrug for inflammation according to claim 3, characterized in that, Step S4 includes the following steps: S4a, compound 6 and N,N-diisopropylethylamine were dispersed in N,N-dimethylformamide to obtain solution 5; S4b, add compound 7 to solution 5, stir the reaction at room temperature, and the reaction time is 12-20h; After the reaction S4c is completed, the probe prodrug is separated and purified by column chromatography. The molar ratio of compound 6, N,N-diisopropylethylamine, and compound 7 is 1:6:2.

5.

8. The method for preparing a ROS-activated probe prodrug for inflammation according to any one of claims 4-7, characterized in that, The column chromatography purification is performed using silica gel column chromatography with hexane and ethyl acetate mixed in different proportions as the solvent system.

9. The method for preparing a ROS-activated probe prodrug for inflammation according to claim 8, characterized in that, The ratio of n-hexane to ethyl acetate is 1-6:1 by volume.

10. A medicament comprising a ROS-activated probe prodrug for inflammation as described in claim 1, for use in inflammation or sepsis.

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