A reactive oxygen species-activated nitrogen mustard prodrug, its preparation method and application
By introducing ROS-sensitive groups into nitrogen mustard compounds to design reactive oxygen species-activated nitrogen mustard prodrugs, the problems of lack of targeting and systemic toxicity of nitrogen mustard drugs have been solved, achieving precise targeting of tumor cells and low toxicity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-21
- Publication Date
- 2026-04-03
AI Technical Summary
Existing nitrogen mustard drugs lack targeting in the treatment of tumors, resulting in severe systemic toxicity and limiting the dosage and therapeutic window. Furthermore, their non-selective action can easily cause adverse reactions such as bone marrow suppression, gastrointestinal reactions, and hair loss.
A reactive oxygen species-activated nitrogen mustard prodrug was designed. By introducing ROS-sensitive groups into the nitrogen mustard compound, it is kept inactive in normal tissues and is only activated in the tumor microenvironment due to increased ROS levels, releasing cytotoxic nitrogen mustard components.
It improves the selectivity of drugs in tumor cells, reduces toxic side effects on normal tissues, enriches the variety of nitrogen mustard antitumor drugs, and exhibits high antitumor activity and low toxicity.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of medicinal chemistry, specifically relating to a reactive oxygen species activated nitrogen mustard prodrug, its preparation method, and its application. Background Technology
[0002] Nitrogen mustard compounds are a classic class of anticancer drugs. Their main antitumor mechanism involves alkylating DNA, inducing DNA double-strand breaks, thereby inhibiting cell division and ultimately leading to apoptosis of tumor cells. Since their antitumor activity was first discovered during World War II, nitrogen mustard drugs have been widely used to treat various cancers, such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, and leukemia. Despite their high antitumor activity, nitrogen mustard compounds lack specific targeting and exhibit significant toxicity to normal cells. This systemic toxicity not only leads to poor patient tolerance but also severely limits the dosage and therapeutic window of nitrogen mustard drugs. Furthermore, the non-selective action of nitrogen mustard drugs easily triggers adverse reactions such as bone marrow suppression, gastrointestinal reactions, and hair loss. Therefore, improving the tumor selectivity of nitrogen mustard compounds and reducing their toxicity to normal tissues has become an important direction in current drug development.
[0003] Reactive oxygen species (ROS) are a class of reactive molecules produced during cellular metabolism, including superoxide anions (O2-). - ), hydrogen peroxide (H2O2) and hydroxyl radicals ( · ROS (Reactive Oxygen Species) and their derivatives (ROS). Under normal physiological conditions, the production and clearance of ROS are in dynamic equilibrium, playing an important role in signal transduction and cellular function regulation. However, in tumor cells, ROS levels are typically significantly elevated. This is due to the combined effects of tumor cell metabolic reprogramming, mitochondrial dysfunction, and inflammatory responses. High levels of ROS in the tumor microenvironment are both a driving force for tumorigenesis and development and a potential target. Excessive ROS can damage DNA, proteins, and lipids, and induce apoptosis. Therefore, drug development strategies based on differential ROS levels can achieve precise targeting of tumors by activating drugs in a high-ROS environment, while avoiding damage to normal tissues. Summary of the Invention
[0004] To address the limitations and side effects of existing nitrogen mustard drug delivery methods during treatment, this invention proposes a prodrug design strategy based on reactive oxygen species (ROS) activation. The core idea of this strategy is to introduce ROS-sensitive groups into nitrogen mustard compounds, keeping them inactive in normal tissues and only activating them in the tumor microenvironment due to elevated ROS levels, releasing cytotoxic nitrogen mustard components. Constructing ROS-activated nitrogen mustard prodrugs not only overcomes the shortcomings of traditional nitrogen mustard drugs but also provides new directions and ideas for the development of antitumor drugs. The nitrogen mustard prodrugs of this invention enrich the structural types of nitrogen mustard prodrugs, improve drug selectivity in tumor cells, and reduce toxic side effects.
[0005] The first objective of this invention is to provide a reactive oxygen species (ROS) activated nitrogen mustard prodrug having the structure shown in Formula I:
[0006]
[0007] Where X represents oxygen and sulfur.
[0008] R1 and R2 are independently selected from hydrogen, alkyl, and haloalkyl;
[0009] R3 is a halogen.
[0010] Another object of the present invention is to provide a method for preparing the reactive oxygen species activated nitrogen mustard prodrug, the method being as follows:
[0011] The amine compound and the base were dissolved in a solvent. Under an ice-water bath, the intermediate M3 dissolved in the solvent was added dropwise. After the addition was complete, the reaction was stirred continuously under nitrogen. The reaction process was monitored using thin-layer chromatography. After the reaction was completed, the reaction product was extracted with an organic solvent, washed with water, dried with anhydrous sodium sulfate, and the solvent was evaporated. The product was then purified by column chromatography to obtain the target product.
[0012] The intermediate M3 is shown in the following formula:
[0013]
[0014] Further, the solvent is an aprotic solvent, specifically dichloromethane or tetrahydrofuran; the base is an inorganic or organic base, including carbonates and bicarbonates; the organic base is triethylamine or dimethylaminopyridine; the amount of base used is molar, with a molar ratio to intermediate M3 of 3:1 to 7:1; the reaction time is 1 to 10 hours, and the reaction temperature of the system is 0 to 5°C.
[0015] Further, the amine compound is 2-chloroethylamine, 2-bromoethylamine, 2-iodoethylamine or its hydrochloride, hydrobromide or hydroiodide; the amount of the amine compound used is in molar quantity, and the molar ratio with intermediate M3 is 1:1 to 5:1.
[0016] Furthermore, during the extraction stage, the amount of organic solvent used is 50-200 mL for every 1 g of intermediate M3 fed.
[0017] Furthermore, the eluent can be a combination of ethyl acetate and n-hexane, ethyl acetate and petroleum ether, and methanol and dichloromethane.
[0018] A third objective of this invention is to use the above-mentioned reactive oxygen species-activated nitrogen mustard prodrug in antitumor drugs.
[0019] A fourth objective of this invention is to provide an antitumor preparation containing the above-mentioned reactive oxygen species activated nitrogen mustard prodrug.
[0020] Beneficial effects of this invention:
[0021] The compounds of this invention can inhibit the toxicity of nitrogen mustard, and only exhibit toxicity after activation; they have a good response to reactive oxygen species, a fast response time, high cell selectivity, and few toxic side effects, thus enriching the variety of nitrogen mustard-based antitumor drugs.
[0022] The results of cytotoxicity experiments showed that the partially reactive oxygen species activated nitrogen mustard prodrug synthesized in this invention was more toxic to human cervical cancer cells and human promyelocytic leukemia cells than to mouse fibroblasts, indicating that the compound of this invention has high antitumor activity and that the nitrogen mustard structure has targeting properties, exhibiting low toxicity to normal cells.
[0023] The above results indicate that the reactive oxygen species-activated nitrogen mustard prodrug synthesized in this invention has potential antitumor activity. Attached Figure Description
[0024] Figure 1 The proton NMR spectrum of compound A1, which has the structure of formula I;
[0025] Figure 2 High-resolution mass spectra of compound A1, which has the structure of formula I;
[0026] Figure 3 The 1H NMR spectrum of compound A2, which has the structure of formula I;
[0027] Figure 4 High-resolution mass spectra of compound A2, which has the structure of formula I;
[0028] Figure 5 The 1H NMR spectrum of compound A3, which has the structure of formula I;
[0029] Figure 6 High-resolution mass spectra of compound A3, which has the structure of formula I;
[0030] Figure 7 The 1H NMR spectrum of compound A4, which has the structure of formula I;
[0031] Figure 8 Mass spectra of compound A4, which has the structure of formula I;
[0032] Figure 9 The fluorescence spectra of compound A1, which has the structure of formula I, before and after its response to hypochlorous acid are shown.
[0033] Figure 10 The kinetic curves of the response of compound A1, which has the structure of formula I, to hypochlorous acid are shown.
[0034] Figure 11 The fluorescence spectra of compound A2, which has the structure of formula I, before and after its response to hypochlorous acid;
[0035] Figure 12 The kinetic curves of the response of compound A2, which has the structure of formula I, to hypochlorous acid are shown.
[0036] Figure 13 The fluorescence spectra of compound A3, which has the structure of formula I, before and after its response to hypochlorous acid;
[0037] Figure 14 The kinetic curves of the response of compound A3, which has the structure of formula I, to hypochlorous acid are shown.
[0038] Figure 15 The fluorescence spectra of compound A4, which has the structure of formula I, before and after its response to hypochlorous acid are shown.
[0039] Figure 16 The kinetic curves of the response of compound A4, which has the structure of formula I, to hypochlorous acid are shown.
[0040] Figure 17 This is a diagram of the cytotoxicity assay for compound A1, which has the structure of formula I. Detailed implementation method:
[0041] The present invention will be further described in detail below with reference to the embodiments. These are merely illustrative examples, and the physical data of the exemplified compounds are consistent with the structures specified for those compounds. However, the examples do not limit the scope of the present invention.
[0042] The general chemical reaction formula of the compounds having the structure of Formula I in this invention is as follows:
[0043]
[0044] The intermediate acyl chloride compound M3 can be prepared according to the literature Chem. Sci., 2018, 9, 495 and J. Mater. Chem. B, 2022, 10, 5211-5217.
[0045] Example 1: Preparation of compound A1 having the structure of formula I
[0046]
[0047] The intermediate acyl chloride M3 is methylene blue acyl chloride, which was prepared according to the reference Chem. Sci., 2018, 9, 495.
[0048] Weigh 0.50 g (1 equivalent) of methylene blue amide chloride into a round-bottom flask and dissolve it in 6 mL of dichloromethane. Weigh 0.55 g (3 equivalents) of 2-chloroethylamine hydrochloride, dissolve it in dichloromethane, and add 600 μL (5 equivalents) of triethylamine to the 2-chloroethylamine hydrochloride. Under nitrogen protection and with the reaction system temperature controlled at 0 °C in an ice-water bath, add 2-chloroethylamine dropwise to the methylene blue amide chloride. After 1.5 h, monitor the reaction for completion using thin-layer chromatography (TLC). Evaporate the solvent, extract with 50 mL of ethyl acetate, wash with water, dry with anhydrous sodium sulfate, evaporate the solvent again, dissolve in dichloromethane, and purify by column chromatography (mobile phase: ethyl acetate and petroleum ether, 1 / 1 ratio). Evaporate the solvent again and slurry with n-hexane for 2 h to obtain product A1 295 mg, yield 50.3%. The 1H NMR spectrum and high-resolution mass spectra of compound A1 are shown below. Figure 1 and Figure 2 .
[0049] Example 2: Preparation of compound A2 having the structure of formula I
[0050]
[0051] The intermediate acyl chloride M3 is methylene blue acyl chloride, which was prepared according to the reference Chem. Sci., 2018, 9, 495.
[0052] Weigh 0.35 g (1 equivalent) of methylene blue amide chloride into a round-bottom flask and dissolve it in 4 mL of dichloromethane. Weigh 0.64 g (3 equivalents) of 2-bromoethylamine hydrobromide and dissolve it in dichloromethane. Weigh 0.64 g (6 equivalents) of sodium carbonate and add it to the 2-bromoethylamine hydrobromide. Under nitrogen protection and with the reaction system temperature controlled at 3 °C in an ice-water bath, add 2-bromoethylamine dropwise to the methylene blue amide chloride. After 1 h, monitor the reaction by thin-layer chromatography (TLC) until it ends. Evaporate the solvent, extract with 30 mL of ethyl acetate, wash with water, dry with anhydrous sodium sulfate, evaporate the solvent again, dissolve in dichloromethane, and purify by column chromatography (mobile phase is ethyl acetate and petroleum ether, initially in a 1 / 4 ratio, gradually increasing to 1 / 1). Evaporate the solvent again and slurry with n-hexane for 2 h to obtain product A2 115 mg, yield 26.4%. The 1H NMR spectrum and high-resolution mass spectra of compound A2 are shown in [reference needed]. Figure 3 and Figure 4 .
[0053] Example 3: Preparation of compound A3 having the structure of formula I
[0054]
[0055] The intermediate acyl chloride M3 is methylene blue acyl chloride, which was prepared according to the reference Chem. Sci., 2018, 9, 495.
[0056] Weigh 0.35 g (1 equivalent) of methylene blue iodide chloride into a round-bottom flask and dissolve it in 4 mL of dichloromethane. Weigh 0.30 g (1 equivalent) of 2-iodoethylamine hydroiodide and dissolve it in dichloromethane. Add 420 μL (3 equivalents) of triethylamine to the 2-iodoethylamine hydroiodide solution. Under nitrogen protection and with the reaction system temperature controlled at 1 °C in an ice-water bath, add 2-iodoethylamine dropwise to the methylene blue iodide chloride. After 1 h, monitor the reaction for completion using thin-layer chromatography (TLC). Evaporate the solvent, extract with 30 mL of ethyl acetate, wash with water, dry with anhydrous sodium sulfate, evaporate the solvent again, dissolve in dichloromethane, and purify by column chromatography (mobile phase: ethyl acetate and petroleum ether, initial ratio 1 / 6, gradually increased to 1 / 1). Evaporate the solvent again and slurry with n-hexane for 2 h to obtain product A3 85 mg, yield 17.6%. The 1H NMR spectrum and high-resolution mass spectra of compound A3 are shown below. Figure 5 and Figure 6 .
[0057] Example 4: Preparation of compound A4 having the structure of formula I
[0058]
[0059] Intermediate acyl chloride M3 is basic blue 3-acyl chloride, as described in the literature.
[0060] Preparations were prepared in J. Mater. Chem. B, 2022, 10, 5211-5217.
[0061] Weigh 0.56 g (1 equivalent) of Basic Blue 3-acyl chloride into a round-bottom flask and dissolve it in 6 mL of dichloromethane. Weigh 0.40 g (2.5 equivalents) of 2-chloroethylamine hydrochloride and dissolve it in dichloromethane. Weigh 1.06 g (7 equivalents) of sodium carbonate and add it to the 2-chloroethylamine hydrochloride. Under nitrogen protection and with the reaction system temperature controlled at 5 °C in an ice-water bath, add 2-chloroethylamine dropwise to Basic Blue 3-acyl chloride. After 2 hours, monitor the reaction by thin-layer chromatography (TLC) to indicate the end of the reaction. After the reaction, evaporate the solvent to dryness, extract with ethyl acetate, wash with water, dry with anhydrous sodium sulfate, evaporate the solvent to dryness, dissolve in dichloromethane, and purify by column chromatography (mobile phase: ethyl acetate and petroleum ether, 1 / 4 ratio). Evaporate the solvent to dryness and slurry with n-hexane for 2 hours to obtain product A4 248 mg, yield 44.6%. The 1H NMR and mass spectra of compound A4 are shown in [reference needed]. Figure 7 and Figure 8 .
[0062] Example 5: Response performance of compound A1 to specific reactive oxygen species
[0063] This embodiment investigates the response performance of compound A1, which has the structure of Formula I, to a specific reactive oxygen species (hypochlorous acid, HOCl).
[0064] Methods: Compound A1 (5 μM) was prepared in 10 mM PBS buffer solution at room temperature, and hypochlorous acid (0, 1, 3, 5, 10 μM) of different concentrations was added to the system. After reacting for 30 minutes, the reaction was measured by fluorescence spectroscopy at an excitation wavelength of 620 nm.
[0065] Experimental results are as follows Figure 9 As shown, when hypochlorous acid (0 μM) was not added to the system, compound A1 showed no fluorescence signal; however, with the increase of hypochlorous acid concentration, chloroethylamine and the fluorophore methylene blue were released into the system, resulting in a significant enhancement of fluorescence emission in the 640-850 nm range. In particular, after adding 10 μM hypochlorous acid, the fluorescence intensity at 686 nm was increased by more than 1100 times compared to the system without hypochlorous acid.
[0066] The results of this experiment show that compound A1 exhibits excellent response to hypochlorous acid.
[0067] Example 6: Reaction kinetic analysis of compound A1 in response to reactive oxygen species
[0068] This embodiment further analyzes the reaction kinetics of compound A1 in response to hypochlorous acid.
[0069] Experimental method: 5 μM of compound A1 was prepared in 10 mM PBS buffer solution, 10 μM hypochlorous acid was added to the system, and the fluorescence intensity change at 686 nm was monitored in real time.
[0070] Reference Figure 10 As shown, 5 μM compound A1 was first added to a buffer solution, at which point the fluorescence intensity was low. Hypochlorous acid was added after 75 seconds, and a rapid increase in fluorescence intensity was observed, reaching equilibrium at 110 seconds, with the fluorescence intensity approaching its maximum value within 30 seconds. These results indicate that compound A1 has a fast response and good kinetic properties.
[0071] In summary, compound A1 exhibits a sensitive and rapid response to hypochlorous acid, demonstrating potential practical application value.
[0072] Example 7: Response performance of compound A2 to specific reactive oxygen species
[0073] This embodiment investigates the response performance of compound A2, which has the structure of Formula I, to a specific reactive oxygen species (hypochlorous acid, HOCl).
[0074] Methods: 5 μM of compound A2 was prepared in 10 mM PBS buffer solution at room temperature, and different concentrations of hypochlorous acid (0, 1, 3, 5, 10 μM) were added to the system. After reacting for 30 minutes, the reaction was measured by fluorescence spectroscopy at an excitation wavelength of 620 nm.
[0075] Experimental results are as follows Figure 11 As shown, when hypochlorous acid (0 μM) was not added to the system, compound A2 showed no fluorescence signal; however, with the increase of hypochlorous acid concentration, ethyl bromide and the fluorophore methylene blue were released into the system, resulting in a significant enhancement of fluorescence emission in the 640-850 nm range. In particular, after adding 10 μM hypochlorous acid, the fluorescence intensity at 686 nm was more than 1100 times stronger than that of the system without hypochlorous acid.
[0076] The results of this experiment show that compound A2 exhibits excellent response performance to hypochlorous acid.
[0077] Example 8: Reaction kinetic analysis of compound A2 in response to reactive oxygen species
[0078] This embodiment further analyzes the reaction kinetics of compound A2 in response to hypochlorous acid.
[0079] Experimental method: 5 μM of compound A2 was prepared in 10 mM PBS buffer solution, 10 μM hypochlorous acid was added to the system, and the fluorescence intensity change at 686 nm was monitored in real time.
[0080] Reference Figure 12As shown, 5 μM compound A2 was first added to the buffer solution, at which point the fluorescence intensity was low. After 40 seconds, hypochlorous acid was added, and a rapid increase in fluorescence intensity was observed, reaching equilibrium at 70 seconds, with the fluorescence intensity approaching its maximum value within 30 seconds. These results indicate that compound A2 has a fast response rate and good kinetic properties.
[0081] In summary, compound A2 exhibits a sensitive and rapid response to hypochlorous acid, demonstrating potential practical application value.
[0082] Example 9: Response performance of compound A3 to specific reactive oxygen species
[0083] This embodiment investigates the response performance of compound A3, which has the structure of Formula I, to a specific reactive oxygen species (hypochlorous acid, HOCl).
[0084] Methods: 5 μM of compound A3 was prepared in 10 mM PBS buffer solution at room temperature, and different concentrations of hypochlorous acid (0, 1, 3, 5, 10 μM) were added to the system. After reacting for 30 minutes, the reaction was measured by fluorescence spectroscopy at an excitation wavelength of 620 nm.
[0085] Experimental results are as follows Figure 13 As shown, when hypochlorous acid (0 μM) was not added to the system, compound A3 showed no fluorescence signal; however, with the increase of hypochlorous acid concentration, iodoethylamine and the fluorophore methylene blue were released from the system, resulting in a significant enhancement of fluorescence emission in the 640-850 nm range. In particular, after adding 10 μM hypochlorous acid, the fluorescence intensity at 686 nm was more than 1400 times stronger than that of the system without hypochlorous acid.
[0086] The results of this experiment show that compound A3 exhibits excellent response to hypochlorous acid.
[0087] Example 10: Reaction kinetic analysis of compound A3 in response to reactive oxygen species
[0088] This embodiment further analyzes the reaction kinetics of compound A3 in response to hypochlorous acid.
[0089] Experimental method: 5 μM of compound A3 was prepared in 10 mM PBS buffer solution, 10 μM hypochlorous acid was added to the system, and the fluorescence intensity change at 686 nm was monitored in real time.
[0090] Reference Figure 14 As shown, 5 μM compound A3 was first added to the buffer solution, at which point the fluorescence intensity was low. Hypochlorous acid was added after 45 seconds, and a rapid increase in fluorescence intensity was observed, reaching equilibrium at 75 seconds, with the fluorescence intensity approaching its maximum value within 30 seconds. These results indicate that compound A3 has a fast response rate and good kinetic properties.
[0091] In summary, compound A3 exhibits a sensitive and rapid response to hypochlorous acid, demonstrating potential practical application value.
[0092] Example 11: Response performance of compound A4 to specific reactive oxygen species
[0093] This embodiment investigates the response performance of compound A4, which has the structure of Formula I, to a specific reactive oxygen species (hypochlorous acid, HOCl).
[0094] Methods: Compound A4 (5 μM) was prepared in 10 mM PBS buffer solution at room temperature, and hypochlorous acid (0, 1, 3, 5, 10 μM) of different concentrations was added to the system. After reacting for 30 minutes, the reaction was measured by fluorescence spectroscopy at an excitation wavelength of 610 nm.
[0095] Experimental results are as follows Figure 15 As shown, when hypochlorous acid (0 μM) was not added to the system, compound A4 showed no fluorescence signal; however, with the increase of hypochlorous acid concentration, chloroethylamine and the fluorophore Basic Blue 3 were released into the system, resulting in a significant enhancement of fluorescence emission in the 640-850 nm range. In particular, after adding 10 μM hypochlorous acid, the fluorescence intensity at 669 nm was more than 1600 times stronger than that of the system without hypochlorous acid.
[0096] The results of this experiment show that compound A4 exhibits excellent response to hypochlorous acid.
[0097] Example 12: Reaction kinetic analysis of compound A4 in response to reactive oxygen species
[0098] This embodiment further analyzes the reaction kinetics of compound A4 in response to hypochlorous acid.
[0099] Experimental method: 5 μM of compound A4 was prepared in 10 mM PBS buffer solution, 10 μM hypochlorous acid was added to the system, and the fluorescence intensity change at 669 nm was monitored in real time.
[0100] Reference Figure 16 As shown, 5 μM compound A4 was first added to a buffer solution, at which point the fluorescence intensity was low. Hypochlorous acid was added after 12 seconds, and a rapid increase in fluorescence intensity was observed, reaching equilibrium after 150 seconds, with the fluorescence intensity approaching its maximum value within 140 seconds. These results indicate that compound A4 has a fast response rate and good kinetic properties.
[0101] In summary, compound A4 exhibits a sensitive and rapid response to hypochlorous acid, demonstrating potential practical application value.
[0102] Example 13: Toxicity analysis of compound A1 against malignant tumors
[0103] The mouse fibroblasts, human cervical cancer cells, and human promyelocytic leukemia cells used in the experiment were all purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee.
[0104] Different concentrations of compound A1 (0-40 μM) with the formula I structure were co-incubated with mouse fibroblasts, human cervical cancer cells, and human promyelocytic leukemia cells for 24 h, and the toxicity of A1 to the three cell types was observed.
[0105] In the experiment, compound A1 was first prepared as a 1 mM stock solution. Then, different amounts of the A1 stock solution were added to the cell culture medium according to different concentration requirements (0, 20, 30, 40 μM). After 24 hours, the effects of different concentrations of A1 on the survival rate of the three cell types were observed. Results are referenced below. Figure 17 As shown, this result indicates that A1 is more toxic to human cervical cancer cells and human promyelocytic leukemia cells than it is to mouse fibroblasts.
[0106] In summary, compound A1 with the formula I structure exhibits high antitumor activity and makes the nitrogen mustard structure targeted, showing low toxicity to normal cells.
[0107] The above description is not limited to the examples above. Technical features not described in this invention can be implemented by or using existing technology, and will not be repeated here. The above embodiments are only used to illustrate the technical solutions of this invention and are not intended to limit this invention. Any changes, modifications, additions or substitutions made by those skilled in the art within the scope of this invention do not depart from the spirit of this invention and should also fall within the scope of protection of the claims of this invention.
Claims
1. A reactive oxygen species-activated nitrogen mustard prodrug, characterized in that, This reactive oxygen species-activated nitrogen mustard prodrug has the structure shown in Formula I: ； Where X represents oxygen and sulfur. R1 and R2 are independently selected from methyl and ethyl; R3 is a halogen.
2. A method for preparing a reactive oxygen species-activated nitrogen mustard prodrug as described in claim 1, characterized in that, The method is as follows: The amine compound and the base were dissolved in a solvent. Under an ice-water bath, the intermediate M3 dissolved in the solvent was added dropwise. After the addition was complete, the reaction was stirred continuously under nitrogen. The reaction process was monitored using thin-layer chromatography. After the reaction was completed, the reaction product was extracted with an organic solvent, washed with water, dried with anhydrous sodium sulfate, and the solvent was evaporated. The product was then purified by column chromatography to obtain the target product. The intermediate M3 is shown in the following formula: 。 3. The preparation method according to claim 2, characterized in that, The solvent is an aprotic solvent, specifically dichloromethane or tetrahydrofuran; the base is an inorganic or organic base, including carbonates and bicarbonates; the organic base is triethylamine or dimethylaminopyridine; the amount of base used is molar, with a molar ratio to intermediate M3 of 3:1 to 7:1; the reaction time is 1 to 10 hours, and the reaction temperature is 0 to 5°C.
4. The preparation method according to claim 2, characterized in that, The amine compound is 2-chloroethylamine, 2-bromoethylamine, 2-iodoethylamine or its hydrochloride, hydrobromide or hydroiodide; the amount of the amine compound is calculated in molar quantity, and the molar ratio with intermediate M3 is 1:1 to 5:
1.
5. The preparation method according to claim 2, characterized in that, During the extraction stage, the amount of organic solvent used is 50-200 mL for every 1 g of intermediate M3 fed.
6. The preparation method according to claim 2, characterized in that, The eluent can be a combination of ethyl acetate and n-hexane, ethyl acetate and petroleum ether, or methanol and dichloromethane.
7. An antitumor agent, characterized in that, The formulation contains the reactive oxygen species activated nitrogen mustard prodrug as described in claim 1.