Active oxygen activated nitrogen mustard prodrug as well as preparation method and application thereof

Abstract

The invention provides an active oxygen activated nitrogen mustard prodrug as well as a preparation method and application thereof, and belongs to the field of medicinal chemistry. An ROS sensitive group is introduced into a nitrogen mustard compound, so that the nitrogen mustard compound is kept in an inactive state in normal tissues and is only activated in a tumor microenvironment due to the rise of the ROS level, and a nitrogen mustard component with cytotoxicity is released. The active oxygen activated nitrogen mustard prodrug provided by the invention not only overcomes the defects of traditional nitrogen mustard drugs, but also provides a new direction and idea for research and development of anti-tumor drugs; according to the nitrogen mustard prodrug, the structure type of the nitrogen mustard prodrug is enriched, the selectivity of the drug in tumor cells is improved, and the toxic and side effects are reduced.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry, and specifically relates to an active oxygen activated nitrogen mustard prodrug and a preparation method and application thereof. Background Art

[0002] Nitrogen mustard compounds are a class of classic anticancer drugs. Their main antitumor mechanism is to alkylate DNA, induce DNA double-strand breaks, thereby inhibiting cell division and ultimately leading to apoptosis of tumor cells. Since nitrogen mustard drugs were first discovered to have antitumor activity during World War II, they have been widely used in the treatment of a variety of tumors, such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, and leukemia. Although nitrogen mustard compounds have high antitumor activity, they lack targeting and are also significantly toxic 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. In addition, the non-selective effects of nitrogen mustard drugs can easily cause adverse reactions such as bone marrow suppression, gastrointestinal reactions, and hair loss. Therefore, how to improve the tumor selectivity of nitrogen mustard compounds and reduce toxicity to normal tissues has become an important direction for current drug research and development.

[0003] Reactive oxygen species (ROS) are a class of active molecules produced during cellular metabolism, including superoxide anions (O2 - ), hydrogen peroxide (H2O2) and hydroxyl radicals ( · OH), etc. Under normal physiological conditions, the generation and clearance of ROS are in a dynamic balance, playing an important role in signal transduction and cell function regulation. However, in tumor cells, ROS levels are usually significantly increased. This is due to the combined effects of tumor cell metabolic reprogramming, mitochondrial dysfunction, and inflammatory response. High levels of ROS in the tumor microenvironment are both a driving force for tumor occurrence and development and a potential target. Excessive ROS can damage DNA, proteins, and lipids and induce cell apoptosis. Therefore, drug development strategies based on differential levels of ROS can achieve precise attacks on tumors by activating drugs in a high ROS environment while avoiding damage to normal tissues. Summary of the invention

[0004] In order to solve the limitations and side effects of the existing nitrogen mustard drug administration methods during the treatment process, the present invention proposes a prodrug design strategy based on active oxygen activation; the core idea of ​​this strategy is to introduce ROS-sensitive groups into nitrogen mustard compounds so that they remain inactive in normal tissues and are only activated by elevated ROS levels in the tumor microenvironment to release cytotoxic nitrogen mustard components. The construction of active oxygen-activated nitrogen mustard prodrugs not only solves the defects of traditional nitrogen mustard drugs, but also provides new directions and ideas for the research and development of anti-tumor drugs. The nitrogen mustard prodrugs of the present invention enrich the structural types of nitrogen mustard prodrugs, improve the selectivity of drugs in tumor cells, and reduce toxic side effects.

[0005] The first object of the present invention is to provide an active oxygen activated nitrogen mustard prodrug having a structure shown in Formula I:

[0006]

[0007] Wherein, X is oxygen or sulfur;

[0008] R1 and R2 are independently selected from hydrogen, alkyl, and haloalkyl;

[0009] R3 is halogen.

[0010] Another object of the present invention is to provide a method for preparing the active oxygen activated nitrogen mustard prodrug, the method being as follows:

[0011] The amine compound and the base are dissolved in a solvent, and the intermediate M3 dissolved in the solvent is added dropwise under an ice-water bath. After the addition is completed, the reaction is continuously stirred under nitrogen; the reaction progress is monitored by thin-layer chromatography. After the reaction is completed, the reaction product is extracted with an organic solvent, washed with water, and dried over anhydrous sodium sulfate. After the solvent is evaporated, it is purified by column chromatography to obtain the target product;

[0012] Among them, the intermediate M3 is shown in the following formula:

[0013]

[0014] Furthermore, the solvent is an aprotic solvent, and the aprotic solvent is dichloromethane or tetrahydrofuran; the base is an inorganic base or an organic base, and the inorganic base includes carbonate and bicarbonate inorganic bases; the organic base is triethylamine or dimethylaminopyridine; the amount of the base is calculated on a molar basis, and the molar ratio of the base to the intermediate M3 is 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] Furthermore, the amine compound is 2-chloroethylamine, 2-bromoethylamine, 2-iodoethylamine or its hydrochloride, hydrobromide, hydroiodide; the amount of the amine compound is calculated on a molar basis, and the molar ratio of the amine compound to the intermediate M3 is 1:1 to 5:1.

[0016] Furthermore, in the extraction stage, the amount of organic solvent used is 50-200 mL corresponding to 1 g of the intermediate M3 feed.

[0017] Furthermore, the eluent may be a combination of ethyl acetate and n-hexane, ethyl acetate and petroleum ether, and methanol and dichloromethane.

[0018] The third object of the present invention is to use the above active oxygen activated nitrogen mustard prodrug as an anti-tumor drug.

[0019] The fourth object of the present invention is to provide an antitumor preparation, which contains the above-mentioned active oxygen-activated nitrogen mustard prodrug.

[0020] Beneficial effects of the present invention:

[0021] The compound of the present invention can inhibit the toxicity of nitrogen mustard and can only show toxicity after being activated; it has a good response effect to active oxygen, a fast response time, high cell selectivity, and small toxic and side effects, enriching the types of nitrogen mustard anti-tumor drugs.

[0022] The results of cytotoxicity experiments show that the partially active oxygen-activated nitrogen mustard prodrugs synthesized by the present invention are more toxic to human cervical cancer cells and human promyelocytic leukemia cells than to mouse fibroblasts, indicating that the compounds of the present invention have higher anti-tumor activity, and the nitrogen mustard structure has targeting, showing low toxicity to normal cells.

[0023] The above results indicate that the reactive oxygen-activated nitrogen mustard prodrug synthesized by the present invention has potential anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of compound A1 having the structure of formula I;

[0025] Figure 2 A high-resolution mass spectrum of compound A1 having the structure of formula 1;

[0026] Figure 3 The hydrogen nuclear magnetic resonance spectrum of compound A2 having the structure of formula 1 is shown below;

[0027] Figure 4 A high-resolution mass spectrum of compound A2 having a structure of formula 1;

[0028] Figure 5 The hydrogen nuclear magnetic resonance spectrum of compound A3 having the structure of formula 1 is shown below;

[0029] Figure 6 A high-resolution mass spectrum of compound A3 having the structure of formula 1;

[0030] Figure 7 The hydrogen nuclear magnetic resonance spectrum of compound A4 having the structure of formula 1 is shown below;

[0031] Figure 8 The mass spectrum of compound A4 having the structure of formula 1 is shown below;

[0032] Fig. 9 : is the fluorescence spectrum of compound A1 having the structure of formula I before and after the reaction with hypochlorous acid;

[0033] Fig.10 is a kinetic curve of the response of compound A1 having the structure of formula 1 to hypochlorous acid;

[0034] Fig.11 : is the fluorescence spectrum of compound A2 having the structure of formula 1 before and after the reaction with hypochlorous acid;

[0035] Fig.12 is a kinetic curve of the response of compound A2 having a structure of formula 1 to hypochlorous acid;

[0036] Fig.13 : is the fluorescence spectrum of compound A3 having the structure of formula 1 before and after the response to hypochlorous acid;

[0037] Fig.14 is a kinetic curve of the response of compound A3 having the structure of formula 1 to hypochlorous acid;

[0038] Fig.15 : is the fluorescence spectrum of compound A4 having the structure of formula 1 before and after the reaction with hypochlorous acid;

[0039] Fig.16 is a kinetic curve of the response of compound A4 having the structure of formula 1 to hypochlorous acid;

[0040] Fig.17 It is a cytotoxicity test diagram of compound A1 having the structure of formula Ⅰ. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below in conjunction with the examples, which are merely exemplary descriptions, and the physical data of the exemplified compounds are consistent with the structures specified by these compounds. However, the examples do not limit the scope of the present invention.

[0042] The chemical reaction formula of the compound having the structure of Formula I of the present invention is as follows:

[0043]

[0044] Among them, the preparation of the intermediate acyl chloride compound M3 can be prepared by referring 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 is prepared according to the reference Chem. Sci., 2018, 9, 495.

[0048] Weigh methylene blue chloride (0.50 g, 1 equivalent) in an eggplant-shaped bottle, add 6 mL of dichloromethane to dissolve; weigh 2-chloroethylamine hydrochloride (0.55 g, 3 equivalents) and dissolve it in dichloromethane and weigh triethylamine (600 μL, 5 equivalents) and add it to 2-chloroethylamine hydrochloride. Control the temperature of the reaction system to 0°C in an ice-water bath. Under nitrogen protection, add 2-chloroethylamine dropwise to methylene blue chloride. After 1.5 hours, the reaction is completed by thin layer chromatography (TLC); evaporate the solvent, extract with 50 ml of ethyl acetate, wash with water, and dry with anhydrous sodium sulfate. After evaporating the solvent, dissolve it in dichloromethane, purify it by column chromatography (the mobile phase is ethyl acetate and petroleum ether, the ratio of the two is 1 / 1), evaporate the solvent, and beat with n-hexane for 2 hours to obtain 295 mg of product A1, with a yield of 50.3%. The nuclear magnetic resonance hydrogen spectrum and high-resolution mass spectrum of compound A1 are shown in Figure 1 and Figure 2 .

[0049] Example 2 Preparation of Compound A2 having Formula I

[0050]

[0051] The intermediate acyl chloride M3 is methylene blue acyl chloride, which is prepared according to the reference Chem. Sci., 2018, 9, 495.

[0052] Weigh methylene blue chloride (0.35 g, 1 equivalent) in an eggplant-shaped bottle, add 4 mL of dichloromethane to dissolve; weigh 2-bromoethylamine hydrobromide (0.64 g, 3 equivalents) and dissolve it in dichloromethane and weigh sodium carbonate (0.64 g, 6 equivalents) and add it to 2-bromoethylamine hydrobromide. The temperature of the reaction system is controlled at 3°C ​​in an ice-water bath. Under nitrogen protection, 2-bromoethylamine is added dropwise to methylene blue chloride. After 1 hour, the reaction is completed by thin layer chromatography (TLC). The solvent is evaporated, extracted with 30 ml of ethyl acetate, washed with water, and dried over anhydrous sodium sulfate. After evaporating the solvent, it is dissolved in dichloromethane and purified by column chromatography (the mobile phase is ethyl acetate and petroleum ether, the initial ratio of the two is 1 / 4, and then gradually increased to 1 / 1). After evaporating the solvent, beat with n-hexane for 2 hours to obtain 115 mg of product A2 with a yield of 26.4%. The nuclear magnetic resonance hydrogen spectrum and high-resolution mass spectrum of compound A2 are shown in Figure 3 and Figure 4 .

[0053] Example 3 Preparation of Compound A3 having Formula I

[0054]

[0055] The intermediate acyl chloride M3 is methylene blue acyl chloride, which is prepared according to the reference Chem. Sci., 2018, 9, 495.

[0056] Weigh methylene blue chloride (0.35 g, 1 equivalent) in an eggplant-shaped bottle, add 4 mL of dichloromethane to dissolve; weigh 2-iodoethylamine hydroiodide (0.30 g, 1 equivalent) and dissolve it in dichloromethane and add triethylamine (420 μL, 3 equivalents) to 2-iodoethylamine hydroiodide. Control the temperature of the reaction system to 1°C in an ice-water bath. Under nitrogen protection, add 2-iodoethylamine dropwise to methylene blue chloride. After 1 hour, the reaction is completed by thin layer chromatography (TLC); evaporate the solvent, extract with 30 ml of ethyl acetate, wash with water, and dry with anhydrous sodium sulfate. After evaporating the solvent, dissolve it in dichloromethane, purify it by column chromatography (the mobile phase is ethyl acetate and petroleum ether, the initial ratio of the two is 1 / 6, and then gradually increase to 1 / 1). Evaporate the solvent and beat with n-hexane for 2 hours to obtain 85 mg of product A3 with a yield of 17.6%. The nuclear magnetic resonance hydrogen spectrum and high-resolution mass spectrum of compound A3 are shown in Figure 5 and Figure 6 .

[0057] Example 4 Preparation of Compound A4 having Formula I

[0058]

[0059] The intermediate acyl chloride M3 is basic blue 3 acyl chloride, which is referred to in the literature

[0060] Prepared by J. Mater. Chem. B, 2022, 10, 5211-5217.

[0061] Weigh basic blue 3 chloride (0.56g, 1 equivalent) in an eggplant-shaped bottle, add 6mL of dichloromethane to dissolve; weigh 2-chloroethylamine hydrochloride (0.40g, 2.5 equivalents) and dissolve it in dichloromethane and weigh sodium carbonate (1.06g, 7 equivalents) and add it to 2-chloroethylamine hydrochloride. Control the temperature of the reaction system to 5°C in an ice-water bath. Under nitrogen protection, add 2-chloroethylamine dropwise to basic blue 3 chloride. After 2h, the reaction is completed by thin layer chromatography (TLC). After the reaction is completed, evaporate the solvent, extract with ethyl acetate, wash with water, and dry with anhydrous sodium sulfate. After evaporating the solvent, dissolve it in dichloromethane, purify by column chromatography (the mobile phase is ethyl acetate and petroleum ether, the ratio of the two is 1 / 4), evaporate the solvent, and beat with n-hexane for 2h to obtain 248mg of product A4 with a yield of 44.6%. The nuclear magnetic resonance hydrogen spectrum and mass spectrum of compound A4 are shown in Figure 7 and Figure 8 .

[0062] Example 5 Response performance of compound A1 to specific reactive oxygen species

[0063] This example studies the response performance of compound A1 having a structure of Formula I to specific active oxygen species (hypochlorous acid, HOCl).

[0064] Method: At room temperature, 5 μM of compound A1 was prepared in 10 mM PBS buffer solution, and different concentrations of hypochlorous acid (0, 1, 3, 5, 10 μM) were added to the system. After reacting for 30 minutes, the system was tested by fluorescence spectrometer with an excitation wavelength of 620 nm.

[0065] The experimental results are as follows Fig. 9 As shown in the figure, when no hypochlorous acid (0 μM) was added to the system, compound A1 had no fluorescence signal; however, as the concentration of hypochlorous acid increased, chloroethylamine and the fluorophore methylene blue were released from the system, resulting in a significant enhancement of the fluorescence emission in the range of 640-850 nm. In particular, after adding 10 μM hypochlorous acid, the fluorescence intensity at 686 nm was enhanced by more than 1100 times compared with the system without hypochlorous acid.

[0066] The experimental results show that compound A1 exhibits excellent response performance to hypochlorous acid.

[0067] Example 6 Reaction kinetics analysis of compound A1's response to reactive oxygen species

[0068] This example 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 change of fluorescence intensity at 686 nm was monitored in real time.

[0070] Reference Fig.10 As shown, 5 μM compound A1 was first added to the buffer solution, at which time the fluorescence intensity was low. Hypochlorous acid was added 75 seconds later, and the fluorescence intensity was observed to increase rapidly, and reached equilibrium at 110 seconds, with the fluorescence intensity approaching the maximum value within 30 seconds. This result shows that compound A1 has a fast response speed and good kinetic properties.

[0071] In summary, compound A1 responds sensitively and rapidly to hypochlorous acid and has potential practical application value.

[0072] Example 7 Response performance of compound A2 to specific reactive oxygen species

[0073] This example studies the response performance of compound A2 having a structure of Formula I to specific active oxygen species (hypochlorous acid, HOCl).

[0074] Method: At room temperature, 5 μM of compound A2 was prepared in 10 mM PBS buffer solution, and different concentrations of hypochlorous acid (0, 1, 3, 5, 10 μM) were added to the system. After reacting for 30 minutes, the system was tested by fluorescence spectrometer with an excitation wavelength of 620 nm.

[0075] The experimental results are as follows Fig.11 As shown in the figure, when no hypochlorous acid (0 μM) was added to the system, compound A2 had no fluorescence signal; however, as the concentration of hypochlorous acid increased, ethylamine bromide and the fluorophore methylene blue were released from the system, resulting in a significant increase in fluorescence emission in the range of 640-850 nm. In particular, after adding 10 μM hypochlorous acid, the fluorescence intensity at 686 nm was enhanced by more than 1100 times compared to the system without hypochlorous acid.

[0076] The experimental results show that compound A2 exhibits excellent response performance to hypochlorous acid.

[0077] Example 8 Reaction kinetics analysis of compound A2's response to reactive oxygen species

[0078] This example 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 change of fluorescence intensity at 686 nm was monitored in real time.

[0080] Reference Fig.12As shown, 5 μM compound A2 was first added to the buffer solution, at which time the fluorescence intensity was low. Hypochlorous acid was added 40 seconds later, and the fluorescence intensity was observed to increase rapidly, reaching equilibrium at 70 seconds, with the fluorescence intensity approaching the maximum value within 30 seconds. This result shows that compound A2 has a fast response speed and good kinetic properties.

[0081] In summary, compound A2 responds sensitively and rapidly to hypochlorous acid and has potential practical application value.

[0082] Example 9 Response performance of compound A3 to specific reactive oxygen species

[0083] This example studies the response performance of compound A3 having a structure of Formula I to specific active oxygen species (hypochlorous acid, HOCl).

[0084] Method: At room temperature, 5 μM compound A3 was prepared in 10 mM PBS buffer solution, and different concentrations of hypochlorous acid (0, 1, 3, 5, 10 μM) were added to the system. After reacting for 30 minutes, the system was tested by fluorescence spectrometer with an excitation wavelength of 620 nm.

[0085] The experimental results are as follows Fig.13 As shown in the figure, when no hypochlorous acid (0 μM) was added to the system, compound A3 had no fluorescence signal; however, as the concentration of hypochlorous acid increased, iodoethylamine and the fluorophore methylene blue were released from the system, resulting in a significant increase in fluorescence emission in the range of 640-850 nm. In particular, after adding 10 μM hypochlorous acid, the fluorescence intensity at 686 nm was enhanced by more than 1400 times compared to the system without hypochlorous acid.

[0086] The experimental results show that compound A3 exhibits excellent response performance to hypochlorous acid.

[0087] Example 10 Reaction kinetics analysis of compound A3's response to reactive oxygen species

[0088] This example 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 change of fluorescence intensity at 686 nm was monitored in real time.

[0090] Reference Fig.14 As shown, 5 μM compound A3 was first added to the buffer solution, at which time the fluorescence intensity was low. Hypochlorous acid was added 45 seconds later, and the fluorescence intensity was observed to increase rapidly, reaching equilibrium at 75 seconds, with the fluorescence intensity approaching the maximum value within 30 seconds. This result shows that compound A3 has a fast response speed and good kinetic properties.

[0091] In summary, compound A3 responds sensitively and rapidly to hypochlorous acid and has potential practical application value.

[0092] Example 11 Response performance of compound A4 to specific reactive oxygen species

[0093] This example studies the response performance of compound A4 having a structure of Formula I to specific active oxygen species (hypochlorous acid, HOCl).

[0094] Method: At room temperature, 5 μM compound A4 was prepared in 10 mM PBS buffer solution, and different concentrations of hypochlorous acid (0, 1, 3, 5, 10 μM) were added to the system. After reacting for 30 minutes, the system was tested by fluorescence spectrometer with an excitation wavelength of 610 nm.

[0095] The experimental results are as follows Fig.15 As shown in the figure, when no hypochlorous acid (0 μM) was added to the system, compound A4 had no fluorescence signal; however, as the concentration of hypochlorous acid increased, chloroethylamine and fluorophore basic blue 3 were released from the system, resulting in a significant enhancement of fluorescence emission in the range of 640-850 nm. In particular, after adding 10 μM hypochlorous acid, the fluorescence intensity at 669 nm was enhanced by more than 1600 times compared with the system without hypochlorous acid.

[0096] The experimental results show that compound A4 exhibits excellent response performance to hypochlorous acid.

[0097] Example 12 Reaction kinetics analysis of compound A4's response to reactive oxygen species

[0098] This example 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 change of fluorescence intensity at 669 nm was monitored in real time.

[0100] Reference Fig.16 As shown, 5 μM compound A4 was first added to the buffer solution, at which time the fluorescence intensity was low. Hypochlorous acid was added 12 seconds later, and then the fluorescence intensity was observed to increase rapidly, reaching equilibrium after 150 seconds, of which the fluorescence intensity was close to the maximum value within 140 seconds. This result shows that compound A4 has a fast response speed and good kinetic properties.

[0101] In summary, compound A4 responds sensitively and rapidly to hypochlorous acid and has potential practical application value.

[0102] Example 13 Analysis of the toxicity of compound A1 on malignant tumors

[0103] The mouse fibroblasts, human cervical cancer cells, and human promyelocytic leukemia cells used in the experiment were purchased from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences.

[0104] Different concentrations of compound A1 (0-40 μM) of formula I were co-incubated with mouse fibroblasts, human cervical cancer cells, and human promyelocytic leukemia cells for 24 hours, and the toxicity of A1 on the three types of cells was observed.

[0105] During the experiment, compound A1 was first prepared into a 1mM stock solution, and then different amounts of A1 stock solution were added to the cell culture medium according to different concentration requirements (0, 20, 30, 40μM). After 24 hours, the effects of different concentrations of A1 on the survival rates of the three types of cells were observed. Fig.17 As shown, the results show that the toxicity of A1 to human cervical cancer cells and human promyelocytic leukemia cells is higher than its toxicity to mouse fibroblasts.

[0106] In summary, compound A1 having the structure of formula I has high anti-tumor activity, and makes the nitrogen mustard structure targeted and exhibits low toxicity to normal cells.

[0107] The above description is not limited to the above examples. Technical features not described in the present invention can be achieved by or by adopting existing technologies, which will not be described herein. The above embodiments are only used to illustrate the technical scheme of the present invention and are not limitations of the present invention. Changes, modifications, additions or substitutions made by ordinary technicians in this technical field within the essential scope of the present invention do not depart from the purpose of the present invention and should also fall within the scope of protection of the claims of the present invention.

Claims

1. An active oxygen activated nitrogen mustard prodrug, characterized in that: The active oxygen activated nitrogen mustard prodrug has a structure shown in Formula 1: Wherein, X is oxygen or sulfur; R1 and R2 are independently selected from hydrogen, alkyl, and haloalkyl; R3 is halogen.

2. A method for preparing an active oxygen activated nitrogen mustard prodrug as claimed in claim 1, characterized in that: Here’s how: The amine compound and the base are dissolved in a solvent, and the intermediate M3 dissolved in the solvent is added dropwise under an ice-water bath. After the addition is completed, the reaction is continuously stirred under nitrogen; the reaction progress is monitored by thin-layer chromatography. After the reaction is completed, the reaction product is extracted with an organic solvent, washed with water, and dried over anhydrous sodium sulfate. After the solvent is evaporated, it is purified by column chromatography to obtain the target product; Among them, 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, and the aprotic solvent is dichloromethane or tetrahydrofuran; the base is an inorganic base or an organic base, and the inorganic base includes carbonate and bicarbonate inorganic bases; the organic base is triethylamine or dimethylaminopyridine; the amount of the base is calculated on a molar basis, and the molar ratio of the base to the intermediate M3 is 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.

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, hydroiodide; the amount of the amine compound is calculated by molar amount, and the molar ratio of the amine compound to the intermediate M3 is 1:1 to 5:

1.

5. The preparation method according to claim 2, characterized in that: In the extraction stage, the amount of organic solvent used is 50-200 mL for 1 g of intermediate M3.

6. The preparation method according to claim 2, characterized in that: As the eluent, a combination of ethyl acetate and n-hexane, ethyl acetate and petroleum ether, and methanol and dichloromethane can be used.

7. Use of the active oxygen activated nitrogen mustard prodrug according to claim 1 as an anti-tumor drug.

8. An anti-tumor preparation, characterized in that: The preparation contains the active oxygen activated nitrogen mustard prodrug according to claim 1.

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