Cobalamin derivatives, their preparation methods, and their application in the synthesis of arsenopyrine.

Abstract

This invention provides a cobalamin derivative, its preparation method, and its application in the synthesis of arsenopyrine, relating to the field of heterocyclic compounds, and more specifically to the field of arsenopyrine synthesis technology. This cobalamin derivative is prepared by linking a 3-amino-3-carboxypropyl (ACP) group to the cobalt atom of cobalamin (CbI), and protecting the 3-amino-3-carboxypropyl group with a 9-fluorenemethoxycarbonyl (Fmoc) and a tert-butyl (tBu) group.

Description

Technical Field

[0001] This invention relates to the field of arsenopyrine synthesis technology, specifically to a cobalamin derivative, its preparation method, and its application in arsenopyrine synthesis. Background Technology

[0002] Arsinothricin (AST), first discovered in rice rhizosphere bacteria, is a non-protein analog of glutamate. It inhibits glutamine synthase (GS) to block bacterial nitrogen metabolism, exhibiting bactericidal effects against a variety of Gram-positive and Gram-negative bacteria. Because AST's mechanism of action differs from traditional antibiotics, it may offer a novel treatment option for multidrug-resistant bacterial infections.

[0003] AST is mainly generated by methylation of its precursor, hydroxyarsenicin (AST-OH), via arsenite (+3 oxidation state) methyltransferase (ArsM). The structural formulas of AST and AST-OH are shown below:

[0004] .

[0005] Among the related technologies, the synthesis method of AST-OH has many defects: the reaction raw materials are toxic, some intermediates are carcinogenic or toxic, and there are also problems such as low product yield, complicated operation process and long reaction time. Summary of the Invention

[0006] In view of this, the present invention provides a cobalamin derivative, its preparation method and its application in the synthesis of arsenopyrine.

[0007] According to a first aspect of the present invention, a cobalamin derivative is provided having the following structure:

[0008] .

[0009] In this context, Fmoc represents 9-fluorenemethyloxycarbonyl, and t-Bu represents tert-butyl.

[0010] A second aspect of the present invention provides a method for preparing the above-mentioned compound, comprising the following steps: reducing hydroxycobalamin to reduce trivalent cobalt in the above-mentioned hydroxycobalamin to monovalent cobalt, thereby obtaining monovalent cobalamin; subjecting tert-butyl 2-(Fmoc-amino)-4-iodobutyrate to the above-mentioned monovalent cobalamin in a first nucleophilic substitution reaction to obtain a cobalamin derivative.

[0011] A third aspect of the present invention provides a method for preparing a hydroxyarsenic trioxide intermediate, comprising the following steps: performing a complexation reaction between reduced glutathione and arsenic acid to obtain an arsenic acid-glutathione complex; performing a free radical reaction between a cobalamin derivative and the above-mentioned arsenic acid-glutathione complex to obtain a compound represented by formula (II); and performing an oxidation reaction on the above-mentioned compound represented by formula (II) to obtain a compound represented by formula (III).

[0012] in, ; ; .

[0013] In this context, Fmoc represents 9-fluorenemethyloxycarbonyl, and t-Bu represents tert-butyl.

[0014] A fourth aspect of the present invention provides a method for preparing hydroxyarsin, comprising: removing a tert-butyl group from the hydroxyl group of the compound represented by formula (III) by a first hydrolysis reaction to obtain the compound represented by formula (IV), wherein the compound represented by formula (IV) is obtained by the above preparation method.

[0015] in, .

[0016] Hydroxyarsenic trioxide is obtained by removing the 9-fluorenemethoxycarbonyl group from the amino group of the compound shown in formula (IV) above through a second hydrolysis reaction.

[0017] A fifth aspect of the present invention provides a method for preparing arsenopyrine, comprising: methylating hydroxyarsenicpyrine to obtain arsenopyrine, wherein the hydroxyarsenicpyrine is obtained by the above preparation method.

[0018] The cobalamin derivative provided in this embodiment of the invention links 3-amino-3-carboxypropyl (ACP) to the cobalt atom of cobalamin (CbI) and protects ACP with 9-fluorenylmethoxycarbonyl (Fmoc) and tert-butyl (tBu). Thus, the cobalamin derivative can generate ACP radicals by light irradiation and transfer the ACP radicals to the arsenite substrate to form C-As bonds with the arsenite substrate, thereby rapidly generating AST-OH.

[0019] This invention provides a green and efficient method for preparing hydroxyarsenic trioxide. The method involves photocatalysis to react a cobalamin derivative with an arsenite substrate via a free radical reaction, generating AST-OH through this novel free radical mechanism. The reaction conditions are mild, requiring no high temperatures (above room temperature), high pressures (above atmospheric pressure), or strong acidic or alkaline conditions. It exhibits high oxygen tolerance, eliminates the need for expensive equipment such as glove boxes, and offers low cost and high yield. It is not limited by enzyme activity pockets and has the potential to synthesize AST-OH analogs. Detailed Implementation

[0020] The embodiments of the present invention will now be described. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the invention. In the following detailed description, numerous specific details are set forth for ease of explanation to provide a full understanding of the embodiments of the invention. However, it will be apparent that one or more embodiments may be practiced without these specific details. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention.

[0021] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. The terms “comprising,” “including,” etc., as used herein indicate the presence of the stated features, steps, operations, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, or components.

[0022] All terms used herein (including technical and scientific terms) have the meanings commonly understood by those skilled in the art, unless otherwise defined. It should be noted that the terms used herein are to be interpreted in a manner consistent with the context of this specification, and not in an idealized or overly rigid way.

[0023] When using expressions such as "at least one of A, B and C", they should generally be interpreted in accordance with the meaning that is commonly understood by those skilled in the art (e.g., "a system having at least one of A, B and C" should include, but is not limited to, a system having A alone, a system having B alone, a system having C alone, a system having A and B, a system having A and C, a system having B and C, and / or a system having A, B and C, etc.).

[0024] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used in this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention; the terms "comprising" and "having," and any variations thereof, in the specification and claims of this invention are intended to cover non-exclusive inclusion. Unless otherwise stated, the terms used in this invention have their commonly known meanings as commonly understood by one of ordinary skill in the art. Unless otherwise stated, the numerical values ​​of the parameters mentioned in this invention can be measured using various measurement methods commonly used in the art (e.g., they can be tested according to the methods given in embodiments of this invention).

[0025] In related technologies, AST-OH can be synthesized through chemical or enzymatic methods.

[0026] The chemical method synthesizes AST-OH via the reaction process shown in Synthetic Route 1. Specifically, sodium arsenite is esterified with 2-chloroethanol under alkaline conditions to generate (2-hydroxyethyl)arsenic acid. The product reacts with hydrochloric acid and SO2 under KI catalysis, reducing As(V) to As(III) while its hydroxyl group undergoes chlorination to generate dichloro(2-hydroxyethyl)arsine. Subsequently, the terminal hydroxyl group is chlorinated with SOCl2 to generate dichloro(2-chloroethyl)arsine. The As(III) of dichloro(2-chloroethyl)arsine is oxidized to As(V) by H2O2 and then undergoes a condensation reaction with diethyl acetaminomalonate and sodium ethoxide at 70°C to generate the malonic acid ester derivative of AST-OH. After deprotection with hydrochloric acid, the racemic final product AST-OH is obtained by cation exchange chromatography. The synthetic route 1 is shown below.

[0027] .

[0028] Enzymatic synthesis of AST-OH can be achieved in vitro using arsinothricin synthase L (ArsL) from the non-classical free radical S-adenosyl-L-methionine enzyme (Radical SAM). ArsL cleaves the C3+ of Radical SAM. γ,Met The -S bond generates a 3-amino-3-carboxypropyl radical, which is then transferred to the arsenite substrate to construct a C-As bond, thereby generating AST-OH.

[0029] Although the two methods mentioned above can synthesize AST-OH, they still have obvious shortcomings.

[0030] The chemical synthesis process has the following drawbacks: it relies on the SO2 / HCl / KI system to reduce pentavalent arsenic, SO2 gas is toxic and irritating to mucous membranes, and the generated intermediate methylarsine iodide (MeAsI2) is a strong carcinogen; the efficiency of chlorination to generate dichloro(2-chloroethyl)arsine is low, with a single-step yield of only 4%, and it generates highly toxic dichloro(2-hydroxyethyl)arsine byproducts; the synthesis process requires multiple column chromatography purifications, including silica gel column chromatography, Dowex ion exchange resin (Dowex resin), and hydroxypropylated cross-linked dextran gel LH-20 (Sephadex LH-20), which is complex, time-consuming, and consumes a huge amount of elution solvent.

[0031] The enzymatic catalysis using ArsL overcomes the drawbacks of chemical methods, such as harsh reaction conditions, long reaction times, and lack of stereoselectivity. However, it also introduces new limitations: ArsL relies on the [4Fe-4S] cluster for catalysis, which is highly sensitive to oxygen, requiring the reaction to be conducted in a glove box, making the experiment difficult and expensive; the efficiency of protein reconstruction of the [4Fe-4S] cluster is only 70%, with some proteins existing in an inactive state lacking the [4Fe-4S] cluster; ArsL achieves a turnover rate of 15 when catalyzing the cleavage of SAM to produce 5'-deoxy-5'-methylthioadenosine, but only 2 when using titanium citrate as a reducing agent to catalyze the final product AST-OH, resulting in low catalytic efficiency and high production costs. This makes it suitable only for obtaining trace amounts of products for basic research, not for mass production. Furthermore, ArsL relies on the C-terminal RCCLKC motif to recognize and bind As(III), and this high selectivity limits the formation of other types of CX bonds besides arsenic, thus lacking the potential for synthesizing AST-OH analogs.

[0032] In the process of realizing this invention, it was discovered that 3-amino-3-carboxypropyl can be linked to the cobalt atom of cobalamin. By utilizing the ability of cobalamin to form carbon-centered free radicals, a greener and more convenient AST-OH synthesis route can be developed, reducing production costs. This is of great significance for the application of AST in the treatment of bacterial infections.

[0033] In view of this, an embodiment of the first aspect of the present invention provides a cobalamin derivative having the structure shown below.

[0034] .

[0035] In this context, Fmoc represents 9-fluorenemethyloxycarbonyl, and t-Bu represents tert-butyl.

[0036] The cobalamin derivative provided in this embodiment of the invention links 3-amino-3-carboxypropyl (ACP) to the cobalt atom of cobalamin (CbI) and protects ACP with 9-fluorenylmethoxycarbonyl (Fmoc) and tert-butyl (tBu). Thus, the cobalamin derivative can generate ACP radicals by light irradiation and transfer the ACP radicals to the arsenite substrate to form C-As bonds with the arsenite substrate, thereby rapidly generating AST-OH.

[0037] Furthermore, the cobalamin derivatives provided in the embodiments of the present invention offer new ideas and insights for the application of cobalamin in photochemistry.

[0038] An embodiment of the second aspect of the present invention provides a method for preparing the above-mentioned compound, including steps S101 and S102.

[0039] In step S101, hydroxycobalamin is reduced to reduce trivalent cobalt to monovalent cobalt, thus obtaining monovalent cobalamin.

[0040] In step S102, tert-butyl 2-(Fmoc-amino)-4-iodobutyrate is subjected to a first nucleophilic substitution reaction with hydroxycobalamin to obtain a cobalamin derivative.

[0041] According to an embodiment of the present invention, step S101, reducing hydroxycobalamin may include: reacting hydroxycobalamin with a reducing agent in a first organic solvent at 20°C to 30°C in the dark for 10 min to 30 min. Exemplarily, hydroxycobalamin and a reducing agent may be reacted in a first organic solvent at a temperature between any two of the following values: 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, or above, in the dark for 10 min, 12 min, 15 min, 18 min, 20 min, 22 min, 25 min, 28 min, 30 min, or above.

[0042] In some embodiments of the present invention, in step S101, the reducing agent may include at least one of sodium borohydride and titanium citrate. The first organic solvent may be methanol.

[0043] According to an embodiment of the present invention, in step S102, the temperature of the first nucleophilic substitution reaction can be 20°C to 30°C, and the time can be 1h to 2h. Exemplarily, the temperature of the first nucleophilic substitution reaction can be any two values ​​between 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, or above, and the time can be any two values ​​between 1h, 1.1h, 1.2h, 1.3h, 1.4h, 1.5h, 1.6h, 1.7h, 1.8h, 1.9h, 2h, or above.

[0044] In some embodiments of the present invention, in step S102, the molar ratio of tert-butyl 2-(Fmoc-amino)-4-iodobutyrate to the first compound can be 2:(3~4). Exemplarily, the molar ratio of tert-butyl 2-(Fmoc-amino)-4-iodobutyrate to the first compound can be 2:3, 2:3.1, 2:3.2, 2:3.3, 2:3.4, 2:3.5, 2:3.6, 2:3.7, 2:3.8, 2:3.9, 2:4, or any range between two of the above ratios.

[0045] For example, hydroxycobalamin (OH-CbI, 1 molar equivalent) and sodium borohydride (10 molar equivalents) can be added to methanol and reacted for 15 min under light-protected and anaerobic conditions. Then, tert-butyl 2-(Fmoc-amino)-4-iodobutyrate (2 molar equivalents) is added and reacted for 1 h under light-protected conditions. The pH of the reaction solution is adjusted to pH=5 using acetic acid to obtain the cobalamin derivative (ACP-CbI-FB).

[0046] In some embodiments of the present invention, the preparation method of tert-butyl 2-(Fmoc-amino)-4-iodobutyrate may include steps S201 to S203.

[0047] In step S201, L-homoserine and fluorenemethoxycarbonylsuccinimide are subjected to an acylation reaction in the first mixed solvent to obtain the compound shown in formula (A).

[0048] .

[0049] In step S202, the compound shown in formula (A) is subjected to a second nucleophilic substitution reaction with tert-butyl-2,2,2-trichloroacetic acid imine ester to obtain the compound shown in formula (B).

[0050] .

[0051] In step S203, the compound shown in formula (B) is subjected to a third nucleophilic substitution reaction with triphenylphosphine, followed by the addition of iodine for halogenation to obtain tert-butyl 2-(Fmoc-amino)-4-iodobutyrate. Tert-butyl 2-(Fmoc-amino)-4-iodobutyrate has the structure shown in formula (C).

[0052] .

[0053] According to an embodiment of the present invention, in step S201, the acylation reaction of L-homoserine and fluorenemethoxycarbonylsuccinimide in the first mixed solvent may include: dissolving L-homoserine and fluorenemethoxycarbonylsuccinimide in the first mixed solvent, cooling to 0°C to 10°C, adding a weak base and reacting for 30 min to 60 min, and then reacting at 20°C to 30°C for 16 h to 24 h. For example, L-homoserine and fluorenemethyloxycarbonyl succinimide can be dissolved in a first mixed solvent, cooled to a temperature between any two values ​​of 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C or above, and then reacted with a weak base for a period of 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min or above. The reaction can then be carried out at a temperature between any two values ​​of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C or above for a period of 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h or above. In the initial stage of acylation, the reaction rate is relatively fast and a lot of heat is released. Too high a temperature is not conducive to product stability. Therefore, the reaction is first carried out at 0℃~10℃ for 30min~60min. After the reaction rate decreases, the reaction is carried out at 20℃~30℃ for 16h~24h, which is conducive to the formation of the compound shown in formula (A).

[0054] In some embodiments of the present invention, in step S201, the molar ratio of L-homoserine, fluorenemethoxycarbonylsuccinimide, and the weak base can be 84:(101~168):(101~168). Exemplarily, the molar ratio of L-homoserine, fluorenemethoxycarbonylsuccinimide, and the weak base can be 84:101:101, 84:101:110, 84:110:110, 84:120:120, 84:130:130, 84:140:140, 84:150:150, 84:160:160, 84:168:168, 84:110:130, 84:130:110, 84:130:150, 84:150:130, or any two of the above ratios.

[0055] In some embodiments of the present invention, in step S201, the first mixed solvent can be a mixture of water and dioxane in a volume ratio of 1:(1~3). Exemplarily, the volume ratio of water to dioxane in the first mixed solvent can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, or any two of the above ratios.

[0056] In some embodiments of the present invention, in step S201, the weak base may include at least one of sodium carbonate and potassium carbonate.

[0057] For example, in step S201, L-homoserine (1 molar equivalent) and fluorenemethoxycarbonylsuccinimide (Fmoc-Osu, 1.2 molar equivalent) can be dissolved in a first mixed solvent of water / dioxane (volume ratio 1:1). After cooling to 0°C, sodium bicarbonate (1.2 molar equivalent) is added, and the reaction is carried out at 0°C for 30 min, followed by reaction at room temperature for 16 h. After removing the dioxane, the aqueous phase is washed with petroleum ether. The pH of the system is adjusted to pH=2 with hydrochloric acid, and then extracted with ethyl acetate. The organic phase is dried with anhydrous sodium sulfate and concentrated under vacuum to remove the solvent, yielding the compound shown in formula (A), i.e., the crude homoserine product with the amino group protected by Fmoc.

[0058] According to an embodiment of the present invention, in step S202, the second nucleophilic substitution reaction of the compound shown in formula (A) with tert-butyl-2,2,2-trichloroacetic acid imine ester may include: dissolving the compound shown in formula (A) in a second mixed solvent, and adding tert-butyl-2,2,2-trichloroacetic acid imine ester dropwise to carry out the reaction.

[0059] In some embodiments of the present invention, in step S202, the temperature of the second nucleophilic substitution reaction can be 20°C to 30°C, and the time can be 16h to 24h. Exemplarily, the temperature of the second nucleophilic substitution reaction can be any two values ​​between 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, or above, and the time can be any two values ​​between 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, or above.

[0060] In some embodiments of the present invention, in step S202, the second mixed solvent is a mixture of dichloromethane and tetrahydrofuran with a volume ratio of (1~3):1. Exemplarily, the volume ratio of dichloromethane to tetrahydrofuran in the second mixed solvent can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, or any two of the above ratios.

[0061] Exemplarily, in step S202, the compound shown in formula (A) can be dissolved in a second mixed solvent of dichloromethane / tetrahydrofuran (volume ratio 4:1), and tert-butyl-2,2,2-trichloroacetic acid imine ester (2 molar equivalents) can be added. The reaction is carried out at room temperature for 16 h. After removing the solvent, the residue is dissolved in ethyl acetate, washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, and dried over anhydrous sodium sulfate. The organic phase is purified by silica gel column chromatography to obtain the compound shown in formula (B), namely homoserine with the amino group protected by Fmoc and the carboxyl group protected by tBu, namely tert-butyl 2-(Fmoc-amino)-4-hydroxybutyrate.

[0062] According to an embodiment of the present invention, in step S203, the compound shown in formula (B) undergoes a third nucleophilic substitution reaction with triphenylphosphine, followed by the addition of iodine for halogenation reaction, which may include: dissolving the compound shown in formula (B) and triphenylphosphine in a second organic solvent under light-proof and oxygen-free conditions, cooling to 0°C to 10°C, adding an organic base and reacting for 30 min to 60 min, then adding iodine and reacting sequentially at 0°C to 10°C for 30 min to 60 min, and at 20°C to 30°C for 6 h to 10 h. For example, under light-protected and oxygen-free conditions, the compound shown in formula (B) and triphenylphosphine can be dissolved in a second organic solvent, cooled to a temperature between any two values ​​of 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, or higher, and then an organic base can be added and reacted for 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, or higher. Iodine can then be added sequentially at 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, or higher. The reaction can proceed within any two of the following temperature ranges: ℃, 6℃, 7℃, 8℃, 9℃, 10℃, or above, for 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, or above; or within any two of the following temperature ranges: 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, or above, for 6 h, 7 h, 8 h, 9 h, 10 h, or above. In the initial stage of the halogenation reaction, the reaction rate is relatively fast and exothermic. Excessively high temperatures are detrimental to product stability. Therefore, it is beneficial to first react at 0℃~10℃ for 30 min~60 min, then, after the reaction rate decreases, react at 20℃~30℃ for 6 h~10 h to promote the formation of tert-butyl 2-(Fmoc-amino)-4-iodobutyrate.

[0063] In some embodiments of the present invention, in step S203, the molar ratio of the compound represented by formula (B), triphenylphosphine, organic base, and iodine can be 85:(102~127):(102~127):(102~127). Exemplarily, the molar ratio of the compound represented by formula (B), triphenylphosphine, organic base, and iodine can be 85:102:102:102, 85:110:110:110, 85:120:120:120, 85:127:127:127, 85:110:120:125, 85:120:125:110, 85:125:120:110, or any two of the above ratios.

[0064] In some embodiments of the present invention, in step S203, the second organic solvent may include at least one of tetrahydrofuran and dichloromethane.

[0065] In some embodiments of the present invention, in step S203, the organic base may include at least one of imidazole, diethylamine, and triethylamine.

[0066] Exemplarily, in step S203, the compound shown in formula (B) (1 molar equivalent), triphenylphosphine (1.2 molar equivalent), and imidazole (1.2 molar equivalent) can be added to tetrahydrofuran and reacted for 30 min under light-protected, oxygen-free, and 0°C conditions; then I2 (1.2 molar equivalent) is added, and the reaction is carried out at 0°C for 30 min, followed by reaction at room temperature for 6 h. After removing the solvent, excess I2 is removed with 10 mL of 10% (w / v) sodium thiosulfate solution, and the aqueous phase is extracted with ethyl acetate and dried over anhydrous sodium sulfate. The organic phase is purified by silica gel column chromatography to obtain tert-butyl 2-(Fmoc-amino)-4-iodobutyrate.

[0067] The third aspect of the present invention provides a method for preparing a hydroxyarsin intermediate, comprising steps S301 to S303.

[0068] In step S301, reduced glutathione and arsenic acid are subjected to a complexation reaction to obtain an arsenic acid-glutathione complex.

[0069] In step S302, the aforementioned cobalamin derivative is subjected to a free radical reaction with the arsenite-glutathione complex to obtain the compound shown in formula (II).

[0070] .

[0071] In this context, Fmoc represents 9-fluorenemethyloxycarbonyl, and t-Bu represents tert-butyl.

[0072] .

[0073] In step S303, the compound shown in formula (II) is subjected to an oxidation reaction to obtain the compound shown in formula (III).

[0074] .

[0075] According to an embodiment of the present invention, step S301, the complexation reaction of reduced glutathione and arsenic acid, may include: adding reduced glutathione to an aqueous solution of arsenic acid, adjusting the pH to 6-7, and reacting at 20°C-30°C under anaerobic conditions for 1-2 hours. Exemplarily, reduced glutathione can be added to an aqueous solution of arsenic acid, adjusting the pH to any two values ​​between 6, 6.2, 6.5, 6.8, 7, or higher, and the temperature to any two values ​​between 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, or higher, and the reaction time to any two values ​​between 1 hour, 1.1 hours, 1.2 hours, 1.3 hours, 1.4 hours, 1.5 hours, 1.6 hours, 1.7 hours, 1.8 hours, 1.9 hours, 2 hours, or higher under anaerobic conditions.

[0076] In some embodiments of the present invention, in step S301, the concentration of the arsenic acid aqueous solution can be 40 mmol / L to 60 mmol / L. Exemplarily, the concentration of the arsenic acid aqueous solution can be 40 mmol / L, 42 mmol / L, 45 mmol / L, 48 mmol / L, 50 mmol / L, 52 mmol / L, 55 mmol / L, 58 mmol / L, 60 mmol / L, or any two of the above values.

[0077] In some embodiments of the present invention, in step S301, the molar ratio of reduced glutathione to the volume ratio of the arsenic acid aqueous solution can be (2~3) mmol:20 mL. Exemplarily, the molar ratio of reduced glutathione to the volume ratio of the arsenic acid aqueous solution can be 2 mmol:20 mL, 2.2 mmol:20 mL, 2.5 mmol:20 mL, 2.8 mmol:20 mL, 3 mmol:20 mL, or any two of the above ratios.

[0078] According to an embodiment of the present invention, in step S302, the free radical reaction can be carried out under anaerobic conditions and white light irradiation of 250W to 500W. Exemplarily, the free radical reaction can be carried out under white light irradiation within the range of anaerobic conditions and any two values ​​between 250W, 300W, 350W, 400W, 450W, 500W, or higher. The reaction between the cobalamin derivative and arsenic trioxide only requires white light irradiation under neutral or weakly acidic conditions (pH 5 to 9), without the need for high temperature, high pressure, or strong acid / alkaline conditions.

[0079] In some embodiments of the present invention, in step S302, the temperature of the free radical reaction can be 20°C to 30°C, and the time can be 6 hours to 10 hours. Exemplarily, the temperature of the free radical reaction can be any two values ​​between 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, and 30°C, and the time can be any two values ​​between 6 hours, 7 hours, 8 hours, 9 hours, and 10 hours. The free radical reaction between ACP-CbI-FB and arsenous acid only requires 6 hours to 10 hours, shortening the reaction time.

[0080] According to an embodiment of the present invention, in step S303, the temperature of the oxidation reaction can be 20°C to 30°C, and the time can be 2h to 3h. Exemplarily, the temperature of the oxidation reaction can be any two values ​​between 220°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, or above, and the time can be any two values ​​between 2h, 2.1h, 2.2h, 2.3h, 2.4h, 2.5h, 2.6h, 2.7h, 2.8h, 2.9h, 3h, or above.

[0081] In some embodiments of the present invention, in step S303, the oxidant used in the oxidation reaction is hydrogen peroxide.

[0082] For example, reduced glutathione (2 molar equivalents) can be added to an aqueous solution of arsenic acid (1 molar equivalent), the pH value adjusted to pH=7 with sodium hydroxide, and the reaction proceeded in the dark for 1 hour. Then, a cobalamin derivative is added, and the reaction proceeds for 6 hours under anaerobic conditions and 300W white light irradiation. The product is purified by preparative high-performance liquid chromatography (HPLC) to obtain a compound of formula (II) containing trivalent arsenic with a protecting group. The compound of formula (II) is then oxidized with hydrogen peroxide to convert trivalent As to pentavalent As, yielding the compound of formula (III).

[0083] An embodiment of the fourth aspect of the present invention provides a method for preparing hydroxyarsin, comprising steps S401 and S402.

[0084] In step S401, the tert-butyl group on the hydroxyl group of the compound shown in formula (III) is removed by a first hydrolysis reaction to obtain the compound shown in formula (IV), wherein the compound shown in formula (III) is obtained by the above preparation method.

[0085] .

[0086] In step S402, the 9-fluorenemethoxycarbonyl group on the amino group of the compound shown in formula (Ⅳ) is removed by a second hydrolysis reaction to obtain hydroxyarsin.

[0087] This invention provides a green and efficient method for preparing hydroxyarsenic trichothecin. The method involves photocatalysis to react a cobalamin derivative with an arsenite substrate via a free radical reaction, generating AST-OH through this novel free radical mechanism. The reaction conditions are mild, requiring no high temperature, high pressure, or strong acid / alkaline conditions. It exhibits high oxygen tolerance, eliminates the need for expensive equipment such as glove boxes, and offers low cost and high yield. It is not limited by enzyme activity pockets and has the potential to synthesize AST-OH analogs.

[0088] According to an embodiment of the present invention, in step S401, removing the tert-butyl group from the hydroxyl group of the compound represented by formula (III) by a first hydrolysis reaction may include: adding the compound represented by formula (III) to an acidic solution and reacting it at 0°C to 10°C for 6 to 10 hours. Exemplarily, the compound represented by formula (III) may be added to an acidic solution and reacted at a temperature between any two of the following values: 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, or above, for 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, or above.

[0089] In some embodiments of the present invention, in step S401, the acidic solution includes at least one of formic acid, acetic acid, trifluoroacetic acid, and hydrochloric acid.

[0090] According to an embodiment of the present invention, in step S402, removing the 9-fluorenemethyloxycarbonyl group from the amino group of the compound represented by formula (Ⅳ) by a second hydrolysis reaction may include: adding the compound represented by formula (Ⅳ) to an alkaline solution and reacting it at 20°C to 30°C for 4 to 6 hours. Exemplarily, the compound represented by formula (Ⅳ) may be added to an alkaline solution and reacted at a temperature between any two of the following values: 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, for 4 hours, 4.2 hours, 4.5 hours, 4.8 hours, 5 hours, 5.2 hours, 5.5 hours, 5.8 hours, 6 hours, or any two of the above values.

[0091] In some embodiments of the present invention, in step S402, the alkaline solution is a methanol solution of diethylamine and / or imidazole.

[0092] For example, the tert-butyl group of the carboxyl group of the compound shown in formula (III) can be removed with formic acid to obtain the compound shown in formula (IV); the compound shown in formula (IV) is purified by preparative HPLC and then the 9-fluorenemethoxycarbonyl group is removed with diethylamine, and then purified by preparative thin-layer chromatography to obtain hydroxyarsin.

[0093] In practical implementation, the preparation method provided by this invention can also be used to synthesize AST-OH analogs.

[0094] An embodiment of the fifth aspect of the present invention provides a method for preparing arsenic trichomycin, including step S501.

[0095] In step S501, hydroxyarsenic trioxide is methylated to obtain arsenic trioxide, which is prepared by the above method.

[0096] The present invention does not particularly limit the method for methylating hydroxyarsin, as long as it achieves the purpose of the present invention. Exemplarily, hydroxyarsin can be methylated using an arsine methyltransferase to generate arsin.

[0097] The present application will now be described in detail with reference to embodiments to facilitate understanding by those skilled in the art. It is important to note that the embodiments are merely illustrative and should not be construed as limiting the scope of protection of the present application. Non-essential improvements and adjustments made to the present application by those skilled in the art based on the above description should still fall within the scope of protection of the present application. Furthermore, any raw materials not described in detail below are commercially available products; any process steps or preparation methods not mentioned in detail are process steps or preparation methods known to those skilled in the art. All raw materials described in this application are obtained commercially, and all materials used in this application are commonly used in the art.

[0098] Example 1

[0099] This embodiment provides a method for preparing hydroxyarsin, as shown in synthetic route formula 2.

[0100] Synthetic route formula 2.

[0101] Specifically, it includes the following steps.

[0102] (1) Synthesis of the compound shown in formula (A)

[0103] L-homoserine (Homo-Ser, 2.0 g, 16.8 mmol) and fluorenemethoxycarbonylsuccinimide (Fmoc-Osu, 6.8 g, 20.2 mmol) were dissolved in 200 mL of a first mixed solvent of water and dioxane (1:1 v / v). The system was cooled to 0 °C, and sodium carbonate (2.1 g, 20.2 mmol) was added. The reaction was carried out at 0 °C for 30 min, then transferred to room temperature. After reacting at room temperature for 16 h, the dioxane solvent was removed by vacuum concentration. The aqueous phase was washed with petroleum ether, and the pH of the solution was adjusted to pH=2 with 12 mol / L hydrochloric acid, during which a large amount of white solid precipitated. The resulting suspension was extracted with ethyl acetate, and the organic phase was treated with anhydrous sodium sulfate and then concentrated under vacuum to obtain the compound shown in formula (A).

[0104] (2) Synthesis of the compound shown in formula (B)

[0105] The compound shown in formula (A) was dissolved in 200 mL of a second mixed solvent of dichloromethane / tetrahydrofuran (v / v 4:1), and tert-butyl-2,2,2-trichloroacetimidate (6.1 mL, 33.6 mmol) was added dropwise. After reacting at room temperature for 16 h, the solvent was removed by vacuum concentration. The resulting solid was dissolved in 100 mL of ethyl acetate, washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, and then concentrated under vacuum. The crude product was purified by column chromatography using petroleum ether / ethyl acetate (v / v 4:1) as the developing solvent. The product was monitored by thin-layer chromatography (TLC) using petroleum ether / ethyl acetate (v / v 1:1) as the developing solvent. f =0.5. The final product was a pale yellow oil, tert-butyl 2-(Fmoc-amino)-4-hydroxybutyrate, the compound shown in formula (B) (2.6 g, 6.5 mmol, yield 38.9%).

[0106] The compound represented by formula (B) obtained above was subjected to liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) analysis, and the results are as follows. Analysis showed that the compound's structure was consistent with that of the compound represented by formula (B).

[0107] LCMS (ESI) C 23 H 28 NO5 + Calculated value [M+H] + 398.1962, actual measurement 398.1911.

[0108] 1H NMR (500 MHz, CDCl3) 7.80 (d, J = 7.5 Hz, 2H), 7.65 (d, J = 7.5 Hz, 2H), 7.44 (t, J = 7.5 Hz, 2H), 7.40 -7.32 (m, 2H), 5.83 (d, J = 7.9 Hz, 1H), 4.61- 4.37 (m, 3H), 4.26 (t, J = 6.9 Hz, 1H), 3.78 - 3.61 (m, 2H), 2.21 (ddt, J =14.4, 9.7, 4.6 Hz, 1H), 1.74 - 1.63 (m, 1H), 1.52 (s, 9H).

[0109] 13 C NMR (126 MHz, chloroform-d) δ 171.76, 156.94, 143.83, 141.30, 127.75, 127.10, 125.11, 120.00, 82.50, 67.15, 58.32, 51.62, 47.16, 35.88, 27.98.

[0110] (3) Synthesis of tert-butyl 2-(Fmoc-amino)-4-iodobutyrate

[0111] Under light-protected and oxygen-free conditions, the compound shown in formula (B) (2.6 g, 6.5 mmol) and triphenylphosphine (2.0 g, 7.8 mmol) were dissolved in 70 mL of tetrahydrofuran. After cooling the solution to 0 °C, imidazole (0.5 g, 7.8 mmol) was added, and the reaction was carried out at 0 °C for 30 min. Then, I2 (1.9 g, 7.8 mmol) was added to the system, and the reaction was carried out at 0 °C for 30 min, followed by a reaction at room temperature for 6 h. After the reaction was completed, the solvent was removed by concentration, and 100 mL of 10% (w / v) sodium thiosulfate solution was added to the resulting solid. After stirring for 30 min, the solid was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The resulting solution was concentrated under vacuum and purified by column chromatography using petroleum ether / ethyl acetate (v / v 10:1). The product was monitored by TLC using petroleum ether / ethyl acetate (v / v 4:1). f =0.5. The final product was a pale yellow solid, 2-(Fmoc-amino)-4-iodobutyrate tert-butyl ester (having the structure shown in formula (C)) (2.7 g, 5.3 mmol, yield 81.5%).

[0112] The tert-butyl 2-(Fmoc-amino)-4-iodobutyrate obtained above was analyzed by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR), and the results are as follows. Analysis showed that the structure was consistent with that of tert-butyl 2-(Fmoc-amino)-4-iodobutyrate.

[0113] LCMS (ESI) C 23 H 26 INNaO4 + Calculated value [M+Na] + 530.0799, actual measurement 530.0789.

[0114] 1 H NMR (500 MHz, chloroform-d) δ 7.77 (d, J = 7.5 Hz, 2H), 7.61 (d, J = 7.4Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.33 (t, J = 7.5 Hz, 1H), 5.58 (d, J = 8.3Hz, 1H), 4.43 (qd, J = 10.6, 7.0 Hz, 2H), 4.32 (td, J = 8.2, 4.8 Hz, 1H), 4.22 (t, J = 7.0 Hz, 1H), 3.15 (tq, J = 9.8, 8.3, 3.9, 3.4 Hz, 2H), 2.50 -2.37 (m, 1H), 2.20 (dtd, J = 14.5, 8.6, 5.9 Hz, 1H), 1.49 (s, 9H).

[0115] 13 C NMR (126 MHz, chloroform-d) δ 170.39, 155.94, 143.62, 141.26, 127.72, 127.05, 125.08, 120.00, 82.73, 66.90, 55.29, 47.15, 37.28, 27.97.

[0116] (4) Synthesis of cobalamin derivatives

[0117] Under anaerobic conditions, hydroxycobalamin (OH-CbI, 190 mg, 0.14 mmol) and sodium borohydride (53 mg, 1.4 mmol) were added to 20 mL of methanol and reacted in the dark for 15 min to obtain a cobalamin derivative. Then, tert-butyl 2-(Fmoc-amino)-4-iodobutyrate (150 mg, 0.28 mmol) was added, and the reaction was carried out in the dark for 1 h. The pH of the reaction solution was adjusted to pH=5 with acetic acid, and the solvent was removed by vacuum concentration to obtain a red solid, i.e., the cobalamin derivative, with a yield of 80%.

[0118] The cobaltamine derivatives prepared above were subjected to liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) detection, and the results are as follows.

[0119] LCMS (ESI) C 85 H 116 CoN 14 O 18 P + Calculated value [M+H] + 1710.7656, actual measurement 1710.7531.

[0120] 1H NMR (800 MHz, Methanol-d4) δ 7.78 (dd, J = 7.7, 3.5 Hz, 2H), 7.70(s, 1H), 7.60 – 7.51 (m, 3H), 7.42 – 7.35 (m, 2H), 7.29 (dt, J = 17.0, 7.4Hz, 2H), 6.86 (s, 1H), 6.51 (d, J = 5.4 Hz, 1H), 4.81 (ddd, J = 8.0, 5.1, 2.9Hz, 1H), 4.66 (q, J = 3.1 Hz, 1H), 4.56 (d, J = 10.7 Hz, 1H), 4.41 (dd, J =9.2, 1.8 Hz, 1H), 4.38 – 4.34 (m, 1H), 4.30 (dd, J = 10.5, 7.0 Hz, 1H), 4.18– 4.07 (m, 2H), 3.95 (dd, J = 8.6, 5.5 Hz, 1H), 3.82 – 3.71 (m, 2H), 3.61 –3.41 (m, 3H), 3.05 (dt, J = 11.7, 6.2 Hz, 1H), 2.78 (d, J = 5.9 Hz, 2H), 2.65– 2.30 (m, 25H), 2.26 (dq, J = 13.9, 7.6, 7.2 Hz, 1 H), 2.20 – 2.07 (m, 3H),2.06 – 1.86 (m, 6H), 1.82 (s, 4H), 1.65 (s, 3H), 1.51 (s, 3H), 1.44 (s, 3H),1.28 (s, 9H), 1.25 – 1.12 (m, 6H), 1.04 (s, 3H), 0.98 (s, 3H).

[0121] Analysis showed that it was consistent with the structural information of cobalamin derivatives.

[0122] (5) Synthesis of the compound shown in formula (II)

[0123] Reduced glutathione (615 mg, 2.0 mmol) was added to an aqueous solution of arsenic trioxide (20 mL, 50 mmol / L), and the pH was adjusted to pH=7 with 5 mol / L sodium hydroxide solution. The reaction was carried out under anaerobic conditions for 1 h to obtain the arsenic trioxide-glutathione complex. A cobalamin derivative was dissolved in 10 mL of methanol and added to the reaction solution of the arsenic trioxide-glutathione complex. The reaction was carried out under anaerobic conditions and irradiated with 300 W white light for 6 h. After removing the precipitate from the reaction solution, the supernatant was purified by preparative HPLC using a reversed-phase C18 column (SHIMADZU C18, 250 mm × 4.6 mm, 5 μm). Mobile phase A was H2O + 1‰ trifluoroacetic acid (TFA), and mobile phase B was CH3CN + 1‰ TFA. Gradient elution was used at a flow rate of 3 mL / min and an injection volume of 5 mL. Detection was performed at dual wavelengths of 254 nm and 361 nm. The elution program was as follows: 0–5 min 20% B, 5–30 min 20–65% B, 30–30.01 min 65–100% B, 30.1–35 min 100% B, 35–35.01 min 100–20% B, and 35.01–46 min 20% B. The collected product was freeze-dried under vacuum to obtain a white solid powder, namely the compound shown in formula (II) (40.2 mg, yield 57%).

[0124] (6) Synthesis of the compound shown in formula (III)

[0125] The compound shown in formula (II) was dissolved in 5 mL of methanol, and then 170 μL of hydrogen peroxide (30% by mass) was added. The reaction was carried out for 2 h. After removing excess hydrogen peroxide by adding 500 μL of toluene, the solvent was removed by vacuum concentration to obtain the compound shown in formula (III).

[0126] (7) Synthesis of the compound shown in formula (Ⅳ)

[0127] Add 5 mL of HCOOH to the compound shown in formula (III), react at 0 °C for 6 h to remove the tert-butyl group on the hydroxyl group, concentrate under vacuum to remove formic acid, and dissolve the residual solid in 5 mL of methanol. Purify by preparative HPLC using reversed-phase C18 column chromatography (SHIMADZ UC18, 250 mm × 4.6 mm, 5 μm). Mobile phase A is H2O + 1‰ TFA, mobile phase B is CH3CN + 1‰ TFA, gradient elution is used, flow rate is 3 mL / min, and elution program is: 0-5 min 15-20% B, 5-30 min 20-50% B, 30-30.01 min 50-100% B, 30.01-35 min 100% B, 35-35.01 min 100-15% B, 35.01-45 min 15% B. The collected product was freeze-dried under vacuum to obtain a white solid powder, namely the compound shown in formula (Ⅳ) (9.0 mg, yield 34%).

[0128] The compound represented by formula (Ⅳ) obtained above was subjected to liquid chromatography-mass spectrometry and nuclear magnetic resonance detection, and the detection results are as follows.

[0129] LCMS (ESI) C 19 H 17 AsNO6 - Calculated value [M+Na] - 430.0277, actual measurement 430.0227.

[0130] 1H NMR (800 MHz, methanol-d4) δ 7.80 (d, J = 7.5 Hz, 2H), 7.68 (dd, J =10.5, 7.8 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H), 7.34 - 7.29 (m, 2H), 4.40 (t, J= 7.4 Hz, 2H), 4.28 (dt, J = 9.4, 5.3 Hz, 1H), 4.23 (t, J = 6.8 Hz, 1H), 2.64- 2.41 (m, 2H), 2.34 (ddt, J = 15.8, 10.7, 5.6 Hz, 1H), 2.11 (dqt, J = 14.4, 10.4, 6.2 Hz, 1H). 13C NMR (800 MHz, CD3OD) δ 172.64, 157.27, 143.93, 141.22, 127.43, 126.77, 124.84, 119.53, 66.53, 53.56, 27.85, 27.33, 23.85.

[0131] Analysis showed that the structure information was consistent with that of the compound shown in formula (Ⅳ).

[0132] (8) Synthesis of hydroxyarsin

[0133] The compound shown in formula (Ⅳ) was dissolved in 3 mL of methanol, and 1.5 mL of diethylamine was added. After reacting for 4 h, the solvent was removed by concentration. The residual solid was dissolved in 5 mL of water, and the pH of the solution was adjusted to pH=7 with hydrochloric acid. The solution was washed with diethyl ether, and the aqueous phase was purified by TLC using i-PrOH / H2O / NH4OH = 5:2:3 as the developing solvent. f =0.4, yielding a white solid product, hydroxyarsin (AST-OH, 3.2 mg, yield 69%).

[0134] The prepared hydroxyarsin was subjected to liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) analysis, and the results are as follows.

[0135] LCMS (ESI) C4H9AsNO4 + Calculated value [M+H] + 209.9057, actual measurement 209.9742.

[0136] 1 H NMR (800 MHz, DO) δ 3.85 (t, J = 5.6 Hz, 1H), 2.36-2.19 (m, 4H).

[0137] Analysis showed that it was consistent with the structural information of hydroxyarsin.

[0138] The embodiments of the present invention have been described above. However, these embodiments are merely illustrative and not intended to limit the scope of the invention. Although various embodiments have been described above, this does not mean that the measures in the various embodiments cannot be used advantageously in combination. Various substitutions and modifications can be made by those skilled in the art without departing from the scope of the invention, and all such substitutions and modifications should fall within the scope of the invention.

Claims

1. A cobalamin derivative, characterized in that, The cobalamin derivative has the structure shown in formula (Ⅰ): ； In this context, Fmoc represents 9-fluorenemethyloxycarbonyl, and t-Bu represents tert-butyl.

2. A method for preparing the cobaltamine derivative according to claim 1, characterized in that, Includes the following steps: Hydroxycobalamin is reduced to reduce trivalent cobalt in hydroxycobalamin to monovalent cobalt, thus obtaining monovalent cobalamin. The monovalent cobalamin was subjected to a first nucleophilic substitution reaction with tert-butyl 2-(Fmoc-amino)-4-iodobutyrate to obtain a cobalamin derivative.

3. The preparation method according to claim 2, characterized in that, The reduction of hydroxycobalamin includes: reacting hydroxycobalamin with a reducing agent in a first organic solvent at 20°C to 30°C in the dark for 10 to 30 minutes; the reducing agent includes at least one of sodium borohydride and titanium citrate; the first organic solvent is methanol. The temperature of the first nucleophilic substitution reaction is 20℃~30℃, and the time is 1h~2h; the molar ratio of tert-butyl 2-(Fmoc-amino)-4-iodobutyrate to hydroxycobalamin is 2:(3~4).

4. The preparation method according to claim 2, characterized in that, The tert-butyl 2-(Fmoc-amino)-4-iodobutyrate was prepared by the following method: L-homoserine and fluorenemethoxycarbonylsuccinimide were acylated in a first mixed solvent to obtain the compound shown in formula (A); The compound shown in formula (A) was subjected to a second nucleophilic substitution reaction with tert-butyl-2,2,2-trichloroacetic acid imine ester to obtain the compound shown in formula (B); The compound shown in formula (B) was subjected to a third nucleophilic substitution reaction with triphenylphosphine, followed by halogenation with iodine to obtain tert-butyl 2-(Fmoc-amino)-4-iodobutyrate. ； 。 5. The preparation method according to claim 4, characterized in that, The acylation reaction of L-homoserine and fluorenemethyloxycarbonyl succinimide in a first mixed solvent includes: dissolving L-homoserine and fluorenemethyloxycarbonyl succinimide in a first mixed solvent, cooling to 0℃~10℃, adding a weak base and reacting for 30min~60min, and then reacting at 20℃~30℃ for 16h~24h; the molar ratio of L-homoserine, fluorenemethyloxycarbonyl succinimide and the weak base is 84:(101~168):(101~168); the first mixed solvent is a mixture of water and dioxane with a volume ratio of 1:(1~3); the weak base includes at least one of sodium carbonate and potassium carbonate; The second nucleophilic substitution reaction of the compound represented by formula (A) with tert-butyl-2,2,2-trichloroacetic acid imine ester comprises: dissolving the compound represented by formula (A) in a second mixed solvent, and adding tert-butyl-2,2,2-trichloroacetic acid imine ester dropwise to carry out the reaction; the temperature of the second nucleophilic substitution reaction is 20℃~30℃, and the time is 16h~24h; the second mixed solvent is a mixture of dichloromethane and tetrahydrofuran in a volume ratio of (1~3):

1. The step of reacting the compound of formula (B) with triphenylphosphine in a third nucleophilic substitution reaction, followed by the addition of iodine for halogenation, comprises: dissolving the compound of formula (B) and triphenylphosphine in a second organic solvent under light-protected and oxygen-free conditions, cooling to 0°C~10°C, adding an organic base and reacting for 30 min~60 min, then adding iodine and reacting sequentially at 0°C~10°C for 30 min~60 min, and at 20°C~30°C for 6 h~10 h; the molar ratio of the compound of formula (B), the triphenylphosphine, the organic base and the iodine is 85:(102~127):(102~127):(102~127); the second organic solvent includes at least one of tetrahydrofuran and dichloromethane; the organic base includes at least one of imidazole, diethylamine and triethylamine.

6. A method for preparing a hydroxyarsin intermediate, characterized in that, Includes the following steps: Reduced glutathione and arsenic acid were complexed to obtain an arsenic acid-glutathione complex. The cobalamin derivative as described in claim 1 is subjected to a free radical reaction with the arsenite-glutathione complex to obtain the compound shown in formula (II); The compound represented by formula (II) is subjected to an oxidation reaction to obtain the compound represented by formula (III); ； ； ； In this context, Fmoc represents 9-fluorenemethyloxycarbonyl, and t-Bu represents tert-butyl.

7. The method for preparing the hydroxyarsin intermediate according to claim 6, characterized in that, The complexation reaction of reduced glutathione and arsenic acid includes: adding reduced glutathione to an aqueous solution of arsenic acid, adjusting the pH to 6-7, and reacting at 20-30°C under anaerobic conditions for 1-2 hours; the concentration of the aqueous solution of arsenic acid is 40 mmol / L-60 mmol / L; the molar ratio of reduced glutathione to the volume of the aqueous solution of arsenic acid is (2-3) mmol: 20 mL. The free radical reaction is carried out under anaerobic conditions and irradiation with 250W~500W white light; the temperature of the free radical reaction is 20℃~30℃, and the time is 6h~10h. The oxidation reaction is carried out at a temperature of 20℃~30℃ for 2h~3h; the oxidant used in the oxidation reaction is hydrogen peroxide.

8. A method for preparing hydroxyarsin, characterized in that, include: The tert-butyl group on the hydroxyl group of the compound of formula (III) as described in claim 6 is removed by a first hydrolysis reaction to obtain the compound of formula (IV), wherein the compound of formula (III) is obtained by the preparation method described in claim 6 or 7; ， ； Hydroxyarsin is obtained by removing the 9-fluorenemethoxycarbonyl group from the amino group of the compound shown in formula (IV) through a second hydrolysis reaction.

9. The method for preparing hydroxyarsin according to claim 8, characterized in that, The removal of the tert-butyl group from the hydroxyl group of the compound represented by formula (III) by the first hydrolysis reaction includes: adding the compound represented by formula (III) to an acidic solution and reacting it at 0°C to 10°C for 6 to 10 hours; wherein the acidic solution includes at least one of formic acid, acetic acid, trifluoroacetic acid, and hydrochloric acid; The removal of the 9-fluorenemethyloxycarbonyl group from the amino group of the compound represented by formula (IV) by the second hydrolysis reaction includes: adding the compound represented by formula (IV) to an alkaline solution and reacting it at 20°C to 30°C for 4 to 6 hours; wherein the alkaline solution is a methanol solution of diethylamine and / or imidazole.

10. A method for preparing arsenic trichomycin, characterized in that, include: Hydroxyarsenic trioxide is methylated to obtain arsenic trioxide, which is obtained by the preparation method according to any one of claims 8 to 9.

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