Preparation method of N-nitroso-vancomycin

Abstract

The invention provides a preparation method of N-nitroso-vancomycin, which comprises the following steps: S1, regulating the pH value of a reaction system to 1.0-4.5 by using a first pH regulator, and adding vancomycin hydrochloride and nitrite into the reaction system according to a molar ratio of 1: (0.25-4); s2, stirring the reaction system at the temperature of 15-30 DEG C to react for 0.3-24 hours, so as to obtain feed liquid; s3, purifying the feed liquid through a high-pressure reversed-phase preparative chromatographic column to obtain a crude product; and step S4, carrying out freeze-drying treatment on the crude product to obtain a finished product of N-nitroso-vancomycin. The preparation method provided by the invention has the advantages that the raw materials are simple and easy to obtain, green and environment-friendly, the safety risk is basically avoided, the large-scale preparation of the N-nitroso-vancomycin can be quickly realized, the related reaction can be carried out at room temperature, the separation and purification method is simple and efficient, and the commercial production of the product is favorably promoted.

Description

Technical Field

[0001] The present invention relates to the field of medicine or medicine quality control, and more particularly, to a method for preparing N-nitroso-vancomycin. Background Art

[0002] Vancomycin Hydrochloride, the structural formula is shown in Formula I below, is a glycopeptide hydrochloride with antibacterial activity produced by Streptomyces orientalis, and is a narrow-spectrum antibiotic effective against Gram-positive cocci and some Gram-positive bacilli. It can effectively kill bacteria through the mechanism of "inhibiting the synthesis of bacterial cell walls (mainly), changing the permeability of bacterial cell membranes and affecting RNA synthesis". Clinically, it is mainly used for the treatment of severe infections caused by penicillin-resistant Staphylococcus aureus, such as pneumonia, endocarditis and sepsis, etc., and also has good curative effects on infections and sepsis caused by hemolytic streptococcus.

[0003]

[0004] In recent years, due to the special toxic effects of nitrosamine impurities, the attention of domestic and foreign to nitrosamine impurities in drugs has been increasing. Nitrosamine drug substance-related impurities (NDSRIs) are a class of specific impurities closely related to the drug active matrix in many drugs, and are usually formed by the nitrosation of APIs (or API fragments) containing secondary or tertiary amines when contacting nitrosating agents (such as nitrites remaining in the preparation of drug excipients). The chemical structure fragment of vancomycin hydrochloride also has reactive sites and has the risk of forming NDSRIs.

[0005] In addition, according to the relevant guiding principles of FDA and ICH M7, it is necessary to conduct a safety risk assessment on NDSRIs (N-nitroso-vancomycin, the structural formula is shown in Formula II) in vancomycin hydrochloride. The FDA official website has recommended its specific AI (Accepted Intake) limit value, and a standard product of N-nitroso-vancomycin is required for further analysis and detection to confirm its risk.

[0006]

[0007] Some preparation methods of nitrosamine substances have been disclosed in the existing literature. Hatt disclosed a method for preparing nitrosamine impurities. In this method, nitrite was added as a nitrosating agent in a dropwise manner (in an acidic environment), and the reaction was carried out in a 70-75 °C water bath for at least 2 h. The obtained target product was purified by rectification, with complex operations and long time consumption (see unsym.-DIMETHYLHYDRAZINE HYDROCHLORIDE[J].Organic Syntheses,1936,16:22); Patent US3975425 disclosed the preparation of nitrosamine impurities using ammonium nitrite as a nitrosating agent (in an acidic environment). This method was preferably carried out in a reaction environment of 0-15 °C, requiring an ice bath or controlling a lower ambient temperature; The preparation method disclosed by A.Y. PAVLOV et al. was relatively complex, and the required materials such as acetone and DMSO were all non-green reagents, with a relatively high safety risk during the preparation process (see SYNTHESIS AND BIOLOGICAL ACTIVITY OF DERIVATIVES OF GLYCOPEPTIDE ANTIBIOTICSEREMOMYCIN AND VANCOMYCIN NITROSATED,ACYLATED OR CARBAMOYLATED AT THE TV-TERMINAL[J].THE JOURNAL OF ANTIBIOTICS,1993,46(11):1731-1739).

[0008] Patent application CN201911086307.7 disclosed a method for preparing vancomycin hydrochloride impurity ImpC. In this method, vancomycin was mixed with a strong acid solution and dichloromethane in an acidic environment, and the reaction was carried out for 16-25 h. After concentration under reduced pressure, filtration, and drying, the crude product of impurity ImpC was obtained, and then the ImpC finished product was obtained by separation using a high-pressure liquid chromatography preparation column.

[0009] In summary, the existing preparation methods of impurity N-nitroso-vancomycin in the prior art have complex operations and high safety risks. Based on this, how to provide a preparation method of matrix nitrosamine impurity (N-nitroso-vancomycin) of vancomycin hydrochloride drug that is simple, easily available, environmentally friendly, and has low safety risks to quickly achieve the large-scale preparation of N-nitroso-vancomycin is an important problem to be solved in this field. Summary of the Invention

[0010] The main purpose of the present invention is to provide a preparation method of N-nitroso-vancomycin to solve the problems of complex operations and low yield in the existing preparation method of N-nitroso-vancomycin.

[0011] To achieve the above object, the present invention provides a method for preparing N-nitroso-vancomycin, comprising:

[0012] Step S1, adjusting the pH of the reaction system to 1.0 - 4.5 with a first pH regulator, and adding vancomycin hydrochloride and nitrite to the reaction system, wherein the molar ratio of vancomycin hydrochloride to sodium nitrite is 1:(0.25 - 4);

[0013] Step S2, stirring and reacting the reaction system at 15 - 30 °C for 0.3 - 24 hours to obtain a feed liquid;

[0014] Step S3, purifying the feed liquid through a high-pressure reverse-phase preparative chromatography column to obtain a crude product;

[0015] Step S4, freeze-drying the crude product to obtain the finished product of N-nitroso-vancomycin.

[0016] Further, in step S1, the reaction system is an aqueous phase system.

[0017] Further, the first pH regulator is selected from one or more of formic acid, trifluoroacetic acid, hydrochloric acid, nitric acid, phosphoric acid, acetic acid, sulfuric acid, and oxalic acid; preferably, the first pH regulator is hydrochloric acid, trifluoroacetic acid, or formic acid.

[0018] Further, in step S1, the nitrite is selected from sodium nitrite and / or potassium nitrite.

[0019] Further, the elution system used in the purification step in step S3 is an acetonitrile-triethylamine elution system.

[0020] Further, the elution system in step S3 includes:

[0021] Mobile phase A: is a 50 - 100% acetonitrile solution;

[0022] Mobile phase B: is a 0.05% - 2% triethylamine aqueous solution;

[0023] And the volume ratio of mobile phase A to mobile phase B is 8:92 - 37:63.

[0024] Further, the elution separation method used in the purification step in step S3 is gradient elution, and the gradient elution is set as:

[0025] Within 0 - 10 min, mobile phase A is 8 - 22%, and mobile phase B is 78 - 92%;

[0026] Within 10 - 30 min, mobile phase A is 13 - 37%, and mobile phase B is 63 - 87%;

[0027] When greater than 30 min, mobile phase A is 23 - 37%, and mobile phase B is 63 - 77%.

[0028] Furthermore, mobile phase B further includes one or more of formic acid, acetic acid, trifluoroacetic acid, hydrochloric acid, phosphoric acid, and nitric acid. Preferably, the pH value of mobile phase B is 2.5 - 4.0.

[0029] Further, the chromatographic column packing used in the purification step in step S3 is C18 or C8; the elution flow rate is 2 - 10 times the column volume / h, and the detection wavelength is 230 - 300 nm.

[0030] Further, the crude product in step S3 is a chromatographic solution with a chromatographic purity > 80%.

[0031] Further, the yield of N-nitroso-vancomycin in step S4 > 50%.

[0032] The preparation method provided by the present invention has simple and easily available raw materials, is green and environmentally friendly, has basically no safety risks, can quickly realize the large-scale preparation of N-nitroso-vancomycin, and the involved reactions can be carried out at room temperature. The separation and purification method is simple and efficient, which is conducive to promoting the commercial production of this impurity. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The specification drawings forming a part of this application are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0034] Figure 1 is the liquid chromatogram of the feed solution obtained in Example 1 of the present invention;

[0035] Figure 2 is the liquid chromatogram of the feed solution obtained in Example 2 of the present invention;

[0036] Figure 3 is the liquid chromatogram of the feed solution obtained in Example 3 of the present invention;

[0037] Figure 4 is the 1 1H-NMR spectrum of the N-nitroso-vancomycin finished product obtained by the present invention;

[0038] Figure 5 is the 13 13C-NMR spectrum of the N-nitroso-vancomycin finished product obtained by the present invention;

[0039] Figure 6 is the HRMS spectrum of the N-nitroso-vancomycin finished product obtained by the present invention;

[0040] Figure 7 is the M + 2H MS simulated comparison spectrum of the N-nitroso-vancomycin finished product obtained by the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0041] It should be noted that, without conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0042] As described in the background art, the existing preparation method of N-nitroso-vancomycin has the problems of complex operation and low yield. To solve the above technical problems, the present invention provides a preparation method of N-nitroso-vancomycin, including:

[0043] Step S1, adjusting the pH of the reaction system to 1.0 - 4.5 with a first pH regulator, and adding vancomycin hydrochloride and nitrite into the reaction system, wherein the molar ratio of vancomycin hydrochloride to sodium nitrite is 1:(0.25 - 4);

[0044] Step S2, stirring and reacting the reaction system at 15 - 30 °C for 0.3 - 24 hours to obtain a liquid material;

[0045] Step S3, purifying the liquid material through a high-pressure reversed-phase preparative chromatography column to obtain a crude product;

[0046] Step S4, performing freeze-drying treatment on the crude product to obtain the finished product of N-nitroso-vancomycin.

[0047] The reaction raw materials used in the preparation method provided by the present invention are economical, safe and easily available. At the same time, the reaction can be carried out at room temperature, and the separation and purification method is simple and efficient, and a large amount of N-nitroso-vancomycin can be prepared within a short experimental period. On the one hand, the obtained N-nitroso-vancomycin can be used as a material for studying the influencing factors of related nitrosamine impurities of vancomycin hydrochloride (such as light, acid, alkali, oxidation, etc.), providing research ideas for improving the product quality of vancomycin hydrochloride API or preparation, and then optimizing its process production parameters; on the other hand, it can be used as a material for animal research on nitrosamine impurities in the matrix of vancomycin hydrochloride (carcinogenicity and mutagenicity research) to further confirm the more accurate intake (AI) limit value for verifying or replacing the current predicted AI value based on carcinogenic potency classification by the FDA; on the third hand, it can be used as a reference standard or control for the analysis and detection of drug matrix nitrosamine impurities in vancomycin hydrochloride raw materials and preparations.

[0048] In the preparation method provided by the present invention, two reaction raw materials, vancomycin hydrochloride and nitrite, are formulated into a mixed solution and stirred and reacted at 15 - 30 °C for 0.3 - 24 hours. The principle of the synthesis reaction is as follows: under acidic conditions, the secondary amine structural fragment in vancomycin hydrochloride undergoes a specific nitrosation reaction with a nitrosating agent (nitrite) to form, and the reaction formula is shown as follows:

[0049]

[0050] Among them, the molar ratio of vancomycin hydrochloride to sodium nitrite is 1:(0.25 - 4). When the above two raw materials participate in the synthesis reaction in this molar ratio, a relatively high yield can be obtained, and at the same time, waste of raw materials is avoided. The reaction temperature is selected to be 15 - 30 °C. Under the condition that the reaction can proceed, there is no safety hazard due to too high temperature, nor will the preparation cost increase due to too low temperature requiring ice bath or additional control of environmental conditions.

[0051] In summary, the preparation method of N-nitroso-vancomycin provided by the present invention has easily available reaction raw materials, does not involve toxic substances, the reaction can be carried out at room temperature, and the separation and purification method is simple and efficient. It can achieve the large-scale preparation of N-nitroso-vancomycin within a relatively short experimental period, and the prepared N-nitroso-vancomycin has a high yield and high purity (chromatographic purity > 80%).

[0052] In many nitrosation reactions, the [NO + donors are generally as follows: nitrite ions, nitrosyl halides, nitrites, dinitrogen trioxide, and dinitrogen tetroxide. Considering that the [NO + donor itself is a highly toxic gas, or corresponding toxic gases are generated during the reaction. If leakage occurs, it is likely to cause poisoning, environmental damage, or affect the smooth progress of the reaction. Based on this, the present invention uses nitrites. Specifically, the nitrite is selected from sodium nitrite and / or potassium nitrite. Compared with other [NO + donors, sodium nitrite and / or potassium nitrite can well avoid the above problems, improve the safety and environmental friendliness of the reaction to a greater extent, and are more suitable for the aqueous reaction system, thereby improving the yield and purity of the obtained product. Since the active reagent of the nitrosation reaction is [NO + , the nitrite ion provided by the nitrite cannot directly carry out an effective reaction and requires other nucleophilic reagents to be activated; while at a lower pH, the nitrite ion can be used as a nucleophilic reagent to carry out the nitrosation reaction. Therefore, it is necessary to adjust the pH value of the reaction system. At the same time, in order to better match the pKa value of the secondary amine fragment in vancomycin, in step S1, a first pH regulator is used to adjust the pH of the reaction system to 1.0 - 4.5 to improve the reaction rate and the yield of the obtained N-nitroso-vancomycin.

[0053] On the basis of the above content, further, the reaction system in step S1 is an aqueous phase system to improve the purity of the obtained product.

[0054] In several typical embodiments, the first pH regulator is selected from one or more of formic acid, trifluoroacetic acid, hydrochloric acid, nitric acid, phosphoric acid, acetic acid, sulfuric acid, and oxalic acid; in order to achieve a more efficient pH adjustment effect and, at the same time, reduce the introduction of impurities to a greater extent, it is preferred that the first pH regulator is hydrochloric acid, trifluoroacetic acid, or formic acid. Compared with other acid-based pH regulators commonly used in the art, in the reaction system involved in the present invention, these three types of acids can reduce the generation of impurities and by-products while adjusting the pH, thereby improving the purity and yield of the obtained N-nitroso-vancomycin.

[0055] In order to better adapt to the polarity and molecular fragments of the obtained N-nitroso-vancomycin, the elution system used in the purification step in step S3 is an acetonitrile-triethylamine elution system, so as to achieve more efficient separation and purification and improve the yield.

[0056] In a preferred embodiment, the elution system in step S3 includes: mobile phase A: a 50-100% acetonitrile solution; mobile phase B: a 0.05%-2% triethylamine aqueous solution. Both the used mobile phase A and mobile phase B are single solvents, rather than mixed solutions prepared from multiple solvents, thereby improving the purity of the obtained product to a greater extent; the volume ratio of the used mobile phase A to mobile phase B is 8:92 to 37:63, so as to better match the polarity of the product N-nitroso-vancomycin, thereby improving the efficiency and yield of separation and purification.

[0057] In order to obtain clearer bands during the purification process, that is, to better separate the product N-nitroso-vancomycin from the unreacted raw materials, thereby improving the purity of the product, while improving the chromatography efficiency and shortening the experimental period, the elution separation method used in the purification step in the present invention is gradient elution. The inventor found through a large number of experiments that when the gradient elution is set as follows: within 0-10 minutes, mobile phase A is 8-22% and mobile phase B is 78-92%; within 10-30 minutes, mobile phase A is 13-37% and mobile phase B is 63-87%; when it is more than 30 minutes, mobile phase A is 23-37% and mobile phase B is 63-77%, the product can be better separated and its loss rate can be reduced, thereby obtaining N-nitroso-vancomycin with higher yield and purity.

[0058] In several typical embodiments, mobile phase B also includes one or more of formic acid, acetic acid, trifluoroacetic acid, hydrochloric acid, phosphoric acid, and nitric acid, so as to adjust the pH value of the system in the purification step, reduce the impurity content to improve the purity; preferably, the pH value of mobile phase B is 2.5-4.0. Under this condition, it is beneficial to protect the nitroso fragment in the obtained product from hydrolysis and removal in a neutral or alkaline environment to a greater extent, and improve the purity and yield of the separated product.

[0059] Further, the chromatographic column packing used in the purification step in Step S3 is C18 or C8. These two packings have high stability and retention performance, can better adapt to the polarity of N-nitroso-vancomycin in the present invention, and can better combine with it, thus facilitating the elution of impurities with weaker binding force and improving the purity of the product; the elution flow rate is 2 - 10 column volumes / h, and the detection wavelength is 230 - 300 nm. Compared with other elution flow rates, a flow rate of 2 - 10 column volumes / h can effectively shorten the experimental period while efficiently separating and purifying the product, thereby increasing the possibility of large-scale commercial application of this preparation method.

[0060] To further improve the purity of the finally obtained N-nitroso-vancomycin finished product, the crude product selected for separation in Step S3 is the chromatography solution with a chromatography purity > 80%. Further, the yield of N-nitroso-vancomycin in Step S4 > 50%.

[0061] Yield calculation formula for nitroso-vancomycin:

[0062]

[0063] Where, mN-nitroso-vancomycin: the mass of the final product N-nitroso-vancomycin;

[0064] mvancomycin: the mass of vancomycin hydrochloride;

[0065] Mvancomycin: the molecular weight of vancomycin hydrochloride 1485.7;

[0066] MN-nitroso-vancomycin: the molecular weight of N-nitroso-vancomycin 1478.3;

[0067] Regarding the calibration of the obtained N-nitroso-vancomycin finished product, the mass balance method is adopted, and the specific chromatographic conditions are as follows:

[0068] Chromatographic column: Waters ACQUITY CSHTM C18 150mm × 2.1mm, 1.7μm;

[0069] Buffer solution: Weigh about 7.0 g of tris(hydroxymethyl)aminomethane, dissolve it in about 950 mL of water, adjust the pH value to 8.0 - 8.3 with 20% glacial acetic acid solution, and dilute it to 1000 mL with water;

[0070] Mobile phase A: Buffer solution: Acetonitrile: Methanol = 93:3:4;

[0071] Mobile phase B: Buffer solution: Acetonitrile: Methanol = 50:10:40;

[0072] Flow rate: 0.30 mL / min;

[0073] Detection wavelength: 280 nm;

[0074] Injection volume: 2 μl;

[0075] Column temperature: 40 °C;

[0076] Autosampler: 5 °C.

[0077] The gradient elution program is shown in the following table:

[0078] Time / min Mobile phase A / % Mobile phase B / % 0 88 12 7 88 12 21 75 25 35 25 75 37 25 75 38 88 12 45 88 12

[0079] The following further describes the present application in detail with specific embodiments, and these embodiments should not be construed as limiting the scope claimed by the present application.

[0080] Unless otherwise defined, all professional terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention.

[0081] Example 1

[0082] A preparation method of N-nitroso-vancomycin:

[0083] Adjust the pH value of the aqueous reaction system to 1.0 with 1 mol / L hydrochloric acid. Under this reaction system, vancomycin hydrochloride and sodium nitrite are prepared at a ratio of 1:4 (molar concentration), where vancomycin hydrochloride is 7.5 g and sodium nitrite is 1.4 g. Stir and react at 25 °C for 1 h to obtain a feed liquid.

[0084] The liquid chromatogram of the feed liquid obtained in Example 1 is shown in Figure 1 .

[0085] Take 40 mL of the feed liquid and separate it on a high-pressure reverse-phase preparative column. The specific steps are as follows:

[0086] Instrument: Chromatographic column: UniSil, 10 - 100, C18, 30 * 250 mm;

[0087] Elution system: Mobile phase A: 80% acetonitrile; Mobile phase B: 0.2% triethylamine aqueous solution, and adjust the pH to 3.2 with phosphoric acid; The gradient elution parameters are shown in the following table:

[0088] Table 1 Gradient elution table

[0089] Time / min Mobile phase A / % Mobile phase B / % 0 10 90 10 15 85 30 25 75

[0090] Column temperature: 25 °C;

[0091] Detection wavelength: 280 nm;

[0092] Flow rate: 17 mL / min;

[0093] Sample loading volume: 40 mL;

[0094] Collect the eluate and lyophilize the obtained white solid to obtain the finished product of N-nitroso-vancomycin. Its 1 1H-NMR, 13 13C-NMR, HRMS and the simulated comparison spectra of M+2H MS are shown in Figure 4 , 5 , 6, and 7 respectively.

[0095] Example 2

[0096] A preparation method of N-nitroso-vancomycin:

[0097] Adjust the pH value of the aqueous reaction system to 2.5 with 1 mol / L hydrochloric acid. Under this reaction system, prepare vancomycin hydrochloride and sodium nitrite at a ratio of 4:1 (molar concentration), where vancomycin hydrochloride is 30.0 g and sodium nitrite is 0.35 g. Stir and react at 25 °C for 24 h to obtain the feed liquid.

[0098] The liquid chromatogram of the feed liquid obtained in Example 2 is shown in Figure 2 .

[0099] Take 50 mL of the feed liquid and separate it on a high-pressure reverse-phase preparative column. The specific steps are as follows:

[0100] Instrument: Chromatographic column: UniSil, 10 - 100, C8, 30 * 250 mm;

[0101] Elution system: Mobile phase A: 80% acetonitrile; Mobile phase B: 0.5% triethylamine aqueous solution, and adjust the pH to 3.5 with phosphoric acid; The gradient elution parameters are shown in Table 1

[0102] Column temperature: 25 °C;

[0103] Detection wavelength: 300 nm;

[0104] Flow rate: 15 mL / min;

[0105] Sample loading volume: 50 mL;

[0106] Collect the eluate and lyophilize the obtained white solid to obtain the finished product of N-nitroso-vancomycin.

[0107] Example 3

[0108] A preparation method of N-nitroso-vancomycin:

[0109] The difference from Example 1 is that the pH value of the aqueous reaction system is adjusted to 4.5, and it is stirred and reacted at 25 °C for 24 h.

[0110] The liquid chromatogram of the feed solution obtained in Example 3 is shown in Figure 3 .

[0111] Example 4

[0112] A preparation method of N-nitroso-vancomycin:

[0113] The difference from Example 1 is that the reaction temperature is 15 °C.

[0114] Example 5

[0115] A preparation method of N-nitroso-vancomycin:

[0116] The difference from Example 1 is that vancomycin hydrochloride and sodium nitrite are prepared at a ratio of 1:2 (molar concentration), where vancomycin hydrochloride is 7.5 g and sodium nitrite is 0.7 g, and the reaction temperature is 30 °C.

[0117] Example 6

[0118] A preparation method of N-nitroso-vancomycin:

[0119] The difference from Example 1 is that the pH regulator of the aqueous reaction system is 2 mol / L hydrochloric acid.

[0120] Example 7

[0121] A preparation method of N-nitroso-vancomycin:

[0122] The difference from Example 1 is that the pH regulator of the aqueous reaction system is acetic acid.

[0123] Example 8

[0124] A preparation method of N-nitroso-vancomycin:

[0125] The difference from Example 1 is as follows.

[0126] Elution system: Mobile phase A: 80% acetonitrile; Mobile phase B: 0.2% aqueous triethylamine solution, and the pH is adjusted to 3.2 with phosphoric acid; The gradient elution parameters are shown in the following table:

[0127] Table 2 Gradient elution table

[0128] Time / min Mobile phase A / % Mobile phase B / % 0 10 90 10 20 80 30 35 65

[0129] Column temperature: 25 °C;

[0130] Detection wavelength: 280 nm;

[0131] Flow rate: 17 mL / min;

[0132] Example 9

[0133] A preparation method of N-nitroso-vancomycin:

[0134] The difference from Example 1 is that in the elution system, mobile phase A is a 70% acetonitrile solution; mobile phase B is a 0.05% aqueous triethylamine solution.

[0135] Example 10

[0136] A preparation method of N-nitroso-vancomycin:

[0137] The difference from Example 1 is that in the elution system, mobile phase A is an 80% acetonitrile solution; mobile phase B is a 2% aqueous triethylamine solution.

[0138] Example 11

[0139] A preparation method of N-nitroso-vancomycin:

[0140] The difference from Example 1 is that in the elution system, the pH value of mobile phase B is 2.5.

[0141] Example 12

[0142] A preparation method of N-nitroso-vancomycin:

[0143] The difference from Example 1 is that in the elution system, the pH value of mobile phase B is 4.5.

[0144] Comparative Example 1

[0145] A preparation method of N-nitroso-vancomycin:

[0146] The difference from Example 1 is that the pH value of the aqueous reaction system is 2.0, vancomycin hydrochloride and sodium nitrite are prepared at a ratio of 5:1 (molar concentration), where vancomycin hydrochloride is 37.5 g and sodium nitrite is 0.35 g. Stir and react at 30 °C for 0.3 h to obtain a feed liquid. Mobile phase B: 1.0% aqueous triethylamine solution, and the pH is adjusted to 4.0 with phosphoric acid;

[0147] Comparative Example 2

[0148] A preparation method of N-nitroso-vancomycin:

[0149] The difference from Example 1 is that the pH value of the aqueous reaction system is 5.0.

[0150] Comparative Example 3

[0151] A preparation method of N-nitroso-vancomycin:

[0152] The difference from Example 1 is that the pH value of the aqueous reaction system is 7.0.

[0153] The weights, yields, and chromatographic purities of the N-nitroso-vancomycin finished products obtained in the above examples and comparative examples are shown in Table 2.

[0154] Table 2 Weights, yields, and chromatographic purities of N-nitroso-vancomycin finished products

[0155]

[0156]

[0157] From the above description, it can be seen that the above embodiments of the present invention achieve the preparation of N-nitroso-vancomycin with high yield and high purity.

[0158] It should be noted that the terms "first", "second", etc. in the specification and claims of this application are used to distinguish similar objects and do not necessarily have to be used to describe a specific order or sequence. It should be understood that such terms can be interchanged under appropriate circumstances so that the embodiments of the present application described herein can be implemented in an order other than those described herein.

[0159] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of N-nitroso-vancomycin, characterized in that, The preparation method includes the following steps: Step S1: Adjust the pH of the reaction system to 1.0 - 4.5 using a first pH regulator, and add vancomycin hydrochloride and nitrite to the reaction system. The molar ratio of vancomycin hydrochloride to sodium nitrite is 1: (0.25～4)； Step S2: Stir and react the reaction system at 15 - 30 °C for 0.3 - 24 hours to obtain a feed liquid; Step S3: Purify the feed liquid through a high-pressure reversed-phase preparative chromatographic column to obtain a crude product; Step S4: Perform freeze-drying on the crude product to obtain the finished product of N-nitroso-vancomycin.

2. The preparation method of N-nitroso-vancomycin according to claim 1, characterized in that, In step S1, the reaction system is an aqueous phase system.

3. The preparation method of N-nitroso-vancomycin according to claim 2, characterized in that, The first pH regulator is selected from one or more of formic acid, trifluoroacetic acid, hydrochloric acid, nitric acid, phosphoric acid, acetic acid, sulfuric acid, and oxalic acid; preferably, the first pH regulator is hydrochloric acid, trifluoroacetic acid, or formic acid.

4. The preparation method of N-nitroso-vancomycin according to any one of claims 1 to 3, characterized in that, The nitrite in step S1 is selected from sodium nitrite and / or potassium nitrite.

5. The preparation method of N-nitroso-vancomycin according to claim 1, characterized in that, The elution system used in the purification step in step S3 is an acetonitrile-triethylamine elution system.

6. The preparation method of N-nitroso-vancomycin according to claim 5, characterized in that, The elution system in step S3 includes: Mobile phase A: a 50 - 100% acetonitrile solution; Mobile phase B: a 0.05% - 2% triethylamine aqueous solution; And the volume ratio of mobile phase A to mobile phase B is 8:92 - 37:

63.

7. The preparation method of N-nitroso-vancomycin according to claim 5 or 6, characterized in that, The elution separation method used in the purification step in step S3 is gradient elution, and the gradient elution is set as follows: within 0 - 10 min, mobile phase A is 8 - 22%, and mobile phase B is 78 - 92%; within 10 - 30 min, mobile phase A is 13 - 37%, and mobile phase B is 63 - 87%; when it is more than 30 min, mobile phase A is 23 - 37%, and mobile phase B is 63 - 77%.

8. The preparation method of N-nitroso-vancomycin according to claim 6, characterized in that, Mobile phase B also includes one or more of formic acid, acetic acid, trifluoroacetic acid, hydrochloric acid, phosphoric acid, and nitric acid. Preferably, the pH value of mobile phase B is 2.5 - 4.

0.

9. The preparation method of N-nitroso-vancomycin according to any one of claims 5 to 8, characterized in that, The chromatographic column packing used in the purification step in step S3 is C18 or C8; the elution flow rate is 2 - 10 times the column volume / h, and the detection wavelength is 230 - 300 nm.

10. The preparation method of N-nitroso-vancomycin according to claim 1, characterized in that, The crude product in step S3 is a chromatographic solution with a chromatographic purity > 80%, and the yield of N-nitroso-vancomycin in step S4 is > 50%.

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