Method for preparing nitrosamine compound

Abstract

The invention relates to the technical field of medicine preparation, in particular to a method for preparing a nitrosamine compound. The method comprises the following steps: S1, carrying out esterification protection by taking a dalbavancin precursor A-40926-B0 as a raw material to obtain an intermediate I; s2, carrying out condensation reaction on the intermediate I and 3-methylaminopropylamine to obtain an intermediate II, and carrying out alkaline hydrolysis to obtain an intermediate III; s3, protecting secondary amine on a parent nucleus of the intermediate III through a protective reagent to obtain an intermediate IV; s4, carrying out nitrosation on the intermediate IV through a nitrosation reagent to obtain an intermediate V; s5, after the intermediate V is subjected to deprotection, dalbavancin demethylated B0 nitrosamine is obtained; the product prepared by the method is high in purity, a reference substance can be provided for quality control of dalbavancin, and important guiding significance is provided for safe medication.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical preparation technology, and specifically to a method for preparing nitrosamine compounds. Background Technology

[0002] Dapavancin is a semi-synthetic glycopeptide antibiotic that exerts its bactericidal effect by disrupting cell wall biosynthesis, making it one of the few antibacterial drugs used in clinical practice on a weekly basis. CN109467592B describes the preparation process of dapavancin: after esterification protection of the dapavancin precursor A-40926, 3-dimethylaminopropylamine and the parent nucleus undergo sequential amidation condensation and deprotection hydrolysis to obtain dapavancin.

[0003] Nitrosamine impurities are classified as substances of concern in the ICH M7(R2) guidance, "Assessing and controlling DNA-reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risks." They are genotoxic substances in some animals, and some have been listed as probable or potential human carcinogens by the International Agency for Research on Cancer (IARC). The National Medical Products Administration's Center for Drug Evaluation (NMPA) issued the "Technical Guidelines for Research on Nitrosamine Impurities in Chemical Drugs (Trial)," which stipulates that the control of nitrosamine impurities in drugs should be based on compliance with ICH M7(R1) requirements, aiming to ensure that the levels of these impurities in active pharmaceutical ingredients (APIs) and finished drug products are below acceptable limits. The FDA released the 18th revision of "Recommended Acceptable Intake Limits for Nitrosamine Drug Substance-Related Impurities (NDSRIs)," further strengthening the regulation of nitrosamine impurities in pharmaceuticals. The guidelines identify nine nitrosamine derivatives of dapavancin and specify that the acceptable intake limit for dapavancin demethyl BO nitrosamine is 1500 ng / day.

[0004] In view of this, the present invention is proposed to control the content of dapavancin demethyl BO nitrosamine in dapavancin raw materials or preparations to a low level that meets safety requirements. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing nitrosamine compounds, which is used to provide impurity reference standards for quality control.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing nitrosamine compounds, comprising the following steps:

[0007] S1: Using Dabavancin precursor A-40926-B0 as raw material, esterification protection was performed to obtain intermediate I; S2: The intermediate I is condensed with 3-methylaminopropylamine to obtain intermediate II, and then intermediate III is obtained by alkaline hydrolysis; S3: Intermediate III is protected by a protecting agent on the secondary amine of the parent nucleus to obtain intermediate IV; S4: Intermediate IV is nitrified by a nitrifying agent to obtain intermediate V; S5: The intermediate V is deprotected to obtain dabavancin demethyl BO nitrosamine; The structural formula of the dabavancin precursor A-40926-B0 is shown in Formula (1), the structural formula of intermediate I is shown in Formula (2), the structural formula of intermediate II is shown in Formula (3), the structural formula of intermediate III is shown in Formula (4), the structural formula of intermediate IV is shown in Formula (5), the structural formula of intermediate V is shown in Formula (6), and the structural formula of dabavancin demethylated B0 nitrosamine is shown in Formula (7). Equation (1); Equation (2); Equation (3); Equation (4); Equation (5); Equation (6); Equation (7).

[0008] The beneficial effects of this invention are as follows: the precursor compound for the synthesis of dabavancin demethyl BO nitrosamine has a complex structure and contains sensitive groups. The reaction process is subject to many impurities and difficult purification, among other technical challenges. Currently, there is no reported method for synthesizing dabavancin demethyl BO nitrosamine.

[0009] This invention provides a method for preparing dapavancin demethyl BO nitrosamine compound, with high product purity, which can provide a reference standard for the quality control of dapavancin and provide important guidance for safe drug use. Attached Figure Description

[0010] Figure 1 This is the hydrogen spectrum of dapavancin demethyl BO nitrosamine in an embodiment of the present invention; Figure 2 This is the carbon spectrum of dapavancin demethyl BO nitrosamine in an embodiment of the present invention; Figure 3 This is the mass spectrum of dapavancin demethyl BO nitrosamine in an embodiment of the present invention; Figure 4 The infrared spectrum of dapavancin demethyl BO nitrosamine in this embodiment of the invention is shown. Detailed Implementation

[0011] To explain in detail the technical content, objectives, and effects of the present invention, the following description is provided in conjunction with the embodiments and accompanying drawings.

[0012] A method for preparing nitrosamine compounds includes the following steps: S1: Using Dabavancin precursor A-40926-B0 as raw material, esterification protection was performed to obtain intermediate I; S2: Intermediate I is condensed with 3-methylaminopropylamine to obtain intermediate II, and then intermediate III is obtained by alkaline hydrolysis; S3: Intermediate III is protected by a protecting agent on the secondary amine on the parent nucleus to obtain intermediate IV; S4: Intermediate IV is nitrified with a nitrifying agent to obtain intermediate V; S5: After deprotection of intermediate V, dabavancin demethyl BO nitrosamine is obtained; The structural formula of Dabavancin precursor A-40926-B0 is shown in Formula (1), the structural formula of intermediate I is shown in Formula (2), the structural formula of intermediate II is shown in Formula (3), the structural formula of intermediate III is shown in Formula (4), the structural formula of intermediate IV is shown in Formula (5), the structural formula of intermediate V is shown in Formula (6), and the structural formula of Dabavancin demethylated B0 nitrosamine is shown in Formula (7). Equation (1); Equation (2); Equation (3); Equation (4); Equation (5); Equation (6); Equation (7).

[0013] As can be seen from the above description, the beneficial effects of the present invention are as follows: Under normal conditions, the formation pathway of dapavancin demethylated BO nitrosamine is as follows: dapavancin BO loses a methyl group from its side chain to form dedapavancin demethylated BO, which then undergoes nitrosation to generate dapavancin demethylated BO nitrosamine. However, the demethylation reaction is difficult to control, making it hard to accurately remove the target methyl group and resulting in numerous reaction impurities. Furthermore, the presence of two secondary amine reaction sites on the substrate during nitrosation makes it difficult to control the nitrosation selectivity, easily leading to the formation of dinitrosamines, thus making it difficult to obtain the target compound. Due to these factors, there is currently no method for preparing dapavancin demethylated BO nitrosamine that meets the requirements, and a compliant reference standard cannot be provided for quality control studies of this impurity.

[0014] Therefore, this invention selects dapavancin precursor A-40926-B0 as the starting material for the directional synthesis of dapavancin demethylated B0. The carboxyl group is protected by esterification, followed by a condensation reaction with 3-methylaminopropylamine. After hydrolysis, dapavancin demethylated B0 is obtained. Dapavancin demethylated B0 is then selectively protected by one of the secondary amines, followed by directional nitrosation of the unprotected secondary amine, and finally deprotection to obtain dapavancin demethylated B0 nitrosamine. The synthetic route is as follows: .

[0015] The entire synthetic route employs multi-step protective controls to manage reaction selectivity, reduce impurity formation, and improve reaction yield. It provides a reference standard for the quality control of dapavancin and offers important guidance for safe drug use.

[0016] Further, the specific steps of S3 are as follows: Dissolve intermediate III in a solvent, add a protective reagent and a base to react, and obtain intermediate IV.

[0017] Further, the specific steps of S3 are as follows: Dissolve intermediate III in a solvent, add a protective reagent and a base to react, then add water and adjust the pH to 7.0, crystallize and filter to obtain intermediate IV.

[0018] As can be seen from the above description, intermediate III selectively protects one of the secondary amines with a protecting agent in order to achieve subsequent directional nitrosation of the unprotected secondary amine.

[0019] Further, the protective reagent in S3 is added at 0~30°C. Preferably, the protective reagent in S3 is added at 0~10°C. More preferably, the protective reagent in S3 is added at 0~5°C.

[0020] Furthermore, the reaction temperature in S3 is 0~30℃, preferably 0~15℃, more preferably 0~10℃, and even more preferably 0~5℃.

[0021] Furthermore, the reaction time in S3 is 1 to 8 hours. Preferably, the reaction time in S3 is 5 to 7 hours.

[0022] Furthermore, the solvent in S3 is selected from one or more of DMSO (dimethyl sulfoxide), DMF (N,N-dimethylformamide), NMP (N-methylpyrrolidone), dioxane, THF (tetrahydrofuran), and acetonitrile.

[0023] Furthermore, the protecting agent in S3 includes one or more of Fmoc-Cl (9-fluorenylmethoxycarbonyl chloride), Fmoc-OSu (9-fluorenylmethoxycarbonyl succinimide), Fmoc-OTCP (9-fluorenylmethoxycarbonyl trichlorophenyl ester), Fmoc-N3 (9-fluorenylmethyl azidoformate), Cbz-Cl (benzyloxycarbonyl chloride), and Alloc-Cl (allyloxycarbonyl chloride), with Fmoc-Cl being preferred.

[0024] Furthermore, the molar ratio of the protective reagent to the dabavancin precursor A-40926-B0 in S3 is (0.5:1) to (1.5:1).

[0025] Furthermore, the base in S3 is selected from one or more of pyridine, triethylamine, DIPEA (N,N-diisopropylethylamine), sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium phosphate, and dipotassium hydrogen phosphate, with sodium bicarbonate being preferred.

[0026] Furthermore, the protecting reagent in S3 is Fmoc-Cl, and the base is selected from sodium bicarbonate.

[0027] Further, the specific steps of S5 are as follows: dissolve intermediate V in a solvent, add a deprotecting agent to react, and obtain dapavancin demethyl BO nitrosamine.

[0028] Further, the specific steps of S5 are as follows: dissolve intermediate V in a solvent, add a deprotecting agent to react, then add water and adjust the pH to 7.0, crystallize and filter to obtain dabavancin demethyl BO nitrosamine.

[0029] As can be seen from the above description, the method of the present invention has a high reaction yield, yielding high-purity dabavancin demethyl BO nitrosamine compound with a purity greater than 96.94%.

[0030] Furthermore, the deprotecting agent in S5 is selected from one or more of piperidine, diethylamine, dimethylamine, morpholine, DIPEA, Pd(PPh3)4-morpholine, and Pd / C-ammonium formate.

[0031] Furthermore, the reaction temperature in S5 is room temperature. The reaction time is 1~5 hours.

[0032] Further, the specific steps of S1 are as follows: add dabavancin precursor A-40926-B0 to isopropanol, add sulfuric acid to react, and obtain intermediate I.

[0033] Further, the specific steps of S1 are as follows: add dabavancin precursor A-40926-B0 to isopropanol, add sulfuric acid to react, add water after the reaction and adjust the pH to neutral, crystallize, filter, and dry to obtain intermediate I.

[0034] As can be seen from the above description, the carboxyl group of the Dabavancin precursor A-40926-B0 in S1 is protected by esterification.

[0035] Furthermore, the reaction temperature of S1 is 5~15℃.

[0036] As can be seen from the above description, if the reaction temperature is too low, the production efficiency will be reduced, while if the reaction temperature is too high, side reactions are likely to occur.

[0037] Furthermore, in S1, the mass-to-volume ratio (g / mL) of A-40926-B0 to isopropanol is 1:(5~10).

[0038] Furthermore, in S1, the molar ratio of A-40926-B0 to concentrated sulfuric acid is 1:(5~15), preferably 1:(8-12).

[0039] Furthermore, the molar ratio of the base of A-40926-B0S2 in S1 to the base of Dabavancin precursor A-40926-B0 in S3 is (1:1) to (1:20), for example, it can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20.

[0040] Further, the specific steps of S2 are as follows: after dissolving intermediate I in a solvent, TBTU, HOBT and 3-methylaminopropylamine are added to react and intermediate II reaction solution is obtained; alkali is added to intermediate II reaction solution to hydrolyze and intermediate III is obtained.

[0041] Further, the specific steps of S2 are as follows: after dissolving intermediate I in a solvent, TBTU, HOBT and 3-methylaminopropylamine are added to react and intermediate II reaction solution is obtained; alkali is added to intermediate II reaction solution for hydrolysis, then water is added and the pH is adjusted to 7.0, crystallization is carried out, filtration is performed, and drying is performed to obtain intermediate III.

[0042] As can be seen from the above description, intermediate I undergoes a condensation reaction with 3-methylaminopropylamine, and after hydrolysis, it is directed to give dabavancin demethyl BO (intermediate III).

[0043] Furthermore, the molar ratio of A-40926-B0S2 of S1 to TBTU, HOBT and 3-methylaminopropylamine of S2 is 1:(1-1.5):(1-1.5):(0.8-1.2).

[0044] Furthermore, the base of S2 is selected from one or more of sodium hydroxide and potassium hydroxide.

[0045] Furthermore, in S2, the molar ratio of 3-methylaminopropylamine to the base is 1:(1.5-2.5).

[0046] Furthermore, in S2, the hydrolysis temperature is 10-30℃, preferably 15-25℃.

[0047] Further, the specific steps of S4 are as follows: dissolve intermediate IV in a solvent, add a nitrosating agent, and then adjust the pH to 2-4 to carry out the reaction to obtain intermediate V.

[0048] Further, the specific steps of S4 are as follows: Dissolve intermediate IV in a solvent, add a nitrosating agent, then adjust the pH to 2-4 to carry out the reaction, then add water and adjust the pH to 7.0, crystallize and filter to obtain intermediate V.

[0049] As can be seen from the above description, S4 can directionally nitrosate unprotected secondary amines to ensure that the final deprotection yields the target product, dabavancin demethyl BO nitrosamine.

[0050] Furthermore, the solvent in S4 is selected from one or more of DMSO, DMF, NMP, dioxane, THF, and acetonitrile.

[0051] Furthermore, the nitrosating agent in S4 is sodium nitrite.

[0052] Furthermore, the nitrifying agent in S4 is 1-2M sodium nitrite.

[0053] Further, the molar ratio of sodium nitrite to dapavancin precursor A-40926-B0 is (1:1) to (20:1). Preferably, the molar ratio of sodium nitrite to dapavancin precursor A-40926-B0 is (1:1) to (5:1).

[0054] Furthermore, the inorganic acid in S4 is one or more of hydrochloric acid, sulfuric acid, and phosphoric acid.

[0055] Furthermore, the deprotecting agent in S4 is selected from diethylamine; preferably, the mass-to-volume ratio (g / ml) of diethylamine to dabavancin precursor A-40926-B0 is (0.5-1.5):1.

[0056] All reagents involved in this invention are commercially available. Dapavancin precursor A-40926-B0 can be obtained commercially or prepared according to the method in CN115160409B.

[0057] The dabavancin precursor A-40926-B0 in the following examples was prepared according to the method in CN115160409B.

[0058] Example 1 of the present invention is a method for preparing nitrosamine compounds, the specific steps of which are as follows: S1: Add dapavancin precursor A-40926-B0 (10g, 80% purity, 4.58mmol, 1eq) and isopropanol (80mL) to a reaction flask. Pre-cool the isopropanol and A-40926 complex suspension in the reaction flask to 5°C, add concentrated sulfuric acid (4.52g, 46mmol, 10eq) dropwise, and react at 10°C. After the reaction is complete (substrate residue less than 5%), add 400mL of water, adjust the pH to 7, crystallize, filter, and dry to obtain intermediate I.

[0059] S2: Add DMF (80 mL) to the reaction flask, add intermediate I to dissolve, add TBTU (1.77 g, 5.52 mmol, 1.2 eq), HOBT (0.76 g, 5.52 mmol, 1.2 eq) and 98 mL (39.20 g, 5.52 mmol, 1 eq) of 3-methylaminopropylamine, react for 5 h to obtain intermediate II reaction solution; then add 9.2 mL of 1 mol / L NaOH (0.37 g, 9.20 mmol, 2 eq) to intermediate II reaction solution, control the temperature at 20 °C, and react for 1 h; after the reaction, add 400 mL of water, adjust the pH to 7, crystallize, filter, and dry to obtain intermediate III.

[0060] S3: Add intermediate III to the reaction flask and dissolve it in 200 mL of dioxane. Add Cbz-Cl (0.78 g, 4.58 mmol, 1.0 eq) and triethylamine (0.93 g, 9.16 mmol, 2.0 eq). React at 30 °C for 4 h. Then add 1000 mL and adjust the pH to 7.0. Crystallize and filter to obtain intermediate IV crystal powder.

[0061] S4: Dissolve intermediate IV in 200 mL of dioxane, add 8.83 mL of 2M sodium nitrite (0.63 g, 9.16 mmol, 2 eq), adjust the pH to 3.0 with hydrochloric acid, react at room temperature for 3 h, then add 1000 mL of water, adjust the pH to 7.0, crystallize and filter to obtain intermediate V crystal powder.

[0062] S5: Dissolve intermediate V in 200 mL of dioxane, add Pb / C (0.8 g, 10% by mass), add ammonium formate (0.58 g, 9.16 mmol, 1 eq), react at room temperature for 4 h, then add 1000 mL of water, adjust the pH to 7.0, crystallize and filter to obtain crude dabavancin demethyl BO nitrosamine.

[0063] S6: The crude product was purified by reversed-phase silica gel chromatography, crystallized, and lyophilized to obtain dabavancin demethyl BO nitrosamine (0.96 g, molar yield 11.5%, purity 95.7%).

[0064] Example 2 of the present invention is a method for preparing nitrosamine compounds, the specific steps of which are as follows: S1: This step is the same as S1 in Example 1.

[0065] S2: This step is the same as S2 in Real-Time Example 1.

[0066] S3: Add intermediate III to the reaction flask and dissolve it in 200 mL of THF. Add Fmoc-Cl (1.18 g, 4.58 mmol, 1 eq) and sodium bicarbonate (3.85 g, 45.81 mmol, 10 eq). React at 30 °C for 1 h. Add 1000 mL of THF and adjust the pH to 7.0. Crystallize and filter to obtain intermediate IV crystal powder.

[0067] S4: Dissolve intermediate IV in 200 mL THF, add 9.16 mL of 2M sodium nitrite (0.63 g, 9.16 mmol, 2 eq), adjust the pH to 3.0 with hydrochloric acid, react at room temperature for 3 h, then add 1000 mL of water, adjust the pH to 7.0, crystallize and filter to obtain intermediate V crystal powder.

[0068] S5: Dissolve intermediate II in 200 mL THF, add 10 mL diethylamine, react at room temperature for 3 h, then add 1000 mL water, adjust the pH to 7.0, crystallize and filter to obtain crude dabavancin demethyl BO nitrosamine.

[0069] S6: The crude product was purified by reversed-phase silica gel chromatography, crystallized, and lyophilized to obtain dabavancin demethyl BO nitrosamine (1.57 g, molar yield 18.8%, 98.1%).

[0070] Example 3 of the present invention is a method for preparing nitrosamine compounds, the specific steps of which are as follows: S1: This step is the same as S1 in Example 1.

[0071] S2: This step is the same as S2 in Example 1.

[0072] S3: Add intermediate III to the reaction flask and dissolve it with 200 mL of THF. Add Alloc-Cl (0.5 g, 4.58 mmol, 1 eq) and DIPEA (0.89 g, 6.87 mmol, 1.5 eq). React at 30 °C for 1 h. Add 1000 mL of THF and adjust the pH to 7.0. Crystallize and filter to obtain intermediate IV crystal powder.

[0073] S4: Dissolve intermediate IV in 200 mL THF, add 9.16 mL of 2M sodium nitrite (0.63 g, 9.16 mmol, 2 eq), adjust the pH to 3.0 with hydrochloric acid, react at room temperature for 3 h, then add 1000 mL of water, adjust the pH to 7.0, crystallize and filter to obtain intermediate V crystal powder.

[0074] S5: Dissolve intermediate V in 200 mL THF, add solvent volume of Pd (Pph3)4 (0.21 g, 0.23 mmol, 0.05 eq), add morpholine (0.80 g, 9.16 mmol, 2 eq), react at room temperature for 4 h, then add 1000 mL water, adjust the pH to 7.0, crystallize and filter to obtain crude dapavancin demethyl BO nitrosamine.

[0075] S6: The crude product was purified by reversed-phase silica gel chromatography, crystallized, and lyophilized to obtain dabavancin demethyl BO nitrosamine (1.10 g, molar yield 13.1%, purity 96.2%).

[0076] Example 4 of the present invention is a method for preparing nitrosamine compounds, the specific steps of which are as follows: S1: This step is the same as S1 in Example 1.

[0077] S2: This step is the same as S2 in Example 1.

[0078] S3: Add intermediate III to the reaction flask and dissolve it in 200 mL of THF. Add Fmoc-Cl (1.18 g, 4.58 mmol, 1 eq) and sodium bicarbonate (3.85 g, 45.81 mmol, 10 eq). React at 15 °C for 3 h. Add 1000 mL of THF and adjust the pH to 7.0. Crystallize and filter to obtain intermediate IV crystal powder.

[0079] S4: This step is the same as step 4 in Example 2.

[0080] S5: This step is the same as step 5 in Example 2.

[0081] S6: The crude product was purified by reversed-phase silica gel chromatography, crystallized, and lyophilized to obtain dabavancin demethyl BO nitrosamine (1.99 g, molar yield 23.7%, purity 97.8%).

[0082] Example 5 of the present invention is a method for preparing nitrosamine compounds, the specific steps of which are as follows: S1: This step is the same as S1 in Example 1.

[0083] S2: This step is the same as S2 in Example 1.

[0084] S3: Add intermediate III to the reaction flask and dissolve it in 200 mL of THF. Add Fmoc-Cl (1.18 g, 4.58 mmol, 1 eq) and sodium bicarbonate (3.85 g, 45.81 mmol, 10 eq). React at 5 °C for 7 h. Add 1000 mL of THF and adjust the pH to 7.0. Crystallize and filter to obtain intermediate IV crystal powder.

[0085] S4: This step is the same as step 4 in Example 2.

[0086] S5: This step is the same as step 5 in Example 2.

[0087] S6: The crude product was purified by reversed-phase silica gel chromatography, crystallized, and lyophilized to obtain dabavancin demethyl BO nitrosamine (2.45 g, molar yield 29.2%, purity 97.7%).

[0088] Example 6 of the present invention is a method for preparing nitrosamine compounds, the specific steps of which are as follows: S1: This step is the same as S1 in Example 1.

[0089] S2: This step is the same as S2 in Example 1.

[0090] S3: Add intermediate III to the reaction flask and dissolve it in 200 mL of THF. Add Fmoc-Cl (1.18 g, 4.58 mmol, 1 eq) and sodium bicarbonate (3.85 g, 45.81 mmol, 10 eq). React at 0 °C for 8 h. Add 1000 mL of THF and adjust the pH to 7.0. Crystallize and filter to obtain intermediate IV crystal powder.

[0091] S4: This step is the same as step 4 in Example 2.

[0092] S5: This step is the same as step 5 in Example 2.

[0093] S6: The crude product was purified by reversed-phase silica gel chromatography, crystallized, and lyophilized to obtain dabavancin demethyl BO nitrosamine (2.43 g, molar yield 29.2%, purity 97.6%).

[0094] Example 7 of the present invention is a method for preparing nitrosamine compounds, the specific steps of which are as follows: S1: This step is the same as S1 in Example 1.

[0095] S2: This step is the same as S2 in Example 1.

[0096] S3: This step is the same as step 3) in Example 5.

[0097] S4: Dissolve intermediate IV in 200 mL THF, add 91.61 mL of 2M sodium nitrite (63.21 g, 91.61 mmol, 20 eq), adjust the pH to 3.0 with hydrochloric acid, react at room temperature for 1 h, add 1000 mL of water, adjust the pH to 7.0, crystallize and filter to obtain intermediate V crystal powder.

[0098] S5: This step is the same as step 5 in Example 2.

[0099] S6: The crude product was purified by reversed-phase silica gel chromatography, crystallized, and lyophilized to obtain dabavancin demethyl BO nitrosamine (2.48 g, molar yield 29.5%, purity 97.5%).

[0100] Example 8 of the present invention is a method for preparing nitrosamine compounds, the specific steps of which are as follows: S1: This step is the same as S1 in Example 1.

[0101] S2: This step is the same as S2 in Example 1.

[0102] S3: This step is the same as step 3) in Example 5.

[0103] S4: This step is the same as step 4 in Example 2.

[0104] S5: Dissolve intermediate II in 200 mL THF, add 40 mL morpholine, react at room temperature for 3 h, then add 1000 mL water, adjust the pH to 7.0, crystallize and filter to obtain crude dabavancin demethyl BO nitrosamine. The steps are the same as those in step 5 of Example 2.

[0105] S6: The crude product was purified by reversed-phase silica gel chromatography, crystallized, and lyophilized to obtain dabavancin demethyl BO nitrosamine (2.07 g, molar yield 24.7%, purity 98.1%).

[0106] Example 9 of the present invention is a method for preparing nitrosamine compounds, the specific steps of which are as follows: S1: This step is the same as S1 in Example 1.

[0107] S2: This step is the same as S2 in Example 1.

[0108] S3: This step is the same as step 3) in Example 5.

[0109] S4: This step is the same as step 4 in Example 2.

[0110] S5: Dissolve intermediate II in 200 mL THF, add 40 mL DIPEA, react at room temperature for 3 h, then add 1000 mL water, adjust the pH to 7.0, crystallize and filter to obtain crude dabavancin demethyl BO nitrosamine. The steps are the same as step 5 in Example 2.

[0111] S6: The crude product was purified by reversed-phase silica gel chromatography, crystallized, and lyophilized to obtain dapavancin demethyl BO nitrosamine (1.56 g, molar yield 18.6%, purity 97.8%).

[0112] The reactions of Examples 1-9 were compared, as shown in Table 1.

[0113] Table 1

[0114] As shown in the table above, by comparing different reaction conditions, different amine protecting reagents-deprotection systems and protecting reaction temperatures have a significant impact on the yield of the final target compound. The preferred protecting reagent is Fmoc-Cl, the preferred deprotection reagent is diethylamine, and the preferred protecting temperature is 0~5℃.

[0115] In summary, the method for preparing nitrosamine compounds provided by this invention proposes for the first time a preparation route for dapavancin demethyl BO nitrosamine compounds. This preparation process can obtain high-purity dapavancin demethyl BO nitrosamine compounds, which can then be used as a reference standard for detecting dapavancin demethyl BO nitrosamine impurities, thereby enabling the monitoring of the dapavancin demethyl BO nitrosamine content in dapavancin.

[0116] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification and drawings, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for preparing nitrosamine compounds, characterized in that, Includes the following steps: S1: Using Dabavancin precursor A-40926-B0 as raw material, esterification protection was performed to obtain intermediate I; S2: The intermediate I is condensed with 3-methylaminopropylamine to obtain intermediate II, and then intermediate III is obtained by alkaline hydrolysis; S3: Intermediate III is protected by a protecting agent on the secondary amine of the parent nucleus to obtain intermediate IV; S4: The intermediate IV is nitrified by a nitrifying agent to obtain intermediate V; S5: The intermediate V is deprotected to obtain dabavancin demethyl BO nitrosamine; The structural formula of the dabavancin precursor A-40926-B0 is shown in Formula (1), the structural formula of intermediate I is shown in Formula (2), the structural formula of intermediate II is shown in Formula (3), the structural formula of intermediate III is shown in Formula (4), the structural formula of intermediate IV is shown in Formula (5), the structural formula of intermediate V is shown in Formula (6), and the structural formula of dabavancin demethylated B0 nitrosamine is shown in Formula (7). Equation (1); Equation (2); Equation (3); Equation (4); Equation (5); Equation (6); Equation (7).

2. The method for preparing nitrosamine compounds according to claim 1, characterized in that, The specific steps of S3 are as follows: Dissolve intermediate III in a solvent, add a protective reagent and a base to react, and obtain intermediate IV.

3. The method for preparing nitrosamine compounds according to claim 2, characterized in that, The protective reagent in S3 includes one or more of Fmoc-Cl, Fmoc-OSu, Fmoc-OTCP, Fmoc-N3, Cbz-Cl, and Alloc-Cl; The base in S3 is selected from one or more of pyridine, triethylamine, DIPEA, sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium phosphate, and dipotassium hydrogen phosphate.

4. The method for preparing nitrosamine compounds according to claim 2, characterized in that, The reaction temperature in S3 is 0~30℃.

5. The method for preparing nitrosamine compounds according to claim 2, characterized in that, The molar ratio of the protective reagent to the dabavancin precursor A-40926-B0 in S3 is (0.5:1) to (1:1).

6. The method for preparing nitrosamine compounds according to claim 1, characterized in that, The specific steps of S5 are as follows: dissolve intermediate V in a solvent, add a deprotecting agent to react, and obtain dabavancin demethyl BO nitrosamine.

7. The method for preparing nitrosamine compounds according to claim 6, characterized in that, The deprotecting agent in S5 is selected from one or more of piperidine, diethylamine, dimethylamine, morpholine, DIPEA, Pd(PPh3)4-morpholine, and Pd / C-ammonium formate.

8. The method for preparing nitrosamine compounds according to claim 1, characterized in that, The specific steps of S1 are as follows: add the Dabavancin precursor A-40926-B0 to isopropanol, add sulfuric acid to react, and obtain intermediate I.

9. The method for preparing nitrosamine compounds according to claim 1, characterized in that, The specific steps of S2 are as follows: after dissolving intermediate I in a solvent, TBTU, HOBT and 3-methylaminopropylamine are added to react and intermediate II reaction solution is obtained; alkali is added to intermediate II reaction solution to perform hydrolysis and intermediate III is obtained.

10. The method for preparing nitrosamine compounds according to claim 1, characterized in that, The specific steps of S4 are as follows: Dissolve intermediate IV in a solvent, add a nitrosating agent, and then adjust the pH to 2-4 to carry out the reaction to obtain intermediate V.

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