A method for preparing oxytocin EP impurity F
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-05-15
- Publication Date
- 2026-08-14
AI Technical Summary
目前文献报道（Int. J. Peptide Rotein Res.22,1983,525-538）缩宫素EP杂质F的合成方法是通过液相多步反应得到的，同时由于结构中有三个伯酰胺，都可以反应，在一定程度上导致反应的选择性变差，产生较多的同分异构体
[0017]有益效果：与现有技术相比，本发明具有如下显著优点：（1）发明只需要一步反应即可得到产物，有效减少了工作量并减少了废液等的产生，符合绿色化学，同时通过氰基化试剂的选择以及比例的调控，以较高的收率得到目标产物，从而避免产生大量其他同分异构体；（2）通过纯化，制备的缩宫素EP杂质F纯度达到97.1914%；（3）合成的缩宫素EP杂质F作为原料药生产过程中的杂质标准品进行对比，对控制原料药过程中的杂质含量起到重要作用；（4）通过合成缩宫素EP杂质F可以进一步对其性质进行研究。
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Abstract
Description
Technical Field
[0001] This invention relates to a method for preparing a compound, and more particularly to a method for preparing oxytocin EP impurity F. Background Technology
[0002] Oxytocin is a peptide hormone secreted by the posterior pituitary gland and synthesized in the paraventricular and supraoptic nuclei of the hypothalamus. It consists of nine amino acids, with cysteine residues at positions "1" and "6" forming a hexapeptide ring structure via disulfide bonds. Clinically, it is widely used in obstetrics and gynecology. Oxytocin stimulates milk production, promotes uterine smooth muscle contraction during labor, and fosters maternal love. Furthermore, it can reduce the levels of stress hormones such as adrenaline in the body, thereby lowering blood pressure. Oxytocin EP impurity F is one of the impurities produced during the production process, and its structure is as follows:
[0003] .
[0004] The synthesized oxytocin EP impurity F can be used as a standard for impurities in the production process of active pharmaceutical ingredients (APIs), playing an important role in controlling the impurity content in the API manufacturing process. Currently, the literature reports (Int. J. Peptide Rotein Res. 22, 1983, 525-538) that the synthesis method of oxytocin EP impurity F involves a multi-step liquid-phase reaction. However, due to the presence of three primary amides in its structure, all of which can react, the selectivity of the reaction is reduced to some extent, resulting in a greater number of isomers. Summary of the Invention
[0005] Purpose of the invention: The present invention aims to provide a method for preparing oxytocin EP impurity F, which shortens the synthesis steps, requires only one reaction to obtain the product, improves the selectivity of amide reaction, and avoids the generation of a large number of other isomers.
[0006] Technical solution: The preparation method of oxytocin EP impurity F according to the present invention includes the following steps: dissolving the raw material oxytocin in a solvent, cooling, adding trifluoroacetic anhydride, then adding triethylamine dropwise, continuing the reaction, removing the solvent after the reaction is completed, then slurrying, filtering to obtain the crude product, and purifying to obtain the final product; the synthetic route is as follows:
[0007] .
[0008] Preferably, the molar ratio of trifluoroacetic anhydride to oxytocin is 1-2:1. Excessive trifluoroacetic anhydride will lead to a significant increase in impurities. More preferably, the molar ratio of trifluoroacetic anhydride to oxytocin is 1-1.5:1.
[0009] Preferably, the molar ratio of triethylamine to oxytocin is 1 to 5:1. Insufficient triethylamine leads to insufficient alkalinity and incomplete cyanation reaction.
[0010] Preferably, the cooling temperature is -5 to 5°C. Lowering the temperature during material addition prevents excessive heat release and thus avoids a violent reaction.
[0011] Preferably, the temperature for continued reaction is 20-40 °C. After the feeding is completed, the temperature is appropriately increased to accelerate the reaction rate.
[0012] Preferably, the solvent is one or more of N,N-dimethylformamide (DMF), tetrahydrofuran (THF), dichloromethane (DCM), and acetonitrile (ACN).
[0013] Preferably, the pulping process uses methyl tert-butyl ether.
[0014] Preferably, the purification involves dissolving the crude product in acetonitrile and an aqueous solution, followed by column purification.
[0015] Preferably, the mobile phase for column purification is a solution of acetonitrile and trifluoroacetic acid.
[0016] Preferably, the column purification process employs a gradient elution with acetonitrile and trifluoroacetic acid solutions. After elution, fractions of the product with a purity greater than 95% are collected.
[0017] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The invention only requires one reaction to obtain the product, which effectively reduces the workload and the generation of waste liquid, etc., which is in line with green chemistry. At the same time, by selecting the cyanidation reagent and controlling the ratio, the target product is obtained with a high yield, thereby avoiding the generation of a large number of other isomers; (2) Through purification, the purity of the prepared oxytocin EP impurity F reaches 97.1914%; (3) The synthesized oxytocin EP impurity F is used as an impurity standard in the production process of raw materials for comparison, which plays an important role in controlling the impurity content in the process of raw materials; (4) The properties of oxytocin EP impurity F can be further studied by synthesizing it. Attached Figure Description
[0018] Figure 1 This is a synthetic route diagram for oxytocin EP impurity F of the present invention;
[0019] Figure 2 The high-performance liquid chromatography (HPLC) spectrum of the product from Example 1 is shown below.
[0020] Figure 3 The hydrogen NMR spectrum of the product from Example 1;
[0021] Figure 4 This is the mass spectrum of the product from Example 1. Detailed Implementation
[0022] The technical solution of the present invention will be further described below with reference to the embodiments.
[0023] Example 1
[0024] The method for preparing oxytocin EP impurity F of the present invention includes the following steps:
[0025] (1) Synthesis of crude oxytocin EP impurity F
[0026] 989 mg of oxytocin (1 mmol) was weighed and dissolved in 10 mL of dry DMF. The solution was added to a round-bottom flask and cooled to 0°C in an ice bath. TFAA (0.25 g, 1.2 mmol) was slowly added, followed by TEA (0.51 g, 5.0 mmol) dropwise over 5 min. The mixture was stirred under a nitrogen atmosphere and allowed to return to room temperature (25°C). Liquid chromatography was performed (flow rate 1 mL / min, detection wavelength 220 nm, analytical column 4.6 x 250 mm, 10 μm C18 column, mobile phase A: pure acetonitrile, mobile phase B: 0.1% TFA solution, elution gradient 10–100%, elution time 20 min, compared with the retention time of the starting material) until the reaction was complete. After removing the solvent under reduced pressure, the mixture was slurried with methyl tert-butyl ether, filtered, and dried to obtain 800 mg of crude product, with a yield of 80.9%.
[0027] (2) High-efficiency preparation of liquid phase purification
[0028] The 800 mg crude product obtained in step (1) was dissolved in 10 mL of acetonitrile and aqueous solution (volume ratio 1:8), and then filtered through a 0.22 μm filter membrane to obtain a pale yellow and clear solution.
[0029] The solution obtained in step (2) was purified by column chromatography under the following conditions:
[0030] Mobile phase: Phase A is acetonitrile, and Phase B is a 0.1% trifluoroacetic acid solution.
[0031] The elution gradient is shown in Table 1.
[0032] Table 1 Elution gradient and time
[0033] time Flow rate mL / min Mobile phase A Mobile phase B 0 50 5 95 5 50 20 80 45 50 60 40 65 50 100 0
[0034] During the elution process, the product with a purity greater than 95% was collected in segments and lyophilized to obtain 400 mg of white solid, with a yield of 40.4%.
[0035] Figure 2The figure shows the high performance liquid chromatography (HPLC) spectrum of impurity F in the product oxytocin EP. The HPLC purity is 97.1914%.
[0036] Figure 3 This is the NMR spectrum of impurity F in oxytocin EP. ¹H NMR (in Dmso-d6, 400 MHz): 9.27 ppm (s, ¹H), 8.71–8.05 ppm (m, 9H), 7.90 ppm (t, ¹H), 7.36 ppm (s, ¹H), 7.13 ppm (m, 4H), 6.87 ppm (s, ¹H), 6.70 ppm (d, 2H), 4.78 ppm (m, ¹H), 4.59–3.86 ppm (m, 7H), 3.63–3.52 p pm(m,4H), 3.41ppm(m,1H), 3.17ppm(m,2H), 3.06-2.93ppm(m,4H), 2.72(m,1H), 2.22ppm(t,2H), 2.04-1.86ppm(m,7H), 1.63-1.49ppm(m,4H), 1.16(m,1H), 0.9-0.82(m,12H) conform to the structure.
[0037] Figure 4 This is the mass spectrum of impurity F in oxytocin EP. Chemical Formula: C 43 H 64 N 12 O 11 S2 [M] + =988.4, [M+Na]:1011.4 was found in the mass spectrum.
[0038] The final product obtained is deoxytocin EP impurity F trifluoroacetate, which can be used as a reference in high performance liquid chromatography without affecting the standardization. If it is necessary to obtain the free product, sodium bicarbonate solution can be added to the aqueous solution to precipitate the product from the water. The free product can be obtained by filtration, thereby removing trifluoroacetic acid.
[0039] Example 2
[0040] The method for preparing oxytocin EP impurity F of the present invention includes the following steps:
[0041] (1) Synthesis of crude oxytocin EP impurity F
[0042] 2 g of oxytocin (2 mmol) was dissolved in 10 mL of dry DMF and added to a round-bottom flask. The flask was cooled to 0°C in an ice bath, and 0.5 g of TFAA (2.38 mmol) was slowly added dropwise over 5 min. Then, 1.0 g of TEA (9.88 mmol) was added dropwise. The mixture was stirred under a nitrogen atmosphere and allowed to return to room temperature (25°C) naturally. The liquid phase was monitored until the reaction was complete. After removing the solvent under reduced pressure, the mixture was slurried with methyl tert-butyl ether, filtered, and dried to obtain 1.8 g of crude product, with a yield of 90.0%.
[0043] (2) High-efficiency preparation of liquid phase purification
[0044] The crude product obtained in step (1) was dissolved in 20 mL of acetonitrile and aqueous solution (volume ratio 1:8), and then filtered through a 0.22 μm filter membrane to obtain a pale yellow and clear solution.
[0045] The solution obtained in step (2) was purified by column chromatography twice (elution gradient as in Example 1). The product with a purity greater than 95% was collected in fractions and lyophilized to obtain 1.1 g of white solid, with a yield of 55%. The yield increased by about 10% compared to Example 1, indicating that the yield can be guaranteed by scale-up feeding.
[0046] The product purity was 96.7399%, and the NMR and mass spectrometry data were basically the same as those in Example 1.
[0047] Example 3
[0048] The method for preparing oxytocin EP impurity F of the present invention includes the following steps:
[0049] (1) Synthesis of crude oxytocin EP impurity F
[0050] 2 g of oxytocin (2 mmol) was dissolved in 10 mL of dry DMF and added to a round-bottom flask. The flask was cooled to 0°C in an ice bath, and 0.63 g of TFAA (3.0 mmol) was slowly added, followed by 1.0 g of TEA (9.88 mmol) dropwise over 5 min. The mixture was stirred under a nitrogen atmosphere and allowed to return to room temperature (25°C). Liquid chromatography was monitored until the reaction was complete. The solvent was then removed under reduced pressure, and the mixture was slurried with methyl tert-butyl ether. After filtration and drying, 1.3 g of crude product was obtained, with a yield of 65%.
[0051] (2) High-efficiency preparation of liquid phase purification
[0052] The crude product obtained in step (1) was dissolved in 20 mL of acetonitrile and aqueous solution (volume ratio 1:8), and then filtered through a 0.22 μm filter membrane to obtain a pale yellow and clear solution.
[0053] The solution obtained in step (2) was purified by column chromatography twice (elution gradient as in Example 1). The product with a purity greater than 95% was collected in fractions and lyophilized to obtain 0.7 g of white solid, with a yield of 35%. The yield decreased by about 10.4% compared to Example 1. This is because the excess cyaniding reagent will cause the remaining primary amides to react in large quantities, resulting in the generation of multiple cyano impurities, which leads to a decrease in yield.
[0054] The product purity was 95.5001%, and the NMR and mass spectrometry data were basically the same as those in Example 1.
[0055] Example 4
[0056] The method for preparing oxytocin EP impurity F of the present invention includes the following steps:
[0057] (1) Synthesis of crude oxytocin EP impurity F
[0058] 2 g of oxytocin (2 mmol) was dissolved in 10 mL of dry DMF. The solution was added to a round-bottom flask and cooled to 0°C in an ice bath. TFAA (0.84 g, 4.0 mmol) was slowly added, followed by TEA (1.0 g, 9.88 mmol) dropwise over 5 min. The mixture was stirred under a nitrogen atmosphere and allowed to return to room temperature (25°C). Liquid chromatography was monitored until the reaction was complete. The solvent was then removed under reduced pressure, and the mixture was slurried with methyl tert-butyl ether. After filtration and drying, 1.5 g of crude product was obtained, with a yield of 75%.
[0059] (2) High-efficiency preparation of liquid phase purification
[0060] The crude product obtained in step (1) was dissolved in 20 mL of acetonitrile and aqueous solution (volume ratio 1:8), and then filtered through a 0.22 μm filter membrane to obtain a pale yellow and clear solution.
[0061] The solution obtained in step (2) was purified by column chromatography twice (elution gradient as in Example 1). The product with a purity greater than 95% was collected in fractions and lyophilized to obtain 0.5 g of white solid with a yield of 25%. The yield decreased by 15.4% compared to Example 1. This was because the excessive cyaniding reagent led to the participation of a large number of other primary amides in the reaction, resulting in more byproducts and more impurities with similar polarity, which reduced the yield.
[0062] The product purity was 96.5051%, and the NMR and mass spectrometry data were basically the same as those in Example 1.
[0063] Comparative Example 1
[0064] 2 g of oxytocin (2 mmol) was dissolved in 10 mL of dry DMF and added to a round-bottom flask. The flask was cooled to 0°C in an ice bath. Oxaloyl chloride (0.13 g, 1.2 mmol) was added dropwise over 1 min. Triphenylphosphine oxide (5.6 mg, 0.01 mmol) was then added. The mixture was stirred under a nitrogen atmosphere and allowed to return to room temperature (25°C). Liquid chromatography was monitored until the reaction was complete. The solvent was then removed under reduced pressure, and the mixture was slurried with methyl tert-butyl ether. After filtration and drying, 0.5 g of crude product was obtained, with a yield of 25%.
[0065] The purification method was the same as in Example 1, yielding 0.1 g of product with a yield of 5% and a purity of 90.32%.
[0066] The yield was lower than in Example 1 because the cyaniding agent reacted poorly with the substrate, contained many impurities, and was difficult to purify.
[0067] Comparative Example 2
[0068] 2 g of oxytocin (2 mmol) was dissolved in 10 mL of dry DMF and added to a round-bottom flask. The flask was cooled to 0°C in an ice bath, and Burgess reagent (0.29 g, 1.2 mmol) was added. The mixture was stirred under a nitrogen atmosphere and allowed to return to room temperature (25°C). Liquid chromatography was monitored until the reaction was complete. The solvent was then removed under reduced pressure, and the mixture was slurried with methyl tert-butyl ether. After filtration and drying, 0.7 g of crude product was obtained, with a yield of 35%.
[0069] The purification method was the same as in Example 1, yielding 0.15 g of product with a yield of 7.5% and a purity of 94.01%.
[0070] The yield was significantly lower than in Example 1 because the cyaniding agent reacted poorly with the substrate, contained many impurities, and was difficult to purify.
Claims
1. A method for preparing oxytocin EP impurity F, characterized in that, The synthetic route includes the following steps: Oxytocin is dissolved in a solvent, cooled, trifluoroacetic anhydride is added, followed by the dropwise addition of triethylamine. The reaction continues until the solvent is removed, then the mixture is slurried, filtered to obtain the crude product, and purified to obtain the final product. 。 2. The method for preparing oxytocin EP impurity F according to claim 1, characterized in that, The molar ratio of trifluoroacetic anhydride to oxytocin is 1~2:
1.
3. The method for preparing oxytocin EP impurity F according to claim 1, characterized in that, The molar ratio of triethylamine to oxytocin is 1~5:
1.
4. The method for preparing oxytocin EP impurity F according to claim 1, characterized in that, The cooling temperature is -5~5℃.
5. The method for preparing oxytocin EP impurity F according to claim 1, characterized in that, The temperature for the continued reaction is 20~40℃.
6. The method for preparing oxytocin EP impurity F according to claim 1, characterized in that, The solvent is one or more of N,N-dimethylformamide, tetrahydrofuran, dichloromethane, and acetonitrile.
7. The method for preparing oxytocin EP impurity F according to claim 1, characterized in that, The pulping process uses methyl tert-butyl ether.
8. The method for preparing oxytocin EP impurity F according to claim 1, characterized in that, The purification process involves dissolving the crude product in acetonitrile and an aqueous solution, followed by column purification.
9. The method for preparing oxytocin EP impurity F according to claim 1, characterized in that, The mobile phase for column purification is an acetonitrile and trifluoroacetic acid solution.
10. The method for preparing oxytocin EP impurity F according to claim 9, characterized in that, The column purification process uses gradient elution with acetonitrile and trifluoroacetic acid solutions.