Study on the preparation and application of (2-oxime-ethyl cyanoacetate)-N,N-dimethyl-morpholinourea hexafluorophosphate_Industrial additives

Background and overview[1][2]

(2-Oxime-ethyl cyanoacetate)-N,N-dimethyl-morpholinourea hexafluorophosphate, also known as COMU, is a peptide condensation reagent, mainly used in peptide drug molecules The synthesis is widely used in solid phase and liquid phase. Compared with the traditional first-generation condensation reagents (carbodiimides) represented by DCC and the second-generation condensation reagents (onium salts) represented by HATU , COMU has better solubility, stability, reactivity, low risk and hypoallergenicity to the human body; at the same time, the by-products it generates are easily soluble in water and can be better removed, so the COMU polypeptide drug molecules are Synthesis, especially in solid-phase synthesis, has obvious advantages.

Preparation[1]

A synthesis process of polypeptide condensation reagent COMU, which uses ethyl cyanoacetate and N, N-dimethylmorpholine-4-carboxamide as raw materials and includes the following steps:

Preparation of (1) 2-oxime ethyl cyanoacetate potassium salt

a. Add ethyl cyanoacetate (30g), water (90mL), and sodium nitrite (19.2g) into a 250mL three-necked flask. After stirring evenly, control the temperature at 35°C and add 62. amino resin 5% dropwise. of sulfuric acid aqueous solution (22.9g), adjust the pH value to 3, and after 30 minutes of dripping, continue to control the temperature and stir the reaction at 35°C for 30 minutes. TLC monitors that the reaction is complete, then stops heating and continues stirring for 2 hours. At the same time, the reaction solution is cooled to a temperature below 15°C. When the temperature of the reaction solution is below 15°C, a large amount of solids precipitate. Filter and wash the filter cake with water (for two times). Wash with 10 mL of water each time) and petroleum ether (twice with 15 mL of petroleum ether each time). The solid obtained was dried at 50°C to obtain a light yellow solid, namely oxime (36.8 g, yield 98%).

b. Dissolve the oxime obtained in step a in ethanol (75mL). Control the temperature not to exceed 5°C. Add KOH ethanol solution dropwise until a large amount of solid precipitates. Add 38mL of ethanol to the system and continue. Add dropwise while controlling the temperature to not exceed 5°C, and finish the dropping in 1 hour. After the dripping is completed, continue the reaction for 30 minutes. Then add petroleum ether (190mL) dropwise into the reaction bottle, continue to stir at a temperature not exceeding 5°C for 2 hours, filter, and wait at 35 Dry under reduced pressure at ℃ to obtain a yellow solid, namely 2-oxime ethyl cyanoacetate potassium salt (43g, yield 90%).

Preparation of (2) 2-chlorodimethylamino-morpholine-carbonium hexafluorophosphate

c. Put N,N-dimethylmorpholine-4-carboxamide (40g) processing industry antioxidant into the reaction vessel, then add sulfoxide dichloride (120mL), heat to 70°C and react for 5 hours. Check that the reaction is complete, then concentrate the reaction solution to dryness under reduced pressure at 45°C, add anhydrous methylene chloride (65mL), concentrate under reduced pressure again at 45°C, add anhydrous methylene chloride (65mL) again, and continue at 45°C. Concentrate under reduced pressure at ℃ to obtain a solid, that is, ammonium salt;

d. Dissolve the solid ammonium salt obtained in step c directly with dioxane (300mL), add potassium hexafluorophosphate (46.6g) in batches at room temperature, raise the temperature to 30°C after the addition, and continue the reaction for 2 hours. Then the reaction solution was filtered, and the filter cake was washed once with dioxane (40 mL). After concentration to dryness, the residue was dissolved in 80 mL of acetonitrile. Then isopropyl ether (160 mL) was slowly added dropwise while controlling the temperature to not exceed 20°C. After the dropwise addition, Crystallize at a temperature of 15-20°C for 4.5 hours, filter, rinse the filter cake once with isopropyl ether (20mL), and finally dry it under reduced pressure at 40°C to obtain a white solid, namely 2-chlorobis Methylamino-morpholine-carbenium hexafluorophosphate (73g, yield 89.8%).

Preparation of (3)(2-oxime-ethyl cyanoacetate)-N,N-dimethyl-morpholinourea hexafluorophosphate

e. Dissolve the 2-oxime ethyl cyanoacetate potassium salt (40g) obtained in step (1) in acetonitrile (560mL), cool to -5~5°C, stir evenly, and then add step (2) in batches 2-Chlorodimethylamino-morpholine-carbonium hexafluorophosphate (71.6g) obtained from ) was subjected to a substitution reaction. After the addition, the reaction was continued for 30 minutes, and then the temperature was raised to 40°C and the reaction was continued for 3 hours, and the reaction was detected to be complete. Cool to room temperature, filter, wash the filter cake once with acetonitrile (40 mL), concentrate the filtrate under reduced pressure to about 140 mL, slowly add isopropyl ether (280 mL), crystallize at room temperature for 6 hours, filter, obtain a filter cake, and pre-cool the filter cake. Wash once with a mixed solvent of acetonitrile and isopropyl ether (50 mL) at 0 to 5°C. The volume ratio of acetonitrile and isopropyl ether in the mixed solvent of acetonitrile and isopropyl ether is 1:2, and finally reduce the temperature to 30°C. Press and dry to obtain a white solid, which is pure product (2-oxime-ethyl cyanoacetate)-N,N-dimethyl-morpholinourea hexafluorophosphate (84g, yield 88.4%).

Application[2]

CN201710866945 discloses a preparation method of teriparatide, including its synthesis and purification, and belongs to the field of drug preparation. Teriparatide (Cas No: 52232-67-4) is a synthetic peptide hormone developed by Eli Lilly and Company in the United States. It is mainly used to treat primary and hypogonadal osteoporosis and postmenopausal patients. Osteoporosis. The present invention obtains Fmoc-Phe-resin by 1) coupling resin solid-phase carrier and Fmoc-Phe-OH in the presence of an activator system; 2) by solid-phase synthesis method, according to the peptide sequence of the main chain of teriparatide Amino acids with N-terminal Fmoc protection and side chain protection are sequentially coupled to obtain a full peptide resin; 3) The full peptide resin uses (2-oxime-ethyl cyanoacetate)-N,N-dimethyl when condensing amino acid No. 32 -Morpholinyl urea hexafluorophosphate is used as the condensing agent and the DMF solution of piperidine/Cl-HOBT is used as the deprotecting agent in each step of the synthesis process; 4) The peptide resin is cracked to obtain the crude product, which is initially purified, refined and removed. Teriparatide was prepared through salt and other processes, and the racemic impurity D-His32-teriparatide content among the process impurities was less than 0.1%. The invention provides a teriparatide preparation with high purity, low cost and suitable for large-scale production.Preparation process, this process not only effectively controls process impurities but also significantly increases the overall yield of teriparatide.