CN108088936B - Impurity obtained in preparation of pidotimod ethyl ester and quality detection method thereof - Google Patents
Impurity obtained in preparation of pidotimod ethyl ester and quality detection method thereof Download PDFInfo
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- CN108088936B CN108088936B CN201711291284.4A CN201711291284A CN108088936B CN 108088936 B CN108088936 B CN 108088936B CN 201711291284 A CN201711291284 A CN 201711291284A CN 108088936 B CN108088936 B CN 108088936B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
Abstract
The invention relates to a quality detection method for preparing pidotimod ethyl ester, which comprises the steps of taking L-thioproline ethyl ester hydrochloride and L-pyroglutamic acid as reactants, and preparing a reaction product containing the pidotimod ethyl ester through free and condensation reactions in sequence, wherein the peak area of a compound A contained in the reaction product is not more than 6.5% and the peak area of a compound B contained in the reaction product is not more than 2.0% through high performance liquid chromatography; the compound A has a structure shown in a formula V, and the compound B has a structure shown in a formula VI. The quality detection method for preparing the pidotimod ethyl ester is beneficial to controlling the medicine quality in the production process.
Description
Technical Field
The invention provides an impurity generated during preparation of pidotimod ethyl ester and a method for applying the impurity to mass analysis, belonging to the technical field of chemical synthesis.
Background
Pidotimod (Pidotimod), developed in the 80S of the 20 th century by the company Poli industria chimca s.p.a, italy, approved for clinical use in 1993, is an artificially synthesized immune enhancer, structurally similar to a dipeptide, chemically known by the name (R) -3- (S) - [ (5-oxo-2-pyrrolidinyl) carbonyl ] -tetrahydro-4-carboxylic acid. It has the advantages of fast distribution and excretion, no accumulation in body, high tolerance, etc. and can promote both non-specific and specific immunoreaction. At present, the traditional Chinese medicine composition is marketed in various countries such as Asia, Europe, America and the like, and is mainly used for treating bacterial (pneumococcus, Escherichia coli, Pseudomonas aeruginosa, proteus and the like) and viral (influenza virus, herpes simplex virus, myocarditis virus, Mengo virus and the like) infection by promoting the immune function of an organism; it can be used together with antibacterial agent for treating recurrent respiratory tract infection, otorhinolaryngological infection, and urinary system infection with suppressed cell immunity concentration. The structural formula is as follows:
the process method adopted for synthesizing the pidotimod generally takes L-thioproline ethyl ester hydrochloride and L-pyroglutamic acid as initial raw materials, the pidotimod ethyl ester is obtained by DCC condensation, and finally the pidotimod is obtained by hydrolysis. The method has the advantages of short synthetic route, reasonable reaction, simple and convenient operation, low energy consumption, easy recovery of reaction solvent, low cost of used reagents, 60 to 64 percent of yield and suitability for industrial production. When the pidotimod is synthesized by the process method, some impurities are inevitably generated.
Any substance that affects the purity of the drug is collectively referred to as an impurity. The research of impurities is an important content of drug development, and comprises selecting a proper analysis method, accurately distinguishing and determining the content of the impurities and determining the reasonable limit of the impurities by integrating the results of pharmaceutical, toxicological and clinical researches. This research is throughout the whole process of drug development, and impurities of drugs are generally classified into three categories according to their physicochemical properties: organic impurities, inorganic impurities and residual solvents. Depending on their origin, impurities can be classified into process impurities (including reactants and reagents, intermediates, by-products, etc., which are not completely reacted in the synthesis), degradation products, impurities mixed from reactants and reagents, and the like. According to the classification of toxicity, the impurities can be classified into toxic impurities and common impurities. Impurities can also be classified according to their chemical structure, such as steroids, alkaloids, geometric isomers, optical isomers, polymers, and the like. Organic impurities include impurities introduced in the process, degradation impurities, etc., and may be known impurities or unknown, volatile or non-volatile. Since the chemical structure of such impurities is generally similar or has a source relationship with the active ingredient, there are substances that may be referred to as related substances.
Disclosure of Invention
The technical problem to be solved by the invention is to provide an impurity obtained in the preparation of pidotimod ethyl ester and a quality detection method for preparing pidotimod ethyl ester, which are beneficial to controlling the quality of medicines in the production process.
The invention provides a technical scheme for solving the technical problems, which comprises the following steps: a quality detection method for preparing pidotimod ethyl ester comprises the steps of taking L-thioproline ethyl ester hydrochloride and L-pyroglutamic acid as reactants, sequentially carrying out free reaction and condensation reaction to obtain a reaction product containing pidotimod ethyl ester, wherein the peak area of a compound A contained in the reaction product is not more than 6.5% and the peak area of a compound B contained in the reaction product is not more than 2.0% as measured by high performance liquid chromatography;
the compound A has a structure shown in a formula V:
the compound B has a structure shown in a formula VI:
adding L-thioproline ethyl ester hydrochloride shown as a formula I and L-pyroglutamic acid shown as a formula II into dichloromethane, dropwise adding a sodium hydroxide solution to carry out a free reaction, then adding a condensing agent to carry out an amide condensation reaction to generate a reaction product containing pidotimod ethyl ester shown as a formula III, wherein the reaction formula is as follows:
the condensing agent is dicyclohexylcarbodiimide, N' -diisopropylcarbodiimide or 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.
The stationary phase of the high performance liquid chromatography takes octadecylsilane chemically bonded silica as a filler, the mobile phase comprises sodium dihydrogen phosphate solution and methanol, the detection wavelength is 210nm, the column temperature is 30 ℃, and the calculation is carried out according to area normalization.
The pH value of the sodium dihydrogen phosphate solution and the methanol is 2.5-3.5, wherein the volume ratio of the methanol to the sodium dihydrogen phosphate solution is 98: 2-45: 55.
The pH of the sodium dihydrogen phosphate solution and methanol was 3.0, wherein the volume ratio of methanol to sodium dihydrogen phosphate solution was 70: 30.
The invention provides a technical scheme for solving the technical problems, which comprises the following steps: an impurity obtained in the preparation of pidotimod ethyl ester, which comprises a compound A and a compound B;
the compound A has a structure shown in a formula V:
the compound B has a structure shown in a formula VI:
the invention provides a technical scheme for solving the technical problems, which comprises the following steps: an application of the impurity obtained in the preparation of the pidotimod ethyl ester as an impurity reference substance in quality detection of the pidotimod ethyl ester.
The invention has the positive effects that: the quality detection method for preparing the pidotimod ethyl ester selects the combination of the new compound A and the compound B generated by the reaction as the impurity reference substance, is used for preparing the quality analysis of the pidotimod ethyl ester, can provide better reference for the selection of the process conditions, and is favorable for controlling the quality of the medicine in the production process. The invention adopts high performance liquid chromatography for separation and detection, and selects proper mobile phase with accurate and high-efficiency result.
Detailed Description
The present invention is described in detail below by way of examples, it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and those skilled in the art can make some insubstantial modifications and adaptations of the present invention based on the above-described disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
Example 1
The quality detection method for preparing pidotimod ethyl ester in the embodiment comprises the steps of adding 1500g of L-thioproline ethyl ester hydrochloride (500 g, 2.53 mol) and dichloromethane into a 3L reaction bottle, cooling to 0-5 ℃, dropwise adding a 20% sodium hydroxide solution (531.16 g, 2.66 mol) while keeping the internal temperature of the solution at 0-10 ℃, keeping the internal temperature of the solution at 8-9 ℃, continuously stirring for 5min, carrying out phase separation, washing an organic phase with 500g of saturated sodium chloride, adding 500g of anhydrous sodium sulfate into the organic phase, drying for 1-2 h, filtering, washing a filter cake with 500g of dichloromethane, pouring the filtrate into another 5L reaction bottle, adding L-pyroglutamic acid (326.56 g, 2.53 mol), stirring for 30min, cooling to 0-5 ℃, dropwise adding a mixed solution of dicyclohexylcarbodiimide (521.88 g, 2.53 mol) and 1000g of dichloromethane, keeping the temperature of 0-10 ℃ during dropwise adding, and after dripping, heating to 15-25 ℃, stirring for 0.5-1 h, filtering, leaching the filter cake with 500g of dichloromethane to obtain a dichloromethane solution containing pidotimod ethyl ester, and concentrating the dichloromethane solution for later use. The reaction formula is as follows:
the concentrated pidotimod ethyl ester solution contains two main impurities, namely compound A and compound B. The mass spectrum detection result is as follows: a compound A: [ M + H ]]+241.32, Compound B: [ M + H ]]+478.36。
The structure of compound a is as follows:
the structure of compound B is as follows:
detecting the oily matter by adopting a high performance liquid chromatography under the detection conditions that a stationary phase uses octadecylsilane chemically bonded silica as a filler, a mobile phase is a sodium dihydrogen phosphate solution and methanol with the pH value of 3.0, wherein the volume ratio of the methanol to the sodium dihydrogen phosphate solution is 70:30, the detection wavelength is 210nm, the column temperature is 30 ℃, the number of theoretical plates is not less than 2000 calculated according to pidotimod, the peak area of the compound A is 4.3272%, and the peak area of the compound B is 0.8136%, so that the detection meets the requirements.
Cooling the dichloromethane solution containing the pidotimod ethyl ester to 0-5 ℃, dropwise adding 7.5% sodium hydroxide solution (1281.53 g, 2.41 mol), keeping the internal temperature of 0-5 ℃ all the time during dropwise adding, keeping the pH value of 9-10 after dropwise adding, keeping the internal temperature, continuously stirring for 1h, carrying out phase separation, extracting the water phase with dichloromethane, combining the dichloromethane phases, and concentrating the dichloromethane phase to obtain the oily substance containing the pidotimod.
Pidotimod ethyl ester shown in the formula III is added with alkali (sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide or potassium hydroxide) in dichloromethane in a dropwise manner to carry out hydrolysis reaction to obtain a reaction product containing pidotimod shown in the formula IV, wherein the reaction formula is as follows:
example 2
The remaining parts of the quality detection method for preparing pidotimod ethyl ester in this example are the same as those in example 1, except that: dicyclohexylcarbodiimide was replaced with equimolar amounts of N, N' -diisopropylcarbodiimide and the pH of the sodium dihydrogen phosphate phosphoric acid solution and methanol was 2.5, where the volume ratio of methanol to sodium dihydrogen phosphate phosphoric acid solution was 60: 40. The reaction product is detected by high performance liquid chromatography, the peak area of the compound A is 5.1123%, and the peak area of the compound B is 1.8637%, which meets the requirement.
Example 3
The remaining parts of the quality detection method for preparing pidotimod ethyl ester in this example are the same as those in example 1, except that: dicyclohexylcarbodiimide was replaced with equimolar amounts of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and the pH of the sodium dihydrogen phosphate phosphoric acid solution and methanol was 3.5, where the volume ratio of methanol to sodium dihydrogen phosphate phosphoric acid solution was 80: 20. The reaction product is detected by high performance liquid chromatography, the peak area of the compound A is 5.9822%, and the peak area of the compound B is 1.5061%, which meets the requirement.
The reagents used in the invention are all purchased products unless specified, and the concentrations are all chemical pure.
It should be understood that the above examples are only for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And such obvious variations or modifications which fall within the spirit of the invention are intended to be covered by the scope of the present invention.
Claims (3)
1. A quality detection method for preparing pidotimod ethyl ester uses L-thioproline ethyl ester hydrochloride and L-pyroglutamic acid as reactants, and prepares a reaction product containing the pidotimod ethyl ester through dissociation and condensation reactions in sequence, and is characterized in that: the peak area of the reaction product containing the compound A is not more than 6.5 percent and the peak area of the reaction product containing the compound B is not more than 2.0 percent as measured by high performance liquid chromatography; the compound A has a structure shown in a formula V:
the compound B has a structure shown in a formula VI:
adding L-thioproline ethyl ester hydrochloride shown as a formula I and L-pyroglutamic acid shown as a formula II into dichloromethane, dropwise adding a sodium hydroxide solution to carry out a free reaction, then adding a condensing agent to carry out an amide condensation reaction to generate a reaction product containing pidotimod ethyl ester shown as a formula III, wherein the reaction formula is as follows:
the condensing agent is dicyclohexylcarbodiimide;
the stationary phase of the high performance liquid chromatography takes octadecylsilane chemically bonded silica as a filler, the mobile phase of the high performance liquid chromatography takes sodium dihydrogen phosphate solution and methanol, the detection wavelength is 210nm, the column temperature is 30 ℃, and the calculation is carried out according to area normalization; wherein the volume ratio of the methanol to the sodium dihydrogen phosphate solution is 98: 2-45: 55.
2. The quality detection method for preparing pidotimod ethyl ester according to claim 1, characterized in that: the pH value of the sodium dihydrogen phosphate solution and the methanol is 2.5-3.5.
3. The quality detection method for preparing pidotimod ethyl ester according to claim 1, characterized in that: the pH value of the sodium dihydrogen phosphate phosphoric acid solution and the methanol is 3.0, wherein the volume ratio of the methanol to the sodium dihydrogen phosphate phosphoric acid solution is 70: 30.
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Citations (3)
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|---|---|---|---|---|
| CN1158620A (en) * | 1994-09-27 | 1997-09-03 | 波利工业化学公司 | Process for the quantitative synthesis of 3-(L-pyrolutamyl)-L-tiazolidine-4-carboxylic acid and derivatives thereof |
| CN102167727A (en) * | 2011-01-29 | 2011-08-31 | 浙江金立源药业有限公司 | Synthesis method of pidotimod |
| CN102952172A (en) * | 2011-08-18 | 2013-03-06 | 北京澳林森科技有限公司 | Pidotimod preparation method |
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- 2017-12-08 CN CN201711291284.4A patent/CN108088936B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1158620A (en) * | 1994-09-27 | 1997-09-03 | 波利工业化学公司 | Process for the quantitative synthesis of 3-(L-pyrolutamyl)-L-tiazolidine-4-carboxylic acid and derivatives thereof |
| CN102167727A (en) * | 2011-01-29 | 2011-08-31 | 浙江金立源药业有限公司 | Synthesis method of pidotimod |
| CN102952172A (en) * | 2011-08-18 | 2013-03-06 | 北京澳林森科技有限公司 | Pidotimod preparation method |
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| HPLC 法监控L-噻唑烷-4-甲酸乙酯生产中的酯化反应;叶爱英等;《化学试剂》;20130531;第35卷(第5期);第478-480页 * |
| 匹多莫德口服液中有关物质的HPLC法测定;毛柯等;《中国医药工业杂志》;20161231;第47卷(第12期);第1568-1573页 * |
| 新型免疫调节剂匹多莫德的合成;杨柳等;《2011 International Symposium on Biomedicine and Engineering》;20111231;第525-526页 * |
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