CN112574062B - Separation and preparation method of carbidopa enantiomer - Google Patents
Separation and preparation method of carbidopa enantiomer Download PDFInfo
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Abstract
The invention discloses a separation preparation method of carbidopa enantiomer, which adopts a counter-current chromatography for separation and comprises the following steps: and (2) adding n-butyl alcohol: ethyl acetate: pouring water into a liquid separator after the water is prepared according to the volume ratio of 4: 0.8-1.2: 4.5-5.5, and respectively obtaining an upper phase and a lower phase; dissolving a chiral reagent in the upper phase, and mixing the obtained chiral reagent solution with the lower phase to obtain a mixed agent; dissolving D, L-carbidopa in the mixture to obtain a D, L-carbidopa solution; injecting the upper phase into a separation column of a countercurrent chromatograph as a stationary phase, injecting the D, L-carbidopa solution into a sample injection ring of the countercurrent chromatograph, starting the countercurrent chromatograph, and injecting the lower phase as a mobile phase to respectively obtain L-carbidopa and D-carbidopa. The purity of the two carbidopa enantiomers separated by the method can reach more than 95%.
Description
Technical Field
The invention belongs to the technical field of resolution research of a racemate of a medicine, and particularly relates to a separation and preparation method of carbidopa enantiomers, in particular to chiral separation of the carbidopa enantiomers by adopting a high-speed counter-current chromatography.
Background
One third of the new drugs developed in the world are chiral drugs, and the different structures of the drug enantiomers (levo and dextro) result in different affinities for the receptor and thus different activities. The difference in enantiomers has three effects on the activity of a drug:
(1) allowing different activities on the levo and dextro sides, e.g., quinine is an antimalarial and quinidine is a treatment for arrhythmia;
(2) the levorotatory activity and the dextrorotatory activity have different differences, for example, the antibacterial activity of levofloxacin is 20 times of that of dextrorotatory activity;
(3) the levo-dextro has different adverse reactions, such as famous reaction stop events, because the levo-dextro is not completely separated in the production process of the medicine, and the dextro has adverse reactions of fetal teratogenesis. Therefore, when a drug is developed as a racemate, two enantiomers must be studied and proved to have no toxic or side effects or low toxic effects before use.
Carbidopa (Carbidopa)) Is a peripheral dopamine decarboxylase inhibitor with stronger effect, white villous crystal; almost no odor. Is often used in combination with levodopa for the treatment of Parkinson's disease and Parkinson's syndrome. Molecular weight is 244.24Da, molecular formula is C10H14N2O4. Carbidopa has two enantiomers, L-carbidopa and D-carbidopa respectively, and the chemical structure is shown in formula I:
few studies are currently being made on chiral resolution methods for such compounds. According to the technical requirements for developing new stereoisomeric medicine and ensuring the safety and effectiveness of the medicine, all optical isomers of chiral starting materials need to be detected. Therefore, establishing a quick and effective method for chiral resolution of D, L-carbidopa is a subject with theoretical and practical significance.
The chromatographic separation method has very excellent resolution capability, and chiral resolution belongs to the hot spot and the leading edge of the chromatographic research field, but is mainly limited in the fields of high-efficiency capillary electrophoresis, capillary gas chromatography and high-efficiency liquid chromatography. Although the resolution of the 3 methods is high, the separation amount is often in the microgram level, and the 3 methods can only be used as an analysis test means. The prepared liquid chromatogram meets the requirement of general preparation and resolution, can be used for pharmacological and toxicological experiments, but the price of a chiral preparation column can reach tens of thousands to tens of thousands yuan, and the service life of the column is seriously influenced by large sample volume; in addition, each column has a limited range of adaptation, which results in high chiral preparation costs, and even relatively small amounts of chiral material are difficult for many researchers to afford. The countercurrent chromatography has the characteristic of preparative resolution, so that the research on chiral separation of the countercurrent chromatography has positive significance.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for splitting an enantiomer of carbidopa so as to deepen the research and utilization of D, L-carbidopa isomers.
The invention takes D, L-carbidopa as a resolution object, takes (R) - (-) -2-amino-1-butanol as a chiral reagent, adds the chiral reagent into an organic phase, and obtains the D-carbidopa and the L-carbidopa by using a counter-current chromatography.
In order to solve the above technical problems, the present invention provides a separation and preparation method of an enantiomer of carbidopa (i.e., a method for separating and preparing carbidopa L-and D-carbidopa from a D, L-carbidopa enantiomer), which adopts a counter-current chromatography for separation, and comprises the following steps:
1) and (3) adding n-butyl alcohol: ethyl acetate: preparing water according to a volume ratio of 4: 0.8-1.2: 4.5-5.5, pouring the water into a liquid separator to form two-phase solvents which are immiscible with each other, and separating to obtain an upper phase and a lower phase respectively;
2) dissolving (R) - (-) -2-amino-1-butanol serving as a chiral reagent in the upper phase to prepare a chiral reagent solution with the chiral reagent concentration of 18-27 mmol/L;
3) mixing the chiral reagent solution obtained in the step 2) with the lower phase obtained in the step 1) according to the volume ratio of 1:1 to obtain a mixture; dissolving D, L-carbidopa in the mixture to obtain a D, L-carbidopa solution, wherein the feed liquid ratio of the D, L-carbidopa to the mixture is 40-60 mg/9-11 mL;
4) injecting the upper phase obtained in the step 1) into a separation column of a countercurrent chromatograph to be used as a stationary phase, injecting the D, L-carbidopa solution obtained in the step 3) into a sample injection ring of the countercurrent chromatograph, starting the countercurrent chromatograph, injecting the lower phase obtained in the step 1) to be used as a mobile phase, wherein the flow rate of the mobile phase is 1.0-3.0 mL/min, respectively collecting components with retention time of 125-165 min and 185-220 min, and correspondingly obtaining L-carbidopa and D-carbidopa (both purified carbidopa) after rotary evaporation (the temperature is 45 +/-5 ℃ to rotary evaporation).
Remarks explanation: and after the mobile phase is injected, continuously distributing the D, L-carbidopa in a counter-current chromatograph, and collecting and combining effluents (collecting components with the retention time of 125-165 min and 185-220 min) by on-line monitoring of an ultraviolet detector.
As an improvement of the separation preparation method of the carbidopa enantiomer of the invention:
in the step 1), n-butanol: ethyl acetate: water to volume ratio of 4:1: 5.
As a further improvement of the separation preparation method of the carbidopa enantiomer of the invention:
in the chiral reagent solution in the step 2), the concentration of the chiral reagent is 24 mmol/L.
As a further improvement of the separation preparation method of the carbidopa enantiomer of the invention:
in the step 3), the feed-liquid ratio of the D, L-carbidopa to the mixture is 50mg/10 mL.
As a further improvement of the separation preparation method of the carbidopa enantiomer of the invention:
in the step 4), the flow rate of the mobile phase is 2.0 mL/min.
The D, L-carbidopa enantiomer has the molecular weight of 244.24Da and the molecular formula of C10H14N2O4The chemical structural formula is shown as the formula (I).
At present, the reason why countercurrent chromatography has not been used for separation and preparation of carbidopa enantiomers at present is that: a proper chiral selection reagent and a countercurrent chromatography two-phase solvent system are not found, and the invention establishes a method for preparing the chiral selective reagent by using n-butyl alcohol: ethyl acetate: water is used as a solvent system, and (R) - (-) -2-amino-1-butanol is used as a chiral selection reagent, so that the baseline separation of the carbidopa enantiomer in the countercurrent chromatography is realized.
The countercurrent chromatography method adopted by the invention can ensure higher peak type resolution, large separation amount, no sample loss and high recovery rate, and the purity of the two separated carbidopa enantiomers can reach more than 95%. And compared with other traditional resolution methods such as a chemical resolution method and a high performance liquid chromatography, the used counter current chromatograph is simpler and cheaper, and the solvent is prepared from common solvents and is cheap, so the method is an economic method for separating and purifying the carbidopa enantiomer.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a high performance liquid chromatography detection chromatogram of a carbidopa racemate mixture;
FIG. 2 is a high-speed countercurrent chromatographic separation profile of a racemic mixture of carbidopa under the experimental conditions of example 1;
FIG. 3 is a HPLC chromatogram of the 125-165 min eluate of FIG. 2, namely L-carbidopa;
FIG. 4 is a high performance liquid chromatography detection chromatogram of the eluate of 185-220 min in FIG. 2, namely D-carbidopa.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
the racemic mixture of carbidopa used in the following cases was analyzed by analytical high performance liquid chromatography area normalization (as shown in fig. 1), and the content of L-carbidopa was 50.9% and the content of D-carbidopa was 48.0%.
The high performance liquid chromatography analysis of the carbidopa racemate mixture and the purified L-type and D-type carbidopa are carried out according to the following high performance liquid chromatography: a chromatographic column: CHIROBIOTICTM T column (chiral analytical column), 4.6mm × 25cm × 5um or equivalent; detection wavelength: UV230 nm; flow rate: 1.0 ml/min; sample introduction amount: 20 ul; column temperature: 30 ℃; operating time: 20 min; mobile phase: 20mmol ammonium acetate buffer (0.31 g ammonium acetate was weighed into 200ml purified water, pH adjusted to 5.3 with acetic acid)/methanol 20/80 (volume ratio), filtered through 0.45um filter membrane, mixed and degassed.
Example 1 a process for the isolation and preparation of L-carbidopa and D-carbidopa from D, L-carbidopa using a counter current chromatograph, the following steps being carried out in order:
1) preparing a two-phase solvent from n-butyl alcohol, ethyl acetate and water according to the volume ratio of 4:1:5, standing and layering the two-phase solvent in a liquid separation container to form two-phase solvents which are insoluble with each other, and separating the two-phase solvents to obtain an upper phase and a lower phase respectively.
2) Weighing chiral reagent (R) - (-) -2-amino-1-butanol, dissolving in 500mL upper phase, and preparing into chiral reagent solution with concentration of 24 mmol/L.
3) Mixing 5ml of chiral reagent solution (containing the upper phase of the chiral reagent) and 5ml of lower phase to obtain a mixture; dissolving 50mg of D, L-carbidopa in the above mixture (10ml) to obtain a carbidopa solution (D, L-carbidopa solution) as a sample solution;
4) the inner diameter of the countercurrent chromatographic column is 2.6mm, and the volume of the column is 310 ml. The upper phase is used as a stationary phase, the lower phase is used as a mobile phase, after the stationary phase is filled in a separation column of the countercurrent chromatograph, a sample solution is injected into a sample injection ring of the countercurrent chromatograph, the countercurrent chromatograph is started, the rotating speed is 800 revolutions, the mobile phase is injected at the flow rate of 2.0ml/min, the flow fraction is monitored by an online ultraviolet detector, the detection wavelength is 230nm, and the separation spectrogram of the countercurrent chromatograph is shown in figure 2.
Collecting and combining the effluent (collecting the components with retention time of 125-165 min and 185-220 min respectively), respectively performing rotary evaporation (at 45 deg.C until rotary drying) to obtain 20.5mg of L-carbidopa and 19.8mg of D-carbidopa respectively with purity of 98.5% and 99.2%, see FIG. 3 and FIG. 4.
Example 2, a method for isolating and preparing L-carbidopa and D-carbidopa from D, L-carbidopa:
the injection flow rate of the mobile phase in step 4) of example 1 was changed from 2.0mL/min to 3.0mL/min, and the rest was identical to example 1. The final L-carbidopa and D-carbidopa have purity of about 95.0%.
Example 3 a method for the isolation of L-carbidopa and D-carbidopa from D, L-carbidopa:
the concentration of chiral reagent (R) - (-) -2-amino-1-butanol in step 2) of example 1 was changed from 24mmol/L to 48mmol/L, and the injection flow rate of the mobile phase was changed from 2.0mL/min to 3.0mL/min, the rest being identical to example 1. The final L-carbidopa and D-carbidopa have purity of about 85.0%.
Comparative example 1-1, the two-phase solvent system consisting of n-butanol, ethyl acetate and water (in a volume ratio of 4:1:5) in example 1 was changed to n-butanol, ethyl acetate and water (in a volume ratio of 8:1:11), and the rest was the same as in example 1.
Comparative example 1-2. the two-phase solvent system consisting of n-butanol, ethyl acetate and water (in a volume ratio of 4:1:5) in example 1 was changed to n-butanol, ethyl acetate and water (in a volume ratio of 2:1:2.5), and the rest was the same as in example 1.
Comparative example 2-1, the two-phase solvent system consisting of n-butanol, ethyl acetate and water (in a volume ratio of 4:1:5) in example 1 was changed to n-hexane, ethyl acetate and water (in a volume ratio of 4:1:5), and the rest was the same as in example 1.
Comparative example 2-2, the two-phase solvent system consisting of n-butanol, ethyl acetate and water (in a volume ratio of 4:1:5) in example 1 was changed to n-butanol, acetone and water (in a volume ratio of 4:1:5), and the rest was identical to example 1.
Comparative examples 2 to 3, the two-phase solvent system consisting of n-butanol, ethyl acetate and water (volume ratio of 4:1:5) in example 1 was changed to n-hexane, acetone and water (volume ratio of 8:3:9), and the rest was the same as in example 1.
Comparative example 3, the addition of the chiral reagent (R) - (-) -2-amino-1-butanol of example 1 was eliminated, and the remainder was the same as in example 1.
Comparative example 4, the chiral reagent of example 1 was changed from (R) - (-) -2-amino-1-butanol to that described in table 1, and the rest was the same as example 1.
The purity of L-carbidopa and D-carbidopa prepared by all the above comparative examples is shown in Table 1.
TABLE 1
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (4)
1. The separation preparation method of carbidopa enantiomer is characterized in that the separation is carried out by adopting a counter-current chromatography, and comprises the following steps:
1) and (3) adding n-butyl alcohol: ethyl acetate: water is prepared according to the volume ratio of 4:1:5 and then poured into a liquid separator to form two-phase solvents which are not mutually soluble, and after separation, an upper phase and a lower phase are respectively obtained;
2) dissolving (R) - (-) -2-amino-1-butanol serving as a chiral reagent in the upper phase to prepare a chiral reagent solution with the chiral reagent concentration of 18-27 mmol/L;
3) mixing the chiral reagent solution obtained in the step 2) with the lower phase obtained in the step 1) according to the volume ratio of 1:1 to obtain a mixture; dissolving D, L-carbidopa in the mixture to obtain a D, L-carbidopa solution, wherein the feed liquid ratio of the D, L-carbidopa to the mixture is 40-60 mg/9-11 mL;
4) injecting the upper phase obtained in the step 1) into a separation column of a countercurrent chromatograph to be used as a stationary phase, injecting the D, L-carbidopa solution obtained in the step 3) into a sample injection ring of the countercurrent chromatograph, starting the countercurrent chromatograph, injecting the lower phase obtained in the step 1) to be used as a mobile phase, wherein the flow rate of the mobile phase is 1.0-3.0 mL/min, respectively collecting components with retention time of 125-165 min and 185-220 min, and correspondingly obtaining L-carbidopa and D-carbidopa after rotary evaporation.
2. The process for the preparation of an enantiomer of carbidopa according to claim 1 wherein:
in the chiral reagent solution in the step 2), the concentration of the chiral reagent is 24 mmol/L.
3. The process for the preparation of carbidopa enantiomers as claimed in claim 2, wherein:
in the step 3), the feed-liquid ratio of the D, L-carbidopa to the mixture is 50mg/10 mL.
4. A method for the preparation of an enantiomer of carbidopa according to any one of claims 1 to 3, characterized in that:
in the step 4), the flow rate of the mobile phase is 2.0 mL/min.
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