CN108707148B - Chiral resolution method of four isomers in racanisodamine - Google Patents

Chiral resolution method of four isomers in racanisodamine Download PDF

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CN108707148B
CN108707148B CN201810596799.3A CN201810596799A CN108707148B CN 108707148 B CN108707148 B CN 108707148B CN 201810596799 A CN201810596799 A CN 201810596799A CN 108707148 B CN108707148 B CN 108707148B
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racanisodamine
crystal
resolving agent
methanol
dissolving
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CN108707148A (en
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杨宝峰
李畅
朱久新
王立波
韩维娜
黄健
王金辉
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Harbin Medical University
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D451/00—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof
    • C07D451/02—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof containing not further condensed 8-azabicyclo [3.2.1] octane or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane; Cyclic acetals thereof
    • C07D451/04—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof containing not further condensed 8-azabicyclo [3.2.1] octane or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane; Cyclic acetals thereof with hetero atoms directly attached in position 3 of the 8-azabicyclo [3.2.1] octane or in position 7 of the 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring system
    • C07D451/06—Oxygen atoms
    • C07D451/10—Oxygen atoms acylated by aliphatic or araliphatic carboxylic acids, e.g. atropine, scopolamine

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Abstract

The invention discloses a chiral resolution method of four isomers in racanisodamine. The invention uses D or L-p-methyl dibenzoyl tartaric acid as resolving agent, and adopts preparative HPLC combined chemical resolving method to resolve racanisodamine. The method has reasonable design, is feasible, has higher product yield and high purity, and the obtained four optical isomers of the anisodamine, namely 6R,2' R, 6R,2' S, 6S and 2' R, have the purity of more than 95 percent, thereby being an effective method for industrially producing the four optical isomers of the anisodamine. The method successfully realizes the resolution of the four optical isomers, can effectively reduce the resolution cost of the four optical isomers of the anisodamine, and has wide social benefit, huge economic benefit and wide application prospect.

Description

Chiral resolution method of four isomers in racanisodamine
Technical Field
The invention relates to a chiral resolution method of four isomers in racanisodamine, which can realize the chiral resolution of four optical isomers in racanisodamine. The invention belongs to the technical field of medicines.
Background
Anisodamine can compete with acetylcholine for antagonizing M receptor, and can be used for treating circulatory disturbance caused by smooth muscle spasm, neuralgia, fulminant meningitis, coccal meningitis, toxic dysentery and vasospasm. The racanisodamine is a synthetic product (654-2) of anisodamine, and comprises four optical isomers, namely 6R,2'S, 6S,2' R, 6R,2'R, 6S,2' S configuration. Relevant animal experiments prove that the 6S and 2' S isomer has the strongest pharmacological activity on the relaxation effect of isolated rat trachea. On the other hand, the 6R,2' S isomer shows the strongest pharmacological activity on the relaxation effect of isolated rat small intestine smooth muscle. Therefore, the racanisodamine is separated into four optical isomers by a chiral resolution method, and the single optical isomer is used for medication, so that the safety and the effectiveness of the medicine can be greatly improved. The patent application with the publication number of CN107991367A attempts to realize the resolution of four optical isomers of anisodamine by adopting a capillary electrophoresis method, but the method has the defects of long time consumption, small preparation amount and the like, is only limited to laboratory preparation, and cannot be applied to industrial large-scale production.
The invention aims to provide a chiral resolution method of four isomers in racanisodamine, which comprises the steps of firstly resolving the racanisodamine into a pair of isomers 654-2-A with the configuration of 6R,2'S +6S,2' R and a pair of isomers 654-2-B with the configuration of 6R,2'R +6S,2' S by preparative High Performance Liquid Chromatography (HPLC). Secondly, taking safe D/L p-methyl dibenzoyl tartaric acid as a resolving agent, and carrying out 3 times of recrystallization to carry out chemical resolution on 654-2-A and 654-2-B, thereby finally obtaining four anisodamine optical isomers with higher purity, namely 6R,2'S, 6S,2' R, 6R,2'R, 6S and 2' S. The chiral resolution of racanisodamine can be realized by the resolution method, the resolution time can be effectively saved, the production cost is reduced, and the method is suitable for industrial production.
Disclosure of Invention
The invention aims to provide a chiral resolution method of racanisodamine, which adopts a method of combining preparative high performance liquid chromatography with chemical resolution and takes safe D/L p-methyl dibenzoyl tartaric acid as a resolving agent to obtain four optical isomers of the racanisodamine with higher purity. Thereby effectively saving the splitting time and reducing the production cost, and being suitable for industrial production.
In order to achieve the purpose, the invention adopts the following technical means:
the invention relates to a chiral resolution method of four isomers in racanisodamine, which comprises the following steps:
step 1, completely dissolving a racanisodamine sample by using an aqueous solution containing 45 percent (v/v) of methanol, and filtering the solution by using a 0.22 mu m microporous filter membrane for later use;
step 2, separating the racanisodamine obtained in the step 1 by adopting preparative HPLC (high performance liquid chromatography) to respectively obtain a racemate 654-2-A and a racemate 654-2-B, wherein the racemate 654-2-A contains two configurations of 6R,2'S and 6S,2' R, and the racemate 654-2-B contains two configurations of 6R,2'R and 6S,2' S;
and 3, splitting the obtained 654-2-A and 654-2-B by a chemical splitting method, wherein D-p-methyl dibenzoyl tartaric acid or L-p-methyl dibenzoyl tartaric acid is used as a splitting agent, and when 654-2-A is split, the adopted solvent system is acetonitrile: the volume ratio of methanol is 15: 1; when 654-2-B is disassembled, the adopted solvent system is ethanol, and needle crystals and granular crystals are respectively formed after 24-48 hours;
step 4, washing, filtering and recrystallizing the precipitated crystals for 3 times;
and 5, reducing the four recrystallized crystals by using a saturated sodium carbonate solution in an ice bath, extracting for 3 times by using dichloromethane with the same volume, combining the extract liquor, and volatilizing the solvent to obtain the four anisodamine optical isomers.
The three-dimensional configuration of four isomers in the synthesis of anisodamine is shown in figure 1.
Preferably, the mobile phase used for HPLC chromatographic separation in step 2 is a mixed solution of methanol, water and diethylamine, wherein the ratio of methanol: water: the volume ratio of diethylamine is 45:55: 0.02; the chromatographic conditions are as follows: the detection wavelength is 210nm, the chromatographic column is an Agilent Prep C18 column, the column temperature is 20 ℃, the sample injection amount is 1mL, and the flow rate is 5 mL/min.
Preferably, the separation of 654-2-A in step 3 is carried out according to the following steps: dissolving a compound 654-2-A in methanol, dissolving a resolving agent D-p-methyl dibenzoyl tartaric acid or L-p-methyl dibenzoyl tartaric acid in acetonitrile, mixing the 654-2-A and the resolving agent after complete dissolution, reacting for 30min at 60 ℃ to generate white flocculent precipitate, filtering, taking filtrate, placing at 4 ℃ to generate a large amount of white needle crystals.
Wherein, the preferable mol ratio of the added resolving agent to 654-2-A to be resolved is 1.5: 1.
preferably, the separation of 654-2-B in step 3 is carried out according to the following steps: dissolving a compound 654-2-B in an ethanol solution, dissolving a resolving agent D-p-methyl dibenzoyl tartaric acid or L-p-methyl dibenzoyl tartaric acid in ethanol, mixing the 654-2-B and the resolving agent after completely dissolving, reacting for 30min at 60 ℃, filtering, and storing the filtrate at 4 ℃ to generate a large amount of granular crystals.
Wherein, the preferable mol ratio of the added resolving agent to 654-2-B to be resolved is 1.5: 1.
preferably, in the step 4, the needle-shaped crystal obtained after the resolution of 654-2-A is filtered, the crystal is washed with pure acetonitrile for 2 times, and the obtained crystal is recrystallized for 3 times under a pure acetonitrile system; filtering granular crystal obtained after 654-2-B resolution, washing the crystal with absolute ethyl alcohol for 2 times, and recrystallizing the obtained crystal for 3 times under an absolute ethyl alcohol system.
Compared with the prior art, the invention has the beneficial effects that:
the invention uses D or L-p-methyl dibenzoyl tartaric acid as resolving agent, and adopts preparative HPLC combined chemical resolving method to resolve racanisodamine. The method has reasonable design, is feasible, has higher product yield and high purity, and the obtained four optical isomers of the anisodamine, namely 6R,2' R, 6R,2' S, 6S and 2' R, have the purity of more than 95 percent, thereby being an effective method for industrially producing the four optical isomers of the anisodamine. The method successfully realizes the resolution of the four optical isomers, can effectively reduce the resolution cost of the four optical isomers of the anisodamine, and has wide social benefit, huge economic benefit and wide application prospect.
Drawings
FIG. 1 shows the structures of four optical isomers in the synthesis of anisodamine;
FIG. 2 is an HPLC chromatogram (210nm) of 654-2-A, 654-2-B and racanisodamine;
FIG. 3 is a chiral HPLC analysis chromatogram (210nm) of 654-2-A;
FIG. 4 is a chiral HPLC analysis chromatogram (210nm) of 654-2-B.
Detailed Description
The present invention discloses a resolution method of racanisodamine, and the technical personnel can appropriately modify the technological parameters by referring to the content in the present document. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
Example 1 chiral resolution of four isomers in racanisodamine
1. Preparation of samples
A racanisodamine sample (sample concentration: 100mg/mL) was completely dissolved in an aqueous solution containing 45% (v/v) methanol, and passed through a 0.22 μm microfiltration membrane for use.
2. HPLC chromatographic separation
Mobile phase: a mixed solution of methanol, water and diethylamine, wherein the ratio of methanol: water: the volume ratio of diethylamine was 45:55: 0.02.
Chromatographic conditions are as follows: the detection wavelength is 210nm, the chromatographic column is an Agilent Prep C18 column (250mm multiplied by 9.4mm), the column temperature is 20 ℃, the sample injection amount is 1mL, and the flow rate is 5 mL/min.
And (3) carrying out HPLC analysis on the separated product under the analysis conditions: mobile phase methanol: water: the diethylamine volume ratio is 50:50:0.02, the chromatographic column is a cosmosil MS-II C18 column, the column temperature is 35 ℃, the detection wavelength is 210nm, the injection volume is 10 muL, the flow rate is 1mL/min, and the analysis result is shown in figure 2. Wherein 654-2-A (purity 97.8%) was prepared in 51.2% yield and 654-2-B (purity 97.2%) in 39.4% yield.
3. Separation of 654-2-A (6R,2'S +6S,2' R) in racanisodamine;
and (3) culturing the crystal: compound 654-2-A (21.71mg) is taken to be dissolved in 100 mul of methanol, 41.18mg of resolved acid D-p-methyl dibenzoyl tartaric acid is dissolved in 1.5mL of acetonitrile, after the two are completely dissolved, the two are mixed and reacted for 30min at the temperature of 60 ℃, a little white flocculent precipitate is generated, and the filtrate is taken after filtration. Placing the mixture at 4 ℃ for 48 hours to generate a large amount of white needle-shaped crystals. The crystals were filtered and washed 2 times with pure acetonitrile.
And (3) treating the crystals: recrystallizing the obtained crystal for 3 times in a 300 mu L pure acetonitrile system, taking a small amount of the crystal, putting the small amount of the crystal into an EP tube, adding a sodium carbonate solution under ice bath, and repeatedly blowing and beating until the crystal is completely dissolved. Extracted 3 times with equal volume of dichloromethane. The dichloromethane layer is taken out after the liquid separation, the extracts are combined, and the sample is numbered 654-2-A1 after the solvent is drained. The sample was dissolved in 500. mu.L of methanol and passed through a 0.22 μm filter and the filtrate was taken for HPLC analysis. Similarly, another anisodamine with another configuration, No. 654-2-A2, can be obtained by using another resolving agent L-p-methyl dibenzoyl tartaric acid and the same method, and the two obtained samples are subjected to HPLC detection by using a chiral chromatographic column. HPLC analysis conditions were n-hexane: ethanol: the volume ratio of diethylamine is 90: 10: 0.1, column model: daicel Chiral cell OX-H (5 μm), flow rate: 1mL/min, column temperature: the results of the analysis at 35 ℃ are shown in FIG. 3.
4. Separation of 654-2-B (6R,2'R +6S,2' S) in racanisodamine;
formation of crystals: dissolving 654-2-B (21.71mg) in 700. mu.l ethanol solution, dissolving 41.20mg of resolving acid L-p-methylbenzoyl tartaric acid in 100. mu.l ethanol solution, mixing the two solutions, reacting at 60 deg.C for 30min, and filtering. The filtrate was stored at 4 ℃ for 24 hours, and a large number of granular crystals appeared, and the crystals were filtered and washed with anhydrous ethanol 2 times.
And (3) treating the crystals: recrystallizing the obtained crystal for 3 times in a 300 mu L absolute ethyl alcohol system, taking a small amount of the crystal, putting the small amount of the crystal into an EP tube, adding a sodium carbonate solution under ice bath, and repeatedly blowing and beating until the crystal is completely dissolved. Extracted 3 times with equal volume of dichloromethane. The dichloromethane layer was taken out by separating the liquids, and the extracts were combined. After the solvent was drained, sample number 654-2-B1. After the methylene chloride had evaporated to dryness, the sample was dissolved in 500. mu.L of methanol and passed through a 0.22 μm filter and the filtrate was taken for HPLC analysis. Similarly, using the resolution reagent L-p-methyl dibenzoyltartaric acid, another configuration of anisodamine, No. 654-2-B2, was obtained in the same manner, and the analytical results are shown in FIG. 4 by HPLC detection of the sample using the above chiral chromatographic column and analytical conditions.
The absolute configurations and purities of the four optical isomers 654-2-A1, 654-2-A2, 654-2-B1 and 654-2-B2 of the obtained racanisodamine are shown in Table 1.
Table 1654-2 yield and purity results for four stereoisomers
Numbering Configuration(s) Purity of
654-2-A1 6S,2'R 98.3％
654-2- A2 6R,2'S 97.5％
654-2-B1 6R,2'R 95.3％
654-2- B2 6S,2'S 98.6％

Claims (3)

1. A chiral resolution method of four isomers in racanisodamine is characterized by comprising the following steps:
step 1, completely dissolving a racanisodamine sample by using an aqueous solution containing 45 percent (v/v) of methanol, and filtering the solution by using a 0.22 mu m microporous filter membrane for later use;
step 2, separating the racanisodamine obtained in the step 1 by adopting preparative HPLC (high performance liquid chromatography) to respectively obtain a racemate 654-2-A and a racemate 654-2-B, wherein the racemate 654-2-A contains two configurations of 6R,2'S and 6S,2' R, and the racemate 654-2-B contains two configurations of 6R,2'R and 6S,2' S;
the mobile phase adopted for HPLC chromatographic separation is a mixed solution of methanol, water and diethylamine, wherein the mass ratio of methanol: water: the volume ratio of diethylamine is 45:55: 0.02; the chromatographic conditions are as follows: the detection wavelength is 210nm, the chromatographic column is an Agilent Prep C18 column, the column temperature is 20 ℃, the sample injection amount is 1mL, and the flow rate is 5 mL/min;
step 3, splitting the obtained 654-2-A and 654-2-B by a chemical splitting method, wherein the 654-2-A is split according to the following steps: dissolving a compound 654-2-A in methanol, dissolving a resolving agent D-p-methyl dibenzoyl tartaric acid or L-p-methyl dibenzoyl tartaric acid in acetonitrile, mixing the 654-2-A and the resolving agent after completely dissolving, reacting for 30min at 60 ℃ to generate white flocculent precipitate, filtering, taking filtrate, placing at 4 ℃ to generate a large amount of white needle crystals; acetonitrile: the volume ratio of methanol is 15: 1;
the resolution of 654-2-B is carried out according to the following steps: dissolving a compound 654-2-B in an ethanol solution, dissolving a resolving agent D-p-methyl dibenzoyl tartaric acid or L-p-methyl dibenzoyl tartaric acid in ethanol, mixing the 654-2-B and the resolving agent after completely dissolving, reacting for 30min at 60 ℃, filtering, storing the filtrate at 4 ℃, and generating a large amount of granular crystals after 24-48 h;
step 4, filtering the needle crystal obtained after the resolution of 654-2-A, washing the crystal with pure acetonitrile for 2 times, and recrystallizing the obtained crystal for 3 times in a pure acetonitrile system; filtering granular crystal obtained after 654-2-B is split, washing the crystal with absolute ethyl alcohol for 2 times, and recrystallizing the obtained crystal for 3 times in an absolute ethyl alcohol system;
and 5, reducing the four recrystallized crystals by using a saturated sodium carbonate solution in an ice bath, extracting for 3 times by using dichloromethane with the same volume, combining the extract liquor, and volatilizing the solvent to obtain the four anisodamine optical isomers.
2. The process of claim 1, wherein the mole ratio of the added amount of resolving agent to 654-2-a to be resolved is 1.5: 1.
3. the process of claim 1, wherein the mole ratio of the added amount of resolving agent to 654-2-B to be resolved is 1.5: 1.
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Title
HPLC分离合成山莨菪碱(654-2)中差向异构体和对映异构体的研究;杨丽敏 等;《2005中国药学杂志岛津杯第七届全国药物分析优秀论文评选交流会》;20130516;第159-161页，尤其是第160页2.1和2.3 *
山莨菪碱4个异构体的分离及其抗胆碱活性比较;孙凯 等;《上海交通大学学报（医学版）》;20110430;第31卷(第4期);第406-410页，尤其是第407页1.2.2和1.2.3，以及第408页2.1 *
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