CN114773332A - Trans isomer of anisodamine and its separation and detection method - Google Patents
Trans isomer of anisodamine and its separation and detection method Download PDFInfo
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- CN114773332A CN114773332A CN202210340806.XA CN202210340806A CN114773332A CN 114773332 A CN114773332 A CN 114773332A CN 202210340806 A CN202210340806 A CN 202210340806A CN 114773332 A CN114773332 A CN 114773332A
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- anisodamine
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- WTQYWNWRJNXDEG-UHFFFAOYSA-N 6-Hydroxy-hyoscyamin Natural products CN1C(C2)CC(O)C1CC2OC(=O)C(CO)C1=CC=CC=C1 WTQYWNWRJNXDEG-UHFFFAOYSA-N 0.000 title claims abstract description 123
- WTQYWNWRJNXDEG-LEOABGAYSA-N anisodamine Chemical compound C1([C@@H](CO)C(=O)O[C@@H]2C[C@H]3[C@@H](O)C[C@@H](C2)N3C)=CC=CC=C1 WTQYWNWRJNXDEG-LEOABGAYSA-N 0.000 title claims abstract description 110
- 238000000926 separation method Methods 0.000 title claims abstract description 50
- DYLIWHYUXAJDOJ-OWOJBTEDSA-N (e)-4-(6-aminopurin-9-yl)but-2-en-1-ol Chemical compound NC1=NC=NC2=C1N=CN2C\C=C\CO DYLIWHYUXAJDOJ-OWOJBTEDSA-N 0.000 title claims abstract description 34
- 238000001514 detection method Methods 0.000 title abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 22
- 239000000178 monomer Substances 0.000 claims abstract description 19
- 238000004808 supercritical fluid chromatography Methods 0.000 claims abstract description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 51
- 238000010828 elution Methods 0.000 claims description 14
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 12
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 claims description 11
- 239000002904 solvent Substances 0.000 claims description 11
- 239000006184 cosolvent Substances 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 7
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 6
- 235000019253 formic acid Nutrition 0.000 claims description 6
- 238000002953 preparative HPLC Methods 0.000 claims description 6
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- 239000012535 impurity Substances 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
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- 239000000126 substance Substances 0.000 description 4
- CPELXLSAUQHCOX-UHFFFAOYSA-N Hydrogen bromide Chemical compound Br CPELXLSAUQHCOX-UHFFFAOYSA-N 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- BMYNFMYTOJXKLE-UHFFFAOYSA-N 3-azaniumyl-2-hydroxypropanoate Chemical compound NCC(O)C(O)=O BMYNFMYTOJXKLE-UHFFFAOYSA-N 0.000 description 2
- 201000009906 Meningitis Diseases 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
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- XTLNYNMNUCLWEZ-UHFFFAOYSA-N ethanol;propan-2-one Chemical compound CCO.CC(C)=O XTLNYNMNUCLWEZ-UHFFFAOYSA-N 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- SYSQUGFVNFXIIT-UHFFFAOYSA-N n-[4-(1,3-benzoxazol-2-yl)phenyl]-4-nitrobenzenesulfonamide Chemical class C1=CC([N+](=O)[O-])=CC=C1S(=O)(=O)NC1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 SYSQUGFVNFXIIT-UHFFFAOYSA-N 0.000 description 2
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- SBTVLCPCSXMWIQ-UHFFFAOYSA-N (3,5-dimethylphenyl) carbamate Chemical compound CC1=CC(C)=CC(OC(N)=O)=C1 SBTVLCPCSXMWIQ-UHFFFAOYSA-N 0.000 description 1
- KZCXMZLEEWLBFE-UHFFFAOYSA-N (3-chloro-4-methylphenyl) carbamate Chemical compound CC1=CC=C(OC(N)=O)C=C1Cl KZCXMZLEEWLBFE-UHFFFAOYSA-N 0.000 description 1
- RKUNBYITZUJHSG-FXUDXRNXSA-N (S)-atropine Chemical compound C1([C@@H](CO)C(=O)O[C@H]2C[C@H]3CC[C@@H](C2)N3C)=CC=CC=C1 RKUNBYITZUJHSG-FXUDXRNXSA-N 0.000 description 1
- RKUNBYITZUJHSG-UHFFFAOYSA-N Hyosciamin-hydrochlorid Natural products CN1C(C2)CCC1CC2OC(=O)C(CO)C1=CC=CC=C1 RKUNBYITZUJHSG-UHFFFAOYSA-N 0.000 description 1
- 208000007101 Muscle Cramp Diseases 0.000 description 1
- 208000005392 Spasm Diseases 0.000 description 1
- 206010047163 Vasospasm Diseases 0.000 description 1
- ZVQOOHYFBIDMTQ-UHFFFAOYSA-N [methyl(oxido){1-[6-(trifluoromethyl)pyridin-3-yl]ethyl}-lambda(6)-sulfanylidene]cyanamide Chemical compound N#CN=S(C)(=O)C(C)C1=CC=C(C(F)(F)F)N=C1 ZVQOOHYFBIDMTQ-UHFFFAOYSA-N 0.000 description 1
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 1
- 229960004373 acetylcholine Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
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- 208000001848 dysentery Diseases 0.000 description 1
- 238000003821 enantio-separation Methods 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 229930005342 hyoscyamine Natural products 0.000 description 1
- 229960003210 hyoscyamine Drugs 0.000 description 1
- 238000006317 isomerization reaction Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 208000004296 neuralgia Diseases 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229960004499 scopolamine hydrobromide Drugs 0.000 description 1
- WTGQALLALWYDJH-MOUKNHLCSA-N scopolamine hydrobromide (anhydrous) Chemical compound Br.C1([C@@H](CO)C(=O)O[C@H]2C[C@@H]3N([C@H](C2)[C@@H]2[C@H]3O2)C)=CC=CC=C1 WTGQALLALWYDJH-MOUKNHLCSA-N 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 210000003437 trachea Anatomy 0.000 description 1
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-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- 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
-
- 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
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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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
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
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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
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- G01N30/60—Construction of the column
- G01N30/6052—Construction of the column body
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- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
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- C07B2200/07—Optical isomers
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- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
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Abstract
The invention belongs to the field of medicine separation and analysis, and particularly relates to a trans-isomer of anisodamine and a separation and detection method thereof. The method has the advantages that the trans-isomer of the racanisodamine can be effectively separated by the combination of HPLC and supercritical chromatography, and then the separation degree is improved by creative chromatographic parameter selection in the HPLC and supercritical chromatography, so that the purity of two trans-isomer monomers of the finally obtained anisodamine is high, and a foundation is laid for large-scale industrial application.
Description
Technical Field
The invention belongs to the field of medicine separation and analysis, and particularly relates to a trans-isomer of anisodamine and a separation and detection method thereof.
Background
Anisodamine can compete with acetylcholine to antagonize 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, which are 6R,2'S, 6S,2' R, 6R,2'R, 6S,2' S configuration. Relevant animal experiments prove that the 6S,2' S isomer shows 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 existing resolution method adopts a capillary electrophoresis method or HPLC to realize the resolution of four optical isomers of anisodamine.
However, the conventional separation method has the defects of low purity of the separated optical isomer and the like, and thus cannot be applied to the requirements of industrial scale production and marketing.
Disclosure of Invention
The invention provides a trans-isomer of anisodamine and a separation and detection method thereof, aiming at solving the technical problems of low purity of the separated optical isomer and the like in the prior separation method in the background technology.
The technical scheme adopted by the invention is as follows:
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. The invention defines the 6R,2'R and 6S,2' S configurations as trans-anisodamine and the 6R,2'S and 6S,2' R configurations as cis-anisodamine.
The invention provides a separation method of trans-isomer of anisodamine, which comprises the following steps:
s1, removing anisodamine, and separating by dynamic preparative HPLC to obtain cis-anisodamine containing 6R,2'S and 6S,2' R and trans-anisodamine containing 6R,2'R and 6S,2' S;
s2, adjusting the pH values of the cis-anisodamine and the trans-anisodamine obtained in the step S1 to 8.5-10, extracting at least 1 time by using chloroform in equal proportion, combining the extracts, and spin-drying to obtain purified cis-anisodamine and trans-anisodamine;
s3, dissolving the trans-anisodamine obtained in the step S2 by using a solvent, then resolving by adopting a supercritical chromatography, and then spinning to obtain 6R,2'R anisodamine and 6S,2' S anisodamine respectively, wherein the specific parameters of the supercritical chromatography are as follows:
and (3) chromatographic column: 30 x 250mm10 μm;
mobile phase: supercritical CO2、N 20 and NH3One of (a) and (b);
cosolvent: methanol and 0.2% of 7mol of NH3The mass of the cosolvent is 15-30% of that of the mobile phase;
cycle time: 10-20 min;
operating time: 20-30 min;
wavelength: 200-240 nm;
back pressure: 90-110 bar;
column temperature: 30-40 ℃;
flow rate of mobile phase: 30-40 ml/min;
total flow rate: 40-50 ml/min.
The invention has the beneficial effects that: through the combination of HPLC and supercritical chromatography, the separation and extraction of the trans-isomer in anisodamine can be smoothly realized, the purity of the extracted trans-isomer monomer is higher than 99%, the purity of the salified anisodamine hydrobromide is still higher than 99%, the purity requirement of Chinese pharmacopoeia can be met and far higher, compared with the purity of the trans-isomer monomer obtained by the isomerization separation of the racemic anisodamine on the market, the purity of the trans-isomer monomer is greatly improved, the quality and the safety of the product are directly improved, and the technical problem in the background technology is solved. The single optical isomer is used for medicine application, so that the safety and the effectiveness of the medicine can be greatly improved.
On the basis of the technical scheme, the invention can be improved as follows.
Further, in step S3, the mobile phase is supercritical CO2。
The beneficial effect of adopting the further scheme is that CO2The supercritical fluid phase condition of the method can lead the separation effect of the trans-isomer in the racanisodamine to be better and realize effective separation.
Further, in step S3, the flow rate of the mobile phase is 33.75ml/min, and the mass of the cosolvent is 25% of the mass of the mobile phase.
The further scheme has the beneficial effects that the appropriate flow rate and the mass ratio of the cosolvent can shorten the peak time and ensure that the racanisodamine can successfully realize the separation of isomers.
Further, in step S3, the solvent is methanol, and the wavelength is 215 nm.
The method has the beneficial effects that the separation degree can be effectively improved by adopting methanol as a solvent, and the resolution of racanisodamine is facilitated.
Further, in step S3, the column temperature was 35 ℃, the chromatography column was CHIRALCEL OX (30 x 250mm10 μm) (Daicel), the cycle time was 13.5min, the run time was 25min, the back pressure was 100bar, and the total flow rate was 45 ml/min.
The method has the advantages that the implementation of the optimal parameters can ensure that the trans-isomer in the racanisodamine is successfully split, the splitting effect is good, and the quality of the product obtained by subsequently using two trans-isomer monomers as raw materials for drug synthesis is directly improved.
Further, in step S1, the chromatographic parameters for the preparative HPLC separation are as follows:
and (3) chromatographic column: DAC80 dynamic preparation separation column;
packing of a chromatographic column: huaban LD-2-C18;
flow rate: 150-220 ml/min;
wavelength: 200-220 nm;
when the elution time is 0-45 min, the mobile phase is a mixture of 0.1% formic acid and methanol in a mass ratio of 95:5, and when the elution time is 45-60 min, the mobile phase is a mixture of 0.1% formic acid and methanol in a mass ratio of 30: 70.
The beneficial effect of adopting above-mentioned further scheme is, can be to better carrying out quality control of split process, promoted the quality that obtains the product after the split.
Further, in step S1, the flow rate was 200ml/min and the wavelength was 210 nm.
The method has the advantages that the trans-isomer of the racanisodamine can be effectively separated through the combination of HPLC and supercritical chromatography, and then the separation degree is improved through creative chromatographic parameter selection in the HPLC and supercritical chromatography, so that the purity of two trans-isomer monomers of the racanisodamine finally obtained is high, and a foundation is laid for large-scale industrial application.
In another aspect, the present invention further provides a method for detecting a pair of trans isomers of anisodamine, comprising the following steps:
taking 6R,2'R anisodamine or 6S,2' S anisodamine, and detecting by LCMS, wherein the parameters of the LCMS are as follows:
a chromatographic column: waters X Bridge C18 column (50mm 4.6mm 3.5 um);
mobile phase: the mobile phase A is 0.01mol/L NH4HCO3The mobile phase B is acetonitrile;
the flow rate is 2mL/min, the column temperature is 40 ℃, when the elution time is 0-1.6 min, the mobile phase B is 5% -95%, and when the elution time is 1.6-3 min, the mobile phase B is 95%.
The method has the beneficial effects that the separated trans-isomer can be successfully and effectively detected through the detection of LCMS, the peak is smoothly formed, and the peak type is good.
In another aspect, the invention also provides a 6R,2' R anisodamine monomer, prepared by the separation method of trans-isomer of anisodamine as described above.
The invention has the beneficial effect that the high-purity 6R,2' R anisodamine monomer can be successfully obtained.
In another aspect, the invention also provides a 6S,2' S anisodamine monomer, prepared by the separation method of trans-isomer of anisodamine as described above.
The invention has the beneficial effect that the high-purity 6S,2' S anisodamine monomer can be successfully obtained.
Drawings
FIG. 1 is a resolution diagram of cis-form anisodamine and trans-form anisodamine according to the present invention;
FIG. 2 is a chromatogram before chiral resolution of trans-anisodamine according to the present invention;
FIG. 3 is a trans-pre-peak chromatogram of the present invention;
FIG. 4 is a trans-late chromatogram of the present invention;
FIG. 5 is a LCMS chromatogram of anisodamine of 6R,2' R of the present invention;
FIG. 6 is a LCMS chromatogram of anisodamine of 6S,2' S according to the present invention;
FIG. 7 is a anisodamine hydrobromide chromatogram of 6R,2' R according to the present invention;
FIG. 8 is a scopolamine hydrobromide chromatogram of 6S,2' S according to the invention;
FIG. 9 is a 6R,2' R ultraviolet spectrum of anisodamine in accordance with the present invention;
FIG. 10 is a 6S,2' S hyoscyamine UV spectrum of the present invention;
FIG. 11 is a separation chromatogram of 6R,2'R anisodamine and 6S,2' S anisodamine in example 1;
FIG. 12 is a 6R,2'R anisodamine and 6S,2' S anisodamine resolution chromatogram of example 7 according to the present invention;
FIG. 13 is a 6R,2'R anisodamine and 6S,2' S anisodamine resolution chromatogram of example 8 according to the present invention;
FIG. 14 is a separation chromatogram of anisodamine at 6R,2'R and anisodamine at 6S,2' S in example 9 of the present invention;
FIG. 15 is a separation chromatogram of anisodamine at 6R,2'R and anisodamine at 6S,2' S in example 10 of the present invention;
FIG. 16 is a separation chromatogram of anisodamine at 6R,2'R and anisodamine at 6S,2' S in example 11 according to the present invention;
FIG. 17 is a separation chromatogram of anisodamine at 6R,2'R and anisodamine at 6S,2' S in example 12 of the present invention;
FIG. 18 is a structural formula of 6R,2'S, 6S,2' R, 6R,2'R, 6S,2' S configuration of the present invention.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
The racanisodamine is a synthetic product (654-2) of anisodamine, and comprises four optical isomers, which are 6R,2'S, 6S,2' R, 6R,2'R, 6S,2' S configuration. The structural formulas of 6R,2'S, 6S,2' R, 6R,2'R, 6S,2' S configuration are shown in figure 18.
Examples 1,
A method for separating trans-isomer of anisodamine comprises the following steps:
s1, taking 14g of racanisodamine, and separating by dynamic preparative HPLC to obtain 7g of cis-anisodamine containing 6R,2'S and 6S,2' R and trans-anisodamine containing 6R,2'R and 6S,2' S in total;
the chromatographic parameters for the separation by preparative HPLC were as follows:
a chromatographic column: DAC80 dynamic preparation separation column;
packing of a chromatographic column: huaban LD-2-C18;
flow rate: 200 ml/min;
wavelength: 210 nm;
when the elution time is 0-45 min, the mobile phase is a mixture of 0.1% formic acid and methanol in a mass ratio of 95:5, and when the elution time is 45-60 min, the mobile phase is a mixture of 0.1% formic acid and methanol in a mass ratio of 30: 70. The obtained separation patterns of cis-anisodamine and trans-anisodamine are shown in FIG. 1.
S2, adjusting the pH of the mixture of the cis-anisodamine and the trans-anisodamine which is obtained in the step S1 and is totally 7g to 9.2, extracting for 5 times by using chloroform in equal proportion, combining the extracts and drying by spinning to obtain 5.33g of purified cis-anisodamine and 5.63g of trans-anisodamine; taking the reverse anisodamine, detecting by HPLC, and obtaining chromatogram before chiral resolution as shown in figure 2, wherein the peak sequences are 6R,2'R and 6S,2' S in the trans isomer of racanisodamine. Thus, as can be seen from fig. 2, trans-anisodamine contains two trans isomers, and the HPLC parameters are as follows:
a chromatographic column: CHIRALCEL OX-3(4.6 x 100mm) chiral chromatography column;
mobile phase: 0.2% of 7mol NH3The methanol of (4);
the flow rate is 2mL/min, the column temperature is 35 ℃, and the detection wavelength is 215 nm.
S3, dissolving 5.63g of trans-anisodamine obtained in the step S2 in 55mL of methanol, and then resolving by supercritical chromatography to obtain a map as shown in figure 11, wherein the map shows that two monomers in the trans-isomer are effectively resolved in figure 11, and the trans-isomer is spun at 35 ℃ to obtain 2.11g of 6R and 2'R anisodamine and 2.08g of 6S and 2' S anisodamine respectively, wherein the specific parameters of the supercritical chromatography are as follows:
a chromatographic column: CHIRALCEL OX (30 × 250mm10 μm) (Daicel);
mobile phase: supercritical CO2Cosolvent: methanol and 0.2% of 7mol of NH3(ii) a The mass of the cosolvent accounts for 25% of the mass of the mobile phase;
cycle time: 10.3 min;
operating time: 20 min;
wavelength: 215 nm;
back pressure: 100 bar;
column temperature: 35 ℃;
flow rate of mobile phase: 38.25 ml/min;
total flow rate: 45 ml/min.
Then, the anisodamine monomer which can obtain 6R,2'R is detected according to the same detection conditions of HPLC in step S2, and as shown in FIG. 3, the trans-propeak in the chromatogram is proved to be the anisodamine of 6R,2' R.
Then, the anisodamine monomer for obtaining 6S,2'S was detected under the same detection conditions as in HPLC in step S2, and as shown in fig. 4, it was confirmed that the trans-end peak in the chromatogram was anisodamine for 6S,2' S.
Examples 2,
The peak of trans isomer of anisodamine obtained by taking 6R,2'R anisodamine obtained in step S3 of example 1 and detecting by LCMS is shown in FIG. 5, and it can be proved from FIG. 5 that 6R,2' R anisodamine is successfully separated in example 1, and the parameters of LCMS are as follows:
a chromatographic column: waters X Bridge C18 column (50mm 4.6mm 3.5 um);
mobile phase: the mobile phase A is 0.01mol/L NH4HCO3The mobile phase B is acetonitrile;
the flow rate is 2mL/min, the column temperature is 40 ℃, and when the elution time is 0-1.6 min, the mobile phase B is 5% -95%; when the elution time is 1.6-3 min, the mobile phase B is 95%.
Examples 3,
The peak of trans isomer of anisodamine obtained by taking 6S,2'S anisodamine obtained in step S3 in example 1 and detecting it by LCMS is shown in fig. 6, and it can be proved from fig. 6 that 6S,2' S anisodamine was successfully isolated in example 1, and the parameters of LCMS are as follows:
a chromatographic column: waters X Bridge C18 column (50mm 4.6mm 3.5 um);
mobile phase: the mobile phase A is 0.01mol/L NH4HCO3The mobile phase B is acetonitrile;
the flow rate is 2mL/min, the column temperature is 40 ℃, and when the elution time is 0-1.6 min, the mobile phase B is 5% -95%; when the elution time is 1.6-3 min, the mobile phase B is 95%.
Examples 4,
The anisodamine monomer of 6R,2'R and the anisodamine monomer of 6S,2' S in example 1 are respectively taken and dissolved in absolute ethyl alcohol, and then the hydrobromic acid is respectively dripped to form salt, and the solid hydrobromide can be obtained by utilizing the solubility difference of the hydrobromic acid in the solvent at different temperatures.
The final 2 isomer hydrobromides were obtained as follows in table 1:
TABLE 1
| Name of isomers | Trans-prepeak | Trans-rear peak |
| Mass (g) | 2.1142 | 2.0810 |
| Mass (g) of hydrobromide | 1.68 | 2.27 |
| Crystallization solvent | Ethanol | Acetone (II) |
| Melting Point (crude,. degree.C.) | 176.7～177.4 | 175.5～176.1 |
Detecting anisodamine hydrobromide by HPLC detection method in Chinese pharmacopoeia to obtain anisodamine hydrobromide chromatogram of 6R,2' R shown in FIG. 7 and anisodamine hydrobromide chromatogram of 6S,2' S shown in FIG. 8, and counting related substances of anisodamine hydrobromide of 6R,2' R according to FIG. 7 as shown in Table 2 below:
TABLE 2
Therefore, as can be seen from table 2 and fig. 7, the salified purity of the 6R,2' R anisodamine is as high as 99.19%, which shows that the separation method of the trans-isomer of anisodamine of the invention has good separation effect and high purification degree, and is suitable for industrial production. The separation purity is far higher than that of the trans-anisodamine monomer obtained by the existing separation method.
According to FIG. 8, the statistics of the relevant substances of 6S,2' S anisodamine hydrobromide are shown in Table 3 below:
TABLE 3
| Name (R) | |
|
|
|
|
Impurity 6 |
| Retention time/min | 5.094 | 9.471 | 10.206 | 10.723 | 13.194 | 24.085 |
| Area% | 0.02％ | 0.18％ | 0.07％ | 0.24％ | 0.31％ | 0.14％ |
Therefore, as can be seen from table 3 and fig. 8, the salified purity of 6S,2' S anisodamine is as high as 99.04%, which proves that the separation method of trans-isomer of anisodamine of the present invention has good separation effect and high purification degree, and is suitable for industrial production. The separation purity of the compound is far higher than that of a trans-anisodamine monomer obtained by the existing separation method.
Examples 5,
The method comprises the steps of preparing a 1mg/ml solution of 6R,2'R anisodamine standard substance by methanol, scanning the solution by an ultraviolet-visible spectrophotometer, recording an ultraviolet spectrogram as shown in figure 9, wherein the ultraviolet spectrogram is shown in figure 9, the 6R,2' R anisodamine has a larger absorption peak near 210nm, and 215nm is selected as the detection wavelength of the method in consideration of the interference of the absorption of the tail end of a solvent, so that the separation degree of the separation method can be effectively improved.
Examples 6,
Taking 6S,2'S anisodamine standard substance to prepare 1mg/ml solution with methanol, scanning with an ultraviolet-visible spectrophotometer, recording an ultraviolet spectrogram as shown in figure 10, and obtaining from figure 10 that 6S,2' S anisodamine has a larger absorption peak near 210nm, and 215nm is selected as the detection wavelength of the invention in consideration of the interference of the absorption of the solvent terminal. The separation degree of the separation method of the invention can be effectively improved.
Examples 7,
The difference from embodiment 1 is that in step S3, the mobile phase is supercritical N2O, the separation chromatogram of 6R,2'R anisodamine and 6S,2' S anisodamine obtained in step S3 is shown in fig. 12, under which chromatographic conditions 6R,2'R anisodamine and 6S,2' S anisodamine could not be separated efficiently.
Example 8,
The difference from embodiment 1 is that, in step S3, the mobile phase is supercritical NH3. The resolution chromatograms of 6R,2'R anisodamine and 6S,2' S anisodamine obtained in step S3 are shown in fig. 13, under which chromatographic conditions 6R,2'R anisodamine and 6S,2' S anisodamine could not be separated effectively.
Examples 9,
The difference from example 1 is that, in step S3, the solvent is ethanol. As shown in fig. 14, the separation chromatogram of 6R,2'R anisodamine and 6S,2' S anisodamine obtained in step S3 shows that under these chromatographic conditions, 6R,2'R anisodamine and 6S,2' S anisodamine were effectively separated, but the peak patterns and the like were poor, and therefore the separation effect was better when the solvent of example 1 was methanol, and the separation effect of example 1 was also better.
Examples 10,
The difference from example 1 is that in step S3, the column temperature was 40 ℃. FIG. 15 shows the resolution chromatograms of 6R,2'R anisodamine and 6S,2' S anisodamine obtained in step S3, under which 6R,2'R anisodamine and 6S,2' S anisodamine were substantially separated efficiently, but the peaks were poor.
Examples 11,
The difference from example 1 is that in step S3, the packing of the column is OZOX, i.e. cellulose-tris (3, 5-dimethylphenylcarbamate). The resolution chromatograms of 6R,2'R anisodamine and 6S,2' S anisodamine obtained in step S3 are shown in fig. 16, under which chromatographic conditions 6R,2'R anisodamine and 6S,2' S anisodamine could not be separated effectively.
Examples 12,
The difference from example 1 is that in step S3, the packing of the column is ODOG, cellulose tris (3-chloro-4-methylphenyl carbamate). The 6R,2'R anisodamine and 6S,2' S anisodamine resolution chromatograms obtained in step S3 are shown in fig. 17, under which chromatographic conditions the 6R,2'R anisodamine and 6S,2' S anisodamine could not be separated efficiently.
In the description herein, the description of the terms "one embodiment," "some embodiments," "specific embodiments," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A method for separating trans-isomer of anisodamine is characterized by comprising the following steps:
s1, removing anisodamine, and separating by dynamic preparative HPLC to obtain cis-anisodamine containing 6R,2'S and 6S,2' R and trans-anisodamine containing 6R,2'R and 6S,2' S;
s2, adjusting the pH values of the cis-anisodamine and the trans-anisodamine obtained in the step S1 to 8.5-10, extracting at least 1 time by using chloroform in equal proportion, combining the extracts, and spin-drying to obtain purified cis-anisodamine and trans-anisodamine;
s3, dissolving the trans-anisodamine obtained in the step S2 by using a solvent, then resolving by adopting a supercritical chromatography, and then spinning to obtain 6R,2'R anisodamine and 6S,2' S anisodamine respectively, wherein the specific parameters of the supercritical chromatography are as follows:
a chromatographic column: 30 x 250mm10 μm;
mobile phase: supercritical CO2、N20 and NH3One of (1);
cosolvent: methanol and 0.2% of 7mol of NH3The mass of the cosolvent is 15-30% of that of the mobile phase;
cycle time: 10-20 min;
operating time: 20-30 min;
wavelength: 200-240 nm;
back pressure: 90-110 bar;
column temperature: 30-40 ℃;
flow rate of mobile phase: 30-40 ml/min;
total flow rate: 40-50 ml/min.
2. The method for separating trans isomers of anisodamine according to claim 1, wherein in step S3 the mobile phase is supercritical CO2。
3. The method for separating a trans isomer of anisodamine according to claim 1, wherein in step S3, the flow rate of the mobile phase is 33.75ml/min and the mass of the co-solvent is 25% of the mass of the mobile phase.
4. The method for separating a trans isomer of anisodamine according to any of the claims 1 to 3, wherein the solvent is methanol and the wavelength is 215nm in step S3.
5. The method for separating trans isomers of anisodamine according to claim 4, wherein in step S3 the column temperature is 35 ℃, the chromatography column is CHIRALCEL OX (30 x 250mm10 μm) (Daicel), the cycle time is 13.5min, the operation time is 25min, the back pressure is 100bar, and the total flow rate is 45 ml/min.
6. The method for separating a trans isomer of anisodamine according to claim 4, wherein the chromatographic parameters for the preparative HPLC separation in step S1 are as follows:
and (3) chromatographic column: DAC80 dynamic preparation separation column;
packing of a chromatographic column: huaban LD-2-C18;
flow rate: 150-220 ml/min;
wavelength: 200-220 nm;
when the elution time is 0-45 min, the mobile phase is a mixture of 0.1% formic acid and methanol in a mass ratio of 95:5, and when the elution time is 45-60 min, the mobile phase is a mixture of 0.1% formic acid and methanol in a mass ratio of 30: 70.
7. The method for separating a trans isomer of anisodamine according to claim 6, wherein the flow rate is 200ml/min and the wavelength is 210nm in step S1.
8. A method for detecting a pair of trans isomers of anisodamine is characterized by comprising the following steps:
taking 6R,2'R anisodamine or 6S,2' S anisodamine, and detecting by LCMS, wherein the parameters of the LCMS are as follows:
a chromatographic column: waters X Bridge C18 column (50mm 4.6mm 3.5 um);
mobile phase: the mobile phase A is 0.01mol/L NH4HCO3The mobile phase B is acetonitrile;
the flow rate is 2mL/min, the column temperature is 40 ℃, when the elution time is 0-1.6 min, the flow phase B is 5% -95%, and when the elution time is 1.6-3 min, the flow phase B is 95%.
9. A6R, 2' R anisodamine monomer, characterized in that it is prepared by the separation method of trans isomer of anisodamine as described in claims 1 to 7.
10. A6S, 2' S anisodamine monomer, characterized in that it is prepared by the separation method of trans isomers of anisodamine according to claims 1 to 7.
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| 张怡评等: "超临界流体色谱分离技术应用研究进展", 《中医药导报》, vol. 18, no. 8, pages 89 - 91 * |
| 钟浩等: "超临界流体色谱法拆分某一生物碱类手性化合物", 《沈阳医学院学报》, vol. 19, no. 6, pages 498 - 504 * |
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