CN111116578B - Preparation method and application of demethyleneberberine - Google Patents

Preparation method and application of demethyleneberberine Download PDF

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CN111116578B
CN111116578B CN201911370091.7A CN201911370091A CN111116578B CN 111116578 B CN111116578 B CN 111116578B CN 201911370091 A CN201911370091 A CN 201911370091A CN 111116578 B CN111116578 B CN 111116578B
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demethyleneberberine
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mao
enzyme
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CN111116578A (en
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陶成
吴正治
陈健
梁少瑜
刘展艳
刘洁人
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Wu Zhengzhi
Shenzhen Jintai Pharmaceutical Technology LP
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Shenzhen Institute of Gerontology
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D455/00—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine
    • C07D455/03—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine containing quinolizine ring systems directly condensed with at least one six-membered carbocyclic ring, e.g. protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs

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Abstract

The invention discloses a preparation method and application of demethyleneberberine, wherein the preparation method comprises the following steps: adding berberine hydrochloride into xylene, adding trifluoromethanesulfonic acid, stirring at room temperature for reaction, adding hydrochloric acid until no yellow precipitate is generated, vacuum-filtering the reaction solution, washing the obtained precipitate, recrystallizing the washed precipitate with ethanol, converting the precipitate into hydrochloride with strong base type anion exchange resin, and concentrating to obtain yellow solid demethyleneberberine. The invention realizes the high-efficiency preparation of the demethyleneberberine, has high yield of a synthetic route, mild conditions, small toxicity of the used reagent, low cost, simple and convenient separation and purification, and can be used for mass preparation. The invention also discovers that the demethyleneberberine has the effect of inhibiting the activity of the MAO-B enzyme for the first time, and shows that the demethyleneberberine also has development potential in the aspect of preventing and treating Parkinson's disease and other diseases caused by abnormal activity of the MAO-B enzyme.

Description

Preparation method and application of demethyleneberberine
Technical Field
The invention relates to the technical field of medicines, and particularly relates to a preparation method and application of demethyleneberberine.
Background
In 1966, the chemical conversion from berberine hydrochloride to demethyleneberberine was first reported by the Cava project group (J.Org.chem.1967,32, 1640-) 1641). From berberine, under the action of 60% sulfuric acid and phloroglucinol, methylene protection is removed to obtain the methylene-removed berberine. Because the product demethyleneberberine is very unstable in a reaction system, the yield is very unstable, and when the reaction is optimal, the yield can reach 30 percent, and the reaction is difficult to repeat, so that the product cannot be obtained frequently or the yield is very low (lower than 10 percent).
In the subsequent research of half a century, the synthesis process of demethyleneberberine has not been broken through, the domestic literature reports the optimization of the transformation, and the method reported by Jinweiping (preparation method of palmatine CN100999622B) requires the use of a large amount of concentrated hydrochloric acid, has severe reaction conditions and poor controllability, and is not suitable for industrial production. Compared with the berberine hydrochloride, the market selling price of the maximum packaged demethyleneberberine is 700 yuan/kg, and the market selling price of the maximum packaged demethyleneberberine is 2090 yuan/10 mg, which also indicates that the preparation difficulty of the demethyleneberberine is very high.
In recent years, biological research on berberine hydrochloride has attracted extensive attention, while research on demethyleneberberine is rare, and related biological research is difficult to carry out mainly due to the extreme difficulty in separating and preparing demethyleneberberine. The research on the biological activity of the demethyleneberberine is relatively few, and only reports on the prevention and treatment of hepatic fibrosis and fatty liver, but no report on the prevention and treatment of Parkinson's disease by inhibiting the activity of MAO-B enzyme.
Disclosure of Invention
In order to solve the problems in the prior art, the embodiment of the invention provides a preparation method and application of demethyleneberberine. The technical scheme is as follows:
in a first aspect, a method for preparing demethyleneberberine comprises the following steps:
adding berberine hydrochloride into xylene, adding trifluoromethanesulfonic acid, stirring at room temperature for reaction, adding hydrochloric acid until no yellow precipitate is generated, vacuum-filtering the reaction solution, washing the obtained precipitate, recrystallizing the washed precipitate with ethanol, converting the precipitate into hydrochloride with strong base type anion exchange resin, and concentrating to obtain yellow solid demethyleneberberine.
Furthermore, the dosage of the berberine hydrochloride is 0.54mol, and the dosage of the trifluoromethanesulfonic acid is 3.17 mmol.
Further, the mixture was stirred at room temperature for 30 min.
Further, 1M hydrochloric acid was added at 0 ℃ until no yellow precipitate was produced.
In a second aspect, the application of demethyleneberberine in preparing MAO-B enzyme inhibiting medicine is provided.
In a third aspect, the application of demethyleneberberine in preparing medicine for treating Parkinson disease is provided.
The technical scheme provided by the embodiment of the invention has the following beneficial effects: the invention realizes the high-efficiency preparation of the demethyleneberberine, and is the most high-efficiency synthesis method aiming at the demethyleneberberine. The synthesis route has high yield, mild conditions, low toxicity of the used reagent, low cost, simple and convenient separation and purification, and can be used for mass preparation. The invention also discovers that the demethyleneberberine has the effect of inhibiting the activity of the MAO-B enzyme for the first time, and shows that the demethyleneberberine also has development potential in the aspect of preventing and treating Parkinson's disease and other diseases caused by abnormal activity of the MAO-B enzyme.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below.
FIG. 1 is a graph showing that demethyleneberberine inhibits MAO-B enzyme activity in the examples of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying drawings.
Examples
Preparation method of demethyleneberberine
Figure BDA0002339444860000031
Berberine hydrochloride (200g, 0.54mol) was added to a 3L three-necked flask, a mechanical stirring device was installed, xylene (1.3L), trifluoromethanesulfonic acid (TfOH, 280mL, 3.17mmol) were further added to the three-necked flask, and the mixture was stirred at room temperature for 30 minutes, and then 1M HCl was added at 0 ℃ until no yellow precipitate was formed. The reaction solution is filtered, the precipitate obtained after filtering is washed by a small amount of water,washing with small amount of petroleum ether, dissolving the precipitate with ethanol, recrystallizing, dissolving the precipitate in acetone/water (volume ratio of 1: 1), and passing through strong base anion exchange resin (Cl)-) Conversion to the hydrochloride salt gave, after concentration, demethyleneberberine (174g, 90%) as a yellow solid.
Rf＝0.4(DCM/MeOH,10:1)；mp:220-222℃；1H NMR(400MHz,DMSO-d6)
δ10.04(s,1H,OH-2),9.81(s,1H,H-8),9.26(s,1H,OH-3),8.74(s,1H,H-13),8.16(d,J＝9.1Hz,1H,H-11),8.04(d,J＝9.1Hz,1H,H-12),7.50(s,1H,H-1),6.80(s,1H,H-4),4.88(t,J＝6.3Hz,2H,H-6),4.08(s,3H,OCH3-10),4.05(s,3H,OCH3-9),3.11(t,J＝6.3Hz,2H,H-5)；13C NMR(100MHz,DMSO)δ150.0(C-10),149.2(C-12),145.6(C-9),145.1(C-3),143.5(C-8),138.3(C-13a),133.3(C-12a),127.2(C-4a),126.7(C-12),123.5(C-11),121.2(C-13b),119.1(C-8a),117.8(C-13),114.9(C-4),112.7(C-1),61.9(OCH3-10),57.1(C-6),55.6(OCH3-9),25.8(C-5)；HRMS(ESI):m/z calcd for C19H18NO4Cl[M-Cl]+324.1230, found 324.1239. purity: 98.4% (high performance liquid chromatography).
Spectrum (1H NMR,13C NMR and HMRS) consistent with literature reports (Li, y.h.; li, y.h.; yang, P.; kong, w.j.; you, x.f.; ren, G.; deng, h.b.; wang, y.m.x.; wang, y.m.x.; jiang, j.d.; song, d.q.bioorg.med.chem.2010,18,6422.).
Application of di-and demethyleneberberine
1. Experimental Material
MAO-B detection kit from Biovision (#797-100) and demethyleneberberine.
2. Experimental methods
Using a BioVision kit, a 96-well blackboard was prepared, and 10. mu.l of demethyleneberberine solution (0.1. mu.M, 1. mu.M, 3. mu.M, 10. mu.M, 30. mu.M, 100. mu.M, 300. mu.M, 1mM, 2mM) and 50. mu.LMAO-B pure enzyme were added to each well, followed by incubation at 37 ℃ for 30 minutes. Then 40. mu.L of substrate reaction solution (containing MAO-B substrate, devilpher and oxiRed) was addedTMProbe), dynamic fluorescence values were measured at Ex/Em-535/587 nm immediately after mixing, and the reaction kinetics were observed over 10-40 min. And (3) drawing by taking the reaction time as an X axis and the fluorescence value as a Y axis, selecting two different time points (T1 and T2) in a linear range, acquiring the fluorescence values (RFU1 and RFU2) corresponding to the time points, and calculating the slope. For example, at 495 s (T1) and 1005 s (T2), the fluorescence values of the control groups (RFU1 and RFU2) are 72500 and 100703, respectively, and the slope is (100703-. Then, taking the logarithm of the concentration of the demethyleneberberine as an X axis and the relative activity of MAO-B as a Y axis, drawing a dose-response curve, and calculating the half inhibitory concentration.
3. Results of the experiment
The demethyleneberberine can significantly inhibit the activity of MAO-B enzyme, and the half Inhibition Concentration (IC) thereof is shown in figure 150) It was 9.06. mu.M.
The invention realizes the high-efficiency preparation of the demethyleneberberine, and is the most high-efficiency synthesis method aiming at the demethyleneberberine. The synthesis route has high yield, mild conditions, low toxicity of the used reagent, low cost, simple and convenient separation and purification, and can be used for mass preparation. The invention also discovers that the demethyleneberberine has the effect of inhibiting the activity of the MAO-B enzyme for the first time, and shows that the demethyleneberberine also has development potential in the aspect of preventing and treating Parkinson's disease and other diseases caused by abnormal activity of the MAO-B enzyme.
The above-mentioned serial numbers of the embodiments of the present invention are merely for description and do not represent the merits of the embodiments. 5
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 (3)

1. A preparation method of demethyleneberberine is characterized by comprising the following steps:
adding berberine hydrochloride into xylene, adding trifluoromethanesulfonic acid, stirring at room temperature for reaction, adding 1M hydrochloric acid at 0 deg.C until no yellow precipitate is generated, vacuum-filtering the reaction solution, washing the obtained precipitate, recrystallizing the washed precipitate with ethanol, dissolving the obtained crystal, converting into hydrochloride with strong base type anion exchange resin, and concentrating to obtain yellow solid demethyleneberberine.
2. The method according to claim 1, wherein the berberine hydrochloride is used in an amount of 0.54mol and the trifluoromethanesulfonic acid is used in an amount of 3.17 mmol.
3. The method of claim 1, wherein the stirring is performed at room temperature for 30 min.
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CN100999522A (en) * 2006-01-09 2007-07-18 金伟平 Preparation process of palmatine
CN106687460A (en) * 2014-07-29 2017-05-17 深圳君圣泰生物技术有限公司 Berberine salts, ursodeoxycholic salts and combinations, methods of preparation and application thereof

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CN109293655A (en) * 2018-02-06 2019-02-01 中国药科大学 Demethyleneberberinehydrochloride hydrochloride crystal form and preparation method thereof

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CN100999522A (en) * 2006-01-09 2007-07-18 金伟平 Preparation process of palmatine
CN106687460A (en) * 2014-07-29 2017-05-17 深圳君圣泰生物技术有限公司 Berberine salts, ursodeoxycholic salts and combinations, methods of preparation and application thereof

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