CN105330563A - Reference compounds for kukoamine B quality control - Google Patents

Reference compounds for kukoamine B quality control Download PDF

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CN105330563A
CN105330563A CN201510655784.6A CN201510655784A CN105330563A CN 105330563 A CN105330563 A CN 105330563A CN 201510655784 A CN201510655784 A CN 201510655784A CN 105330563 A CN105330563 A CN 105330563A
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compound
dissolved
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layer chromatography
react completely
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CN105330563B (en
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董凯
韩冬
王琳琳
张存彦
孙长海
姚小青
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Tianjin Chase Sun Pharmaceutical Co Ltd
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    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/32—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton containing six-membered aromatic rings
    • C07C235/34—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton containing six-membered aromatic rings having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
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    • G01N30/02—Column chromatography
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    • 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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    • 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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    • G01N2030/027—Liquid chromatography
    • 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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Abstract

The present invention relates to four reference compounds for kukoamine B quality control, and preparation methods thereof, and a quality control method for controlling kukoamine B quality with the four reference compounds.

Description

For the reference compound in Kukoamine B quality control
Technical field
The invention belongs to medical art, being specifically related to a kind of Kukoamine B detection method and for detecting the reference compound in Kukoamine B.
Background technology
Kukoamine B (KukoamineB, chemical structure is as shown in Equation 1):
Natural product Kukoamine B (KukoamineB) is the alkaloid compound of separation and Extraction from Root-bark of Chinese Wolfberry first such as ShinjiFunayama.Kukoamine B effectively can cause pyemic bacterial pathogens associated molecule-lipopolysaccharide (endotoxin/lipopolysaccharide by antagonism, and DNA of bacteria (CpGDNA) LPS), excellent activity is shown to treatment Sepsis, and effect is obviously better than positive control medicine, thus has good patent medicine prospect.
The easy in preparation and put procedure of Kukoamine B makes its related substance increase because of the change of the conditions such as acid, alkali, oxidation.Kukoamine B is not mixed Quality Research at present, study report in great detail also not about Kukoamine B impurity source.In order to better study the pharmaceutical properties of Kukoamine B, improve the drug quality of Kukoamine B.Experimenter has carried out separation, enrichment to the related substance of Kukoamine B, has collected respectively, has then carried out structural identification.In order to obtain more substantial impurity sample, experimenter analyzes its structure, is then studied its synthetic route.
In experimentation, we find, Kukoamine B is more unstable, in unshielded alkaline aqueous solution situation, and the impurity that as easy as rolling off a log generation is a large amount of.As the amido linkage that causes as easy as rolling off a log under the effect compared with strong alkaline condition or hydroamidase ruptures, thus cause the combination of multiple active group.
For this reason, we study by the impurity produced in process of production to Kukoamine B, and according to the feature of impurity, design a kind of detection method of content of Kukoamine B.
The synthetic route of the open Kukoamine B of Chinese patent CN201010539028.4 is as follows:
Summary of the invention
An object of the present invention is, provide a kind of reference compound 1 for detecting Kukoamine B content, its structure is as follows:
The present invention also provides the preparation method of reference compound 1, and synthetic route is as follows:
Concrete, the preparation method of reference compound 1 of the present invention, comprises the following steps:
The triethylamine (0.22mol) of 24.1g compound 0 (0.1mol), 31.4ml is dissolved in the methylene dichloride of 400ml drying, by 3,4-dibenzyloxy benzene propionyl chloride dry methylene chloride 200ml dissolves, lower slowly instillation is stirred in ice bath, after dropping terminates, ice bath reacts about 1h, monitors reaction end by thin-layer chromatography, until react completely, concentrated organic solvent, add dilute hydrochloric acid 500ml, ethyl acetate 400ml × 3 extract, saturated common salt water washing, anhydrous sodium sulfate drying, suction filtration, concentrate to obtain crude product, column chromatography, obtains sterling compound B-11.
46.8g compound B-11 (0.08mol) is dissolved in 30ml tetrahydrofuran (THF), add saturated methanolic ammonia solution 800ml and RaneyNi4.3g, after draining air, under the hydrogen pressure of about 3MPa, stirring reaction 12 ± 2h under 50 DEG C of conditions in autoclave, reaction end is monitored by thin-layer chromatography, until react completely, suction filtration, concentrate to obtain crude product, column chromatography, obtains sterling compd B 2, yellow oil.
The triethylamine (0.22mol) of 41.2g compd B 2 (0.7mol), 31.4ml is dissolved in the methylene dichloride of 400ml drying, by 3,4-dibenzyloxy benzene propionyl chloride dry methylene chloride 200ml dissolves, lower slowly instillation is stirred in ice bath, after dropping terminates, ice bath reacts about 1h, monitors reaction end by thin-layer chromatography, until react completely.Concentrated organic solvent, add dilute hydrochloric acid 500ml, ethyl acetate 400ml × 3 extract, saturated common salt water washing, anhydrous sodium sulfate drying, suction filtration, concentrate to obtain crude product, column chromatography, obtains sterling compd B 3.
93.3g compd B 3 (0.1mol) is dissolved in 600ml methylene dichloride, slowly adds the ethereal HCI solution that 300ml is saturated under stirring at room temperature, reacts 6 ± 2h under ice bath, reaction end is monitored by thin-layer chromatography, until react completely, concentrated, suction filtration, obtains product Compound B4.
66.6g compd B 4 (0.08mol) is dissolved in 400ml methyl alcohol, vinyl cyanide (13.6ml is instilled under stirring at room temperature, methyl alcohol (100ml) solution 0.20mol), after dropping terminates, room temperature reaction 5 ± 2h, reaction end is monitored, until react completely by thin-layer chromatography.Concentrate to obtain compd B 5.
The triethylamine (0.22mol) of 62g compd B 5 (0.7mol), 77ml is dissolved in the methylene dichloride of 400ml drying, by 3,4-dibenzyloxy benzene propionyl chloride dry methylene chloride 200ml dissolves, lower slowly instillation is stirred in ice bath, after dropping terminates, ice bath reacts about 1h, monitors reaction end by thin-layer chromatography, until react completely.Concentrated organic solvent, adds dilute hydrochloric acid 500ml, ethyl acetate 400ml × 3 extracts, saturated common salt water washing, anhydrous sodium sulfate drying, suction filtration, concentrate to obtain crude product.Column chromatography, obtains sterling compound B-26.
61.5g compound B-26 (0.05mol) is dissolved in 60ml tetrahydrofuran (THF), add saturated methanolic ammonia solution 800ml and RaneyNi4.3g, after draining air, under the hydrogen pressure of about 3MPa, stirring reaction 12 ± 2h under 50 DEG C of conditions in autoclave, reaction end is monitored, until react completely by thin-layer chromatography.Suction filtration (diatomite drainage), concentrate to obtain crude product, column chromatography, obtains sterling compd B 7, yellow oil.
The triethylamine (0.044mol) of 49g compd B 7 (0.04mol), 7ml is dissolved in the methylene dichloride of 200ml drying, by 3,4-dibenzyloxy benzene propionyl chloride dry methylene chloride 200ml dissolves, lower slowly instillation is stirred in ice bath, after dropping terminates, ice bath reacts about 1h, monitors reaction end by thin-layer chromatography, until react completely.Concentrated organic solvent, adds dilute hydrochloric acid 200ml, ethyl acetate 400ml × 3 extracts, saturated common salt water washing, anhydrous sodium sulfate drying, suction filtration, concentrate to obtain crude product.Column chromatography, obtains sterling compd B 8.
47g compd B 8 (0.03mol) is dissolved in 30ml tetrahydrofuran (THF), adds 700ml methyl alcohol, and after 4.8g10% palladium/carbon, drain air, under hydrogen normal pressure, room temperature reaction 15 ± 2h, monitors reaction end by thin-layer chromatography, until react completely.Filter, vacuum-drying obtains compound 1.
An object of the present invention is, provide a kind of reference compound 2 for detecting Kukoamine B content, its structure is as follows:
The present invention also provides the preparation method of reference compound 2, and synthetic route is as follows:
Concrete, the preparation method of reference compound 2 of the present invention, comprises the following steps:
12g compd B 7 (0.01mol) is dissolved in 10ml tetrahydrofuran (THF), adds 100ml methyl alcohol, and after 1.2g10% palladium/carbon, drain air, under hydrogen normal pressure, room temperature reaction 15 ± 2h, monitors reaction end by thin-layer chromatography, until react completely.Filter, vacuum-drying obtains compound 2.
An object of the present invention is, provide a kind of reference compound 3 for detecting Kukoamine B content, its structure is as follows:
The present invention also provides the preparation method of reference compound 3, and synthetic route is as follows:
Concrete, the preparation method of reference compound 3 of the present invention, comprises the following steps:
The triethylamine (0.11mol) of 38g Compound C 2 (0.1mol), 15.7ml is dissolved in the methylene dichloride of 200ml drying, 0.1molCBZCl dry methylene chloride 200ml is dissolved, lower slowly instillation is stirred in ice bath, after dropping terminates, ice bath reacts about 1h, reaction end is monitored, until react completely by thin-layer chromatography.Concentrated organic solvent, ethyl acetate 400ml extracts, anhydrous sodium sulfate drying, suction filtration, concentrates to obtain crude product.Column chromatography, obtains sterling Compound C 3.
51.3g Compound C 3 (0.1mol) is dissolved in 100ml methylene dichloride, slowly adds the ethereal HCI solution that 300ml is saturated under stirring at room temperature, reacts 6 ± 2h, monitor reaction end, until react completely by thin-layer chromatography under ice bath.Concentrated, suction filtration, obtains product Compound C4.
33g Compound C 4 (0.08mol) is dissolved in 100ml methyl alcohol, vinyl cyanide (13.6ml is instilled under stirring at room temperature, methyl alcohol (100ml) solution 0.20mol), after dropping terminates, room temperature reaction 5 ± 2h, reaction end is monitored, until react completely by thin-layer chromatography.Concentrate to obtain Compound C 5.
The triethylamine (0.22mol) of 28g Compound C 5 (0.06mol), 31.4ml is dissolved in the methylene dichloride of 400ml drying, by 0.06mol3,4-dibenzyloxy benzene propionyl chloride dry methylene chloride 200ml dissolves, lower slowly instillation is stirred in ice bath, after dropping terminates, ice bath reacts about 1h, monitors reaction end by thin-layer chromatography, until react completely.Concentrated organic solvent, adds dilute hydrochloric acid 500ml, ethyl acetate 400ml × 3 extracts, saturated common salt water washing, anhydrous sodium sulfate drying, suction filtration, concentrate to obtain crude product.Column chromatography, obtains sterling Compound C 6.
48g Compound C 6 (0.06mol) is dissolved in 40ml tetrahydrofuran (THF), add saturated methanolic ammonia solution 800ml and RaneyNi10g, after draining air, under the hydrogen pressure of about 3MPa, stirring reaction 12 ± 2h under 50 DEG C of conditions in autoclave, reaction end is monitored, until react completely by thin-layer chromatography.Suction filtration, concentrates to obtain crude product.Column chromatography, obtains sterling Compound C 7, yellow oil.
8.1g Compound C 7 (0.01mol) is dissolved in 10ml tetrahydrofuran (THF), adds 100ml methyl alcohol, and after 1g10% palladium/carbon, drain air, under hydrogen normal pressure, room temperature reaction 15 ± 2h, monitors reaction end by thin-layer chromatography, until react completely.Filter, vacuum-drying obtains compound 3.
An object of the present invention is, provide a kind of reference compound 4 for detecting Kukoamine B content, its structure is as follows:
The present invention also provides the preparation method of reference compound 4, and synthetic route is as follows:
Concrete, the preparation method of reference compound 4 of the present invention, comprises the following steps:
The triethylamine (0.22mol) of 35.4g compd B 2 (0.06mol), 31.4ml is dissolved in the methylene dichloride of 400ml drying, 0.06molCBZCl dry methylene chloride 200ml is dissolved, lower slowly instillation is stirred in ice bath, after dropping terminates, ice bath reacts about 1h, reaction end is monitored, until react completely by thin-layer chromatography.Concentrated organic solvent, ethyl acetate 400ml extracts, anhydrous sodium sulfate drying, suction filtration, concentrates to obtain crude product.Column chromatography, obtains sterling Compound D 1.
72.4g Compound C 3 (0.1mol) is dissolved in 100ml methylene dichloride, slowly adds the ethereal HCI solution that 300ml is saturated under stirring at room temperature, reacts 6 ± 2h, monitor reaction end, until react completely by thin-layer chromatography under ice bath.Concentrated, suction filtration, obtains product Compound D2.
50g Compound D 2 (0.08mol) is dissolved in 100ml methyl alcohol, vinyl cyanide (13.6ml is instilled under stirring at room temperature, methyl alcohol (100ml) solution 0.20mol), after dropping terminates, room temperature reaction 5 ± 2h, monitors reaction end (silica-gel plate: GF by thin-layer chromatography 254, developping agent: methylene dichloride: methyl alcohol=10:1), until react completely.Concentrate to obtain compound d3.
The triethylamine (0.22mol) of 41g compound d3 (0.06mol), 31.4ml is dissolved in the methylene dichloride of 400ml drying, by 0.06mol3,4-dibenzyloxy benzene propionyl chloride dry methylene chloride 200ml dissolves, lower slowly instillation is stirred in ice bath, after dropping terminates, ice bath reacts about 1h, monitors reaction end by thin-layer chromatography, until react completely.Concentrated organic solvent, adds dilute hydrochloric acid 500ml, ethyl acetate 400ml × 3 extracts, saturated common salt water washing, anhydrous sodium sulfate drying, suction filtration, concentrate to obtain crude product.Column chromatography, obtains sterling Compound D 4.
61g Compound D 4 (0.06mol) is dissolved in 40ml tetrahydrofuran (THF), add saturated methanolic ammonia solution 800ml and RaneyNi10g, after draining air, under the hydrogen pressure of about 3MPa, stirring reaction 12 ± 2h under 50 DEG C of conditions in autoclave, reaction end is monitored, until react completely by thin-layer chromatography.Suction filtration, concentrate to obtain crude product, column chromatography, obtains sterling Compound C 7, yellow oil.
10g Compound D 5 (0.01mol) is dissolved in 10ml tetrahydrofuran (THF), adds 100ml methyl alcohol, and after 1.2g10% palladium/carbon, drain air, under hydrogen normal pressure, room temperature reaction 15 ± 2h, monitors reaction end by thin-layer chromatography, until react completely.Filter, vacuum-drying obtains compound 4.
The present invention also aims to, provide 4 kinds of reference compounds of the present invention detecting the application in Kukoamine B content.
Kukoamine B detection method of content of the present invention, comprises the following steps:
The preparation of need testing solution: it is appropriate that precision takes Kukoamine B, puts in 100ml measuring bottle, add strong base solution 70ml, ultrasonic 10 minutes and constantly jolting Kukoamine B is dissolved, let cool, with solvent cut to scale, shake up, filter with the filter membrane of 0.45 μm, subsequent filtrate is as need testing solution;
Reference substance stock solution: accurate Weigh Compound 1, compound 2, compound 3 and compound 4 reference substance are in right amount each, also dilutes the solution made containing 45 μ g in every 1ml, as impurity reference substance stock solution with dissolve with methanol;
Kukoamine B reference substance solution: it is appropriate that precision takes Kukoamine B reference substance, solubilizing agent dissolves and dilutes the solution made containing 45 μ g in every 1ml, product stock solution in contrast; Precision measures impurity reference substance stock solution and each 5ml of reference substance stock solution respectively, puts in 50ml measuring bottle, with solvent cut to scale, shakes up, in contrast product solution.
Chromatographic condition:
According to China's coastal port two annex V D high effective liquid chromatography for measuring
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are weighting agent; Be moving phase with 0.02mol/L ammonium dihydrogen phosphate-acetonitrile (600:470 is 2.4 ~ 3.5 by phosphoric acid adjust ph after mixing), column temperature: 40 DEG C, flow velocity is 1.0ml/min; Determined wavelength is 252nm.Kukoamine B peak and compound 1, compound 2, compound 3 and the peak-to-peak resolution of compound 4 all should be greater than 6.
Flow velocity: 1.2ml/min.
Precision measures reference substance solution and each 15 μ l of need testing solution, respectively injection liquid chromatography, and record color atlas is to 12 times of main peak retention time.Kukoamine B peak and compound 1, compound 2, compound 3 and the peak-to-peak resolution of compound 4 all should be greater than 6.
The noun occurred in the present invention is made an explanation:
CBZCl: chloroformic acid benzyl ester
RaneyNi: Raney's nickel
Compound 1,2 and 3 of the present invention, as the reference compound detecting Kukoamine B quality, has very important significance, can improve the accuracy of Kukoamine B quality examination further, improves the quality standard of product, ensures Drug safety.
Through test, our find, detection method of the present invention has that precision is high, good stability and the feature such as reproducible.
Figure of description
Fig. 1: embodiment 5 high-efficient liquid phase chromatogram.
Embodiment
By following specific embodiment, the present invention is further illustrated in the present invention, but not as restriction.
The synthesis of compound 1-compound 4 is the professional that be familiar with of more conventional chemical reaction to this specialty and can finds suitable synthetic route by the expertise of oneself.
Synthesis, the Compound C 2 of compound 0 are prepared with the synthesis of compound 3,4-dibenzyloxy benzene propionyl chloride: synthesize with reference to CN201010539028.4.
Embodiment 1: the preparation of compound 1
The triethylamine (0.22mol) of 24.1g compound 0 (0.1mol), 31.4ml is dissolved in the methylene dichloride of 400ml drying, by 3,4-dibenzyloxy benzene propionyl chloride dry methylene chloride 200ml dissolves, lower slowly instillation is stirred in ice bath, after dropping terminates, ice bath reacts about 1h, monitors reaction end (silica-gel plate: GF by thin-layer chromatography 254, developping agent: methylene dichloride: methyl alcohol=10:1, inspects under ultraviolet lamp), until react completely.Concentrated organic solvent, adds dilute hydrochloric acid 500ml, ethyl acetate 400ml × 3 extracts, saturated common salt water washing, anhydrous sodium sulfate drying, suction filtration, concentrate to obtain crude product.Column chromatography (eluent: sherwood oil: ethyl acetate=1:2), obtains sterling compound B-11, 1hNMR (400MHz, DMSO): 7.19 (10H) 6.61 (1H), 6.57 (1H), 6.52 (1H), 5.20 (4H) 3.57 (2H), 3.20 (2H), 2.83 (2H), 2.67 (2H), 2.51 (2H), 1.55 (4H), 1.40 (9H).
46.8g compound B-11 (0.08mol) is dissolved in 30ml tetrahydrofuran (THF), add saturated methanolic ammonia solution 800ml and RaneyNi4.3g, after draining air, under the hydrogen pressure of about 3MPa, in autoclave, stirring reaction 12 ± 2h under 50 DEG C of conditions, monitors reaction end (silica-gel plate: GF by thin-layer chromatography 254, developping agent: methylene dichloride: methyl alcohol=10:1, inspects under ultraviolet lamp), until react completely.Suction filtration (diatomite drainage), concentrates to obtain crude product.Column chromatography (eluent: methylene dichloride: methyl alcohol=10:1), obtains sterling compd B 2, yellow oil, 1hNMR (400MHz, DMSO): 7.19 (10H), 6.61 (1H), 6.57 (1H), 6.52 (1H), 5.20 (4H), 3.20 (4H), 2.96 (2H), 2.83 (2H), 2.65 (2H), 2.51 (2H), 1.81 (2H), 1.55 (4H), 1.40 (9H).
The triethylamine (0.22mol) of 41.2g compd B 2 (0.7mol), 31.4ml is dissolved in the methylene dichloride of 400ml drying, by 3,4-dibenzyloxy benzene propionyl chloride dry methylene chloride 200ml dissolves, lower slowly instillation is stirred in ice bath, after dropping terminates, ice bath reacts about 1h, monitors reaction end (silica-gel plate: GF by thin-layer chromatography 254, developping agent: methylene dichloride: methyl alcohol=10:1, inspects under ultraviolet lamp), until react completely.Concentrated organic solvent, adds dilute hydrochloric acid 500ml, ethyl acetate 400ml × 3 extracts, saturated common salt water washing, anhydrous sodium sulfate drying, suction filtration, concentrate to obtain crude product.Column chromatography (eluent: sherwood oil: ethyl acetate=1:2), obtains sterling compd B 3, 1hNMR (400MHz, DMSO): 7.19 (20H), 6.61 (2H), 6.57 (2H), 6.52 (2H), 5.20 (8H), 3.20 (6H), 2.96 (2H), 2.83 (4H), 2.51 (4H), 1.81 (2H), 1.55 (4H), 1.40 (9H).
93.3g compd B 3 (0.1mol) is dissolved in 600ml methylene dichloride, slowly adds the ethereal HCI solution that 300ml is saturated under stirring at room temperature, reacts 6 ± 2h, monitor reaction end (silica-gel plate: GF by thin-layer chromatography under ice bath 254, developping agent: methylene dichloride: methyl alcohol=10:1, inspects under ultraviolet lamp), until react completely.Concentrated, suction filtration, obtains product Compound B4, 1hNMR (400MHz, DMSO): 7.19 (20H), 6.61 (2H), 6.57 (2H), 6.52 (2H), 5.20 (8H), 3.20 (6H), 2.65 (2H), 2.83 (4H), 2.51 (4H), 1.81 (2H), 1.55 (4H).
66.6g compd B 4 (0.08mol) is dissolved in 400ml methyl alcohol, vinyl cyanide (13.6ml is instilled under stirring at room temperature, methyl alcohol (100ml) solution 0.20mol), after dropping terminates, room temperature reaction 5 ± 2h, monitors reaction end (silica-gel plate: GF by thin-layer chromatography 254, developping agent: methylene dichloride: methyl alcohol=10:1), until react completely.Concentrate to obtain compd B 5, 1hNMR (400MHz, DMSO): 7.19 (20H), 6.61 (2H), 6.57 (2H), 6.52 (2H), 5.20 (8H), 3.20 (6H), 2.92 (2H), 2.83 (4H), 2.55 (2H), 2.53 (2H), 2.51 (4H), 1.81 (2H), 1.55 (2H), 1.41 (2H).
The triethylamine (0.22mol) of 62g compd B 5 (0.7mol), 77ml is dissolved in the methylene dichloride of 400ml drying, by 3,4-dibenzyloxy benzene propionyl chloride dry methylene chloride 200ml dissolves, lower slowly instillation is stirred in ice bath, after dropping terminates, ice bath reacts about 1h, monitors reaction end (silica-gel plate: GF by thin-layer chromatography 254, developping agent: methylene dichloride: methyl alcohol=10:1, inspects under ultraviolet lamp), until react completely.Concentrated organic solvent, adds dilute hydrochloric acid 500ml, ethyl acetate 400ml × 3 extracts, saturated common salt water washing, anhydrous sodium sulfate drying, suction filtration, concentrate to obtain crude product.Column chromatography (eluent: sherwood oil: ethyl acetate=1:2), obtains sterling compound B-26, 1hNMR (400MHz, DMSO): 7.19 (30H), 6.61 (3H), 6.57 (3H), 6.52 (3H), 5.20 (12H), 3.20 (8H), 3.57 (2H), 2.83 (6H), 2.67 (2H), 2.51 (6H), 1.81 (2H), 1.55 (4H).
61.5g compound B-26 (0.05mol) is dissolved in 60ml tetrahydrofuran (THF), add saturated methanolic ammonia solution 800ml and RaneyNi4.3g, after draining air, under the hydrogen pressure of about 3MPa, in autoclave, stirring reaction 12 ± 2h under 50 DEG C of conditions, monitors reaction end (silica-gel plate: GF by thin-layer chromatography 254, developping agent: methylene dichloride: methyl alcohol=10:1, inspects under ultraviolet lamp), until react completely.Suction filtration (diatomite drainage), concentrates to obtain crude product.Column chromatography (eluent: methylene dichloride: methyl alcohol=10:1), obtains sterling compd B 7, yellow oil, 1hNMR (400MHz, DMSO): 7.19 (30H), 6.61 (3H), 6.57 (3H), 6.52 (3H), 5.20 (12H), 3.20 (10H), 3.57 (2H), 2.83 (6H), 2.67 (2H), 2.51 (6H), 1.81 (4H), 1.55 (4H).
The triethylamine (0.044mol) of 49g compd B 7 (0.04mol), 7ml is dissolved in the methylene dichloride of 200ml drying, by 3,4-dibenzyloxy benzene propionyl chloride dry methylene chloride 200ml dissolves, lower slowly instillation is stirred in ice bath, after dropping terminates, ice bath reacts about 1h, monitors reaction end (silica-gel plate: GF by thin-layer chromatography 254, developping agent: methylene dichloride: methyl alcohol=10:1, inspects under ultraviolet lamp), until react completely.Concentrated organic solvent, adds dilute hydrochloric acid 200ml, ethyl acetate 400ml × 3 extracts, saturated common salt water washing, anhydrous sodium sulfate drying, suction filtration, concentrate to obtain crude product.Column chromatography (eluent: sherwood oil: ethyl acetate=1:2), obtains sterling compd B 8, 1hNMR (400MHz, DMSO): 7.19 (40H), 6.61 (4H), 6.57 (4H), 6.52 (4H), 5.20 (16H), 3.20 (12H), 2.83 (8H), 2.51 (8H), 1.81 (4H), 1.55 (4H).
47g compd B 8 (0.03mol) is dissolved in 30ml tetrahydrofuran (THF), adds 700ml methyl alcohol, after 4.8g10% palladium/carbon, drains air, and room temperature reaction 15 ± 2h under hydrogen normal pressure monitors reaction end (silica-gel plate: GF by thin-layer chromatography 254, developping agent: propyl carbinol: water: acetic acid=4:1:1, inspects under ultraviolet lamp), until react completely.Filter, vacuum-drying obtains compound 1, 1hNMR (400MHz, DMSO): 6.51 (8H), 6.42 (4H), 3.20 (12H), 2.83 (8H), 2.51 (8H), 1.81 (6H), 1.55 (4H).
Embodiment 2: the preparation of compound 2
12g compd B 7 (0.01mol) is dissolved in 10ml tetrahydrofuran (THF), adds 100ml methyl alcohol, after 1.2g10% palladium/carbon, drains air, and room temperature reaction 15 ± 2h under hydrogen normal pressure monitors reaction end (silica-gel plate: GF by thin-layer chromatography 254, developping agent: propyl carbinol: water: acetic acid=4:1:1, inspects under ultraviolet lamp), until react completely.Filter, vacuum-drying obtains compound 2, 1hNMR (400MHz, DMSO): 6.51 (6H), 6.42 (3H), 3.20 (10H), 2.83 (6H), 2.65 (2H), 2.51 (6H), 1.81 (4H), 1.55 (4H).
Embodiment 3: the preparation of compound 3
The triethylamine (0.11mol) of 38g Compound C 2 (0.1mol), 15.7ml is dissolved in the methylene dichloride of 200ml drying, 0.1molCBZCl dry methylene chloride 200ml is dissolved, lower slowly instillation is stirred in ice bath, after dropping terminates, ice bath reacts about 1h, monitors reaction end (silica-gel plate: GF by thin-layer chromatography 254, developping agent: methylene dichloride: methyl alcohol=10:1), until react completely.Concentrated organic solvent, ethyl acetate 400ml extracts, anhydrous sodium sulfate drying, suction filtration, concentrates to obtain crude product.Column chromatography, obtains sterling Compound C 3, 1hNMR (400MHz, DMSO): 7.19 (10H), 5.34 (4H), 2.96 (8H), 1.81 (2H), 1.55 (4H), 1.40 (9H).
51.3g Compound C 3 (0.1mol) is dissolved in 100ml methylene dichloride, slowly adds the ethereal HCI solution that 300ml is saturated under stirring at room temperature, reacts 6 ± 2h, monitor reaction end (silica-gel plate: GF by thin-layer chromatography under ice bath 254, developping agent: methylene dichloride: methyl alcohol=10:1, inspects under ultraviolet lamp), until react completely.Concentrated, suction filtration, obtains product Compound C4, 1hNMR (400MHz, DMSO): 7.19 (10H), 5.34 (4H), 2.96 (6H), 2.65 (2H), 1.81 (2H), 1.55 (4H).
33g Compound C 4 (0.08mol) is dissolved in 100ml methyl alcohol, vinyl cyanide (13.6ml is instilled under stirring at room temperature, methyl alcohol (100ml) solution 0.20mol), after dropping terminates, room temperature reaction 5 ± 2h, monitors reaction end (silica-gel plate: GF by thin-layer chromatography 254, developping agent: methylene dichloride: methyl alcohol=10:1), until react completely.Concentrate to obtain Compound C 5, 1hNMR (400MHz, DMSO): 7.19 (10H), 5.34 (4H), 2.96 (8H), 2.55 (4H), 1.81 (2H), 1.55 (2H), 1.41 (2H).
The triethylamine (0.22mol) of 28g Compound C 5 (0.06mol), 31.4ml is dissolved in the methylene dichloride of 400ml drying, by 0.06mol3,4-dibenzyloxy benzene propionyl chloride dry methylene chloride 200ml dissolves, lower slowly instillation is stirred in ice bath, after dropping terminates, ice bath reacts about 1h, monitors reaction end (silica-gel plate: GF by thin-layer chromatography 254, developping agent: methylene dichloride: methyl alcohol=10:1, inspects under ultraviolet lamp), until react completely.Concentrated organic solvent, adds dilute hydrochloric acid 500ml, ethyl acetate 400ml × 3 extracts, saturated common salt water washing, anhydrous sodium sulfate drying, suction filtration, concentrate to obtain crude product.Column chromatography (eluent: sherwood oil: ethyl acetate=1:2), obtains sterling Compound C 6, 1hNMR (400MHz, DMSO): 7.19 (20H), 6.61 (1H), 6.57 (1H), 6.52 (1H), 5.34 (4H), 5.20 (4H), 3.57 (2H), 3.20 (2H), 2.96 (6H), 2.83 (2H), 2.67 (2H), 2.51 (2H), 1.81 (2H), 1.55 (4H).
48g Compound C 6 (0.06mol) is dissolved in 40ml tetrahydrofuran (THF), add saturated methanolic ammonia solution 800ml and RaneyNi10g, after draining air, under the hydrogen pressure of about 3MPa, in autoclave, stirring reaction 12 ± 2h under 50 DEG C of conditions, monitors reaction end (silica-gel plate: GF by thin-layer chromatography 254, developping agent: methylene dichloride: methyl alcohol=10:1, inspects under ultraviolet lamp), until react completely.Suction filtration (diatomite drainage), concentrates to obtain crude product.Column chromatography (eluent: methylene dichloride: methyl alcohol=10:1), obtains sterling Compound C 7, yellow oil, 1hNMR (400MHz, DMSO): 7.19 (20H), 6.61 (1H), 6.57 (1H), 6.52 (1H), 5.34 (4H), 5.20 (4H), 3.20 (4H), 2.96 (6H), 2.83 (2H), 2.67 (2H), 2.51 (2H), 1.81 (4H), 1.55 (4H).
8.1g Compound C 7 (0.01mol) is dissolved in 10ml tetrahydrofuran (THF), adds 100ml methyl alcohol, after 1g10% palladium/carbon, drains air, and room temperature reaction 15 ± 2h under hydrogen normal pressure monitors reaction end (silica-gel plate: GF by thin-layer chromatography 254, developping agent: propyl carbinol: water: acetic acid=4:1:1, inspects under ultraviolet lamp), until react completely.Filter, vacuum-drying obtains compound 3, 1hNMR (400MHz, DMSO): 6.51 (2H), 6.42 (1H), 3.20 (4H), 2.83 (2H), 2.67 (4H), 2.55 (4H), 2.51 (2H), 1.81 (2H), 1.67 (2H), 1.55 (2H), 1.41 (2H).
Embodiment 4: the preparation of compound 4
The triethylamine (0.22mol) of 35.4g compd B 2 (0.06mol), 31.4ml is dissolved in the methylene dichloride of 400ml drying, 0.06molCBZCl dry methylene chloride 200ml is dissolved, lower slowly instillation is stirred in ice bath, after dropping terminates, ice bath reacts about 1h, monitors reaction end (silica-gel plate: GF by thin-layer chromatography 254, developping agent: methylene dichloride: methyl alcohol=10:1), until react completely.Concentrated organic solvent, ethyl acetate 400ml extracts, anhydrous sodium sulfate drying, suction filtration, concentrates to obtain crude product.Column chromatography, obtains sterling Compound D 1, 1hNMR (400MHz, DMSO): 7.19 (15H), 6.61 (1H), 6.57 (1H), 6.52 (1H), 5.34 (2H), 5.20 (4H), 3.20 (4H), 2.96 (2H), 2.83 (2H), 2.65 (2H), 2.51 (2H), 1.81 (2H), 1.55 (4H), 1.40 (9H).
72.4g Compound C 3 (0.1mol) is dissolved in 100ml methylene dichloride, slowly adds the ethereal HCI solution that 300ml is saturated under stirring at room temperature, reacts 6 ± 2h, monitor reaction end (silica-gel plate: GF by thin-layer chromatography under ice bath 254, developping agent: methylene dichloride: methyl alcohol=10:1, inspects under ultraviolet lamp), until react completely.Concentrated, suction filtration, obtains product Compound D2, 1hNMR (400MHz, DMSO): 7.19 (15H), 6.61 (1H), 6.57 (1H), 6.52 (1H), 5.34 (2H), 5.20 (4H), 3.20 (4H), 2.96 (2H), 2.83 (2H), 2.65 (2H), 2.51 (2H), 1.81 (2H), 1.55 (4H).
50g Compound D 2 (0.08mol) is dissolved in 100ml methyl alcohol, vinyl cyanide (13.6ml is instilled under stirring at room temperature, methyl alcohol (100ml) solution 0.20mol), after dropping terminates, room temperature reaction 5 ± 2h, monitors reaction end (silica-gel plate: GF by thin-layer chromatography 254, developping agent: methylene dichloride: methyl alcohol=10:1), until react completely.Concentrate to obtain compound d3, 1hNMR (400MHz, DMSO): 1HNMR (400MHz, DMSO): 7.19 (15H), 6.61 (1H), 6.57 (1H), 6.52 (1H), 5.34 (2H), 5.20 (4H), 3.20 (4H), 2.96 (2H), 2.92 (2H), 2.83 (2H), 2.51 (2H), 2.55 (4H), 1.81 (2H), 1.55 (2H), 1.41 (2H).
The triethylamine (0.22mol) of 41g compound d3 (0.06mol), 31.4ml is dissolved in the methylene dichloride of 400ml drying, by 0.06mol3,4-dibenzyloxy benzene propionyl chloride dry methylene chloride 200ml dissolves, lower slowly instillation is stirred in ice bath, after dropping terminates, ice bath reacts about 1h, monitors reaction end (silica-gel plate: GF by thin-layer chromatography 254, developping agent: methylene dichloride: methyl alcohol=10:1, inspects under ultraviolet lamp), until react completely.Concentrated organic solvent, adds dilute hydrochloric acid 500ml, ethyl acetate 400ml × 3 extracts, saturated common salt water washing, anhydrous sodium sulfate drying, suction filtration, concentrate to obtain crude product.Column chromatography (eluent: sherwood oil: ethyl acetate=1:2), obtains sterling Compound D 4, 1hNMR (400MHz, DMSO): 7.19 (25H), 6.61 (2H), 6.57 (2H), 6.52 (2H), 5.34 (2H), 5.20 (8H), 3.20 (6H), 2.96 (2H), 3.57 (2H), 2.83 (4H), 2.51 (4H), 2.67 (2H), 1.81 (2H), 1.55 (4H).
61g Compound D 4 (0.06mol) is dissolved in 40ml tetrahydrofuran (THF), add saturated methanolic ammonia solution 800ml and RaneyNi10g, after draining air, under the hydrogen pressure of about 3MPa, in autoclave, stirring reaction 12 ± 2h under 50 DEG C of conditions, monitors reaction end (silica-gel plate: GF by thin-layer chromatography 254, developping agent: methylene dichloride: methyl alcohol=10:1, inspects under ultraviolet lamp), until react completely.Suction filtration (diatomite drainage), concentrates to obtain crude product.Column chromatography (eluent: methylene dichloride: methyl alcohol=10:1), obtains sterling Compound C 7, yellow oil, 1hNMR (400MHz, DMSO): 7.19 (25H), 6.61 (2H), 6.57 (2H), 6.52 (2H), 5.34 (2H), 5.20 (8H), 3.20 (8H), 2.96 (2H), 3.57 (2H), 2.83 (4H), 2.51 (4H), 2.67 (2H), 1.81 (2H), 1.55 (4H).
10g Compound D 5 (0.01mol) is dissolved in 10ml tetrahydrofuran (THF), adds 100ml methyl alcohol, after 1.2g10% palladium/carbon, drains air, and room temperature reaction 15 ± 2h under hydrogen normal pressure monitors reaction end (silica-gel plate: GF by thin-layer chromatography 254, developping agent: propyl carbinol: water: acetic acid=4:1:1, inspects under ultraviolet lamp), until react completely.Filter, vacuum-drying obtains compound 4, 1hNMR (400MHz, DMSO): 1hNMR (400MHz, DMSO): 6.51 (4H), 6.42 (2H), 3.20 (8H), 2.83 (4H), 2.51 (4H), 2.65 (4H), 1.81 (4H), 1.55 (4H).
Embodiment 5 compound 1, compound 2, compound 3 and compound 4 in contrast product are detecting the application in Kukoamine B medicine.
The preparation of need testing solution: precision takes Kukoamine B appropriate (being about 0.15g), put in 100ml measuring bottle, add strong base solution 70ml, ultrasonic 10 minutes and constantly jolting Kukoamine B is dissolved, let cool, with solvent cut to scale, shake up, filter with the filter membrane of 0.45 μm, subsequent filtrate is as need testing solution;
Reference substance stock solution: accurate Weigh Compound 1, compound 2, compound 3 and compound 4 reference substance are in right amount each, also dilutes the solution made containing 45 μ g in every 1ml, as impurity reference substance stock solution with dissolve with methanol;
Kukoamine B reference substance solution: it is appropriate that precision takes Kukoamine B reference substance, solubilizing agent dissolves and dilutes the solution made containing 45 μ g in every 1ml, product stock solution in contrast; Precision measures impurity reference substance stock solution and each 5ml of reference substance stock solution respectively, puts in 50ml measuring bottle, with solvent cut to scale, shakes up, in contrast product solution.
Chromatographic condition:
Measure according to high performance liquid chromatography (China's coastal port two annex V D).
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are weighting agent; Be moving phase with 0.02mol/L ammonium dihydrogen phosphate-acetonitrile (600:470 is 2.4 ~ 3.5 by phosphoric acid adjust ph after mixing), column temperature: 40 DEG C, flow velocity is 1.0ml/min; Determined wavelength is 252nm.Kukoamine B peak and compound 1, compound 2, compound 3 and the peak-to-peak resolution of compound 4 all should be greater than 6.
Flow velocity: 1.2ml/min.
Precision measures reference substance solution and each 15 μ l of need testing solution, respectively injection liquid chromatography, and record color atlas is to 12 times of main peak retention time.Kukoamine B peak and compound 1, compound 2, compound 3 and the peak-to-peak resolution of compound 4 all should be greater than 6.
Test-results color atlas is shown in accompanying drawing 1, and wherein compound 2 retention time is 4.462min, and compound 1 retention time is 8.424min, and compound 3 retention time is 14.081min, and compound 4 retention time is 15.334min, and Kukoamine B retention time is 10.967.

Claims (10)

1. for detecting the reference compound of Kukoamine B content, it is characterized in that, reference compound 1 possesses following structure,
2. for detecting the reference compound of Kukoamine B content, it is characterized in that, reference compound 2 possesses following structure,
3. for detecting the reference compound of Kukoamine B content, it is characterized in that, reference compound 3 possesses following structure,
4. for detecting the reference compound of Kukoamine B content, it is characterized in that, reference compound 4 possesses following structure,
5. reference compound according to claim 1, is characterized in that, the preparation method of reference compound 1 is as follows:
The triethylamine of 24.1g compound 0,31.4ml is dissolved in the methylene dichloride of 400ml drying, by 3,4-dibenzyloxy benzene propionyl chloride dry methylene chloride 200ml dissolves, lower slowly instillation is stirred in ice bath, after dropping terminates, ice bath reacts about 1h, monitors reaction end by thin-layer chromatography, until react completely, concentrated organic solvent, adds dilute hydrochloric acid 500ml, ethyl acetate 400ml × 3 extracts, saturated common salt water washing, anhydrous sodium sulfate drying, suction filtration, concentrates to obtain crude product, column chromatography, obtain sterling compound B-11
46.8g compound B-11 is dissolved in 30ml tetrahydrofuran (THF), adds saturated methanolic ammonia solution 800ml and RaneyNi4.3g, after draining air, under the hydrogen pressure of about 3MPa, in autoclave, stirring reaction 12 ± 2h under 50 DEG C of conditions, monitors reaction end by thin-layer chromatography, until react completely, suction filtration, concentrate to obtain crude product, column chromatography, obtains sterling compd B 2, yellow oil
The triethylamine of 41.2g compd B 2,31.4ml is dissolved in the methylene dichloride of 400ml drying, by 3,4-dibenzyloxy benzene propionyl chloride dry methylene chloride 200ml dissolves, lower slowly instillation is stirred in ice bath, after dropping terminates, ice bath reacts about 1h, monitors reaction end by thin-layer chromatography, until react completely, concentrated organic solvent, adds dilute hydrochloric acid 500ml, ethyl acetate 400ml × 3 extracts, saturated common salt water washing, anhydrous sodium sulfate drying, suction filtration, concentrates to obtain crude product, column chromatography, obtain sterling compd B 3
93.3g compd B 3 is dissolved in 600ml methylene dichloride, slowly adds the ethereal HCI solution that 300ml is saturated under stirring at room temperature, reacts 6 ± 2h, monitor reaction end by thin-layer chromatography under ice bath, until react completely, concentrated, suction filtration, obtains product Compound B4,
66.6g compd B 4 is dissolved in 400ml methyl alcohol, the appropriate methanol solution containing vinyl cyanide of instillation under stirring at room temperature, and drip after terminating, room temperature reaction 5 ± 2h, monitors reaction end by thin-layer chromatography, until react completely, concentrates to obtain compd B 5,
The triethylamine of 62g compd B 5,77ml is dissolved in the methylene dichloride of 400ml drying, is dissolved by 3,4-dibenzyloxy benzene propionyl chloride dry methylene chloride 200ml, stir lower slowly instillation in ice bath, drip after terminating, ice bath reacts about 1h, reaction end is monitored by thin-layer chromatography, until react completely, concentrated organic solvent, add dilute hydrochloric acid 500ml, ethyl acetate 400ml × 3 extract, saturated common salt water washing, anhydrous sodium sulfate drying, suction filtration, concentrates to obtain crude product, column chromatography, obtain sterling compound B-26
61.5g compound B-26 is dissolved in 60ml tetrahydrofuran (THF), adds saturated methanolic ammonia solution 800ml and RaneyNi4.3g, after draining air, under the hydrogen pressure of about 3MPa, in autoclave, stirring reaction 12 ± 2h under 50 DEG C of conditions, monitors reaction end by thin-layer chromatography, until react completely, suction filtration, concentrate to obtain crude product, column chromatography, obtains sterling compd B 7, yellow oil
The triethylamine of 49g compd B 7,7ml is dissolved in the methylene dichloride of 200ml drying, is dissolved by 3,4-dibenzyloxy benzene propionyl chloride dry methylene chloride 200ml, stir lower slowly instillation in ice bath, drip after terminating, ice bath reacts about 1h, reaction end is monitored by thin-layer chromatography, until react completely, concentrated organic solvent, add dilute hydrochloric acid 200ml, ethyl acetate 400ml × 3 extract, saturated common salt water washing, anhydrous sodium sulfate drying, suction filtration, concentrates to obtain crude product, column chromatography, obtain sterling compd B 8
47g compd B 8 is dissolved in 30ml tetrahydrofuran (THF), adds 700ml methyl alcohol, and after 4.8g10% palladium/carbon, drain air, under hydrogen normal pressure, room temperature reaction 15 ± 2h, monitors reaction end by thin-layer chromatography, until react completely, filter, vacuum-drying obtains compound 1.
6. reference compound according to claim 1, is characterized in that, the preparation method of reference compound 2 is as follows:
12g compd B 7 is dissolved in 10ml tetrahydrofuran (THF), adds 100ml methyl alcohol, and after 1.2g10% palladium/carbon, drain air, under hydrogen normal pressure, room temperature reaction 15 ± 2h, monitors reaction end by thin-layer chromatography, until react completely.Filter, vacuum-drying obtains compound 2.
7. reference compound according to claim 1, is characterized in that, the preparation method of reference compound 3 is as follows:
The triethylamine of 38g Compound C 2,15.7ml is dissolved in the methylene dichloride of 200ml drying, is dissolved by 0.1molCBZCl dry methylene chloride 200ml, stirs lower slowly instillation in ice bath, after dropping terminates, ice bath reacts about 1h, monitors reaction end by thin-layer chromatography, until react completely, concentrated organic solvent, ethyl acetate 400ml extracts, anhydrous sodium sulfate drying, suction filtration, concentrates to obtain crude product, column chromatography, obtain sterling Compound C 3
51.3g Compound C 3 is dissolved in 100ml methylene dichloride, slowly adds the ethereal HCI solution that 300ml is saturated under stirring at room temperature, reacts 6 ± 2h, monitor reaction end by thin-layer chromatography under ice bath, until react completely, concentrated, suction filtration, obtains product Compound C4,
33g Compound C 4 (0.08mol) is dissolved in 100ml methyl alcohol, instills the appropriate methanol solution containing vinyl cyanide under stirring at room temperature, drips after terminating, room temperature reaction 5 ± 2h, monitors reaction end by thin-layer chromatography, until react completely, concentrate to obtain Compound C 5
The triethylamine of 28g Compound C 5,31.4ml is dissolved in the methylene dichloride of 400ml drying, by 0.06mol3,4-dibenzyloxy benzene propionyl chloride dry methylene chloride 200ml dissolves, lower slowly instillation is stirred in ice bath, after dropping terminates, ice bath reacts about 1h, monitors reaction end by thin-layer chromatography, until react completely, concentrated organic solvent, adds dilute hydrochloric acid 500ml, ethyl acetate 400ml × 3 extracts, saturated common salt water washing, anhydrous sodium sulfate drying, suction filtration, concentrates to obtain crude product, column chromatography, obtain sterling Compound C 6
48g Compound C 6 is dissolved in 40ml tetrahydrofuran (THF), adds saturated methanolic ammonia solution 800ml and RaneyNi10g, after draining air, under the hydrogen pressure of about 3MPa, in autoclave, stirring reaction 12 ± 2h under 50 DEG C of conditions, monitors reaction end by thin-layer chromatography, until react completely, suction filtration, concentrate to obtain crude product, column chromatography, obtains sterling Compound C 7, yellow oil
8.1g Compound C 7 is dissolved in 10ml tetrahydrofuran (THF), adds 100ml methyl alcohol, and after 1g10% palladium/carbon, drain air, under hydrogen normal pressure, room temperature reaction 15 ± 2h, monitors reaction end by thin-layer chromatography, until react completely, filter, vacuum-drying obtains compound 3.
8. reference compound according to claim 1, is characterized in that, the preparation method of reference compound 4 is as follows:
The triethylamine of 35.4g compd B 2,31.4ml is dissolved in the methylene dichloride of 400ml drying, is dissolved by 0.06molCBZCl dry methylene chloride 200ml, stirs lower slowly instillation in ice bath, after dropping terminates, ice bath reacts about 1h, monitors reaction end by thin-layer chromatography, until react completely, concentrated organic solvent, ethyl acetate 400ml extracts, anhydrous sodium sulfate drying, suction filtration, concentrates to obtain crude product, column chromatography, obtain sterling Compound D 1
72.4g Compound C 3 is dissolved in 100ml methylene dichloride, slowly adds the ethereal HCI solution that 300ml is saturated under stirring at room temperature, reacts 6 ± 2h, monitor reaction end by thin-layer chromatography under ice bath, until react completely, concentrated, suction filtration, obtains product Compound D2,
50g Compound D 2 is dissolved in 100ml methyl alcohol, and instill the appropriate methanol solution containing vinyl cyanide under stirring at room temperature, drip after terminating, room temperature reaction 5 ± 2h, monitors reaction end by thin-layer chromatography, until react completely, concentrates to obtain compound d3,
The triethylamine of 41g compound d3,31.4ml is dissolved in the methylene dichloride of 400ml drying, by 0.06mol3,4-dibenzyloxy benzene propionyl chloride dry methylene chloride 200ml dissolves, lower slowly instillation is stirred in ice bath, after dropping terminates, ice bath reacts about 1h, monitors reaction end by thin-layer chromatography, until react completely, concentrated organic solvent, adds dilute hydrochloric acid 500ml, ethyl acetate 400ml × 3 extracts, saturated common salt water washing, anhydrous sodium sulfate drying, suction filtration, concentrates to obtain crude product, column chromatography, obtain sterling Compound D 4
61g Compound D 4 is dissolved in 40ml tetrahydrofuran (THF), adds saturated methanolic ammonia solution 800ml and RaneyNi10g, after draining air, under the hydrogen pressure of about 3MPa, in autoclave, stirring reaction 12 ± 2h under 50 DEG C of conditions, monitors reaction end by thin-layer chromatography, until react completely, suction filtration, concentrate to obtain crude product, column chromatography, obtains sterling Compound C 7, yellow oil
10g Compound D 5 is dissolved in 10ml tetrahydrofuran (THF), adds 100ml methyl alcohol, and after 1.2g10% palladium/carbon, drain air, under hydrogen normal pressure, room temperature reaction 15 ± 2h, monitors reaction end by thin-layer chromatography, until react completely, filter, vacuum-drying obtains compound 4.
9. the reference compound described in claim 1-4 is as the application detected in Kukoamine B content.
10. a detection method of content for Kukoamine B, is characterized in that, comprises the following steps:
The preparation of need testing solution: it is appropriate that precision takes Kukoamine B, puts in 100ml measuring bottle, add strong base solution 70ml, ultrasonic 10 minutes and constantly jolting Kukoamine B is dissolved, let cool, with solvent cut to scale, shake up, filter with the filter membrane of 0.45 μm, subsequent filtrate is as need testing solution;
Reference substance stock solution: accurate Weigh Compound 1, compound 2, compound 3 and compound 4 reference substance are in right amount each, also dilutes the solution made containing 45 μ g in every 1ml, as impurity reference substance stock solution with dissolve with methanol;
Kukoamine B reference substance solution: it is appropriate that precision takes Kukoamine B reference substance, solubilizing agent dissolves and dilutes the solution made containing 45 μ g in every 1ml, product stock solution in contrast; Precision measures impurity reference substance stock solution and each 5ml of reference substance stock solution respectively, puts in 50ml measuring bottle, with solvent cut to scale, shakes up, in contrast product solution,
Chromatographic condition:
According to China's coastal port two annex V D high effective liquid chromatography for measuring
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are weighting agent; Be moving phase with 0.02mol/L ammonium dihydrogen phosphate-acetonitrile (600:470 is 2.4 ~ 3.5 by phosphoric acid adjust ph after mixing), column temperature: 40 DEG C, flow velocity is 1.0ml/min; Determined wavelength is 252nm, and Kukoamine B peak and compound 1, compound 2, compound 3 and the peak-to-peak resolution of compound 4 all should be greater than 6,
Flow velocity: 1.2ml/min,
Precision measures reference substance solution and each 15 μ l of need testing solution, respectively injection liquid chromatography, and record color atlas is to 12 times of main peak retention time, and Kukoamine B peak and compound 1, compound 2, compound 3 and the peak-to-peak resolution of compound 4 all should be greater than 6.
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