CN107286162A - Heterocycle matrine derivative and its preparation method and application - Google Patents
Heterocycle matrine derivative and its preparation method and application Download PDFInfo
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- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed systems contains four or more hetero rings
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Abstract
The invention discloses a kind of heterocycle matrine derivative and its preparation method and application.The heterocycle matrine derivative structural formula such as formula（Ⅰ）It is shown.Obtain two kinds of matrine derivatives has obvious inhibitory action to tumour cell or insect cell, art technology is filled up not enough, researched and developed available for antineoplastic or insecticide, therefore the present invention provides candidate compound for the new antitumor or insecticidal activity matrine derivative of exploitation, has great importance to the medicinal active and agricultural active of full-scale development matrine.Invention further provides the preparation method of described two derivatives, it is that indoles or cyclohexylamine heterocycle are introduced in the key mapping of lead compound matrine 13, significantly improves the bioactivity of matrine, preparation method mild condition is simple and easy to apply.（Ⅰ）.
Description
Technical field
The present invention relates to compound and its separation and preparation technology field, derive more particularly, to a kind of heterocycle matrine
Thing and its preparation method and application.
Background technology
For a long time, malignant tumour turns into one of principal disease of serious harm human life and quality of life, turns into
The primary cause of the death of the mankind, moreover, as expanding economy and people are to the ineffective of environmental protection, the fatal rate of malignant tumour also exists
Constantly increase.The World Health Organization predicts, to the year two thousand twenty, will there is 20,000,000 de novo malignancy cases, wherein death toll reaches
12000000, and the overwhelming majority will occur in developing country.For the treatment of malignant tumour, natural products and its derivative medicine
Play an important role.It is reported that between 1981 to 2008, the antineoplastic in natural products source accounts for listing antineoplastic
More than the 60% of thing, moreover, new type natural product and its derivative are also constantly increasing as quantity shared by new antineoplastic
Plus.
Matrine was separated and confirmed from kuh-seng plant by botanist first in 1958, belonged to a class unique
Alkaloid, scientific name is western pyridine alkaloid in quinoline, at present, chemical composition of the domestic and international many scholars to matrine compound, medicine
Reason is acted on and clinical practice has been carried out more detailed research, it is found that matrine compound has significant anti-swollen
The multiple biological activities such as knurl, anti-inflammatory, analgesia, antiviral.The matrine molecular structure unique because having and the bioactivity enriched,
Become the important lead compound of developing new drug thing.Structural modification can be carried out to the prototype structure of natural active compound
Modification is transformed, and improves shortcoming, improves activity, solves the problems, such as the influence drug effect that original structure is present.The D rings 13,14- of matrine
Position be it is important modify key mapping, it is modified, a series of tools antitumor, anti-inflammatories, antibacterial, analgesia isoreactivity can be obtained and spread out
It is biological.
Botanical pesticide belongs to a major class of environment friendly agricultural, and with action target diversity, no Environmental Residues are pacified to people and animals
Congruent advantage.Analyze plant chemical ingredient, seek the native compound with agricultural biological activity, initiative novel green agricultural chemicals into
One of main flow for current pesticide developing.Matrine is also a kind of traditional biological agricultural chemicals, and it is to bollworm (Heliothis
Armigera), brown paddy plant hopper (Nilaparvata lugens), cabbage caterpillar (Pierisrapae Linne), diamondback moth
Etc. (Plutellaxylostella) 20 various pests have toxic action, also have to various plants pathogenic bacteria and preferably suppress to make
With；Single matrine is higher than part chemical pesticide with the insecticidal effect of other mixed pesticides, and mixture can significantly improve kuh-seng
The drug effect and insecticidal spectrum of alkali.The basic research of the agricultural aspect of current matrine is limited only to the toxicity of the test of pesticide effectiveness and form aspect
Analysis；Application and development is focused primarily upon extracts and separating matrine-like compound from various plants, then should with crude extract form
For agricultural production, or with its pesticide species with the development commercialization of other mixed pesticides, these applications are more extensive, fail
Matrine is fully demonstrated as the value of important botanical pesticide, therefore, the efficient new matrine agricultural chemicals of exploitation is current
Vital task.In recent years, as computer aided technique introduces pesticide molecule design, the exploitation paces of novel agrochemical are accelerated,
Achieve notable achievement.With these technologies using active components of plants as lead compound, using its activity mechanism as theoretical foundation,
Structural modification and transformation are carried out to lead compound, a series of efficient eco-friendly pesticides, such as pyrethroid class, cigarette have been developed
Bases, AVM etc..Matrine is the excellent lead compound for formulating novel environment friendly agricultural chemicals, but currently for agricultural evil
Worm, using matrine as lead compound, structural modification, the research of the new reactive derivative of screening are not yet carried out.
The content of the invention
The technical problem to be solved in the present invention is obtained newly through structural modification by lead compound of matrine for existing
The technical deficiency of reactive derivative there is provided a kind of heterocycle matrine derivative.
Another technical problem to be solved by the present invention is that providing the preparation method of the heterocycle matrine derivative.
A present invention also another technical problem to be solved is to provide the application of the heterocycle matrine derivative.
The purpose of the present invention is achieved by the following technical programs：
A kind of heterocycle matrine derivative is provided, shown in its structure such as formula (I)：
Wherein, the heterocyclic group is indolesOr cyclohexylamine
Invention also provides the preparation method of the matrine derivative, matrine D rings contain lactam structure,
13,14 are saturated bond, and performance is stable, and the difficulty for introducing group is relatively large, the present invention creatively with 13,14 bit strips not
The matrine derivative sophocarpine of saturation double bond is raw material, is successfully introduced into two heterocyclic radicals of indoles and cyclohexylamine respectively at its 13
Group, modification two heterocyclic radical matrine derivatives of synthesis, obtains crystal, then carry out single crystal diffraction and structure point by recrystallization
Analysis, it is respectively indyl matrine (derivative II Ind-Mat) and the cyclohexylamino matrine (Cyc- of derivative III to determine it
Mat).Structural formula is respectively as shown in formula (II) or formula (III)：
The present invention provides indyl matrine (Ind-Mat) preparation method and comprised the following steps：
S01. prepared by crude product：Logical N2Under the conditions of, sophocarpine, indoles, cesium fluoride and petroleum ether are added, heating keeps permanent
Temperature, is added dropwise tetramethoxy-silicane, reaction；After reaction terminates, slightly cool down, add dichloromethane, filtered after stirring, filtrate boils off molten
Crude product is obtained after agent.
Preferably, the indyl matrine (Ind-Mat) can obtain described by following purified crystals operating procedure
Derivative crystal：
S02. isolate and purify：Using silica gel column chromatography, with 200~300 mesh silica gel as stationary phase, mobile phase is acetic acid
After the completion of ethyl ester and alcohol mixture, wet method dress post, post activation is crossed with mobile phase repeatedly；Sealing, mobile phase immersion is stood overnight；
Sample introduction and elution：Using loading after a small amount of eluant, eluent dissolved samples, sample is fully penetrated into after silicagel column, adds elution
Agent starts elution.Often once, boil off is used to detect outflow 5mL fraction collections after most of solvent；
Detection：Target product begins to flow out time and eluant, eluent body in TLC thin-layer chromatography tracing detection Analyze ＆ separate liquid
Product, object elution finishes time and eluting solvent volume, merges that target product is begun to flow out and elution was finished between time point
All elution samples, concentration；
S03. recrystallize：
The product of concentration obtained by step S02 is dissolved with recrystallization solvent, volatilize to obtain grease, suction out grease, added
Continue to volatilize after solvent, ultrasonic dissolution, repeat 2~3 times, obtain target derivative (II).
It is further preferred that mobile phase described in step S02 is ethyl acetate：Ethanol is 10 according to volume ratio:1 mixing
Thing.
It is further preferred that the recrystallization solvent described in step S03 is n-hexane and absolute ethyl alcohol is according to volume ratio
6：1 mixture.
Present invention simultaneously provides cyclohexylamino matrine (Cyc-Mat) preparation method, comprise the following steps：
S11. prepared by crude product：Constant temperature oil bath adds under magnetic stirring after in proportion mixing sophocarpine, cyclohexylamine and water
Thermal response, after reaction terminates, is slightly cooled down, and adds dichloromethane, is filtered after stirring, and filtrate, which boils off, obtains crude product after solvent.
Preferably, the cyclohexylamino matrine (Cyc-Mat) can obtain institute by following purified crystals operating procedure
State derivative crystal：
S12. isolate and purify：Using silica gel column chromatography, with 200~300 mesh silica gel as stationary phase, mobile phase is acetic acid
After the completion of ethyl ester and alcohol mixture, wet method dress post, post activation is crossed with mobile phase repeatedly；Sealing, mobile phase immersion is stood overnight；
Sample introduction and elution：Using loading after a small amount of eluant, eluent dissolved samples, sample is fully penetrated into after silicagel column, adds elution
Agent starts elution, often flows out 5mL fraction collections once, and boil off is used to detect after most of solvent；
Detection：Target product begins to flow out time and eluant, eluent body in TLC thin-layer chromatography tracing detection Analyze ＆ separate liquid
Product, object elution finishes time and eluting solvent volume, merges that target product is begun to flow out and elution was finished between time point
All elution samples, concentration；
S13. by the sample of concentration obtained by step S12, dissolved with solvent, normal temperature evaporation of solvent obtains crystal, obtain mesh
Mark derivative III.
It is further preferred that mobile phase described in step S12 is ethyl acetate：Ethanol is 1 according to volume ratio:8 mixture.
It is further preferred that solvent described in step S13 is petroleum ether.
It is specifically to prepare with antitumor activity medicine present invention also offers the application of the heterocycle matrine derivative
Application in terms of thing or agricultural chemicals.
Antitumor activity application has good especially in terms of applied to the medicine for preparing anti-human cervical cancer cell lines
Effect.Further, derivative I nd-Mat has notable under the conditions of 0.32~0.64mmol/L dose concentrations to tumour cell
Inhibitory action, and show substantially ageing, Cyc-Mat is to the increase of the inhibitory action of cell with the time under same concentrations
And strengthen, strengthen within identical action time with the increase of concentration.Derivative Cyc-Mat is dense in 0.8~1.6mmol/L dosage
There is significant inhibitory action under the conditions of degree to tumour cell, and show it is substantially ageing, Cyc-Mat pairs under same concentrations
The inhibitory action of cell strengthens with the increase of time, strengthens within identical action time with the increase of concentration,.
Preferably, the application mainly application and preparation prepared in terms of agricultural chemicals is in the eco-friendly pesticide of preventing and treating agricultural pests
Candidate compound.The insect includes but is not limited to bollworm (Heliothis armigera), brown paddy plant hopper
(Nilaparvata lugens), cabbage caterpillar (PierisrapaeLinne), diamondback moth (Plutellaxylostella), meadow
Beet armyworm (S.frugperda) etc.
Further, in terms of applied to Spodopterafrugiperda (S.frugperda) Sf9 cell lines are suppressed.Derivative I nd-Mat
The Apoptosis can be substantially induced in 2.5mM concentration, 3mM concentration versus cells breed growth inhibition ratio more than 80%.
1.2mM Cyc-Mat are to Sf9 cell inhibitory rates more than 60%.Indoles is introduced in the key mapping of matrine 13 and two kinds of cyclohexylamine are miscellaneous
Ring, can significantly improve its insect cell inhibitory activity.
Beneficial effects of the present invention：
The invention discloses two kinds of new heterocyclic radical matrine derivatives, the derivative has well antitumor and killed
Worm activity, obtain two kinds of matrine derivatives has obvious inhibitory action to tumour cell or insect cell, has filled up this
Art deficiency, is researched and developed available for antineoplastic or insecticide, therefore the present invention is to develop newly antitumor or kill
Worm activity matrine derivative provides candidate compound, has weight to the medicinal active and agricultural active of full-scale development matrine
The meaning wanted.
It is demonstrated experimentally that 0.32~0.64mmol/L derivative I nd-Mat has good inhibitory action to tumour cell, and
Show substantially it is ageing, Cyc-Mat strengthens with the increase of time the inhibitory action of cell under same concentrations；Identical
Strengthen in action time with the increase of concentration.0.8~1.6mmol/L derivative Cyc-Mat has good to tumour cell
Inhibitory action, and show substantially ageing, Cyc-Mat is to the increase of the inhibitory action of cell with the time under same concentrations
And strengthen；Strengthen within identical action time with the increase of concentration.
The derivative, which has, to be suppressed using Spodopterafrugiperda (S.frugperda) Sf9 cell lines as the agricultural of Typical Representative
Active aspect.Derivative I nd-Mat can substantially induce the Apoptosis, 3mM concentration versus cells propagation in 2.5mM concentration
Growth inhibition ratio is more than 80%.1.2mM Cyc-Mat are to Sf9 cell inhibitory rates more than 60%.Introduced in the key mapping of matrine 13
Two kind heterocycles of indoles and cyclohexylamine, can significantly improve its insect cell inhibitory activity.
It is in the key mapping of lead compound matrine 13 invention further provides the preparation method of described two derivatives
Upper introducing indoles or cyclohexylamine heterocycle, significantly improve the bioactivity of matrine, preparation method mild condition is simple and easy to apply.
Brief description of the drawings
The crystal habit figure of Fig. 1 derivative indyl matrines (Ind-Mat).
The MS analysis results of Fig. 2 derivative indyl matrines (Ind-Mat).
The monocrystalline of Fig. 3 derivative indyl matrines (Ind-Mat) derives result.
The monocrystalline of Fig. 4 derivative cyclohexylamino matrines (Cyc-Mat) derives result.
The crystal habit figure of Fig. 5 cyclohexylaminos matrine (Cyc-Mat).
Fig. 6 derivatives influence on Hela229 cells microscopic morphology.
Fig. 7 derivatives influence on Sf9 cells microscopic morphology.
Embodiment
The present invention is further illustrated with reference to specific embodiment.Following being given for example only property of embodiment explanations, it is impossible to manage
Solve as limitation of the present invention.Unless stated otherwise, the reagent used in following embodiments is that conventional purchased in market or commercial sources are obtained
The reagent obtained, unless stated otherwise, the method and apparatus used in following embodiments are method commonly used in the art and set
It is standby.
It is prepared by the indyl matrine (Ind-Mat) of embodiment 1
S01. crude product is prepared
100mL three-necked flasks, are passed through N2After 5min, sophocarpine 1.23g, indoles 0.585g, cesium fluoride 0.76g and stone are added
Oily ether 12mL.Adjust N2Start glycerine oil bath heating after flow velocity, 5min, keep 80 DEG C of constant temperature, 0.5mL tetramethoxy-silicanes are added dropwise
Alkane；React and stop heating after 6h.2~3mL can be supplemented when quantity of solvent is maintained at 4~5mL deficiencies in course of reaction.Reaction terminates
Afterwards, slightly cool down, add 10mL dichloromethane, filtered after stirring, filtrate, which boils off, obtains crude product after solvent.
S02. isolate and purify and structural characterization
Crude product obtained by step S01 is isolated and purified using silica gel column chromatography.
(1) separation condition
Stationary phase：200~300 mesh silica gel 230mL；
Screen eluent system：TLC methods screen eluting solvent system, and it is acetic acid second finally to determine pillar layer separation mobile phase
Ester：Ethanol=10:1.
Fill post and activation：After the completion of wet method dress post, post activation is crossed with mobile phase repeatedly；Sealing, mobile phase immersion was stood
Night.
Sample introduction and elution：Using loading after a small amount of eluant, eluent dissolved samples, sample is fully penetrated into after silicagel column, adds elution
Agent starts elution.Often once, boil off is used to detect outflow 5mL fraction collections after most of solvent.Detection：TLC thin-layer chromatographys with
Target product begins to flow out time and eluant, eluent volume in track detection and analysis separating liquid, and object elution finishes time and elution
Solvent volume.Merge target product and begin to flow out and elute all elution samples finished between time point, concentrate.
(2) recrystallization and structural characterization
Recrystallisation solvent system：N-hexane：Absolute ethyl alcohol=6：1.
Concentrating sample, dissolved with 1.5~2mL recrystallization solvents, be transferred to glass sample bottle, bottleneck is sealed with filter paper, logical
Volatilization is opened wide under wind cupboard.Stand overnight rear bottom of bottle occur crystallizing and yellow oil (bottle domestic demand leaves certain quantity of fluid, can not wave
It is dry), grease is suctioned out with capillary, adds after solvent, ultrasonic dissolution and continues to volatilize, 2~3 processes is repeated and can obtain form
Preferable crystal.TLC thin-layer chromatographys, which are further detected, confirms that obtained sample is synthetic product sterling, takes partial crystals sample to enter
Row mass spectrum (LC-MS) is analyzed and monocrystalline deriveding analysis.Characterization result confirms that indolyl radical has been introduced into the key mapping of matrine 13, and obtains
Target derivative (II).Shown in the crystal habit as accompanying drawing 1 of target derivative (II), as shown in Figure 2, monocrystalline spreads out MS analysis results
Life the results are shown in Table shown in 1 and Fig. 3.
The Data Collection of table 1 and processing
It is prepared by the cyclohexylamino matrine (Cyc-Mat) of embodiment 2
S11. crude product is prepared
50mL single-necked flasks, add 60 DEG C of oil of constant temperature under sophocarpine 0.62g, cyclohexylamine 0.55mL, water 4mL, magnetic agitation
Bath heating 6 hours.After reaction terminates, slightly cool down, add 10mL dichloromethane, filtered after stirring, filtrate boils off and obtained slightly after solvent
Product.
S12. isolate and purify and structural characterization
Crude product obtained by step S11 is used into silica gel column chromatography.
(1) separation condition
Stationary phase：200~300 mesh silica gel 230mL；
Screen eluent system：TLC methods screen eluting solvent system, and it is acetic acid second finally to determine pillar layer separation mobile phase
Ester：Ethanol=1：8.
Fill post and activation：After the completion of wet method dress post, post activation is crossed with mobile phase repeatedly；Sealing, mobile phase immersion was stood
Night.
Sample introduction and elution：Using loading after a small amount of eluant, eluent dissolved samples, sample is fully penetrated into after silicagel column, adds elution
Agent starts elution.Often once, boil off is used to detect outflow 5mL fraction collections after most of solvent.
Detection：Target product begins to flow out time and eluant, eluent body in TLC thin-layer chromatography tracing detection Analyze ＆ separate liquid
Product, object elution finishes time and eluting solvent volume.Merge that target product is begun to flow out and elution was finished between time point
All elution samples, concentration.
(2) recrystallization and structural characterization
Recrystallisation solvent system：Petroleum ether
Concentrating sample, with 1.5~2mL petroleum ether dissolutions, glass sample bottle is transferred to, bottleneck is sealed with filter paper, in fume hood
Middle normal temperature evaporation of solvent obtains crystal.TLC thin-layer chromatographys, which are further detected, confirms that obtained sample is synthetic product sterling, takes
Partial crystals sample carries out LC-MS analyses and monocrystalline deriveding analysis.Confirm that indolyl radical has been introduced into the key mapping of matrine 13, and obtain
Target derivative III.As shown in Figure 5, monocrystalline derives the results are shown in Table shown in 2 and Fig. 4 the crystal habit of derivative III.
Table 2:Data Collection and processing
The two derivative Analysis on Biological Activity of the invention of embodiment 3
1. two derivatives are tested to tumour Hela229 cell inhibitory activities
(1) experimental program：
Test compound：Lead compound matrine (Mat) before modification；Compound experiment raw material, matrine derivative Chinese scholartree
Fruit alkali (Sop)；Two kinds of new matrine derivative indyl matrines (derivative II Ind-Mat) and cyclohexylamino of the invention are bitter
Join alkali (Cyc-Mat of derivative III).
Cell culture：For the human cervical carcinoma cell Hela229 of examination, 10% hyclone is added with MEM basal mediums,
5% carbon dioxide, the cell culture incubator cellar culture of 37 DEG C and appropriate humidity.
MTT detects cell viability：Selection cell in good condition, adjustment cell suspension density to 8 × 104~1 × 105Individual/
ML, is inoculated into 96 orifice plates, the influence of the medicine cell proliferation such as Mat with every μ L of hole 100, then is inoculated with after 24h, and experiment is divided to two
Group：Control group and agent-feeding treatment group, setting 24,48, tri- time points of 72h, every group of each time point each four repetitions of concentration are arrived
After time point, to corresponding hole, MTT solution is added with every μ L of hole 10, lucifuge is during which noted, in discarding training after incubator culture 4h
Nutrient solution, adds DMSO with every μ L of hole 150, and gently concussion is that bluish violet crystallization first a ceremonial jade-ladle, used in libation is completely dissolved at room temperature, about 15min；By 96 holes
It is put into ELIASA, sets wavelength as 429mm, determines and record the absorbance (OD values) of corresponding aperture.Cell inhibitory rate (%)=
(1-ODExperimental group/ODControl group) × 100%.
Inverted phase contrast microscope (IPCM) morphologic observation：The cell for selecting form good, adjustment cell density to 8 × 104
~1 × 105According to experimental design after individual/mL, the cell suspension added into 6 orifice plates with every hole 1mL after dilution, 24h, at dosing
Reason, carries out IPCM fixed point observations in 0,24,48,72h respectively, takes photo record cellular morphology.
(2) result and analysis
Observed from IPCM microscopic features, cellular control unit form is in shuttle row, and refractivity is good, 24,48h be in it is vigorous
Vegetative state (Fig. 6-A, B).Induced respectively with final concentration 1.6,0.64,1.6mmol/L Mat, Ind-Mat and Cyc-Mat
Cell 24 and 48h, are compared with control group, and different degrees of change, wherein Ind-Mat and Cyc-Mat occur for three processing cellular morphologies
Treatment group, cellular morphology change is especially pronounced, and cell quantity gradually tails off, and attached cell form diminishes, and finally disintegrate the (figure that disappears
6-E, F, G, H).Mat treatment group cellular morphologies have a certain impact, but intensity of variation does not have Ind-Mat and Cyc-Mat treatment groups
Significantly, cell remains to continue to breed growth (Fig. 6-C, D).MTT cell viability 0.32~0.64mmol/L of analysis shows Ind-
Mat has different degrees of inhibitory action to tumour cell, and shows substantially ageing, and Cyc-Mat is to thin under same concentrations
The inhibitory action of born of the same parents strengthens with the increase of time；Strengthen within identical action time with the increase of concentration, 0.64mmol/L,
72h inhibiting rates are handled up to 83.83%, than 0.8mmol/L concentration Mat and Sop treatment groups 7.91% and 14.46% significantly
Increase (being shown in Table shown in 3,4,5).0.8~1.6mmol/L Cyc-Mat also have different degrees of inhibitory action to tumour cell, and
Show substantially it is ageing, Cyc-Mat strengthens with the increase of time the inhibitory action of cell under same concentrations；Identical
Strengthen in action time with the increase of concentration, 0.8mmol/L, processing 72h inhibiting rates compare same concentrations up to 66.32%
The 7.91% of Sop, Mat treatment group and 14.46% increases considerably (table 3,4,6), it is seen that introduce Yin in the key mapping of matrine 13
Two heterocycles of diindyl and cyclohexylamine, can significantly improve its antitumor activity.In Fig. 6, A-B：CK, 24h and 48h；C-D:
2.5mmol/L Matrine handle 24h and 48h；E-F:2.5mmol/L Ind-Mat handle 24h and 48h；G-H：1.2mmol/L
Cyc-Mat handles 24h and 48h.
The various concentrations Mat of table 3 influences on growth of tumour cell
The Sop of the various concentrations of table 4 influences on growth of tumour cell
The Ind-Mat of the various concentrations of table 5 influences on growth of tumour cell
The Cyc-Mat of the various concentrations of table 6 influences on growth of tumour cell
Two derivatives of the invention of embodiment 4 are to Insect cells Sf9 inhibitory activity
(1) experimental program：
Two derivative various insects cells of the invention have good inhibitory activity.It is greedy with meadow below to exempt to repeat
Illustrated exemplified by noctuid (S.frugperda).
Test compound：Lead compound matrine (Mat) before modification；Compound experiment raw material, matrine derivative Chinese scholartree
Fruit alkali (Sop)；Two kinds of new matrine derivative indyl matrines (derivative II Ind-Mat) and cyclohexylamino of the invention are bitter
Join alkali (Cyc-Mat of derivative III).
Cell culture：It is Spodopterafrugiperda (S.frugperda) Sf9 cell lines for examination insect cell, with containing 10% tire ox
The Insect culture medium of serum is placed in the conventional adhere-wall culture of 27 DEG C of constant temperature of cell culture incubator.Cell culture medium main component includes
Grace ' s insects dehydrated medium (Gibco companies), hyclone living, yeast extract, peptone, lactoalbumin hydrolysate, cell
Secondary Culture.
MTT detects cell viability：Selection cell in good condition, adjustment cell suspension density to 8 × 104~1 × 105Individual/
ML, is inoculated into 96 orifice plates, the influence of the medicine cell proliferation such as Mat with every μ L of hole 100, then is inoculated with after 24h, and experiment is divided to two
Group：Control group and agent-feeding treatment group, setting 24,48, tri- time points of 72h, every group of each time point each four repetitions of concentration are arrived
After time point, to corresponding hole, MTT solution is added with every μ L of hole 10, lucifuge is during which noted, in discarding training after incubator culture 4h
Nutrient solution, adds DMSO with every μ L of hole 150, and gently concussion is that bluish violet crystallization first a ceremonial jade-ladle, used in libation is completely dissolved at room temperature, about 15min；By 96 holes
It is put into ELIASA, sets wavelength as 429mm, determines and record the absorbance (OD values) of corresponding aperture.Cell inhibitory rate (%)=
(1-ODExperimental group/ODControl group) × 100%.
Inverted phase contrast microscope (IPCM) morphologic observation：The cell for selecting form good, adjustment cell density to 8 × 104
~1 × 105According to experimental design after individual/mL, the cell suspension added into 6 orifice plates with every hole 1mL after dilution, 24h, at dosing
Reason, carries out IPCM fixed point observations in 0,24,48,72h respectively, takes photo record cellular morphology.
(2) result and analysis
Understood by IPCM microscopic morphologies observation (Fig. 7, A-H) and cytoactive detection (table 7~10) result, cellular control unit
Form is rounded, and refractivity is good, 24,48h be in vigorous vegetative state (Fig. 7, A, B), below concentration 3mM Mat and Sop pairs
Sf9 cell growths have certain promotion, and cell state is good (Fig. 7, C, D), 3~4.5mM propagation suppressions certain to cells show
Make and use, inhibiting rate is less than 20%.And the Ind-Mat that 13 key mappings are introduced after indyl modification can be obvious in 2.5mM concentration
Inducing cell apoptosis, induces 24h, and cell floats, around produces a large amount of apoptotic bodies, show notable apoptosis feature, 48h
Afterwards, most cells have been floated, disintegrate dead (Fig. 7-E, F), and 3mM concentration versus cells breed growth inhibition ratio more than 80%.
1.2mM Cyc-Mat are to Sf9 cell inhibitory rates more than 60%, and microscopic morphology observation finds that Cyc-Mat induction Sf9 cells 24h is big
Part still cell attachment, but occur after vacuolation, 48h, the adherent death in situ of disintegrating of most cells, but cell peripheral is not a large amount of
There is apoptotic body (Fig. 7-G, H), it is dead that autophagy occurs for Cyc-Mat induction Sf9 cells.It can be seen that being introduced in the key mapping of matrine 13
Two kind heterocycles of indoles and cyclohexylamine, can significantly improve its insect cell inhibitory activity.In Fig. 7, A-B：CK, 24h and 48h；
C-D:2.5mmol/L Matrine handle 24h and 48h；E-F:2.5mmol/L Ind-Mat handle 24h and 48h；G-H：
1.2mmol/L Cyc-Mat handle 24h and 48h.
Growth effects of the Mat of the various concentrations of table 7 to insect cell
Growth effects of the Sop of the various concentrations of table 8 to insect cell
Growth effects of the table 9Ind-Mat to insect cell
Growth effects of the Cyc-Mat of the various concentrations of table 10 to insect cell
Claims (10)
1. a kind of heterocycle matrine derivative, it is characterised in that its structure such as formula（Ⅰ）It is shown：
（Ⅰ）,
Wherein, the heterocyclic group isOr。
2. the preparation method of matrine derivative described in claim 1, it is characterised in that with 13,14 bit strip unsaturated double-bonds
Matrine derivative sophocarpine is raw material, and heterocyclic group is introduced at its 13, and modification synthesis obtains heterocyclic radical matrine derivative.
3. the preparation method of matrine derivative according to claim 2, it is characterised in that the preparation method also includes point
From purifying and re-crystallization step；Described isolate and purify uses silica gel column chromatography.
4. the preparation method of matrine derivative according to Claims 2 or 3, it is characterised in that the derivative is indoles
Base matrine, structural formula such as formula（Ⅱ）It is shown：
（Ⅱ）；
Preparation method comprises the following steps：
S01. prepared by crude product：Logical N2Under the conditions of, sophocarpine, indoles, cesium fluoride and petroleum ether are added, heating keeps constant temperature, drop
Plus tetramethoxy-silicane, reaction；After reaction terminates, slightly cool down, add dichloromethane, filtered after stirring, filtrate is boiled off after solvent
Obtain crude product；
S02. isolate and purify：Using silica gel column chromatography, with 200~300 mesh silica gel as stationary phase, mobile phase is ethyl acetate
And alcohol mixture, after the completion of wet method dress post, post activation is crossed with mobile phase repeatedly；Sealing, mobile phase immersion is stood overnight；
Sample introduction and elution：Using loading after a small amount of eluant, eluent dissolved samples, sample is fully penetrated into after silicagel column, is added eluant, eluent and is opened
Begin to elute, often flow out 5mL fraction collections once, boil off is used to detect after most of solvent；
Detection：Target product begins to flow out time and eluant, eluent volume, mesh in TLC thin-layer chromatography tracing detection Analyze ＆ separate liquid
Mark thing elution finishes time and eluting solvent volume, merges target product and begins to flow out and elute and finishes owning between time point
Elution samples, concentration；
S03. recrystallize：
The product of concentration obtained by step S02 is dissolved with recrystallization solvent, volatilize to obtain grease, suctions out grease, add solvent,
Continue to volatilize after ultrasonic dissolution, repeat 2~3 times, obtain target derivative（Ⅱ）.
5. the preparation method of matrine derivative according to claim 4, it is characterised in that mobile phase described in step S02 is
Ethyl acetate：Ethanol is 10 according to volume ratio:1 mixture；The recrystallization solvent is n-hexane and anhydrous described in step S03
Ethanol is 6 according to volume ratio：1 mixture.
6. the preparation method of matrine derivative according to Claims 2 or 3, it is characterised in that characterized in that, described spread out
Biology is cyclohexylamino matrine, and structural formula is respectively such as formula（Ⅲ）It is shown：
（Ⅲ）；
The preparation method comprises the following steps：
S11. prepared by crude product：Constant temperature oil bath is heated instead under magnetic stirring after in proportion mixing sophocarpine, cyclohexylamine and water
Should, after reaction terminates, slightly cool down, add dichloromethane, filtered after stirring, filtrate, which boils off, obtains crude product after solvent；
S12. isolate and purify：Using silica gel column chromatography, with 200~300 mesh silica gel as stationary phase, mobile phase is ethyl acetate
And alcohol mixture, after the completion of wet method dress post, post activation is crossed with mobile phase repeatedly；Sealing, mobile phase immersion is stood overnight；
Sample introduction and elution：Using loading after a small amount of eluant, eluent dissolved samples, sample is fully penetrated into after silicagel column, is added eluant, eluent and is opened
Begin to elute, often flow out 5mL fraction collections once, boil off is used to detect after most of solvent；
Detection：Target product begins to flow out time and eluant, eluent volume, mesh in TLC thin-layer chromatography tracing detection Analyze ＆ separate liquid
Mark thing elution finishes time and eluting solvent volume, merges target product and begins to flow out and elute and finishes owning between time point
Elution samples, concentration；
S13. by the sample of concentration obtained by step S12, dissolved with solvent, normal temperature evaporation of solvent obtains crystal, obtain target and spread out
Biology III.
7. the preparation method of matrine derivative according to claim 6, it is characterised in that mobile phase described in step S12 is
Ethyl acetate：Ethanol is 1 according to volume ratio:8 mixture；Solvent described in step S13 is petroleum ether.
8. any one of claim 1 to the 7 heterocycle matrine derivative is being prepared with anti-tumor activity medicine or agricultural chemicals
The application of aspect.
9. application according to claim 8, it is characterised in that in terms of anti-human cervical cancer cell lines medicine is prepared.
10. application according to claim 8, it is characterised in that applied to the eco-friendly pesticide for preparing preventing and treating agricultural pests
Candidate compound；It is preferably applied to prepare preventing and treating bollworm（Heliothis armigera）, brown paddy plant hopper（Nilaparvata lugens）, cabbage caterpillar（Pierisrapae Linne）, diamondback moth（Plutella xylostella）Or Spodopterafrugiperda（S. frugperda）Eco-friendly pesticide candidate compound.
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| CN108299435A (en) * | 2018-01-29 | 2018-07-20 | 广西大学 | 13- ethanol amine matrine derivatives prepare and its application |
| CN109928972A (en) * | 2019-03-19 | 2019-06-25 | 中国医学科学院药用植物研究所 | A kind of matrine derivative and its application in drug |
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