CN108084295A - A kind of Beta-cyclodextrin-based nitric oxide donors and preparation method thereof - Google Patents
A kind of Beta-cyclodextrin-based nitric oxide donors and preparation method thereof Download PDFInfo
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- CN108084295A CN108084295A CN201710930549.4A CN201710930549A CN108084295A CN 108084295 A CN108084295 A CN 108084295A CN 201710930549 A CN201710930549 A CN 201710930549A CN 108084295 A CN108084295 A CN 108084295A
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
- C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/716—Glucans
- A61K31/724—Cyclodextrins
Abstract
The invention discloses a kind of Beta-cyclodextrin-based nitric oxide donors, as shown in formula (I), and disclose the preparation method of above-mentioned Beta-cyclodextrin-based nitric oxide donors, preparation method is：By the reaction product dissolving of amidized cyclodextrin and N acetyl penicilliamine thiolactones in organic solvent, add in nitroso spy's butyl ester, after the reaction was complete under certain condition, precipitated with poor solvent to get to Beta-cyclodextrin-based nitric oxide donors.Beta-cyclodextrin-based nitric oxide donors prepared by the present invention can be widely applied in drug development, and subcutaneous, muscle or intravenous injection is such as made, for the treatment of angiocardiopathy, tumour etc., can obtain good therapeutic effect, have application value.
Description
Technical field
The invention belongs to medicinal chemistry arts, and in particular to a kind of cyclodextrin nitric oxide donors and preparation method thereof.
Background technology
Nitric oxide (NO) is the in vivo important messenger molecule of biology and effector molecule, it can participate in a variety of lifes in life entity
The adjusting of function is managed, plays extremely important function in vivo.As nitric oxide takes part in regulation of blood vessels, neurotransmission, inflammation
With the processes such as immune response.Nitric oxide can induce macrophage, cardiac muscle cell, vascular smooth muscle cells, islet cells etc.
Apoptosis.In addition, substantial amounts of research shows the in close relations of nitric oxide and tumour, the nitric oxide of low concentration can promote tumour
Growth, and the nitric oxide of high concentration can inhibit the growth of tumour.
In order to overcome internal nitric oxide generation deficiency or nitric oxide signal transduction abnormal, the research of nitric oxide donors
Receive the extensive concern of researcher.Nitric oxide donors drug plays in nitric oxide production research and the treatment of clinical disease
Positive effect, it can quick release high concentration nitric oxide in physiological conditions, play nitric oxide production physiological action, have become
For one of the hot spot of biomedical sector research and forward position.Nitric oxide donors can extend nitric oxide production half-life period and
Carrier as NO realizes transport in vivo.According to the difference for the atom being connected with nitric oxide releasing position, nitric oxide donors
Six major classes can be divided into：O-NO donors, C-NO donors, N-NO donors, S-NO donors, heterocycle NO donors and transition metal NO donors.
However, the building-up process of most of nitric oxide donors is comparatively laborious, and condition is more harsh, big if desired for 5
Air pressure nitric oxide gases at high pressure participate in reaction etc..In addition, major part NO donors are highly unstable, half-life period generally arrives in the several seconds
A few hours etc., this causes the storage of NO donors and biomedical applications very big puzzlement.On the other hand, nitric oxide exists
In vivo half-life period only has 3-6 seconds, and dilation angle only has 40-200 μm, this requires nitric oxide donors must the accurate transmission arrive
Target site competence exertion acts on.
Preferable nitric oxide donors, nitric oxide will not release in transportational process, after target site is reached,
Nitric oxide spontaneous can steadily release.How by the designs of nitric oxide donors, nitric oxide transportational process is realized
In do not discharge and reach quick release after targeting moiety, be the key scientific problems of nitric oxide donors drug design.
Cyclodextrin is one that amylose generates under the cyclodextrin glycosyltransferase effect generated by bacillus
The general name of series of annular oligosaccharide receives in the fields such as catalysis, separation, food and drug and greatly payes attention to and extensively should
With.In field of medicaments, cyclodextrin can effectively increase some water-soluble undesirable drugs solubility in water and solution rate,
It improves the stability of drug and bioavilability, reduces the stimulation of drug and toxic side effect and make medicament slow release and improver
Type.In addition, cyclodextrin has been used as solubilizing agents for drugs to be ratified by FDA (Food and Drug Adminstration) (FDA), it is to human body use
Safety.Therefore, the research and development of Beta-cyclodextrin-based drug have great application prospect.
The content of the invention
The present invention provides using cyclodextrin as raw material, synthesize a kind of stability it is good, can be by cell high-efficient endocytosis, Targeting delivery
Nitric oxide production Beta-cyclodextrin-based nitric oxide donors solve the problems, such as that traditional nitric oxide donors stability is poor, half-life short.
A kind of Beta-cyclodextrin-based nitric oxide donors, shown in general structure such as formula (I)：
Wherein n be integer, 1≤n≤3；
R is-(CH2)2-、-(CH2)3-、-(CH2)4-、-(CH2)5-、-(CH2)6-、-(CH2)2O(CH2)2-、-(CH2)2O
(CH2)2O(CH2)2-、-CH2PhCH2-、-(CH2)2Ph(CH2)2-、-CH(CH3)CH2-、-CH(CH3)CH2CH2-、-(CH2)2NH
(CH2)2-、-(CH2)2NH(CH2)2NH(CH2)2-、-(CH2)2NH(CH2)2NH(CH2)2NH(CH2)2-、-CH2CH=CHCH2- in
One kind.
The present invention also provides the preparation method of above-mentioned Beta-cyclodextrin-based nitric oxide donors, preparation method is simple, reaction item
Part is mild, easy.
A kind of preparation method of Beta-cyclodextrin-based nitric oxide donors, comprises the following steps：
(1) by cyclodextrin (the hydroxy activated method reference literature of cyclodextrin of tolysulfonyl chlorine activation
Macromol.Rapid Commun.2011,32,397.), in organic solvent, 80 DEG C are added with stirring diamine and tie up for dissolving
Sour agent carries out nucleophilic substitution, when reaction 24~72 is small, after complete reaction, is precipitated in poor solvent three times, after filtering
Filter residue puts drying in vacuum drying oven, obtains white solid aminocyclodextrin；
(2) by Beracilline dissolving in organic solvent, it is added with stirring acetic anhydride, at -20~80 DEG C, reaction 2~48
Hour, after the reaction was complete, post processing obtains N- acetyl penicilliamine thiolactones；
(3) by amidized cyclodextrin in organic solvent, N- acetyl penicilliamine thiolactones are slowly added into, treat it
All after dissolving, when reaction 1~48 is small at 0~60 DEG C, after the reaction was complete, precipitated in poor solvent three times, obtained solid
It puts dry in vacuum drying oven；
(4) the solid product dissolving obtained step (3) in organic solvent, adds in nitroso spy's butyl ester, -20~80
When reaction 2~72 is small at DEG C, after the reaction was complete, being precipitated in poor solvent three times, filtered filtration residue puts drying in vacuum drying oven,
Obtain the Beta-cyclodextrin-based nitric oxide donors of white solid.
The chemical equation of the Beta-cyclodextrin-based nitric oxide donors is as follows：
The cyclodextrin is one kind in alpha-cyclodextrin, beta-cyclodextrin or gamma-cyclodextrin.
The structural formula of the diamine is NH2-R-NH2, the wherein definition of R is identical with formula (I).
The acid binding agent is one kind in triethylamine or 4-dimethylaminopyridine.
The organic solvent includes N,N-dimethylformamide (DMF), dichloromethane, chloroform, toluene, pyridine or diformazan
Base sulfoxide.
The poor solvent includes acetonitrile, ether, methanol or n-hexane.
Post processing described in step (2) includes vacuum distillation, extraction and recrystallization.
The organic solvent for extraction includes dichloromethane or chloroform.
The recrystallization is hexane with poor solvent.
The dosage of reaction raw materials does not have specific requirement there is no stringent restriction in the present invention, is counted generally according to chemical reaction
Amount ratio is reacted.
The dosage of organic solvent, acid binding agent, can be according to the dosage tune of reaction raw materials there is no stringent restriction in the present invention
It is whole：The dosage of organic solvent, which is subject to, can dissolve raw material, and the dosage of acid binding agent is carried out with that can promote to react to target product direction
Subject to.
Compared with prior art, the present invention has advantageous effect following prominent：
1) the Beta-cyclodextrin-based nitric oxide donors stability that prepared by the present invention is good, and physiological environment half-life is long, and in water
Middle dissolubility is good；
2) Beta-cyclodextrin-based nitric oxide donors prepared by the present invention can be by the effective endocytosis of cell, and only reduces in the cell
Quick release nitric oxide under environment, and it is very slow extracellularly to discharge nitric oxide；
3) In vitro cell experiment shows that Beta-cyclodextrin-based nitric oxide donors prepared by the present invention can effectively facilitate endothelial cell
Growth and inhibit the growth of smooth muscle cell simultaneously, beneficial to cardiovascular quick endothelialization；
4) In vitro cell experiment shows that Beta-cyclodextrin-based nitric oxide donors prepared by the present invention can effectively inhibit tumour cell
Multiplication.
The present invention also provides Beta-cyclodextrin-based nitric oxide donors as Beta-cyclodextrin-based drug in angiocardiopathy and tumour
Subcutaneous, muscle or intravenous injection is such as made in application process in therapy field, for the treatment of angiocardiopathy, tumour etc.,
Good therapeutic effect can be obtained.
Beta-cyclodextrin-based nitric oxide donors prepared by the present invention can be widely applied in drug development, are had and are promoted and applied
Value.
Description of the drawings
Fig. 1 is the nuclear magnetic resonance spectroscopy of the beta-cyclodextrin for the tolysulfonyl chlorine activation that the embodiment of the present invention 1 obtains.
Fig. 2 is the nuclear magnetic resonance spectroscopy for the amination beta-cyclodextrin that the embodiment of the present invention 1 obtains.
Fig. 3 is the nuclear magnetic resonance spectroscopy for the N- acetyl group penicillamine thiolactones that the embodiment of the present invention 1 obtains.
Fig. 4 is that the nuclear-magnetism for N- acetamido penicillanates element acyl-triethylene tetramine group-beta-cyclodextrin that the embodiment of the present invention 1 obtains is total to
The hydrogen that shakes is composed.
Fig. 5 is the nuclear magnetic resonance spectroscopy for the beta-cyclodextrin base nitric oxide donors that the embodiment of the present invention 1 obtains.
Fig. 6 is the Beta-cyclodextrin-based nitric oxide donors Human Umbilical Vein Endothelial Cells and smooth muscle of various concentration in application examples 1 of the present invention
The column comparison diagram that cell Proliferation influences, wherein upper figure is the block diagram that β-CD-NO promote endothelial cell proliferation, figure below is inhibition
The block diagram of smooth muscle cell growth.
Fig. 7 is the Beta-cyclodextrin-based nitric oxide donors Human Umbilical Vein Endothelial Cells and smooth muscle of various concentration in application examples 2 of the present invention
The fluorescent microscopy images that cell Proliferation influences.
Fig. 8 is that the Beta-cyclodextrin-based nitric oxide donors of various concentration in application examples 3 of the present invention influence tumor cell proliferation
Block diagram.
Fig. 9 discharges nitric oxide production song in different environments for 4 cyclodextrin base nitric oxide donors of application examples of the present invention
Line comparison diagram.
Specific embodiment
For a further understanding of the present invention, with reference to embodiment to a kind of Beta-cyclodextrin-based nitric oxide provided by the invention
The preparation method of donor is specifically described, but the present invention is not limited to these embodiments, personnel are in the present invention for the field technology
The non-intrinsically safe modifications and adaptations made under core guiding theory, still fall within protection scope of the present invention.
Embodiment 1
60g beta-cyclodextrins are suspended in 500mL water, 7g sodium hydroxides saturated solution to solution is added dropwise dropwise in 0 DEG C is in
Clear shape；It is kept for 0 DEG C, 15g paratoluensulfonyl chlorides is dissolved in 10mL acetonitriles, instill above-mentioned solution dropwise, at room temperature instead
Answer 2 it is small when；It is placed in 4 DEG C overnight, filters solid and recrystallized 3 times with water, obtain the beta-cyclodextrin of tolysulfonyl chlorine activation, nuclear-magnetism
Hydrogen is composed referring to Fig. 1.
The beta-cyclodextrin of 1.5g tolysulfonyl chlorine activations is dissolved in 20mL DMF, 80 DEG C is heated to, sequentially adds
3.5mL triethylene tetramines and 2mL triethylamines；React 48 it is small when after, with ether precipitation three times, white solid in vacuum drying oven do
It is dry, amination beta-cyclodextrin is obtained, nucleus magnetic hydrogen spectrum is referring to Fig. 2.
3.5g Beracillines are dissolved in 20mL dimethyl sulfoxide (DMSO)s, are cooled to 0 DEG C, 15mL acetic anhydride is added dropwise dropwise
Dimethyl sulphoxide solution, be maintained at -20 DEG C reaction 48 it is small when, after the reaction was complete, solvent is removed with Rotary Evaporators, by residue
Product disperses in methylene chloride again, and organic layer is extracted three times with water；Finally, revolving removes solvent, recrystallizes in hexane
N- acetyl penicilliamine thiolactones are can obtain, nucleus magnetic hydrogen spectrum is referring to Fig. 3.
By 0.5g aminations beta-cyclodextrin in room-temperature dissolution in 15mL toluene, 0.15g N- acetyl penicilliamine sulphur is added in
Lactone, when stirring 1 is small at 0 DEG C, with acetonitrile precipitation three times, dry white solid product, nucleus magnetic hydrogen spectrum ginseng in vacuum drying oven
See Fig. 4.
By 0.6g white solid products obtained in the previous step in room-temperature dissolution in 15mL DMF, 0.5mL nitrosos are added in
Special butyl ester, when stirring 72 is small at -20 DEG C, white solid is dry in vacuum drying oven, you can obtains β-ring with methanol extraction three times
Dextrin base nitric oxide donors, nucleus magnetic hydrogen spectrum is referring to Fig. 5.
Embodiment 2
60g alpha-cyclodextrins are suspended in 500mL water, 7g sodium hydroxides saturated solution to solution is added dropwise dropwise in 0 DEG C is in
Clear shape；It is kept for 0 DEG C, 15g paratoluensulfonyl chlorides is dissolved in 10mL acetonitriles, instill above-mentioned solution dropwise, at room temperature instead
Answer 2 it is small when；It is placed in 4 DEG C overnight, filters solid and recrystallized 3 times with water, obtain the alpha-cyclodextrin of tolysulfonyl chlorine activation.
The alpha-cyclodextrin of 3g tolysulfonyl chlorine activations is dissolved in 25mL DMF, 80 DEG C is heated to, sequentially adds 3mL
Ethylenediamine and 2mL4- dimethylamino naphthyridines, reaction 24 it is small when after, with acetonitrile precipitation three times, white solid in vacuum drying oven do
It is dry, obtain amination alpha-cyclodextrin.
5g Beracillines are dissolved in 20mL DMF, the DMF solution of 15mL acetic anhydride is added dropwise dropwise, is maintained at 80 DEG C
It is lower reaction 2 it is small when, after the reaction was complete, with Rotary Evaporators remove solvent, resultant product is dispersed in chloroform again, organic layer
With water extraction three times, finally, revolving removes solvent, and recrystallization can obtain N- acetyl penicilliamine thiolactones in hexane.
By 0.7g aminations alpha-cyclodextrin in room-temperature dissolution in 55mL pyridines, 0.25g N- acetyl penicilliamine sulphur is added in
Lactone, when stirring 48 is small at 60 DEG C, white solid is dry in vacuum drying oven with acetonitrile precipitation three times.
By 0.2g white solids obtained in the previous step in room-temperature dissolution in 15mL DMF, 0.2mL nitroso spy's fourths are added in
Ester, when stirring 2 is small at 80 DEG C, white solid is dry in vacuum drying oven, you can obtains alpha-cyclodextrin base with acetonitrile precipitation three times
Nitric oxide donors.
Embodiment 3
60g alpha-cyclodextrins are suspended in 500mL water, 7g sodium hydroxides saturated solution to solution is added dropwise dropwise in 0 DEG C is in
Clear shape；It is kept for 0 DEG C, 15g paratoluensulfonyl chlorides is dissolved in 10mL acetonitriles, instill above-mentioned solution dropwise, at room temperature instead
Answer 2 it is small when；It is placed in 4 DEG C overnight, filters solid and recrystallized 3 times with water, obtain the alpha-cyclodextrin of tolysulfonyl chlorine activation.
By the cyclodextrin of 3g tolysulfonyl chlorine activations in 25mL DMF, 80 DEG C are heated to, sequentially adds 3mL
NH2-CH2CH=CHCH2-NH2With 2mL triethylamines, reaction 36 it is small when after, white solid is in vacuum drying oven with methanol extraction three times
Middle drying obtains amination alpha-cyclodextrin.
15g Beracillines are dissolved in 20mL toluene, the toluene solution of 150mL acetic anhydride is added dropwise dropwise, is maintained at 60
When reaction 20 is small at DEG C, after the reaction was complete, solvent is removed with Rotary Evaporators, resultant product is dispersed in chloroform again, is had
Machine layer is extracted three times with water, and finally, revolving can obtain N- acetyl penicilliamine thiolactones except solvent, in hexane recrystallization.
By 1.7g aminations alpha-cyclodextrin in room-temperature dissolution in 25mL pyridines, 0.3g N- acetyl penicilliamine sulphur is added in
Lactone, when stirring 20 is small at 50 DEG C, with ether precipitation three times, white solid is dry in vacuum drying oven.
By 0.1g white solids obtained in the previous step in room-temperature dissolution in 15mL dichloromethane, 0.1mL nitrosos are added in
Special butyl ester, when stirring 22 is small at 45 DEG C, white solid is dry in vacuum drying oven, you can obtains α-ring with acetonitrile precipitation three times
Dextrin base nitric oxide donors.
Embodiment 4
50g gamma-cyclodextrins are suspended in 500mL water, 7g sodium hydroxides saturated solution to solution is added dropwise dropwise in 0 DEG C is in
Clear shape；It is kept for 0 DEG C, 15g paratoluensulfonyl chlorides is dissolved in 10mL acetonitriles, instill above-mentioned solution dropwise, at room temperature instead
Answer 2 it is small when；It is placed in 4 DEG C overnight, filters solid and recrystallized 3 times with water, obtain the gamma-cyclodextrin of tolysulfonyl chlorine activation.
By the cyclodextrin of 4.2g tolysulfonyl chlorine activations in 25mL DMF, 80 DEG C are heated to, sequentially adds 3mL
Butanediamine and 2mL triethylamines；React 48 it is small when after, with n-hexane precipitation three times, white solid is dry in vacuum drying oven, obtains
Amination gamma-cyclodextrin.
15g Beracillines are dissolved in 50mL pyridines, the pyridine solution of 45mL acetic anhydride is added dropwise dropwise, is maintained at 50
When reaction 12 is small at DEG C, after the reaction was complete, solvent is removed with Rotary Evaporators, resultant product is dispersed in dichloromethane again
In, organic layer is extracted three times with water, and finally, revolving removes solvent, and recrystallization can obtain N- acetyl penicilliamines in hexane
Thiolactone.
By 2.7g aminations gamma-cyclodextrin in room-temperature dissolution in 25mL pyridines, 0.45g N- acetyl penicilliamines are added in
Thiolactone, when stirring 24 is small at 30 DEG C, white solid is dry in vacuum drying oven with methanol extraction three times.
By 0.4g white solids obtained in the previous step in room-temperature dissolution in 30mL dichloromethane, 0.6mL nitrosos are added in
Special butyl ester, when stirring 24 is small at 40 DEG C, white solid is dry in vacuum drying oven, you can obtains γ-ring with acetonitrile precipitation three times
Dextrin base nitric oxide donors.
Embodiment 5
50g gamma-cyclodextrins are suspended in 500mL water, 7g sodium hydroxides saturated solution to solution is added dropwise dropwise in 0 DEG C is in
Clear shape；It is kept for 0 DEG C, 15g paratoluensulfonyl chlorides is dissolved in 10mL acetonitriles, instill above-mentioned solution dropwise, at room temperature instead
Answer 2 it is small when；It is placed in 4 DEG C overnight, filters solid and recrystallized 3 times with water, obtain the gamma-cyclodextrin of tolysulfonyl chlorine activation.
The gamma-cyclodextrin of 4g tolysulfonyl chlorine activations is dissolved in 25mL DMF, 80 DEG C is heated to, sequentially adds
3mL diethylenetriamines and 2mL 4-dimethylaminopyridine, reaction 72 it is small when after, white solid is in vacuum with acetonitrile precipitation three times
It is dry in baking oven, obtain amination gamma-cyclodextrin.
3.5g Beracillines are dissolved in 100mL DMF, the pyridine solution of 20mL acetic anhydride is added dropwise dropwise, is maintained at 0
When reaction 24 is small at DEG C, after the reaction was complete, solvent is removed with Rotary Evaporators, resultant product is dispersed in dichloromethane again
In, organic layer is extracted three times with water, and finally, revolving removes solvent, and recrystallization can obtain N- acetyl penicilliamines in hexane
Thiolactone.
By 2.2g aminations gamma-cyclodextrin in room-temperature dissolution in 25mL chloroforms, 1.2g N- acetyl penicilliamine sulphur is added in
Lactone, when stirring 36 is small at 37 DEG C, with ether precipitation three times, white solid is dry in vacuum drying oven.
By 0.2g white solids obtained in the previous step in room-temperature dissolution in 30mL DMF, 0.5mL nitroso spy's fourths are added in
Ester, when stirring 48 is small at 70 DEG C, with n-hexane precipitation three times, white solid is dry in vacuum drying oven, you can obtains γ-ring paste
Smart base nitric oxide donors.
Application examples 1
To study the influence of the growth of Beta-cyclodextrin-based nitric oxide donors Human Umbilical Vein Endothelial Cells and smooth muscle cell, cell is carried out
It is proliferated performance testing experiment：
When endothelial cell HUVEC grows to 80%~90% fusion, digestion centrifugation is carried out to it, culture medium disperses, with 6 ×
103The density of cells/well is inoculated into 24 porocyte culture plates, when adherency 4 is small after, it is every 12 it is small when be separately added into 1mL and contain
The cell culture medium of 0 μM, 5 μM, 10 μM Beta-cyclodextrin-based nitric oxide donors, wherein 0 μM of Beta-cyclodextrin-based nitric oxide donors is thin
Born of the same parents' culture medium is PBS control group；After cultivating 1 day and 3 days respectively, original fluid, PBS cleaning 3 times, with containing 4% poly first are abandoned in suction
The PBS room temperatures of aldehyde fix 20 minutes, to F-actin (F-actin) of Tissue Culture Plate surface adhesion endothelial cell HUVEC
Immunofluorescence dyeing is carried out with nucleus, and the sample after fluorescent staining carries out taking pictures under 200 inverted microscopes of Axiovert point
Analysis carries out the cell on fluorescence photo under the auxiliary of ImageJ softwares the statistics of cell density.
Smooth muscle cell is operated as above.
The result is shown in Fig. 6, Beta-cyclodextrin-based nitric oxide donors have smooth muscle cell significant inhibitory action, and simultaneously can
Promote the multiplication of endothelial cell, and its bioactivity is positively correlated with the concentration added in.
Application examples 2
To study the influence of the growth of Beta-cyclodextrin-based nitric oxide donors Human Umbilical Vein Endothelial Cells and smooth muscle cell, we are by
Chrotoplast and smooth muscle cell co-culture.
Co-culture experiments use CDB131 culture mediums, and Cell tracker are adopted before cell seeding and are marked：It will
The endothelial cell and smooth muscle of fusion digest respectively, Cell tracker blue CMAC mark endothelial cells, Cell
Tracker orange CMTMR mark smooth muscle cell；Marked endothelial cell and smooth muscle cell equivalent are mixed afterwards
It closes, obtains two kinds of identical mixed cell suspensions of cell concentration, and with 6 × 103Cells/well is inoculated in 24 hole cell culture
In plate, when adherency 4 is small after, it is every 12 it is small when be separately added into 1mL and contain 0 μM, 5 μM, 10 μM of Beta-cyclodextrin-based nitric oxide donors, wherein
The cell culture medium of 0 μM of Beta-cyclodextrin-based nitric oxide donors is PBS control group；After under fluorescence microscope when culture 24 is small respectively
Photographic analysis.
Fluorescent microscopy images are shown in Fig. 7, with the increase of the concentration of Beta-cyclodextrin-based nitric oxide donors, the number of endothelial cell
Amount dramatically increases, and the quantity of smooth muscle cell substantially reduces.
Application examples 3
To study influence of the Beta-cyclodextrin-based nitric oxide donors to Cytostatic to tumor cell, we use tetramethyl nitrogen azoles
Blue colorimetric method (MTT) evaluates antiproliferative activity of the Beta-cyclodextrin-based nitric oxide donors to tumour cell.
Using human lung carcinoma cell line A549 as model cell, A549 cells are seeded in 96 orifice plates with every 6000 density in hole
It is interior, it is 200 μ L per pore volume, is grouped according to different drug concentrations, culture plate is placed in incubator and trains by every group of 5 multiple holes
Support, 24 it is small when after, culture medium is suctioned out, to every hole add in 200 μ L various concentrations Beta-cyclodextrin-based nitric oxide donors.At 37 DEG C
It is lower culture 24 it is small when after, add in 20 μ L MTT, continue culture 4 it is small when, culture medium is suctioned out, add in 150 μ L DMSO, concussion
5min is detected the absorption value in each hole at 490nm with microplate reader and calculates the survival rate of cell.
The result is shown in Fig. 8, Beta-cyclodextrin-based nitric oxide donors can significantly inhibit the multiplication of tumour cell by MTT, has good
Antitumor activity.
Application examples 4
To study the stability of Beta-cyclodextrin-based nitric oxide donors, stability test experiment is carried out：
With reduced glutathione (GSH) come the reproducibility environment of analog cell, by Beta-cyclodextrin-based nitric oxide donors point
Different solution (A is not added to:Phosphate buffer solution PBS；B:2 μM of GSH solution；C:5mM GSH solution；D:10mM GSH
Solution) in, the concentration of final solution cyclodextrin base nitric oxide donors is 2mM, then measures nitric oxide production rate of release.
The results are shown in Figure 9, and Beta-cyclodextrin-based nitric oxide donors are in the phosphate buffer solution PBS of simulation physiological environment
(curve A) hardly is discharged, shows its excellent physiological environment stability；Beta-cyclodextrin-based nitric oxide donors are outside analog cell
It is discharged in 2 μM of GSH solution of microenvironment very slowly (curve B)；Beta-cyclodextrin-based nitric oxide donors reproducibility ring in the cell
Nitric oxide energy quick release (curve C and D) in 5mM or 10mM high concentrations GSH solution in border.
Claims (10)
1. a kind of Beta-cyclodextrin-based nitric oxide donors, shown in general structure such as formula (I)：
Wherein n be integer, 1≤n≤3；
R is-(CH2)2-、-(CH2)3-、-(CH2)4-、-(CH2)5-、-(CH2)6-、-(CH2)2O(CH2)2-、-(CH2)2O(CH2)2O
(CH2)2-、-CH2PhCH2-、-(CH2)2Ph(CH2)2-、-CH(CH3)CH2-、-CH(CH3)CH2CH2-、-(CH2)2NH(CH2)2-、-
(CH2)2NH(CH2)2NH(CH2)2-、-(CH2)2NH(CH2)2NH(CH2)2NH(CH2)2-、-CH2CH=CHCH2- in one kind.
2. a kind of preparation method of Beta-cyclodextrin-based nitric oxide donors according to claim 1, comprises the following steps：
(1) by the cyclodextrin of tolysulfonyl chlorine activation in organic solvent, diamine and acid binding agent are added with stirring, into
Row nucleophilic substitution after the reaction was complete, precipitates in poor solvent, filters to take filter residue and puts vacuum drying oven drying, obtains amino
Change cyclodextrin；
(2) by Beracilline dissolving in organic solvent, acetic anhydride is added with stirring, carries out sulfydryl lactonization reaction, reacted
Quan Hou, post processing obtain N- acetyl penicilliamine thiolactones；
(3) aminocyclodextrin is dissolved in organic solvent, adds in N- acetyl penicilliamine thiolactones, carry out ring-opening reaction,
It after the reaction was complete, is precipitated in poor solvent, filters to take filter residue and put vacuum drying oven drying, obtain solid product；
(4) the solid product dissolving obtained step (3) in organic solvent, adds in nitroso spy's butyl ester, carries out sulfydryl nitrous
Change reaction, after the reaction was complete, precipitated in poor solvent, filter to take filter residue and put vacuum drying oven drying, obtain a Beta-cyclodextrin-based oxygen
Change nitrogen donor.
3. the preparation method of Beta-cyclodextrin-based nitric oxide donors according to claim 2, which is characterized in that in step (1),
The cyclodextrin is one kind in alpha-cyclodextrin, beta-cyclodextrin or gamma-cyclodextrin.
4. the preparation method of Beta-cyclodextrin-based nitric oxide donors according to claim 2, which is characterized in that in step (1),
The structural formula of the diamine is NH2-R-NH2, the wherein definition of R is identical with formula (I).
5. the preparation method of Beta-cyclodextrin-based nitric oxide donors according to claim 2, which is characterized in that in step (1),
The acid binding agent is one kind in triethylamine or 4-dimethylaminopyridine.
6. the preparation method of Beta-cyclodextrin-based nitric oxide donors according to claim 2, which is characterized in that described is organic
Solvent includes N,N-dimethylformamide, dichloromethane, chloroform, toluene, pyridine or dimethyl sulfoxide (DMSO).
7. the preparation method of Beta-cyclodextrin-based nitric oxide donors according to claim 2, which is characterized in that described is bad
Solvent includes acetonitrile, ether, methanol or n-hexane.
8. the preparation method of Beta-cyclodextrin-based nitric oxide donors according to claim 2, which is characterized in that step (1)
Reaction condition is 80 DEG C, when reaction 24~72 is small；The reaction condition of step (2) is：- 20~80 DEG C, when reaction 2~48 is small；Step
Suddenly the reaction condition of (3) is：0~60 DEG C, when reaction 1~48 is small；The reaction condition of step (4) is：- 20~80 DEG C, reaction 2~
72 it is small when.
9. the preparation method of Beta-cyclodextrin-based nitric oxide donors according to claim 2, which is characterized in that in step (2),
Post processing includes vacuum distillation, extraction and recrystallization.
10. Beta-cyclodextrin-based nitric oxide donors according to claim 1 as Beta-cyclodextrin-based drug in angiocardiopathy and
Application in therapeutic field of tumor.
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| CN113396967A (en) * | 2021-06-17 | 2021-09-17 | 浙江工商大学 | Preparation method of natural nano multi-effect preservative and application of natural nano multi-effect preservative in preservation of cauliflower products |
| CN113412854A (en) * | 2021-06-17 | 2021-09-21 | 浙江工商大学 | Natural nano biological composite preservative, preparation method thereof and application thereof in fresh keeping of giant salamander meat |
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