CN110251685A - Taxol-berberine Nano medication synthetic method and application - Google Patents
Taxol-berberine Nano medication synthetic method and application Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention discloses a kind of taxol-berberine Nano medication synthetic methods to realize the self assembly of coupling drug, obtain anticancer nano drug by designing a taxol containing disulfide bond-berberine coupling drug molecule.Taxol provided by the invention-berberine Nano medication GSH response is sensitive, can be realized drug and improves drug utilization efficiency in the quick release of tumor locus, and embodies significant inhibition of cancer cell effect to a variety of cancer cells.
Description
Technical field
The present invention relates to medicine synthesis techniques, and in particular to a kind of taxol-berberine Nano medication of GSH response and its
Preparation method and application.
Background technique
Cancer death rate just becomes to be on the rise in worldwide.Many scientific workers are in resisting cancer work
It is made that outstanding contribution.In numerous treatment methods, chemical medicinal treatment is still main cancer treatment method.However, big
Most anticancer drugs are just faced with that solubility is low, side effect is big and the obstacles such as multidrug resistance before reaching focal area.For
The above-mentioned problem of solution, administration nano-drug administration system such as micella, liposome, dendritic macromole and vesica etc. receive extensive pass
Note.However, preparing for nano-carrier is extremely complex, the degradation of nano-carrier, metabolism and excretion can cause apparent toxicity problem.
Therefore, nano-drug preparation of the exploitation without toxic solvent or nano-carrier is extremely important.
Taxol is a kind of natural products of the complexity separated and extracted from yewtree bark, can be thin by blocking
Born of the same parents normally divide, and cancer cell is inhibited to increase, and are the fiest-tire medications of current clinical chemical drug therapy.Clinical research has confirmed, purple
China fir alcohol is primarily adapted for use in oophoroma, breast cancer and non-small cell lung cancer, and some other pernicious swollen to the cancer of the esophagus, head and neck cancer etc.
Tumor also has certain curative effect.However, the water solubility of taxol is poor, to cause administration difficult, this day is significantly limited
Right activated product is directed to the treatment use of tumour.Clinically using Emulsifier EL-60 and ethyl alcohol as cosolvent, however it is poly-
Ethylene oxide castor oil may cause patient and generate allergic reaction, moreover, the use of Emulsifier EL-60 reduces taxol
Anticancer activity.Meanwhile taxol has stronger toxic side effect, is also easy to produce drug resistance, and can not penetrate due to poor selectivity
The defects of blood-brain barrier.
Berberine is a kind of water-soluble morphinane alkaloid, is had been found in addition to good anti-microbial effect
Except, moreover it is possible to effectively antagonize diarrhea, diabetes, metabolic syndrome, Stein-Leventhal syndrome, coronary artery disease, hyperlipidemia,
Fat and fatty liver diseases.In recent years, many research discovery coptiss are known as good antitumous effect, its property of can choose
Ground is accumulated in tumour cell mitochondria, induces cancer cell mitochondria permeability that transformation cause occurs by reducing mitochondrial membrane potential
Make cancer cell death.
Reduction response type pharmaceutical carrier is between molecule by having the disulfide bond of reduction responsiveness to connect to form nano junction
The drug delivery system of structure.Disulfide bond is highly stable under the environment such as the normal body temperature of human body, pH and oxidation, in a certain amount of gluathione
Generation sulfydryl is reduced in the presence of the reducing agents such as fabk polypeptide (GSH) or dithiothreitol (DTT) (DTT).Cancer cell it is inside and outside there is exactly
Oxidation-reduction potential, intracellular glutathione concentrations (0.5~10mmol/L) are extracellular glutathione concentrations (2~20 μ
Mol/L) 200 times or more, extracellular glutathione concentrations are not enough to Reduction of Disulfide.Therefore, containing the nanometer of disulfide bond
Drug is restored after entering target cell by GSH, and the disulfide bonds of connection generate sulfydryl, to effectively and rapidly discharge drug.
This patent is based on hydrophobic drug taxol and is connected with hydrophilic medicament berberine by disulfide bond, in water certainly
Assembling forms the carrier-free Nano medication with tumor-targeting, to reach antitumor action, it is often more important that solve and receive
The many clinical safety problems of meter Zai Ti bring.
Summary of the invention
Goal of the invention: in view of the above problem existing for existing treatment of cancer and pharmaceutical carrier, the application first purpose is
A kind of taxol-berberine coupled to Nano drug is provided；The second object of the present invention is that providing taxol-berberine coupling receives
The preparation method of rice drug；The third object of the present invention is to provide the application of taxol-berberine coupled to Nano drug.
Technical solution: taxol of the present invention-berberine Nano medication synthetic method includes the following steps:
(1) Halomine is taken, demethyl berberine is obtained under elevated temperature in vacuo；
(2) it removes methyl berberine to react with bromoethanol, obtains the demethyl berberine of ethoxy substitution；
(3) the demethyl berberine that ethoxy replaces is dissolved in methanol, the methanol solution of sodium borohydride is added, reacted
The ethoxy berberine restored；
(4) it takes the ethoxy berberine of reduction and dithiodipropionic acid to be used as at 4-dimethylaminopyridine (DMAP) to be catalyzed
Agent, dicyclohexylcarbodiimide (DCC) obtain dithiodipropionic acid berberine as reaction under the conditions of condensing agent；
(5) dithiodipropionic acid berberine reacts to obtain taxol-berberine coupling drug precursor with taxol；
(6) taxol-berberine coupling drug precursor reacts to obtain taxol-Huang with N- bromo-succinimide (NBS)
Even plain coupling drug；
(7) in organic solvent by taxol-berberine coupling drug dissolution, it drips in poor solvent water, ultrasound, then
Organic solvent is dried up to get taxol-berberine Nano medication is arrived.
Wherein, in step (3), first the demethyl berberine that ethoxy replaces is dissolved in methanol, is then slowly added into
The methanol solution of sodium borohydride reacts 12~24 hours ethoxy berberines restored under ice bath.Wherein, the ethoxy
The mass ratio of the material of substituted demethyl berberine and sodium borohydride is 1:1~2.
In step (4), first successively by dithiodipropionic acid, N, N'- dicyclohexylcarbodiimide and 4-dimethylaminopyridine
It is dissolved in pyridine, is stirred under ice bath inert gas conditions and obtain within 15~30 minutes the first mixed solution, then mixed to first
It is slowly added to the ethoxy berberine dichloromethane solution of reduction in solution, is reacted 24~48 hours under condition of ice bath and obtains two sulphur
For dipropionic acid berberine.
In step (4), the amount of the substance of dithiodipropionic acid is 1~2 times of the ethoxy berberine of reduction.
In step (4), the amount of the substance of DCC is 1~2 times of the ethoxy berberine of reduction.
In step (5), the ratio between amount of substance of dithiodipropionic acid berberine and taxol is 1:1.
Further, in step (5), using 4-dimethylaminopyridine as catalyst, N, N'- dicyclohexylcarbodiimide is made
For condensing agent, wherein the amount of the substance of DCC is 1~2 times of dithiodipropionic acid berberine.
In step (6), taxol-berberine coupling drug precursor is dissolved in chloroformic solution, after stirring 30min, is then delayed
The slow chloroformic solution that NBS is added, the reaction was continued for 24 hours.
In step (6), the ratio between amount of substance of taxol-berberine coupling drug precursor and NBS is 1:1~1.2.
In step (7), reaction temperature is room temperature~50 DEG C.
In step (7), organic solvent is selected from acetone, isopropanol, methanol, ethyl alcohol, pyridine, dimethyl sulfoxide or N, N- diformazan
Base formamide.
In step (7), ultrasonic temperature is room temperature, ultrasonic power is 300W, the time of ultrasound is 10~60min.
The Nano medication that is prepared according to the above method and its application in anticancer drug is being prepared also of the invention
In protection scope.Wherein, the tumour includes lung cancer and liver cancer.
It is assembled in GSH response type taxol-berberine Nano medication altogether the utility model has the advantages that the present invention prepares gained, hydrophobic medicine
Object is taxol, and hydrophilic drugs are berberine, using taxol-berberine molecule special construction, both improves dewatering medicament
Water solubility, and the molecule newly obtained is made to be capable of forming nanoparticle in aqueous solution, to improve the passive target of drug
Property, drug effect is enhanced, possible side effect is reduced.Novel drugs molecule, as a result determines designed by the present invention, is easy to carry out weight
Multiple and characterization, and preferable therapeutic effect.
Detailed description of the invention
Fig. 1 is the hydrogen spectrum for the berberine that ethoxy replaces；
Fig. 2 is the high resolution mass spectrum for the berberine that ethoxy replaces；
Fig. 3 is the hydrogen spectrum of the ethoxy berberine of reduction；
Fig. 4 is the high resolution mass spectrum of the ethoxy berberine of reduction；
Fig. 5 is that the hydrogen of dithiodipropionic acid berberine is composed；
Fig. 6 is the high resolution mass spectrum of dithiodipropionic acid berberine；
Fig. 7 is taxol-berberine coupling drug precursor hydrogen spectrum；
Fig. 8 is taxol-berberine coupling drug precursor high resolution mass spectrum；
Fig. 9 is taxol-berberine coupling drug hydrogen spectrum；
Figure 10 is taxol-berberine coupling drug high resolution mass spectrum；
Figure 11 is taxol-berberine coupled to Nano drug transmission electron microscope shape appearance figure；
Figure 12 is taxol-berberine coupled to Nano drug grain size distribution；
Figure 13 is vitro cytotoxicity (MTT) figure to A549 of taxol-berberine coupled to Nano drug and taxol,
Abscissa is drug concentration in figure, and ordinate is cancer cell survival rate；
Figure 14 be taxol-berberine coupled to Nano drug at 37 DEG C in PBS, containing/do not contain 10mM paddy Guang
The accumulative release of drug in sweet peptide (GSH) is schemed, and abscissa is the time in figure, and ordinate is the drug of accumulative release.
Specific embodiment
To have a clear understanding of technical solution of the present invention, below by drawings and examples, technical solution of the present invention is done
It is described in further detail.The person that is not specified actual conditions in embodiment, carries out according to conventional conditions or manufacturer's recommended conditions.
Reagents or instruments used without specified manufacturer is the conventional products that can be obtained by commercially available purchase.
Embodiment 1 synthesizes demethyl berberine
Halomine 10g is added into 250mL round-bottomed flask, is 30mmHg in vacuum degree, is heated under the conditions of 190 DEG C
The solid powder of 60min, yellow gradually become dark red powder, obtain demethyl berberine crude product.Column chromatography (eluant, eluent:
Chloroform/methanol 20/1) separation, obtain kermesinus demethyl berberine powder 6.92g, yield 80%.
Embodiment 2 synthesizes the berberine that ethoxy replaces
Demethyl berberine 10g (31.14mmol), the acetonitrile prepared in embodiment 1 is added into 250mL round-bottomed flask
(CH3CN) 20mL, potassium carbonate 5.16g (37.37mmol) stir 30min at 50 DEG C, and bromoethanol 4.67g is then added
(37.37mmol), for 24 hours, column chromatographs (washing and dehydrating integrated machine: chloroform/methanol 20/1) separation to temperature rising reflux, obtains the Huang of yellow ethoxy substitution
Even plain powder 12.2g, yield 88%.1H NMR (600MHz, MeOD) δ 9.95 (s, 1H), 8.62 (s, 1H), 8.05 (d, J=
9.1Hz, 1H), 8.00 (d, J=9.0Hz, 1H), 7.59 (s, 1H), 6.93 (s, 1H), 6.13 (s, 2H), 4.99-4.93 (m,
2H),4.54–4.50(m,2H),4.12(s,3H),4.00–3.96(m,2H),3.31–3.26(m,2H)；ESI-MS m/z[M+
H]+=366.13315.Fig. 1 and Fig. 2 is respectively the hydrogen spectrogram and high resolution mass spectrum figure for the berberine that ethoxy replaces, it was demonstrated that
Prepare compound success.
The ethoxy berberine of the synthesis reduction of embodiment 3
Ethoxy berberine 10g (22.47mmol), the methanol prepared in embodiment 2 is added into 250mL round-bottomed flask
(CH3OH) 20mL is stirred under ice bath, and sodium borohydride (NaBH is then added4) (1.28g, 33.71mmol) methanol solution, reaction
12h, the ethoxy berberine crude product restored, column chromatograph (eluant, eluent: chloroform/methanol 20/1) separation, obtain White Reduction
Ethoxy berberine powder 8.29g, yield 95%.1H NMR (600MHz, CDCl3) δ 6.88 (d, J=8.4Hz, 1H),
6.79 (d, J=8.4Hz, 1H), 6.72 (s, 1H), 6.59 (s, 1H), 5.91 (s, 2H), 4.26 (d, J=15.5Hz, 1H),
4.16-4.12 (m, 1H), 4.04 (dd, J=10.3,5.8Hz, 1H), 3.88-3.81 (m, 5H), 3.57-3.51 (m, 2H),
3.22 (dd, J=15.9,3.3Hz, 1H), 3.17 (dd, J=10.4,4.7Hz, 1H), 3.10 (d, J=11.2Hz, 1H), 2.82
(dd, J=15.5,11.6Hz, 1H), 2.63 (dd, J=21.8,10.6Hz, 2H)；ESI-MS m/z [M+H] +=
370.16589.Fig. 3 and Fig. 4 is respectively the hydrogen spectrogram and high resolution mass spectrum figure of the ethoxy berberine restored, it was demonstrated that preparation should
Compound success.
Embodiment 4 synthesizes dithiodipropionic acid berberine
The dithiodipropionic acid 2.84g (13.54mmol) synthesized in embodiment 3, catalysis are added into 100mL round-bottomed flask
The 4-dimethylaminopyridine (DMAP) of amount, dicyclohexylcarbodiimide (DCC) (2.80g, 13.54mmol), pyridine (pydine)
20mL, nitrogen protection stir 30min under ice bath, be added into reaction system reduction ethoxy berberine (5g,
Dichloromethane solution 13.54mmol), continuation react 48h under ice bath, obtain dithiodipropionic acid berberine crude product, subtract
Solvent pyridine is distilled off in pressure, and then column chromatography (eluant, eluent: chloroform/methanol=20/1) separation, obtains the dithio dipropyl of reduction
Sour berberine powder 4.18g, yield 55%.1H NMR(600MHz,MeOD)δ7.03(s,2H),6.91(s,1H),6.71(s,
1H), 5.96 (s, 2H), 4.47 (s, 1H), 4.42-4.37 (m, 2H), 4.35-4.30 (m, 2H), 3.86 (d, J=3.7Hz,
3H), 3.72 (s, 1H), 3.64 (dd, J=17.0,4.3Hz, 1H), 3.37 (s, 1H), 3.22 (s, 1H), 3.11-3.09 (m,
1H), 3.07-2.99 (m, 2H), 2.96 (dd, J=13.5,6.2Hz, 4H), 2.82 (t, J=6.9Hz, 2H), 2.66 (t, J=
7.0Hz, 2H), 2.59 (t, J=6.1Hz, 1H)；ESI-MS m/z [M+H] +=562.15688.Fig. 5 and Fig. 6 is respectively two sulphur
For the hydrogen spectrogram and high resolution mass spectrum figure of dipropionic acid berberine, it was demonstrated that prepare compound success.
Embodiment 5 synthesizes dithiodipropionic acid berberine
The dithiodipropionic acid 5.69g (27.08mmol) synthesized in embodiment 3, catalysis are added into 100mL round-bottomed flask
The 4-dimethylaminopyridine (DMAP) of amount, dicyclohexylcarbodiimide (DCC) (2.80g, 13.54mmol), pyridine (pydine)
20mL, nitrogen protection stir 30min under ice bath, be added into reaction system reduction ethoxy berberine (5g,
Dichloromethane solution 13.54mmol), continuation react 48h under ice bath, obtain dithiodipropionic acid berberine crude product, subtract
Solvent pyridine is distilled off in pressure, and then column chromatography (eluant, eluent: chloroform/methanol=20/1) separation, obtains the dithio dipropyl of reduction
Sour berberine powder 4.79g, yield 63%.
Embodiment 6 synthesizes dithiodipropionic acid berberine
The dithiodipropionic acid 5.69g (27.08mmol) synthesized in embodiment 3, catalysis are added into 100mL round-bottomed flask
The 4-dimethylaminopyridine (DMAP) of amount, dicyclohexylcarbodiimide (DCC) (2.80g, 27.08mmol), pyridine (pydine)
20mL, nitrogen protection stir 30min under ice bath, be added into reaction system reduction ethoxy berberine (5g,
Dichloromethane solution 13.54mmol), continuation react 48h under ice bath, obtain dithiodipropionic acid berberine crude product, subtract
Solvent pyridine is distilled off in pressure, and then column chromatography (eluant, eluent: chloroform/methanol=20/1) separation, obtains the dithio dipropyl of reduction
Sour berberine powder 6.08g, yield 80%.
7 taxol biosynthesis of embodiment-berberine coupling drug precursor
Into 100mL round-bottomed flask be added embodiment 6 in synthesize dithiodipropionic acid berberine 1.7g (2.97mmol),
The 4-dimethylaminopyridine (DMAP) of catalytic amount, dicyclohexylcarbodiimide (DCC) (0.61g, 2.97mmol), pyridine
(pydine) 10mL, nitrogen protection stir 30min under ice bath, into reaction system be added taxol (PTX) (2.54g,
Dichloromethane solution 2.97mmol), continuation react 48h under ice bath, obtain taxol-berberine coupling drug precursor and slightly produce
Product, vacuum distillation remove solvent pyridine, and then column chromatography (eluant, eluent: chloroform/methanol=50/1) separation, obtains taxol-coptis
Plain coupling drug precursor 2.94g, yield 71%.1H NMR (600MHz, CDCl3) δ 8.14 (d, J=7.6Hz, 2H), 7.75 (d,
J=7.2Hz, 2H), 7.61 (t, J=7.4Hz, 1H), 7.55-7.48 (m, 3H), 7.44-7.37 (m, 6H), 7.34 (d, J=
7.5Hz, 1H), 7.06 (s, 1H), 6.87 (d, J=8.4Hz, 1H), 6.78 (d, J=8.3Hz, 1H), 6.72 (s, 1H), 6.58
(d, J=6.5Hz, 1H), 6.29 (s, 1H), 6.24 (t, J=8.7Hz, 1H), 5.98 (d, J=9.0Hz, 1H), 5.91 (s,
2H), 5.68 (d, J=7.1Hz, 1H), 5.53 (s, 1H), 4.97 (d, J=9.0Hz, 1H), 4.44 (t, J=8.5Hz, 1H),
4.38 (t, J=9.4Hz, 2H), 4.32 (d, J=8.5Hz, 1H), 4.26 (dd, J=17.7,14.3Hz, 2H), 4.22-4.16
(m, 2H), 3.81 (d, J=10.5Hz, 4H), 3.51 (s, 2H), 3.22 (d, J=15.0Hz, 1H), 3.12 (d, J=24.4Hz,
2H), 2.91 (t, J=7.0Hz, 2H), 2.87 (d, J=5.1Hz, 2H), 2.85-2.75 (m, 4H), 2.65 (d, J=14.5Hz,
2H), 2.54 (d, J=14.7Hz, 2H), 2.45 (s, 3H), 2.40-2.33 (m, 1H), 2.22 (s, 3H), 2.19-2.13 (m,
1H),1.94(s,3H),1.91–1.81(m,2H),1.68(s,3H),1.23(s,3H),1.13(s,3H)；ESI-MS m/z[M+
H] +=1397.47749.Fig. 7 and Fig. 8 is respectively taxol-berberine coupling drug precursor hydrogen spectrogram and high resolution mass spectrum
Figure, it was demonstrated that prepare the compound successfully.
8 taxol biosynthesis of embodiment-berberine coupling drug precursor
Into 100mL round-bottomed flask be added embodiment 6 in synthesize dithiodipropionic acid berberine 1.7g (2.97mmol),
The 4-dimethylaminopyridine (DMAP) of catalytic amount, dicyclohexylcarbodiimide (DCC) (1.22g, 5.94mmol), pyridine
(pydine) 10mL, nitrogen protection stir 30min under ice bath, into reaction system be added taxol (PTX) (2.54g,
Dichloromethane solution 2.97mmol), continuation react 48h under ice bath, obtain taxol-berberine coupling drug precursor and slightly produce
Product, vacuum distillation remove solvent pyridine, and then column chromatography (eluant, eluent: chloroform/methanol=50/1) separation, obtains taxol-coptis
Plain coupling drug precursor 3.52g, yield 85%.
9 taxol biosynthesis of embodiment-berberine coupling drug
Taxol-berberine coupling drug precursor the 2g synthesized in embodiment 8 is added into 100mL round-bottomed flask
(1.43mmol), chloroform (CHCl3) 20mL, 30min is stirred at room temperature, and N- bromo-succinimide then is added to reaction system
(NBS) chloroformic solution of (254.52mg, 1.43mmol), continues the reaction at room temperature for 24 hours, and obtains taxol-berberine coupling medicine
Object crude product, column chromatograph (eluant, eluent: chloroform/methanol=10/1) separation, obtain taxol-berberine coupling drug 1.09g, yield
52%.1H NMR (600MHz, MeOD) δ 9.75 (s, 1H), 8.70 (s, 1H), 8.10 (d, J=8.4Hz, 3H), 8.00 (d, J=
9.1Hz, 1H), 7.79 (d, J=8.5Hz, 2H), 7.67 (d, J=8.6Hz, 1H), 7.65 (s, 1H), 7.60 (d, J=7.9Hz,
2H), 7.52 (d, J=7.5Hz, 1H), 7.47-7.43 (m, 6H), 7.26 (t, J=7.2Hz, 1H), 6.94 (s, 1H), 6.40
(s, 1H), 6.10 (s, 2H), 6.01 (d, J=10.1Hz, 1H), 5.78 (d, J=6.8Hz, 1H), 5.61 (d, J=7.2Hz,
1H), 5.46 (d, J=6.7Hz, 1H), 4.97 (d, J=7.9Hz, 1H), 4.94-4.91 (m, 2H), 4.67 (t, J=4.0Hz,
2H), 4.56 (d, J=8.2Hz, 2H), 4.29 (d, J=11.0Hz, 1H), 4.16 (s, 2H), 4.10 (s, 3H), 3.76 (d, J=
7.2Hz, 1H), 3.27-3.24 (m, 2H), 2.86 (d, J=6.3Hz, 2H), 2.81 (dd, J=14.5,8.1Hz, 4H), 2.67
(t, J=6.8Hz, 2H), 2.37 (s, 3H), 2.13 (s, 3H), 1.87 (s, 2H), 1.76 (dd, J=14.6,4.6Hz, 2H),
1.63 (s, 3H), 1.28 (s, 3H), 1.12 (s, 3H), 1.09 (s, 3H), 0.90 (t, J=7.0Hz, 1H)；ESI-MS m/z[M+
H] +=1393.44253.Fig. 9 and Figure 10 is respectively taxol-berberine coupling drug hydrogen spectrogram and high resolution mass spectrum figure, card
It is illustrated and prepares compound success.
10 taxol biosynthesis of embodiment-berberine coupling drug
Taxol-berberine coupling drug precursor the 2g synthesized in embodiment 8 is added into 100mL round-bottomed flask
(1.43mmol), chloroform (CHCl3) 20mL, 30min is stirred at room temperature, and N- bromo-succinimide then is added to reaction system
(NBS) chloroformic solution of (306.14mg, 1.72mmol), continues the reaction at room temperature for 24 hours, and obtains taxol-berberine coupling medicine
Object crude product, column chromatograph (eluant, eluent: chloroform/methanol=10/1) separation, obtain taxol-berberine coupling drug 0.94g, yield
45%.
11 taxol biosynthesis of embodiment-berberine coupling drug
Taxol-berberine coupling drug precursor the 2g synthesized in embodiment 8 is added into 100mL round-bottomed flask
(1.43mmol), chloroform (CHCl3) 20mL, 30min is stirred at room temperature, and N- bromo-succinimide then is added to reaction system
(NBS) chloroformic solution of (254.52mg, 1.43mmol), the reaction was continued at 60 DEG C for 24 hours, obtains taxol-berberine coupling medicine
Object crude product, column chromatograph (eluant, eluent: chloroform/methanol=10/1) separation, obtain taxol-berberine coupling drug 1.46g, yield
70%.
Embodiment 12 prepares taxol-berberine Nano medication
The taxol synthesized in Example 11-berberine coupling drug 2mg is dissolved in 1mL acetone, then drips to 10mL
Deionized water in and stir, at room temperature with 300W power ultrasound 30min, blow the organic solvent of the inside off, obtain taxol-Huang
Even plain Nano medication.
Embodiment 13 prepares taxol-berberine Nano medication
The taxol synthesized in Example 11-berberine coupling drug 2mg is dissolved in 1mL acetone, then drips to 10mL
Deionized water in and stir, at room temperature with 300W power ultrasound 45min blow off the inside organic solvent, obtain taxol-Huang
Even plain Nano medication.
Embodiment 14 prepares taxol-berberine Nano medication
The taxol synthesized in Example 11-berberine coupling drug 2mg is dissolved in 1mL acetone, then drips to 10mL
Deionized water in and stir, at room temperature with 300W power ultrasound 60min blow off the inside organic solvent, obtain taxol-Huang
Even plain Nano medication.
Embodiment 15 prepares taxol-berberine Nano medication
The taxol synthesized in Example 11-berberine coupling drug 2mg is dissolved in 1mL isopropanol, is then dripped to
It in the deionized water of 10mL and stirs, blows the organic solvent of the inside off with 300W power ultrasound 60min at room temperature, obtain Japanese yew
Alcohol-berberine Nano medication.
Embodiment 16 prepares taxol-berberine Nano medication
The taxol synthesized in Example 11-berberine coupling drug 2mg is dissolved in 1mL methanol, then drips to 10mL
Deionized water in and stir, at room temperature with 300W power ultrasound 60min blow off the inside organic solvent, obtain taxol-Huang
Even plain Nano medication.
17 taxols of embodiment-berberine Nano medication relevant characterization
Transmission electron microscope measures the form of 16 nano particle of embodiment, as the result is shown the equal display bar of all nano particles
Shape structure.Dynamic light scattering (DLS) method detects 16 nano particle diameter of embodiment, and all nano particles are averaged grain as the result is shown
Diameter is 100~250nm.Figure 11 is its nano particle form, and in regular club shaped structure, Figure 12 is dynamic light scattering method measurement
Taxol-berberine nano particle average grain diameter, value 165nm.
18 mtt assay cancer cell viability experiment of example
A549 human lung carcinoma cell inoculum concentration is inoculated in 96 well culture plates, 5%CO2, cultivate for 24 hours in 37 DEG C of incubators after, often
Taxol-berberine Nano medication solution, each 100 μ L of paclitaxel solution of the embodiment 16 of various concentration is added in hole, makes final
Drug concentration be respectively 0.625,1.25,2.5,5,10,20,40 μM, continue culture for 24 hours；It is separately added into the MTT of 50 μ L, after
Continue in 5%CO2, continue to cultivate 4h in 37 DEG C of incubators, discard culture medium, the DMSO of 150 μ L be added, is shaken on plate shaker
Even, microplate reader 495nm read plate calculates cell inhibitory rate according to the absorbance value measured.Data are expressed as average ± SD (n=
3)。
As a result, it has been found that (as shown in figure 13), after 24 hours of incubation, taxol-berberine Nano medication (IC50=1.29 μ
M taxol (IC) is better than to the inhibitory effect of A54950=1.55 μM).
19 taxols of embodiment-berberine Nano medication release experiment
Taxol and berberine are measured from the release in Nano medication by dialysis.By 16 taxols of embodiment-coptis
Plain Nano medication is diluted with phosphate buffer solution (PBS), generates 60 μM of solution.Take the solution of 1mL into bag filter (MwCO:
2000) it, is placed in the PBS of 30mL or the PBS buffer solution containing 10mM glutathione and dialyses 55 hours.When scheduled
Between take the sample of 2mL for fluorescence detection (excitation wavelength: 380nm) from bag filter, and supplement the PBS of 2mL.
As a result, it has been found that (as shown in figure 14), taxol-berberine Nano medication in the solution without glutathione very
Stablize.In contrast, in the presence of glutathione, there is 50% or so drug quick release in 10 hours.
Claims (10)
1. a kind of taxol-berberine Nano medication synthetic method, which comprises the steps of:
(1) Halomine is taken, demethyl berberine is obtained under elevated temperature in vacuo；
(2) it removes methyl berberine to react with bromoethanol, obtains the demethyl berberine of ethoxy substitution；
(3) the demethyl berberine that ethoxy replaces is dissolved in methanol, the methanol solution of sodium borohydride is added, reaction obtains
The ethoxy berberine of reduction；
(4) take reduction ethoxy berberine and dithiodipropionic acid in 4-dimethylaminopyridine as catalyst, N, bis- ring of N'-
Hexyl carbodiimide obtains dithiodipropionic acid berberine as reaction under the conditions of condensing agent；
(5) dithiodipropionic acid berberine reacts to obtain taxol-berberine coupling drug precursor with taxol；
(6) taxol-berberine coupling drug precursor reacts to obtain taxol-berberine coupling medicine with N- bromo-succinimide
Object；
(7) in organic solvent by taxol-berberine coupling drug dissolution, it drips in poor solvent water, then ultrasound dries up
Organic solvent to get arrive taxol-berberine Nano medication.
2. synthetic method according to claim 1, which is characterized in that in step (3), the reaction carries out under ice bath,
The mass ratio of the material of demethyl berberine and sodium borohydride that the ethoxy replaces is 1:1~2.
3. synthetic method according to claim 1, which is characterized in that in step (4), first successively by dithiodipropionic acid,
N, N'- dicyclohexylcarbodiimide and 4-dimethylaminopyridine are dissolved in pyridine, stir 15 under ice bath inert gas conditions
The first mixed solution is obtained within~30 minutes, then is slowly added to the ethoxy berberine methylene chloride of reduction into the first mixed solution
Solution reacts 24~48 hours under condition of ice bath and obtains dithiodipropionic acid berberine.
4. synthetic method according to claim 1, which is characterized in that in step (4), the substance of the dithiodipropionic acid
Amount be 1~2 times of ethoxy berberine of reduction.
5. synthetic method according to claim 1, which is characterized in that in step (4), the N, N'- dicyclohexyl carbon two
The amount of imines substance is 1~2 times of the ethoxy berberine of reduction.
6. synthetic method according to claim 1, which is characterized in that in step (5), the dithiodipropionic acid berberine
It is 1:1 with the ratio between the amount of substance of taxol.
7. synthetic method according to claim 1, which is characterized in that in step (6), taxol-berberine coupling drug
Precursor is dissolved in chloroformic solution, after stirring 30~60min, is then slowly added into the chloroformic solution of N- bromo-succinimide, after
24~48h of continuous reaction.
8. synthetic method according to claim 1, which is characterized in that in step (6), the taxol-berberine coupling
The ratio between amount of substance of prodrug and N- bromo-succinimide is 1:1~1.2.
9. synthetic method according to claim 1, which is characterized in that in step (7), organic solvent is selected from acetone, isopropyl
Alcohol, methanol, ethyl alcohol, pyridine, dimethyl sulfoxide or N,N-dimethylformamide.
10. any one of claim 1-9 preparation gained Nano medication is preparing the application in anticancer drug.
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