CN106588902B - A kind of taxol anticancer drug, preparation method and application - Google Patents
A kind of taxol anticancer drug, preparation method and application Download PDFInfo
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- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 title claims abstract description 28
- 229930012538 Paclitaxel Natural products 0.000 title claims abstract description 27
- 229960001592 paclitaxel Drugs 0.000 title claims abstract description 27
- 239000002246 antineoplastic agent Substances 0.000 title claims abstract description 22
- 229940041181 antineoplastic drug Drugs 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- IGHBXJSNZCFXNK-UHFFFAOYSA-N 4-chloro-7-nitrobenzofurazan Chemical compound [O-][N+](=O)C1=CC=C(Cl)C2=NON=C12 IGHBXJSNZCFXNK-UHFFFAOYSA-N 0.000 claims abstract description 27
- 239000003960 organic solvent Substances 0.000 claims abstract description 20
- 238000006243 chemical reaction Methods 0.000 claims abstract description 5
- 239000012535 impurity Substances 0.000 claims abstract description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000008367 deionised water Substances 0.000 claims description 7
- 229910021641 deionized water Inorganic materials 0.000 claims description 7
- 238000000502 dialysis Methods 0.000 claims description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 6
- 238000004108 freeze drying Methods 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 5
- 238000013019 agitation Methods 0.000 claims description 2
- 239000003560 cancer drug Substances 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 39
- 230000005284 excitation Effects 0.000 abstract description 8
- 210000004881 tumor cell Anatomy 0.000 abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 39
- 239000000523 sample Substances 0.000 description 11
- 230000015572 biosynthetic process Effects 0.000 description 9
- 238000003786 synthesis reaction Methods 0.000 description 9
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 7
- 239000003814 drug Substances 0.000 description 6
- 238000005303 weighing Methods 0.000 description 6
- 230000000259 anti-tumor effect Effects 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 4
- 230000009194 climbing Effects 0.000 description 4
- 238000004043 dyeing Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 3
- 235000016408 Podocarpus macrophyllus Nutrition 0.000 description 3
- 244000162450 Taxus cuspidata Species 0.000 description 3
- 235000009065 Taxus cuspidata Nutrition 0.000 description 3
- 239000000090 biomarker Substances 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000012800 visualization Methods 0.000 description 3
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 102000004243 Tubulin Human genes 0.000 description 2
- 108090000704 Tubulin Proteins 0.000 description 2
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- 238000011161 development Methods 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 238000010534 nucleophilic substitution reaction Methods 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- MQLACMBJVPINKE-UHFFFAOYSA-N 10-[(3-hydroxy-4-methoxyphenyl)methylidene]anthracen-9-one Chemical compound C1=C(O)C(OC)=CC=C1C=C1C2=CC=CC=C2C(=O)C2=CC=CC=C21 MQLACMBJVPINKE-UHFFFAOYSA-N 0.000 description 1
- -1 Alkaloid compound Chemical class 0.000 description 1
- 201000004384 Alopecia Diseases 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 238000000919 Fourier transform infrared map Methods 0.000 description 1
- 238000004566 IR spectroscopy Methods 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 206010028116 Mucosal inflammation Diseases 0.000 description 1
- 201000010927 Mucositis Diseases 0.000 description 1
- 206010033372 Pain and discomfort Diseases 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 241001149649 Taxus wallichiana var. chinensis Species 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 231100000360 alopecia Toxicity 0.000 description 1
- 230000000118 anti-neoplastic effect Effects 0.000 description 1
- 239000003005 anticarcinogenic agent Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000004700 cellular uptake Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 229930004069 diterpene Natural products 0.000 description 1
- 150000004141 diterpene derivatives Chemical class 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000000119 electrospray ionisation mass spectrum Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- JYVHOGDBFNJNMR-UHFFFAOYSA-N hexane;hydrate Chemical compound O.CCCCCC JYVHOGDBFNJNMR-UHFFFAOYSA-N 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 210000000578 peripheral nerve Anatomy 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
- C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
The present invention provides a kind of taxol anticancer drug, and structural formula is shown in structural formula 1 shown in structural formula 1.Be the following steps are included: step 1 the present invention also provides above-mentioned taxol anticancer drug preparation method: by PTX and NBD-Cl, hybrid reaction obtains PTX-NBD mixed liquor in organic solvent；Step 2: removing the impurity in the PTX-NBD mixed liquor, be lyophilized, obtain PTX-NBD, the PTX-NBD is taxol anticancer drug.The present invention also provides above-mentioned PTX-NBD in the application for marking or showing that PTX is distributed in living cells.PTX-NBD of the invention has the characteristics that price is low, excitation energy is low, colour developing is strong, biological compatibility is preferable and highly selective marked tumor cell.
Description
Technical field
The present invention relates to drug labelling field, in particular to a kind of taxol anticancer drug, preparation method and application.
Background technique
Taxol (Paclitaxel, PTX), No. CAS is 33069-62-4, relative molecular weight 853.92, diterpene biology
Alkaloid compound is a kind of natural anti-cancer drugs extracted from Chinese yew, belongs to non-specific cell cycle antineoplastic,
Antitumor test is wide.
PTX is white crystalline body powder, is insoluble in water and n-hexane, and it is organic to be soluble in methanol, acetonitrile, chloroform, acetone etc.
Solvent.
The mechanism of action of PTX: micro-pipe is a kind of important composition ingredient of eukaryocyte, it is sub- single by two similar polypeptides
The micro-pipe dimer that position is constituted is formed.Under normal circumstances, there are dynamic equilibrium between micro-pipe and tubulin dimer.PTX
As a kind of novel breast cancer, it can make to lose this dynamic equilibrium therebetween, induction and promotion tubulin polymerization suppression
Depolymerization processed keeps tubulin to stablize.These effects cause cell that cannot form spindle and spindle when carrying out mitosis
Silk, effectively inhibits cell division and proliferation, to play antitumor action.
The medicine clinic is mainly used for treating oophoroma and breast cancer, it can also be used to treat lung cancer, colorectal cancer, incidence tumor,
Melanoma, lymthoma, brain tumor.But it is disliked with general toxicity, especially lasting bone marrow suppression and peripheral nerve toxicity
The heart, vomiting, diarrhea, alopecia, mucositis, muscle arthrosis pain and discomfort etc. also have generation.In recent years, people have been developed very in succession
More PTX novel pharmaceutical formulations only eliminate cancer cell to reach, and minimize the purpose influenced on normal cell simultaneously.Although these
Though novel pharmaceutical formulation has larger improvement in some aspects, but also is difficult to eliminate its high poison to normal cell, tissue, organ
Property.
Pharmacological development, half of generation will largely be promoted by allowing a kind of drug to visualize in cell or even living body
The fast development of fluorescence microscope and fluorescent marker method provides new approaches for drug labelling since discipline.Currently, tracking PTX is thin
The most basic thinking of born of the same parents and the motor behavior in living body be by the covalent site for being connected to drug of suitable fluorogen, it is selected
Fluorogen is typically all expensive fluorescein isothiocynate (Fluorescein isothiocyanate, FITC).So
Filter out it is cheap, being covalently attached with PTX, being suitble to that laser confocal microscope use, can be in living cells and dynamic
Visual fluorescence probe is extremely necessary in object.
Therefore, it is necessary to invent it is a kind of facilitate tracking and low-cost taxol anticancer drug, preparation method and application.
Summary of the invention
It is an object of the invention to overcome drawbacks described above, provide it is a kind of facilitate tracking and low-cost taxol anticarcinogen
Object, preparation method and application.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention are as follows:
The present invention provides a kind of taxol anticancer drug, and structural formula structural formula is shown in structural formula 1
The present invention also provides a kind of preparation methods of above-mentioned taxol anticancer drug, comprising the following steps:
Step 1: by PTX and NBD-Cl, hybrid reaction obtains PTX-NBD mixed liquor in organic solvent；
Step 2: removing the impurity in the PTX-NBD mixed liquor, be lyophilized, obtain PTX-NBD, the PTX-NBD is Japanese yew
Alcohol anticancer drug.
The NBD-Cl be chloro- 7- nitro benzo -2- oxa- -1, the 3- diazole of 4-, the chloro- 7- nitrobenzofurazan of alias 4-,
No. CAS is 10199-89-0, English name 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole or NBDchloride.
A kind of application of above-mentioned taxol anticancer drug, the PTX-NBD are marking or are showing that PTX is distributed in living cells
Application.
The beneficial effects of the present invention are: it is obtained by introducing fluorescence probe NBD-Cl in the structure of taxol (PTX)
PTX-NBD (PTX conjugates), and then realize visualization of the PTX in cell and living body, facilitate tracking, with respect to PTX-FITC
Speech, PTX-NBD price of the invention is low, excitation energy is low, colour developing is strong, biological compatibility is preferable；The preparation of PTX-NBD of the present invention
Method, synthesis technology is simple, easy to operate；Experimental result confirms that PTX-NBD of the invention can be used to mark or show PTX in work
Be distributed in cell, have the characteristics that highly selective marked tumor cell, slow down PTX to normal cell, tissue, organ shadow
It rings, provides cheap, simple and direct, intuitive biological detection reagent for the antitumor research of PTX；The NBD-CL fluorescence probe and DID,
DIR has good biocompatibility, has potential application in LASER Excited Fluorescence biomarker field.
Detailed description of the invention
Fig. 1 is the electrospray ionization mass spectrum (Electrospray of NBD-CL, PTX and PTX-NBD in the embodiment of the present invention 5
Ionization mass spectrometry, ESI-MS) map: (a) fluorescence probe NBD-Cl, (b) PTX, (c) PTX-NBD.
Fig. 2 is the mixture and PTX-NBD of PTX, NBD-Cl, PTX and NBD-Cl in the embodiment of the present invention 5) infrared light
Compose (FTIR) map；(a) PTX, (b) fluorescence probe NBD-Cl, (c) mixture of PTX and NBD-Cl, (d) PTX-NBD.
Fig. 3 is the mixture and PTX-NBD of PTX, NBD-Cl, PTX and NBD-Cl in the embodiment of the present invention 5) infrared light
Compose (FTIR) map；(a) PTX, (b) fluorescence probe NBD-Cl, (c) mixture of PTX and NBD-Cl, (d) PTX-NBD.
Fig. 4 is the images that PTX-NBD carries out after dyeing 0.5h to Hela cell in the embodiment of the present invention 5, (a)
DAPI: excitation wavelength 405nm, phosphor collection wavelength is 420-460nm；(b) NBD, excitation wavelength 458nm, phosphor collection
Wavelength is 500-580nm；(c) light field picture；(d) Merge1:DAPI and NBD is superimposed picture；(e) Merge2:DAPI, NBD and
Light field is superimposed picture.
Fig. 5 is the images that PTX-NBD carries out after dyeing 4h to Hela cell in the embodiment of the present invention 5, (a) DAPI:
Excitation wavelength is 405nm, and phosphor collection wavelength is 420-460nm；(b) NBD: excitation wavelength 458nm, phosphor collection wavelength is
500-580nm；(c) light field picture；(d) Merge1:DAPI and NBD is superimposed picture；(e) Merge2:DAPI, NBD and light field are folded
Add picture.
Specific embodiment
In order to describe the technical content, the structural feature, the achieved object and the effect of this invention in detail, below in conjunction with embodiment
And attached drawing is cooperated to be explained in detail.
The most critical design of the present invention is: fluorescence probe NBD-CL synthesizes PTX- by nucleophilic substitution with PTX
NBD。
For NBD as fluorogen unlike fluorescein is so ripe well known, the reactivity of NBD-Cl is not so good as FITC, people from this field
Member is not readily conceivable that, therefore, never has relevant report with more cheap NBD-Cl label taxol, while NBD-Cl marks Japanese yew
The reaction condition of alcohol needs voluntarily to grope and optimize.
Fig. 1-5 is please referred to, the present invention provides a kind of taxol anticancer drug, and structural formula is shown in structural formula 1
The principle of the present invention are as follows: fluorescence probe NBD-CL synthesizes PTX-NBD by nucleophilic substitution with PTX, by PTX-
NBD is for marking or showing that PTX is distributed in living cells.
As can be seen from the above description, the beneficial effects of the present invention are: by introducing fluorescence in the structure of taxol (PTX)
Probe NBD-Cl obtains PTX-NBD, and then realizes visualization of the PTX in cell and living body, facilitates tracking, opposite PTX-
For FITC, PTX-NBD price of the invention is low, excitation energy is low, colour developing is strong, biological compatibility is preferable.
The present invention also provides a kind of preparation methods of above-mentioned taxol anticancer drug, comprising the following steps:
Step 1: by PTX and NBD-Cl, hybrid reaction obtains PTX-NBD mixed liquor in organic solvent；
Step 2: removing the impurity in the PTX-NBD mixed liquor, be lyophilized, obtain PTX-NBD, the PTX-NBD is Japanese yew
Alcohol anticancer drug.
As can be seen from the above description, the beneficial effects of the present invention are: the preparation method of PTX-NBD of the present invention, synthesis technology
Simply, easy to operate.
Further, the concrete operations of the step 1 are as follows: accurate weighing a certain amount of PTX and NBD, and be dissolved in
In solvent, room temperature magnetic agitation 22-26h preferably for 24 hours obtains pistac PTX-NBD mixed liquor.
Further, the concrete operations of the step 2 are as follows: deionized water is added in the PTX-NBD mixed liquor, uses
The bag filter of 1000DA molecular weight carries out dialysis and removes unreacted NBD-CL and organic solvent, and the PTX-NBD purified freezes
It is dry, obtain faint yellow PTX-NBD.
Further, the molar ratio of the PTX and NBD-Cl is 1: 1~4.
Further, the organic solvent is one of methanol, acetonitrile, chloroform or acetone or a variety of.
Further, the organic solvent is methanol.
The present invention also provides above-mentioned PTX-NBD in the application for marking or showing that PTX is distributed in living cells.
As can be seen from the above description, the beneficial effects of the present invention are: this PTX-NBD of the invention can be used to mark or show
PTX is distributed in living cells, has the characteristics that highly selective marked tumor cell, slows down PTX to normal cell, tissue, device
The influence of official provides cheap, simple and direct, intuitive biological detection reagent for the antitumor research of PTX；The NBD-CL fluorescence probe
There is good biocompatibility with DID, DIR, had potential application in LASER Excited Fluorescence biomarker field
Further, the living cells is HeLa cell.
Embodiment 1
(synthesis of PTX-NBD (a)): weighing PTX, NBD (molar ratio are as follows: 1:4), be placed in round bottom beaker, is added appropriate
Organic solvent methanol, room temperature magnetic force are stirred to react for 24 hours, be can be obtained pistac PTX-NBD solution and are added wherein appropriate
Deionized water carries out dialysis using the bag filter of 1000DA molecular weight and removes unreacted NBD-CL and organic solvent methanol, obtains
To the PTX-NBD of purifying, freeze-drying obtains yellow solid.Yield: 90%.
Example 2
(synthesis of PTX-NBD (b)): weighing PTX, NBD (molar ratio are as follows: 1:3), be placed in round bottom beaker, is added appropriate
Organic solvent methanol, room temperature magnetic force are stirred to react for 24 hours, be can be obtained pistac PTX-NBD solution and are added wherein appropriate
Deionized water carries out dialysis using the bag filter of 1000DA molecular weight and removes unreacted NBD-CL and organic solvent methanol, obtains
To the PTX-NBD of purifying, freeze-drying obtains yellow solid.Yield: 92%.
Example 3
(synthesis of PTX-NBD (c)): weighing PTX, NBD (molar ratio are as follows: 1:2), be placed in round bottom beaker, is added appropriate
Organic solvent methanol, room temperature magnetic force are stirred to react for 24 hours, be can be obtained pistac PTX-NBD solution and are added wherein appropriate
Deionized water carries out dialysis using the bag filter of 1000DA molecular weight and removes unreacted NBD-CL and organic solvent methanol, obtains
To the PTX-NBD of purifying, freeze-drying obtains yellow solid.Yield: 88%.
Example 4
(synthesis of PTX-NBD (d)): weighing PTX, NBD (molar ratio are as follows: 1:1), be placed in round bottom beaker, is added appropriate
Organic solvent methanol, room temperature magnetic force are stirred to react for 24 hours, be can be obtained pistac PTX-NBD solution and are added wherein appropriate
Deionized water carries out dialysis using the bag filter of 1000DA molecular weight and removes unreacted NBD-CL and organic solvent methanol, obtains
To the PTX-NBD of purifying, freeze-drying obtains yellow solid.Yield: 91%.
Example 5
Fig. 1 to Fig. 5 is please referred to, (synthesis of PTX-NBD (e)): weighing PTX, NBD (molar ratio are as follows: 1:1.2), be placed in circle
In the beaker of bottom, appropriate organic solvent methanol is added, room temperature magnetic force is stirred to react for 24 hours, can be obtained pistac PTX-NBD solution
And appropriate amount of deionized water is added wherein, using the bag filter of 1000DA molecular weight carry out dialysis remove unreacted NBD-CL and
Organic solvent methanol, the PTX-NBD purified, freeze-drying obtain yellow solid.Yield: 92%.
Preferably, in the PTX-NBD synthesis technology in examples detailed above, the molar ratio of the PTX and NBD-CL are as follows: 1:1.2,
The molecular weight of synthesized PTX-NBD is that 1017.8 (the ESI-MS map of NBD-CL, PTX, PTX-NBD are shown in Fig. 1, ESI-MS figure
The abscissa of spectrum indicates mass-to-charge ratio value, and ordinate indicates relative abundance)；The infrared spectroscopy of (PTX, PTX-NBD and PTX-NBD)
(FTIR) map is shown in that Fig. 2 and Fig. 3, the abscissa of FTIR map indicate wave number (cm-1)。
The culture of Hela cell: HeLa cell strain adhere-wall culture is in including in 10% hyclone nutrient solution, at 37 DEG C,
5%CO2Saturated humidity incubator in cultivate, every 2~3d change liquid pass on 1 time.Logarithmic phase, contact pin culture: 1. are grown into cell
Coverslip is impregnated into 30min in dehydrated alcohol, is put into disposable 35mm culture dish after alcolhol burner drying；2. 100ml is thin
Cell in born of the same parents' bottle is washed three times with PBS, is digested 3-5 minutes with 0.25% pancreatin of 1ml, and culture medium is carefully poured out, and is added a small amount of
Fresh culture piping and druming uniformly, after cell count, leaves the cell of proper density, and culture medium is added to required volume, and (control is thin
The final concentration of 1x10 of born of the same parents5), it is seeded in the culture dish for including coverslip, is put into CO2It is cultivated in incubator, keeps cell climbing sheet raw
It is long.
Cellular uptake of the Hela cell to PTX-NBD (e): inoculated cell climbing sheet is washed three times with PBS, dilute with PBS
The DAPI solution of the 250nM released is in CO2Staining cell 0.5h in incubator, the cell climbing sheet after dyeing take out, wash away unbonded
Extra dye liquor, then with diluted 2 μM of PBS of PTX-NBD solution of the present invention in CO2Staining cell is distinguished in incubator
0.5h and 4h.Cell climbing sheet after dyeing takes out, and washes away unbonded extra dye liquor, and cell growth is covered in glass slide down
On, under laser confocal microscope observe cell color position, fluorescence distribution and brightness change etc., as a result see respectively Fig. 4 and
Fig. 5.
As a result, it has been found that PTX-NBD fluorescence in living cells is not quenched, but with the extension of incubation time, fluorescence is strong
Degree enhances therewith, or shows that living cells enhances the intake of PTX drug therewith, this is of the present invention from the one hand confirming
PTX-NBD can highly selective marked tumor cell.
In conclusion taxol anticancer drug, preparation method and application provided by the invention, by taxol
(PTX) fluorescence probe NBD-Cl is introduced in structure and obtains PTX-NBD, and then realizes visualization of the PTX in cell and living body,
Facilitate tracking, for opposite PTX-FITC, PTX-NBD price of the invention is low, excitation energy is low, colour developing is strong, biological compatibility
Preferably；The preparation method of PTX-NBD of the present invention, synthesis technology is simple, easy to operate；Experimental result confirmation, PTX-NBD of the invention
It can be used to mark or show that PTX to be distributed in living cells, have the characteristics that highly selective marked tumor cell, slow down PTX to just
The influence of normal cell, tissue, organ provides cheap, simple and direct, intuitive biological detection reagent for the antitumor research of PTX；Institute
Stating NBD-CL fluorescence probe and DID, DIR has good biocompatibility, has in LASER Excited Fluorescence biomarker field latent
Application value.
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair
Equivalent structure or equivalent flow shift made by bright specification and accompanying drawing content is applied directly or indirectly in other relevant skills
Art field, is included within the scope of the present invention.
Claims (6)
1. a kind of taxol anticancer drug, which is characterized in that its structural formula is shown in structural formula 1
2. a kind of preparation method of taxol anticancer drug described in claim 1, which comprises the following steps:
Step 1: by PTX and NBD-Cl, hybrid reaction obtains PTX-NBD mixed liquor in organic solvent；
Step 2: removing the impurity in the PTX-NBD mixed liquor, be lyophilized, obtain PTX-NBD, the PTX-NBD is anti-for taxol
Cancer drug.
3. the preparation method of taxol anticancer drug according to claim 2, which is characterized in that the step 1 it is specific
Operation are as follows: PTX and NBD are dissolved in organic solvent, room temperature 22~26h of magnetic agitation obtains PTX-NBD mixed liquor.
4. the preparation method of taxol anticancer drug according to claim 2, which is characterized in that the step 2 it is specific
Operation are as follows: deionized water is added in the PTX-NBD mixed liquor, carries out dialysis removal using the bag filter of 1000DA molecular weight
Unreacted NBD-Cl and organic solvent, freeze-drying, obtain PTX-NBD.
5. the preparation method of taxol anticancer drug according to claim 2, which is characterized in that the PTX and NBD-Cl
Molar ratio be 1: 1~4.
6. the preparation method of taxol anticancer drug according to claim 2, which is characterized in that the organic solvent is first
One of alcohol, acetonitrile, chloroform or acetone are a variety of.
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| "LHRHa 靶向紫杉醇脂质体的制备及体外抗肿瘤作用";陈佳，等;《重庆医科大学学报》;20120428;第37卷(第4期);298-301 |
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