CN108467439A - A kind of preparation method of water-soluble paclitaxel anticancer drug - Google Patents
A kind of preparation method of water-soluble paclitaxel anticancer drug Download PDFInfo
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- CN108467439A CN108467439A CN201810242891.XA CN201810242891A CN108467439A CN 108467439 A CN108467439 A CN 108467439A CN 201810242891 A CN201810242891 A CN 201810242891A CN 108467439 A CN108467439 A CN 108467439A
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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
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
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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
- A61K31/335—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
- 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
- A61K47/56—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/08—Drugs for disorders of the urinary system of the prostate
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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 preparation methods of water-soluble paclitaxel anticancer drug, belong to field of medicaments.The PTX prodrugs of the present invention have similar antitumor activity than clinical medicine PTX, can slowly hydrolyze in vivo, effectively reduce toxicity in vivo.The PTX prodrugs of the present invention have preferable dissolubility in water.The PTX prodrug targetings of the present invention increased.The present invention modifies hyaluronic acid with acid anhydrides, then is connected to taxol, which shows good injection safety, self aggregation characteristic and Drug loading capacity.
Description
Technical field
The present invention relates to a kind of preparation methods of water-soluble paclitaxel anticancer drug, belong to field of medicaments.
Background technology
Cancer, which is seriously endangered, coerces the health and life of the mankind, and the lethality of cancer rises year by year at present.Therefore tumour is pre-
Anti- and treatment arduous task, chemotherapeutics is the most effective means for treating tumour at present.
Taxol alias taxol, taxol, Paclitaxe, Paclitaxe (structure such as formula 1), is the classic day having now been found that
Right anticancer drug is clinically widely used for the treatment of breast cancer, oophoroma and part head and neck cancer and lung cancer.Taxol is made
For a diterpene alkaloid class compound with active anticancer, novel complicated chemical constitution, extensively significant biology
Active, completely new unique mechanism of action, in great shortage natural resources make it receive botanist, chemist, pharmacologist, molecule
The very big favor of biologist becomes anticancer star and research emphasis that the second half in 20th century attracts worldwide attention.
Taxol is the four bad diterpene-kind compound of one kind extracted from the bark of Pacific Ocean Chinese yew, is the first choice of anticancer
One of drug.The antitumor mechanism of taxol is：Promote microtubule polymerization in the cell mitogen phase and inhibits microtubule depolymerization, to
Inhibit cell mitogen, make cellular retention in G2 the and M phases, achievees the purpose that inhibit tumour growth.Taxol is slightly water-soluble
Drug, limits its clinical extensive use, and the taxol injection clinically used at present is(6mg/mL PTX/ are poly-
Ethylene oxide castor oil and absolute ethyl alcohol 1：1 mixed solution), however the Emulsifier EL-60 in its vehicle components can cause seriously
The toxic side effects such as allergic reaction.Suitable carrier is selected, the solubility of drug is increased, reduces the toxicity of drug delivery system, is to need
The matter of utmost importance to be solved.Taxol lacks active targeting, equally has lethal effect to normal cell, is carried using suitable
Body makes drug targeting in tumour cell, increases concentration of the drug in tumor locus, can improve drug effect, reduce dosage, together
When reduce drug adverse reaction.
The trial of serious side effects that overcomes done in the past is included in various carriers such as polymer conjugate, liposome,
Taxol is prepared in polymer micelle, lotion and nanosphere.Paclitaxel liposome has class eucaryotic cell structure, into human body in it is main
The autoimmune function of body is activated by reticuloendothelial system phagocytic, is changed the internal distribution for being encapsulated drug, is made drug master
It to be put aside in the histoorgans such as liver, spleen, lung and marrow, to improve therapeutic index.But still face some problems.Such as its stabilization
Property be not resolved, the shelf-life is shorter, and shelf appeal is high.Albumin combination type taxol be it is a kind of using human serum albumin as
The novel taxol albumin of pharmaceutical carrier and stabilizer is freeze-dried, eliminates and the relevant adverse reaction of organic solvent, raising
Safety when using Paclitaxel Chemotherapy and dose-effect relationship.But the price limit of injection albumin combination type taxol costliness
It is in clinical application.Drug-polymer conjugate has the advantages that significant, performance compared with conventional polymer nano-carrier
Go out high medicament contg, solubility is good in water, increases human body medicine half-life period, enhances antitumor action.Although various water-soluble
Property synthetic polymer have been widely used for the conjugated of hydrophobic drug, but natural with intrinsic cell-specific binding ability being deposited
Polymer there is great potential as target specific drug carrier.
The hyaluronic acid (HA) for the natural polysaccharide being made of N- acetyl group-d-glucosamine and D-Glucose aldehydic acid is special with cell
Anisotropic surface marker such as CD44 and RHAMM have very strong affinity.HA plays an important role on biological function, such as stablizes
With tissue ECM, cell adherence and movement and mediated cell proliferation and differentiation are adjusted.HA in the tumour of many types also with
Angiogenesis is closely related, and wherein HA receptors (CD44 and RHAMM) are a large amount of on the surface is overexpressed.Therefore, there is high transfer to live
The malignant cell of property typically exhibits the combination and intake of the enhancing of HA.HA and its derivative have been widely used as various therapeutic agents
Targeting specific drug delivery vehicle.Hyaluronic acid can be coupled as pharmaceutical polymers with taxol, form prodrug.
The prodrug of existing report and the combination of hyaluronic acid are mostly focused on after being modified hyaloplasmic COOH and drug
Precursor is connected, and the carboxylic moiety of hyaluronic acid is the recognition site of hyaluronic acid receptor CD44, but the steric hindrance at this is larger, no
Easily it is connect with taxol.The existing method to HA hydroxyl modifications is mostly carried out with alkyl succinic anhydride or methacrylic anhydride
Esterification, but reaction condition is not easy to control.And the degree of substitution (DS) of MeHA is relatively low, and the value range of DS is also relatively narrow, certain
In degree, esterification deficiency is to lead to the unappeasable one of the main reasons of MeHA hydrogel mechanical performances.
Invention content
To solve the above-mentioned problems, the present invention is by hyaluronic acid and diacid anhydride reactant, to saturating at the hydroxyl of hyaluronic acid
Bright matter acid is modified, and the hyaluronic acid after modification can be combined with receptor CD44 and is coupled with taxol drug.And this
The dicarboxylic anhydride method of modifying of invention has high substituted degree and high-performance.
The first purpose of the invention is to provide a kind of preparation method of taxol (PTX) prodrug, the method is
The hydroxyl that hyaluronic acid is modified by dicarboxylic anhydride, then couples with taxol drug again.
In one embodiment of the invention, the structural formula of the paclitaxel prodrug is as follows：
In one embodiment of the invention, the method the specific steps are：
(1) in the presence of solvent, by hyaluronic acid and dicarboxylic anhydride according to certain mol proportion, reaction generates hyaluronic acid and derives
Object；
(2) derivatives of hyaluronic acids occurs esterification with taxol, generates taxol drug in the presence of condensing agent
Precursor.
In one embodiment of the invention, the solvent in the step (1) is formamide, dimethylformamide, N-
One or both of methyl pyrrolidone, dimethyl sulfoxide (DMSO), 1,3- dimethyl-2-imidazolinones or hexamethylphosphoramide with
Upper combination.
In one embodiment of the invention, the ratio of the hyaluronic acid and solvent is 1:70~100 (m/v).
In one embodiment of the invention, in the step (1) dicarboxylic anhydride be maleic anhydride, phthalic anhydride,
Succinic anhydride or anhydride diethylene glycol.
In one embodiment of the invention, the molar ratio of the hyaluronic acid and dicarboxylic anhydride is 1:4~4:1.
In one embodiment of the invention, it is 40~60 DEG C that the reaction condition of the step (1), which is reaction temperature, instead
It is 4~6h between seasonable, is cooled to room temperature after reaction, freeze-drying is centrifuged after being cleaned with solvent.
In one embodiment of the invention, in the step (2) condensing agent be N, N '-dicyclohexylcarbodiimides,
1- (3- dimethylamino-propyls) -3- ethyl carbodiimides, hexafluorophosphoric acid benzotriazole -1- bases-oxygroup tripyrrole alkyl, tripyrrole
Alkane base phosphonium bromide hexafluorophosphate, 4-dimethylaminopyridine, I-hydroxybenzotriazole, O- benzotriazole-tetramethylurea hexafluoro
One or more of phosphate or O- benzotriazole-N, N, N', N'- tetramethylurea tetrafluoro boric acid combine.
In one embodiment of the invention, it is 30~50 DEG C that the reaction condition of the step (2), which is reaction temperature, instead
It is 1~3 day between seasonable.
Second object of the present invention is to provide the paclitaxel prodrug that the method is prepared.
Third object of the present invention is to provide application of the paclitaxel prodrug in preparing medicine for prostate disease.
In one embodiment of the invention, the paclitaxel prodrug is led to by hydrophilic radical and taxol drug
The ester bond connection that can be broken is crossed, directly discharges antineoplastic component by way of hydrolysis in vivo conducive to premise drug.
Paclitaxel prodrug provided by the invention can be dissolved in the amphiphilic surfactants such as tween can dissolve again
Yu Shui, this considerably increases the application ranges of taxol drug.
Taxol drug anti-cancer drug compounds provided by the invention, have high anti-cancer activity and toxicity is lower, described
Compound can not only be dissolved in water, but also can form nano particle in aqueous solution, have drug slow release function.
The water-soluble paclitaxel medical compounds of the present invention, contains anticancer drug active part and hydrophilic segment, anticarcinogen
Object active part is the medical compounds molecule taxol for having active anticancer, and hydrophilic segment is that water-soluble hyaluronic acid derives
Object.They form the anti-cancer drug compounds of the present invention with ester bond covalent bond.
Beneficial effects of the present invention：
(1) PTX prodrugs of the invention have similar antitumor activity than clinical medicine PTX, can delay in vivo
Slow hydrolysis, effectively reduces toxicity in vivo.
(2) PTX prodrugs of the invention have preferable dissolubility in water.
(3) PTX prodrug targetings of the invention increased.
(4) present invention modifies hyaluronic acid with acid anhydrides, then is connected to taxol, which shows good injection safety
Property, self aggregation characteristic and Drug loading capacity.
Specific embodiment
Embodiment 1：
The preparation of maleic acid-derivatives of hyaluronic acids
1.0g hyaluronic acids are added in the formamide of 80mL, are stirred strongly at 50 DEG C；By maleic acid (hyaluronic acid：
Maleic acid molar ratio=0.4：1.6、0.6：1.4、0.8：1.2、1：1、1.2：0.8、1.4：0.6、1.6：0.4) it is dissolved in 20mL
It in formamide, is added in hyaluronic acid solution, is cooled to room temperature solution after reacting 5h.Solution after cooling is dissolved in nothing
It is strong to stir under water-ethanol, precipitation.It cleans precipitation repeatedly with absolute ethyl alcohol, 3h is centrifuged under the conditions of 11000r/min.Decantation, and
With distillation water dissolution.Above-mentioned solution ph is adjusted to 8~9 with 2molNaOH solution.With deionized water dialysis 24 hours.-63℃
For 24 hours, -112 DEG C are dried in vacuo 48h for lower freezing.Yield：75~90%.
The preparation of paclitaxel prodrug 1
Maleic acid-derivatives of hyaluronic acids powder 600mg is dissolved in the anhydrous DMSO of 5mL, different mol ratio is added
DCC and DMAP.Solution is stirred 1 hour to activate the carboxyl of maleic acid-derivatives of hyaluronic acids.To not same amount taxol (10
And 20mg) be dissolved in the anhydrous DMSO of 1mL, and using syringe in dry N2Under be slowly added into above-mentioned solution.Then
Mixture is stirred 2 days at 40 DEG C.Use dialysis membrane (MWCO：3500Da) by acquired solution with DMSO dialyse 1 day, spend from
Sub- water dialysis deionized water, to remove unreacted taxol.It collects paclitaxel prodrug 1 and is lyophilized 3 days and is analyzed.
Yield：65%~88%.
In acetonitrile/water (50:50, v/v) in mixed solution, by measuring PTX on conjugate in the UV absorbances of 227nm
Drugloading rate (weight %).Minimum drugloading rate is 10.2%, and highest drugloading rate is 22.7%.Exist between drugloading rate and water solubility
Contradiction, drugloading rate is higher, water-soluble lower.It is compared with taxol, the water-soluble significant raising of paclitaxel prodrug 1, solubility
275mg/mL is risen to from 162mg/mL.The excellent water solubility of paclitaxel prodrug 1 is attributed to the carboxylic for keeping hyaluronic acid
Hydrochlorate without any modification hyaluronic acid chain it is highly hydrophilic.Since it is with good water solubility, highest is selected to carry
The conjugate of dose 22.7% is used for further experiment.
With taxol control, after free paclitaxel is handled 48 hours, is measured by CCK-8 and determine versus cell vigor.With
Free paclitaxel is compared, and paclitaxel prodrug 1 shows dose-dependent cytotoxicity to HepG2 and A549 cell lines,
And show excellent antitumor effect.The excellent cytotoxicity of paclitaxel prodrug 1, which may be attributed to, passes through hyalomitome
The cellular uptake of acid-receptor mediated endocytosis enhancing, because all to over-express HA identifiable for HepG2 and A549 cells
CD44 receptors.In addition, the cytotoxicity of paclitaxel prodrug 1 is related with drug load.For example, with the increase that PTX is loaded,
Observe the paclitaxel prodrug 1 modified with dicarboxylic anhydride with higher cytotoxicity.Drugloading rate is high, the purple of good water solubility
China fir alcohol prodrug 1 more likely improves antitumor drug effect.
Embodiment 2：
The preparation of phthalic anhydride-derivatives of hyaluronic acids
1.0g hyaluronic acids are added in the dimethyl sulfoxide (DMSO) of 80mL, are stirred strongly at 50 DEG C；By phthalic anhydride
(hyaluronic acid：Phthalic anhydride molar ratio=0.4：1.6、0.6：1.4、0.8：1.2、1：1、1.2：0.8、1.4：0.6、
1.6：0.4) it is dissolved in 20mL dimethyl sulfoxide (DMSO)s, is added in hyaluronic acid solution, be cooled to room temperature solution after reacting 5h.
Solution after cooling is dissolved under absolute methanol, strong to stir, precipitation.Clean precipitation, 11000r/ repeatedly with absolute methanol
3h is centrifuged under the conditions of min.Distillation water dissolution is used in combination in decantation.Above-mentioned solution ph is adjusted to 8~9 with 2mol solution of potassium carbonate.With
Deionized water dialysis 24 hours.It is freezed at -63 DEG C for 24 hours, -112 DEG C of vacuum drying 48h.Yield：70~91%.
The preparation of paclitaxel prodrug 2
Phthalic anhydride-derivatives of hyaluronic acids powder 600mg is dissolved in the anhydrous DMSO of 5mL, difference is added and rubs
The DCC and DMAP of your ratio.Solution is stirred 1 hour to activate the carboxyl of phthalic anhydride-derivatives of hyaluronic acids.It will not
Same amount taxol (10 and 20mg) is dissolved in the anhydrous DMSO of 1mL, and is slowly added at dry N 2 using syringe
It states in solution.Then mixture is stirred 2 days at 40 DEG C.Use dialysis membrane (MWCO：3500Da) by acquired solution DMSO
Dialysis 1 day, with deionized water dialysis deionized water, to remove unreacted taxol and dmPEG.Collect paclitaxel prodrug
It 2 and is lyophilized and 3 days and is analyzed.Yield：60~85%.
In acetonitrile/water (50:50, v/v) in mixed solution, by measuring PTX on conjugate in the UV absorbances of 227nm
Drugloading rate (weight %).Minimum drugloading rate is 10.1%, and highest drugloading rate is 21.3%.Exist between drugloading rate and water solubility
Contradiction, drugloading rate is higher, water-soluble lower.It is compared with taxol, the water-soluble significant raising of paclitaxel prodrug 2, solubility
268mg/mL is risen to from 159mg/mL.The excellent water solubility of paclitaxel prodrug 2 is attributed to the carboxylic for keeping hyaluronic acid
Hydrochlorate without any modification hyaluronic acid chain it is highly hydrophilic.Since it is with good water solubility, highest is selected to carry
The conjugate of dose 21.3% is used for further experiment.
With taxol control, after free paclitaxel is handled 48 hours, is measured by CCK-8 and determine versus cell vigor.With
Free paclitaxel is compared, and paclitaxel prodrug 2 shows dose-dependent cytotoxicity to HepG2 and A549 cell lines,
And show excellent antitumor effect.The excellent cytotoxicity of paclitaxel prodrug 1, which may be attributed to, passes through hyalomitome
The cellular uptake of acid-receptor mediated endocytosis enhancing, because all to over-express HA identifiable for HepG2 and A549 cells
CD44 receptors.In addition, the cytotoxicity of paclitaxel prodrug 2 is related with drug load.For example, with the increase that PTX is loaded,
Observe the paclitaxel prodrug 2 modified with dicarboxylic anhydride with higher cytotoxicity.Drugloading rate is high, the purple of good water solubility
China fir alcohol prodrug 2 more likely improves antitumor drug effect.
Embodiment 3:
The preparation of succinic anhydride-derivatives of hyaluronic acids
1.0g hyaluronic acids are added in the formamide of 80mL, are stirred strongly at 50 DEG C；By succinic anhydride (hyalomitome
Acid：Succinic anhydride molar ratio=0.4：1.6、0.6：1.4、0.8：1.2、1：1、1.2：0.8、1.4：0.6、1.6：0.4) it is dissolved in
It in 20mL formamides, is added in hyaluronic acid solution, is cooled to room temperature solution after reacting 5h.Solution after cooling is dissolved
It is strong to stir under absolute ethyl alcohol, precipitation.It cleans precipitation repeatedly with absolute ethyl alcohol, 3h is centrifuged under the conditions of 11000r/min.Incline
Distillation water dissolution is used in combination in analysis.Above-mentioned solution ph is adjusted to 8~9 with 2molNaOH solution.With deionized water dialysis 24 hours.-
It is freezed at 63 DEG C for 24 hours, -112 DEG C of vacuum drying 48h.Yield：70~85%.
The preparation of paclitaxel prodrug 3
Succinic anhydride-derivatives of hyaluronic acids powder 600mg is dissolved in the anhydrous DMSO of 5mL, different mol ratio is added
DCC and DMAP.Solution is stirred 1 hour to activate the carboxyl of succinic anhydride-derivatives of hyaluronic acids.To not same amount Japanese yew
Alcohol (10 and 20mg) is dissolved in the anhydrous DMSO of 1mL, and using syringe in dry N2Under be slowly added into above-mentioned solution.
Then mixture is stirred 2 days at 40 DEG C.Use dialysis membrane (MWCO：3500Da) acquired solution DMSO is dialysed 1 day, is used
Deionized water dialysis deionized water, to remove unreacted taxol.It collects paclitaxel prodrug 3 and is lyophilized 3 days and carries out
Analysis.Yield：60~85%.
In acetonitrile/water (50:50, v/v) in mixed solution, by measuring PTX on conjugate in the UV absorbances of 227nm
Drugloading rate (weight %).Minimum drugloading rate is 11.2%, and highest drugloading rate is 21.7%.Exist between drugloading rate and water solubility
Contradiction, drugloading rate is higher, water-soluble lower.It is compared with taxol, the water-soluble significant raising of paclitaxel prodrug 3, solubility
270mg/mL is risen to from 168mg/mL.The excellent water solubility of paclitaxel prodrug 3 is attributed to the carboxylic for keeping hyaluronic acid
Hydrochlorate without any modification hyaluronic acid chain it is highly hydrophilic.Since it is with good water solubility, highest is selected to carry
The conjugate of dose 21.7% is used for further experiment.
With taxol control, after free paclitaxel is handled 48 hours, is measured by CCK-8 and determine versus cell vigor.With
Free paclitaxel is compared, and paclitaxel prodrug 3 shows dose-dependent cytotoxicity to HepG2 and A549 cell lines,
And show excellent antitumor effect.The excellent cytotoxicity of paclitaxel prodrug 1, which may be attributed to, passes through hyalomitome
The cellular uptake of acid-receptor mediated endocytosis enhancing, because all to over-express HA identifiable for HepG2 and A549 cells
CD44 receptors.In addition, the cytotoxicity of paclitaxel prodrug 3 is related with drug load.For example, with the increase that PTX is loaded,
Observe the paclitaxel prodrug 3 modified with dicarboxylic anhydride with higher cytotoxicity.Drugloading rate is high, the purple of good water solubility
China fir alcohol prodrug 3 more likely improves antitumor drug effect.
Embodiment 4:
The preparation of anhydride diethylene glycol-derivatives of hyaluronic acids
1.0g hyaluronic acids are added in the formamide of 80mL, are stirred strongly at 50 DEG C；By anhydride diethylene glycol (hyalomitome
Acid：Succinic anhydride molar ratio=0.4：1.6、0.6：1.4、0.8：1.2、1：1、1.2：0.8、1.4：0.6、1.6：0.4) it is dissolved in
It in 20mL formamides, is added in hyaluronic acid solution, is cooled to room temperature solution after reacting 5h.Solution after cooling is dissolved
It is strong to stir under absolute ethyl alcohol, precipitation.It cleans precipitation repeatedly with absolute ethyl alcohol, 3h is centrifuged under the conditions of 11000r/min.Incline
Distillation water dissolution is used in combination in analysis.Above-mentioned solution ph is adjusted to 8~9 with 2molNaOH solution.With deionized water dialysis 24 hours.-
It is freezed at 63 DEG C for 24 hours, -112 DEG C of vacuum drying 48h.Yield：72~84%.
The preparation of paclitaxel prodrug 4
Anhydride diethylene glycol-derivatives of hyaluronic acids powder 600mg is dissolved in the anhydrous DMSO of 5mL, is added different moles
The DCC and DMAP of ratio.Solution is stirred 1 hour to activate the carboxyl of anhydride diethylene glycol-derivatives of hyaluronic acids.To not same amount
Taxol (10 and 20mg) is dissolved in the anhydrous DMSO of 1mL, and using syringe in dry N2Under be slowly added into it is above-mentioned molten
In liquid.Then mixture is stirred 2 days at 40 DEG C.Use dialysis membrane (MWCO：3500Da) by acquired solution DMSO dialysis 1
It, with deionized water dialysis deionized water, to remove unreacted taxol.It collects paclitaxel prodrug 4 and is lyophilized 3 days simultaneously
It is analyzed.Yield：63~82%.
In acetonitrile/water (50:50, v/v) in mixed solution, by measuring PTX on conjugate in the UV absorbances of 227nm
Drugloading rate (weight %).Minimum drugloading rate is 11.3%, and highest drugloading rate is 22.5%.Exist between drugloading rate and water solubility
Contradiction, drugloading rate is higher, water-soluble lower.It is compared with taxol, the water-soluble significant raising of paclitaxel prodrug 4, solubility
272mg/mL is risen to from 167mg/mL.The excellent water solubility of paclitaxel prodrug 4 is attributed to the carboxylic for keeping hyaluronic acid
Hydrochlorate without any modification hyaluronic acid chain it is highly hydrophilic.Since it is with good water solubility, highest is selected to carry
The conjugate of dose 22.5% is used for further experiment.
With taxol control, after free paclitaxel is handled 48 hours, is measured by CCK-8 and determine versus cell vigor.With
Free paclitaxel is compared, and paclitaxel prodrug 4 shows dose-dependent cytotoxicity to HepG2 and A549 cell lines,
And show excellent antitumor effect.The excellent cytotoxicity of paclitaxel prodrug 1, which may be attributed to, passes through hyalomitome
The cellular uptake of acid-receptor mediated endocytosis enhancing, because all to over-express HA identifiable for HepG2 and A549 cells
CD44 receptors.In addition, the cytotoxicity of paclitaxel prodrug 4 is related with drug load.For example, with the increase that PTX is loaded,
Observe the paclitaxel prodrug 1 modified with dicarboxylic anhydride with higher cytotoxicity.Drugloading rate is high, the purple of good water solubility
China fir alcohol prodrug 4 more likely improves antitumor drug effect.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not limited to the present invention, any to be familiar with this skill
The people of art can do various change and modification, therefore the protection model of the present invention without departing from the spirit and scope of the present invention
Enclosing be subject to what claims were defined.
Claims (10)
1. a kind of preparation method of paclitaxel prodrug, which is characterized in that the method is to modify hyalomitome by dicarboxylic anhydride
The hydroxyl of acid, then couples with taxol drug again.
2. according to the method described in claim 1, it is characterized in that, the structural formula of the paclitaxel prodrug is as follows：
3. according to the method described in claim 1, it is characterized in that, the method the specific steps are：
(1) in the presence of solvent, by hyaluronic acid and dicarboxylic anhydride according to certain mol proportion, reaction generates derivatives of hyaluronic acids；
(2) in the presence of condensing agent with taxol esterification occurs for derivatives of hyaluronic acids, before generating taxol drug
Body.
4. according to the method described in claim 3, it is characterized in that, the solvent in the step (1) is formamide, dimethyl methyl
One kind in amide, N-Methyl pyrrolidone, dimethyl sulfoxide (DMSO), 1,3- dimethyl-2-imidazolinones or hexamethylphosphoramide
Or two or more combinations.
5. according to the method described in claim 3, it is characterized in that, dicarboxylic anhydride is maleic anhydride, adjacent benzene two in the step (1)
Formic anhydride, succinic anhydride or anhydride diethylene glycol；The molar ratio of the hyaluronic acid and dicarboxylic anhydride is 1:4~4:1.
6. according to the method described in claim 3, it is characterized in that, it is 40 that the reaction condition of the step (1), which is reaction temperature,
~60 DEG C, the reaction time is 4~6h, is cooled to room temperature after reaction, and freeze-drying is centrifuged after being cleaned with solvent.
7. according to the method described in claim 3, it is characterized in that, condensing agent is N, N '-dicyclohexyl carbon in the step (2)
Diimine, 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides, hexafluorophosphoric acid benzotriazole -1- bases-oxygroup tripyrrole alkane
Base, tripyrrole alkane base phosphonium bromide hexafluorophosphate, 4-dimethylaminopyridine, I-hydroxybenzotriazole, O- benzotriazole-tetramethyl
One or more of base urea hexafluorophosphoric acid ester or O- benzotriazole-N, N, N', N'- tetramethylurea tetrafluoro boric acid group
It closes.
8. according to the method described in claim 3, it is characterized in that, it is 30 that the reaction condition of the step (2), which is reaction temperature,
~50 DEG C, the reaction time is 1~3 day.
9. the paclitaxel prodrug that any the method for claim 1~8 is prepared.
10. application of the paclitaxel prodrug described in claim 9 in preparing medicine for prostate disease.
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