CN103897176B

CN103897176B - Anthracycline antibiotic is coupled non-linear segmented copolymer and its preparation method and application

Info

Publication number: CN103897176B
Application number: CN201210595214.9A
Authority: CN
Inventors: 张雅珍; 李铁力; 白毅
Original assignee: Individual
Current assignee: Individual
Priority date: 2012-12-24
Filing date: 2012-12-24
Publication date: 2018-08-17
Anticipated expiration: 2032-12-24
Also published as: CN103897176A

Abstract

The invention discloses a new class of compound anthracycline antibiotics to be coupled non-linear segmented copolymer and preparation method and purposes.It is more better on treatment choroid and retinal disease than the drug of other forms or medicament that the anthracycline antibiotic is coupled non-linear segmented copolymer.

Description

Anthracycline antibiotic is coupled non-linear segmented copolymer and its preparation method and application

Technical field

The invention discloses a new class of compound anthracycline antibiotics to be coupled non-linear segmented copolymer, and prepares Method and purposes.

Background technology

Anthracycline antibiotic is a kind of antimitotic and the drug of great cytotoxicity.Doxorubicin can successfully inhibit Several Kinds of Malignancy, including acute leukemia, lymthoma, soft tissue and osteosarcoma, children malignant tumors and adult solid's tumor.Its The mechanism of action is that drug can penetrate into cell, is combined with chromosome.Planar rings in medicines structure be inserted between base-pair from And it is combined to form compound with DNA, severe jamming DNA synthesis, DNA dependent rna synthesis and protein synthesis.But pass through the machine The drug concentration that system generates tumor locus in the Doxorubicin concentration ratio clinical treatment needed for anti-proliferative effect wants high.In addition anthracene nucleus Class antibiotic is also related with redox, may participate in and cytotoxic compound is obtained by the reaction, such as peroxide, active hydrogen-oxygen Base and hydrogen peroxide etc..The site of action of anthracycline antibiotic may be in cell membrane, and anthracycline antibiotic is for various diseases at present Disease pharmacodynamic action not yet reach common understanding in scientific circles, for each disease mechanism of action all without general character, need into The scientific research of one step is explained.

Anthracycline antibiotic is widely used at present, but generally requires to be become salt, such as how soft ratio in Point of View of Clinical Star, common medicinal form are doxorubicin hydrochlorides, i.e., need to become hydrochloride, because Doxorubicin solubility itself is very low, are not converted At the form of salt, can not apply.And after becoming hydrochloride, the pharmacodynamics function of Doxorubicin declines to a great extent, therefore this gives clinic Using bringing very big difficulty.

Anthracycline antibiotic another disadvantage is that due to strong cytotoxicity itself, the toxicity of itself is very big, Often there is apparent bone marrow suppression within 10 days or so after use, using after a week, you can obviously gastrointestinal tract is not or not performance Good reaction and cardiac toxic, therefore must could be applied after accurate calculation when medication, and the half-life period of this medicine is very short, and it is same to be answered Limited to bringing.

When anthracene nucleus medicament is applied in eye, dosage is very sensitive with medicine efficacy relation, anthracycline antibiosis of the invention Element is coupled non-linear segmented copolymer, the new construction containing anthracycline antibiotic of preparation can be made to dissolve in water, very well Control drug delivery amount, and then reach treatment chorioido-retinal disorders or illness.

It is bis- (to carboxyl benzene oxygen with poly- 1,6- when treating choroid and retinal disease in final product prepared by this patent Base) nanoparticle of the simply wrapped Doxorubicin of hexane-polyethylene glycol is compared, and the effect of drugs of this patent new product is completely super The nanometer or microball preparation of the simply wrapped Doxorubicin of more poly- bis- (to the carboxyphenoxy) hexane-polyethylene glycol of 1,6-, drug are controlled Therapeutic effect is very good.

Invention content

Present disclosure is as follows：

The invention discloses the non-linear segmented copolymers that anthracycline antibiotic shown in following formula I is coupled, and structure is such as Under：

Wherein R1, R2, R3, R4, R5 respectively stand alone as H, OH, CH₃、CH₂OH or OCH₃, R6 H, OH, CH₃、CH₂OH、 OCH₃、COCH₃Or COCH₂OH, R7, R8 respectively stand alone as H, OH, CH₃、OCH₃、COCH₃Or OR9, wherein R9 are pyridyl group, furans Base, pyrrole radicals, thienyl or pyranose.R1, R2, R3, R4 respectively stand alone as H, OH or OCH preferably wherein₃, R5 OH, R6 For H, OH, COCH₃Or COCH₂OH, R7, R8 respectively stand alone as OH, CH₃、OCH₃、COCH₃, the integer between n=1-20.More preferably Wherein R1 is H or OCH₃, R2 H, R3, R4 and R5 are OH, R6 COCH₃Or COCH₂OH, R7, R8 respectively stand alone as OH, CH₃ Or OR9, wherein R9 are pyranose.PEG refers to polyethylene glycol, refers specifically to-C₂H₄(OC₂H₄)_W, the integer between wherein W=1-500, It is preferred that 1-300.

The polymer is respectively：

1) R1 is OCH₃, R2 H, R3, R4 and R5 are OH, R6 COCH₂OH, R7 CH₃, R8 OH, n=1-200 it Between integer (preferably n=1-100)；

2) R1 is OCH₃, R2 H, R3, R4 and R5 are OH, R6 COCH₂OH, R7 CH₃, R8 OR9, R9 are pyrans Base, the integer (preferably n=1-100) between n=1-200；

3) R1 is OCH₃, R2 H, R3, R4 and R5 are OH, R6 COCH₃, R7 CH₃, between R8 OH, n=1-200 Integer (preferably n=1-100)；

4) R1, R2 H, R3, R4 and R5 are OH, R6 COCH₃, R7 CH₃, the integer between R8 OH, n=1-200 (preferably n=1-100).

The preparation method of present copolymer, it is characterised in that：

1) compound A is obtained with methoxy poly (ethylene glycol) amine and citric acid reactions；

2) polymer B, wherein n=1- is obtained by the reaction in bis- (to the carboxyphenoxy) hexanes of compound A and 1,6- of acetylation 200 integer, preferably 1-100；

3) final product is obtained by the reaction with anthracycline antibiotic C in polymer B；

It is preferred that wherein anthracycline antibiotic C be selected from Doxorubicin, adriamycin, Epi-ADM, pirarubicin, daunorubicin, Daunoblastin, idarubicin, Aclarubicin, daunomycins, aclacinomycin or carminomycin.Wherein step 1) optionally employs Solvent, the solvent are selected from：Benzene, toluene, pyridine, tetrahydrofuran, chloroform, carbon tetrachloride, dichloromethane, dicyclohexyl carbon two are sub- It is one or more in amine；Step 3) optionally employs solvent, and the solvent is selected from methanol, ethyl alcohol, dichloromethane, chloroform, tetrachloro Change one or more in carbon, dimethyl sulfoxide (DMSO), n,N-Dimethylformamide.

The preparation therapy of the present invention is specific as follows：

1) bis- (to the carboxyphenoxy) hexanes of 1,6- are flowed back in acetic anhydride, 1, the 6- for forming acetylation is bis- (to carboxyl Phenoxy group) hexane (can also be commercially available)；

2) methoxy poly (ethylene glycol) amine mixes in a solvent with citric acid dissolving, and reaction at room temperature overnight, is dried to obtain Compound A；

3) bis- (to the carboxyphenoxy) hexanes of the 1 of acetylation, 6- are mixed with compound A, are reacted at 100-200 DEG C, Reaction time 20min to 8h；After reaction mixture cooling, washing is dried to obtain polymer B；

4) by anthracycline antibiotic C and polymer B as 3-48 hours in solvent, ultrasonic reaction obtains after 1-20 minutes Formulas I polymer, then homogenizer high-speed stirred 1-10 minutes in subzero 30 DEG C of 0-, rotation volatilization obtain crude product, post-process Obtain the nanometer formulation of finished product Formulas I polymer.

Its reaction equation is as follows：

The polymer containing anthracycline antibiotic structure that the present invention obtains is easy to dissolve in water, and its half-life period Extend much than anthracycline antibiotic, such noval chemical compound solves the application limitation of anthracene nucleus medicament.

The disease or illness that final product prepared by this patent can treat or prevent relate generally to treatment choroid and view Film age-related disease and center and branch retinal vein occlusion class disease.

The polymer containing anthracycline antibiotic structure that the present invention obtains is easy to dissolve in water, and its half-life period Extend much than anthracycline antibiotic, such noval chemical compound solves the application limitation of anthracene nucleus medicament.It is prepared in this patent Final product (be claim one in medicines structure nanometer formulation) treatment eye disease comparison in, this patent is new The medication effect of polymer is very good, surmount completely-by the nanometer formulation of polymer wrapped Doxorubicin.

Description of the drawings：

The nuclear magnetic resonance map of the final product of Fig. 1 embodiments 1.

Bis- (to the carboxyphenoxy) hexane-glycol copolymers of Fig. 2 embodiments 1,1,6- directly wrap up Doxorubicin nanometer The drug accumulative releasing degree and time chart of grain and Doxorubicin ordinary preparation.

Fig. 3 directly wraps up nanoparticle-associated doxorubicin, and solubility compares in the water of embodiment 1-4

Specific implementation mode

Invention is further described in detail for specific embodiment below, but the present invention not only limits to following embodiment.

It is as follows to prepare embodiment：

Embodiment 1

1) mixture reflux of bis- (to carboxyphenoxy) the hexane 25g of 1,6- in 250ml acetic anhydrides, to form acetylation Bis- (to the carboxyphenoxy) hexanes of 1,6-；

2) methoxy poly (ethylene glycol) amine 3.8g, citric acid 80mg, dicyclohexylcarbodiimide 164mg and pyridine 9mg mixing, It is stirred at room temperature overnight；Then it is washed, and is dried under vacuum with ether, obtain polymer；

3) by 1) step and 2) step product mix be put into flask, decompression melting polymerisation 1 hour at 170 DEG C； Object to be polymerized is cooled to room temperature to be dissolved with chloroform, and petroleum ether and drying is used in combination；

4) the 800mg polymer of Doxorubicin and step 3 is put into 10ml dimethyl sulphoxide solutions 48 hours；Then it sets Enter in baking oven 1 hour；Ultrasound 20 minutes；Homogenizer high-speed stirred 2 minutes, is then put into 5% cholic acid in subzero 10-20 degree It is stirred 2 hours for 500 turns in solution；Freeze-drying is to get final product after being collected by centrifugation.

Embodiment 2

1) mixture reflux of bis- (to carboxyphenoxy) the hexane 20g of 1,6- in 500ml acetic anhydrides, to form acetylation Bis- (to the carboxyphenoxy) hexanes of 1,6-；

2) methoxy poly (ethylene glycol) amine 3g, citric acid 45mg, dicyclohexylcarbodiimide 130mg and pyridine 5mg mixing, It is stirred overnight at room temperature；Then it is washed, and is dried under vacuum with ether, obtain polymer；

3) by 1) step and 2) step product mix be put into flask, decompression melting polymerisation 1 hour at 180 DEG C； Object to be polymerized is cooled to room temperature to be dissolved with chloroform, and petroleum ether and drying is used in combination；

4) pirarubicin and polymer 900mg are put into the solution mixed by 5ml ethyl alcohol and 15ml dichloromethane；So It is placed in baking oven 24 hours afterwards；Ultrasound 2 minutes；In subzero 10-20 degree, homogenizer high-speed stirred 2 minutes, product is put into 3% Cholic acid solution in 400 turns stir 3 hours；Freeze-drying is to get final product after being collected by centrifugation.

Embodiment 3

1) mixture reflux of bis- (to carboxyphenoxy) the hexane 28g of 1,6- in 300ml acetic anhydrides, to form acetylation Bis- (to the carboxyphenoxy) hexanes of 1,6-；

2) methoxy poly (ethylene glycol) amine 2g, citric acid 28mg, dicyclohexylcarbodiimide 100mg and pyridine 3.5mg mixing 10ml chloroforms are added, are stirred at room temperature overnight；Then it is washed, and is dried under vacuum with ether, obtain polymer；

3) by 1) step and 2) step product mix be put into flask, decompression melting polymerisation 1 hour at 175 DEG C； Object to be polymerized is cooled to room temperature to be dissolved with chloroform, and petroleum ether and drying is used in combination；

4) the 1000mg polymer of Aclarubicin and step 3 is put into and is mixed by 13ml dichloromethane and 8ml dimethyl sulfoxide (DMSO)s In the solution of conjunction；Then it is placed in 24 hours dry in baking oven；Ultrasound 7 minutes；The homogenizer high-speed stirred 5 in subzero 20-30 degree Minute, product is put into 2% cholic acid solution and stirs 2.5 hours for 500 turns；Freeze-drying is to get final product after being collected by centrifugation.

Embodiment 4

1) mixture reflux of bis- (to carboxyphenoxy) the hexane 30g of 1,6- in 500ml acetic anhydrides, to form acetylation Bis- (to the carboxyphenoxy) hexanes of 1,6-；

2) methoxy poly (ethylene glycol) amine 4g, citric acid 65mg, the mixing of dicyclohexylcarbodiimide 190mg, 6mg pyridine, add Enter 20ml dichloromethane, is stirred at room temperature overnight；Then it is washed, and is dried under vacuum with ether, obtain polymer；

4) the 600mg polymer of daunorubicin and step 3) is put into mixed by 10ml dichloromethane and 10mlDMF it is molten In liquid；Then it is placed in baking oven 48 hours；Ultrasound 3 minutes；The homogenizer high-speed stirred 7 minutes in subzero 10 degree, product is put into It is stirred 3 hours for 600 turns in 2% poly-vinyl alcohol solution；Freeze-drying is to get final product after being collected by centrifugation.

Effect experiment is as follows：

By the embodiment 1-4 samples prepared and 1, bis- (to the carboxyphenoxy) hexane-glycol copolymers of 6- directly wrap The nanoparticle pharmaceutical group (chemical coupling reaction not occurring, anthracycline antibiotic does not have structure change) for the Doxorubicin wrapped up in, anthracene nucleus Class antibiotic ordinary preparation (powder-injection) carry out respectively stability test, drug release in vitro experiment, in water solubility test with And the drug action experiment of choroid cambium.

Stability test：

By the sample and 1 of 1 group of preparation of embodiment, bis- (to the carboxyphenoxy) hexanes of 6--glycol copolymer package The nanoparticle pharmaceutical group of Doxorubicin, Doxorubicin ordinary preparation take same amount (in terms of Doxorubicin) to measure absorbance value respectively and put Enter in 20 degree of incubators 3 months, then takes out and measure 480 nanometers of lower absorbance values, as a result visible embodiment 1-4 groups and ordinary preparation The absorbance value of the Doxorubicin of group is front and back without change, and the nanoparticle group absorbance wrapped up declines 33%.

Drug release in vitro is tested：

By 1 group of embodiment, the nanometer of the Doxorubicin of 1,6- bis- (to carboxyphenoxy) hexanes-glycol copolymer package Grain medicine group and Doxorubicin ordinary preparation component also known as take the drug of equivalent, and (in terms of Doxorubicin, every group how soft containing 10mg Than star), then each group drug after being impregnated with PBS buffer solution, is shaken in shaking table under 37 degrees Celsius as in test tube, timing After sampling, the content of drug is measured under 480 nanometers of ultraviolet specrophotometer, and the medicament contg percentage of release is calculated after recording Than doing releasing curve diagram, abscissa is the time (day), and ordinate is the percentage of release.See Fig. 1, it is seen that embodiment release Drug is more permanent, keeps drug half-life longer.

Solubility test in water：

By embodiment 1-4 groups, the Doxorubicin of 1,6- bis- (to carboxyphenoxy) hexanes-glycol copolymer package is received Grain of rice medicine group and Doxorubicin ordinary preparation component also known as take the drug of equivalent, and (in terms of anthracycline antibiotic, every group contains 100mg anthracycline antibiotics), it is respectively put into test tube, with 10mlPBS buffer solutions and shakes, dissolved form is observed after static Condition.3 minutes and 20 minutes dissolved states of record, it is as a result as follows.

1 solubility of table compares and (see Fig. 3, from left to right, directly wraps up nanoparticle-associated doxorubicin (layering), embodiment 1-4)

Inhibiting effect of the drug to tela chorioidea's hyperplasia：

Male rat 56 is taken, is randomly divided into 7 groups, i.e. bis- (to the carboxyphenoxy) hexane-ethylene glycol of control group, 1,6- are total The nanoparticle pharmaceutical group (chemical coupling reaction not occurring, Doxorubicin does not have structure change) of the Doxorubicin of polymers package, it is real Apply a 1-4 groups, Doxorubicin general formulation group.Each group number of animals is 8.It is experimental eye at a glance that every rat, which randomly selects, Another eye is control eye.The equal 10 μ g drugs of intraocular injection of each group or the load medicine durative action preparation containing 10 μ g drugs, right in addition to control group According to group to isometric PBS solution.

With laser irradiation rat eye, there is bubble to generate or (ring) mark with light sometimes with hyporrhea after light is solidifying and hit Broken Bruch films, are denoted as available point.After laser photocoagulation, each group drug is injected to rat right eye eyeball.14d after light is solidifying, tissues observed Hyperplasia area simultaneously carries out histological examination.As a result as follows：

2 retina of table and tela chorioidea's hyperplasia area compare (unit .mm²)

Compared with control group group^*p<0.05,^**p<0.01, compared with ordinary preparation group^#p<0.05,^##p<0.01

Upper table the result shows that, it is larger that hyperplasia area occurs for control group retina and choroid, and each treatment group is respectively not Hyperplasia area is reduced with limit.Each embodiment group can subtract simultaneously to be shown to the experiment of rat ocular tissue hyperplasia area Small lesion retina and choroidal hyperblastosis area, but effect difference, wherein the embodiment 1-4 groups being coupled It is better.

The result of histological examination is that it is disorderly to coagulate visible outer retina and train of thought membrane structure at spot for control group light under light microscopic Random, retinal pigment epithelium chants network film cambium hyperplasia, is free with inflammatory cell infiltration.Medicine group and control group phase Than cambium is rarely found and less with serosa circumscripta detachment of retina；Embodiment group is simply wrapped more compared to high polymer Soft more notable without the nanoparticle pharmaceutical group effect of coupling reaction than star, the compound after coupling can substantially reduce train of thought Membrane tissue hyperplasia.Histological examination shows nanoparticle pharmaceutical group of the embodiment group than ordinary preparation group to the treatment of lesion more Thoroughly, it plays a role simultaneously to the retina of lesion and choroid.

Claims

1. the non-linear segmented copolymer for the anthracycline antibiotic coupling being shown below, structure are as follows：

Wherein R1, R2, R3, R4, R5 respectively stand alone as H, OH, CH₃、CH₂OH or OCH₃, R6 H, OH, CH₃、CH₂OH、OCH₃、 COCH₃Or COCH₂OH, R7, R8 respectively stand alone as H, OH, CH₃、OCH₃、COCH₃Or the integer between OR9, n=1-200；Wherein R9 is pyridyl group, furyl, pyrrole radicals, thienyl or pyranose, and PEG refers to polyethylene glycol, refers specifically to-C₂H₄(OC₂H₄)_W, In_WInteger between=1-500.

2. the copolymer of claim 1, wherein R1, R2, R3, R4 respectively stand alone as H, OH or OCH₃, R5 OH, R6 H, OH, COCH₃Or COCH₂OH, R7, R8 respectively stand alone as OH, CH₃、OCH₃、COCH₃, the integer between n=1-200, wherein_W=1-500 Between integer.

3. the copolymer of claim 1, wherein R1 are H or OCH₃, R2 H, R3, R4 and R5 are OH, R6 COCH₃Or COCH₂OH, R7, R8 respectively stand alone as OH, CH₃Or the integer between OR9, n=1-200；Wherein R9 is pyranose.

4. the copolymer of claim 1, is respectively：

1) R1 is OCH₃, R2 H, R3, R4 and R5 are OH, R6 COCH₂OH, R7 CH₃, between R8 OH, n=1-200 Integer；

2) R1 is OCH₃, R2 H, R3, R4 and R5 are OH, R6 COCH₂OH, R7 CH₃, R8 OR9, R9 are pyranose, n Integer between=1-200；

3) R1 is OCH₃, R2 H, R3, R4 and R5 are OH, R6 COCH₃, R7 CH₃, whole between R8 OH, n=1-200 Number；

4) R1, R2 H, R3, R4 and R5 are OH, R6 COCH₃, R7 CH₃, the integer between R8 OH, n=1-200.

5. the copolymer of claim 1, structure are as follows：

6. such as the preparation method of any one of claim 1-5 copolymers, it is characterised in that：

2) polymer B is obtained by the reaction in bis- (to the carboxyphenoxy) hexanes of compound A and 1,6- of acetylation, wherein n=1-200's Integer；

3) final copolymer is obtained by the reaction with anthracycline antibiotic C in polymer B；

7. the preparation method described in claim 6, wherein anthracycline antibiotic C are selected from Doxorubicin, Epi-ADM, the soft ratio of pyrrole Star, daunorubicin, idarubicin, Aclarubicin, carminomycin.

8. the method for claim 6 or 7, wherein step 1) optionally employ solvent, the solvent is selected from：Benzene, toluene, pyridine, four It is one or more in hydrogen furans, chloroform, carbon tetrachloride, dichloromethane, dicyclohexylcarbodiimide；Step 3) optionally employs molten Agent, the solvent is in methanol, ethyl alcohol, dichloromethane, chloroform, carbon tetrachloride, dimethyl sulfoxide (DMSO), n,N-Dimethylformamide It is one or more.

9. the purposes of any one of claim 1-5 copolymers, to prepare the age-related disease for the treatment of choroid and retina And the purposes in treatment center and branch retinal vein occlusion class disease medicament.

10. the purposes of any one of claim 1-5 copolymers is treated to prepare in age-related macular degeneration disease medicament Purposes.