CN103910870B

CN103910870B - Anthracycline antibiotic is coupled non-linear segmented copolymer and its preparation method and application

Info

Publication number: CN103910870B
Application number: CN201210595882.1A
Authority: CN
Inventors: 张雅珍; 李铁力; 白毅
Original assignee: Individual
Current assignee: Individual
Priority date: 2012-12-28
Filing date: 2012-12-28
Publication date: 2018-08-17
Anticipated expiration: 2032-12-28
Also published as: CN103910870A

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 eye 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 disease related with intraocular neovascularization or illness.

In final product prepared by this patent when treat ocular angiogenesis and bis- (to the carboxyphenoxy) hexanes-of poly- 1,6- The nanoparticle of the simply wrapped Doxorubicin of polyethylene glycol is compared, and the effect of drugs of this patent new product surmounts poly- 1,6- completely The nanometer or microball preparation of the simply wrapped Doxorubicin of bis- (to carboxyphenoxy) hexane-polyethylene glycol, medication effect are non- Chang Hao.

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 the integer between OR9, n=1-20, It is preferred that n=1-10；Wherein R9 is pyridyl group, furyl, pyrrole radicals, thienyl or pyranose.R1, R2, R3, R4 preferably wherein 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=1-20.More preferable wherein R1 is H or OCH₃, R2 H, R3, R4 and R5 are OH, and R6 is COCH₃Or COCH₂OH, R7, R8 respectively stand alone as OH, CH₃Or the integer (preferably n=1-100) between OR9, n=1-200；Its Middle R9 is pyranose.PEG refers to polyethylene glycol, i.e.-C₂H₄(O C₂H₄)_W, the integer between wherein W=1-500, preferably 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).

Most preferred configuration is as follows：

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

1) compound A is obtained by the reaction with methoxy poly (ethylene glycol) amine and 2- hydroxysuccinic acids；

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；

Compound A；

Polymer B；

Anthracycline antibiotic C.

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 method 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 the dissolving of 2- hydroxysuccinic acids, and reaction at room temperature overnight, is done It is dry 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 2h；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, the ultrasonic reaction 1- in subzero 30 DEG C of 0- After twenty minutes, then homogenizer high-speed stirred 1-10 minutes, rotation volatilization obtained crude product, and the rear frozen dried that centrifuges obtains whole production The suitable formulations of 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 retina and choroid disease Disease, it is characterised in that the disease or illness for the treatment of are in eyes of patients.It is characterized in that, the retinal and choroidal disease is Age-related macular degeneration disease, proliferative diabetic retinopathy, proliferative vitreoretinopathy, premature regard Retinopathy, pathological myopia, it is assumed that ocular histoplasmosis's syndrome, branch retinal vein occlusion remaining, retinal centre are quiet Arteries and veins blocks, Branch Retinal Artery obstruction, central retinal artery occlusion etc..

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, surmounts-directly wrapped up by polymer the nanometer formulation of Doxorubicin completely.

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.

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 500ml acetic anhydrides, to form acetylation Bis- (to the carboxyphenoxy) hexanes of 1,6-；

2) methoxy poly (ethylene glycol) amine 3.5g, 2- hydroxysuccinic acid 51mg, dicyclohexylcarbodiimide 165mg and pyridine 8mg is mixed, and 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 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) polymer of Doxorubicin and step 3 is put into 20ml dimethyl sulphoxide solutions 48 hours；Then merging is dried 3 hours in case；In subzero 10-20 degree, ultrasound 15 minutes；Homogenizer high-speed stirred 1 minute, be then put into 5% cholic acid it is molten It is stirred 2 hours for 400 turns in liquid；Freeze-drying is to get final product after being collected by centrifugation.

Embodiment 2

1) mixture reflux of bis- (to carboxyphenoxy) the hexane 30g of 1,6- in 300ml acetic anhydrides, to form acetylation Bis- (to the carboxyphenoxy) hexanes of 1,6-；

2) methoxy poly (ethylene glycol) amine 3g, 2- hydroxysuccinic acid 29mg, dicyclohexylcarbodiimide 130mg and pyridine 5mg Mixing, 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 150 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 the polymer are put into the solution mixed by 5ml ethyl alcohol and 5ml dichloromethane；Then it is placed in 24 hours in baking oven；In subzero 10-20 degree, ultrasound 2 minutes；Then homogenizer high-speed stirred 2 minutes, product is put into 3% It is stirred 2 hours for 400 turns in cholic acid solution；Freeze-drying is to get final product after being collected by centrifugation.

Embodiment 3

1) mixture reflux of bis- (to carboxyphenoxy) the hexane 17g of 1,6- in 500ml acetic anhydrides, to form acetylation Bis- (to the carboxyphenoxy) hexanes of 1,6-；

2) methoxy poly (ethylene glycol) amine 2g, 2- hydroxysuccinic acid 26mg, dicyclohexylcarbodiimide 90mg and pyridine 4mg are mixed It closes and 10ml chloroforms is added, be 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) polymer of Aclarubicin and step 3 is put into mixed by 10ml dichloromethane and 6ml dimethyl sulfoxide (DMSO)s it is molten In liquid；Then it is placed in 1 hour dry in baking oven；1 minute ultrasonic in subzero 20-30 degree, then homogenizer high-speed stirred 5 is divided Clock, product are put into 2% cholic acid solution and stir 4 hours for 400 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 350ml acetic anhydrides, to form acetylation Bis- (to the carboxyphenoxy) hexanes of 1,6-；

2) methoxy poly (ethylene glycol) amine 4g, 2- hydroxysuccinic acid 47mg, dicyclohexylcarbodiimide 160mg, 4mg pyridine are mixed It closes, 20ml dichloromethane is added, is stirred at room temperature overnight；Then it is washed, and is dried under vacuum with ether, is polymerize Object；

4) polymer of daunorubicin and step 3) is put into the solution mixed by 10ml dichloromethane and 10mlDMF； Then it is placed in baking oven 48 hours；Subzero 10 degree in 0-, ultrasound 10 minutes；Then homogenizer high-speed stirred 10 minutes, product is put Enter in 2% cholic acid solution and stirs 2.5 hours for 800 turns；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 choroidal neovascularization.

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

Inhibiting effect of the drug to choroidal neovascularization：

Male rat 35 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 5.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 observes blood vessel Hyperplasia area simultaneously carries out histological examination.As a result as follows：

2 retina of table and choroidal neovascularization area compare (unit：mm²N=5)

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 blood vessel hyperplasia area occurs for control group retina and choroid, each therapeutic component Not different limits reduce blood vessel hyperplasia area.Each embodiment group is shown to the experiment of rat ocular vascular proliferation area It can reduce lesion retina and choroidal blood vessel hyperplasia area simultaneously, but effect difference, wherein the reality being coupled It is better to apply a 1-4 groups.

The result of histological examination is, it is breakdown to coagulate visible Bruch films at spot for control group light under light microscopic, outer retina and Choroid structure disturbance, retinal pigment epithelium, choroidal neovascularization hyperplasia, free companion's inflammatory cell infiltration.Drug Compared with the control group, new vessels are rarely found and less with serosa circumscripta detachment of retina for group；Embodiment group is poly- compared to high The simply wrapped Doxorubicin of object acts on more notable, the compound energy after coupling without the nanoparticle pharmaceutical group of coupling reaction Substantially reduce choroidal neovascularization.Histological examination shows nanoparticle pharmaceutical group of the embodiment group than ordinary preparation group to disease The treatment of stove is more thorough, and the retina and choroid to lesion play a role simultaneously.

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, Integer between middle W=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 CH_3,R8 is OH, between n=1-200 Integer；

2) R1 is OCH₃, R2 H, R3, R4 and R5 are OH, R6 COCH₂OH, R7 CH_3,R8 is OR9, and R9 is 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；

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 polymer, purposes is to prepare disease at treatment eye choroid and retina Purposes in drug.

10. the purposes of claim 9, purposes is the purposes in the drug for prepare treatment age-related macular degeneration disease.