CN103897177B

CN103897177B - Anthracycline antibiotic is coupled non-linear segmented copolymer and its preparation method and application

Info

Publication number: CN103897177B
Application number: CN201210595215.3A
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: 2017-08-29
Anticipated expiration: 2032-12-24
Also published as: CN103897177A

Abstract

Non-linear segmented copolymer, and preparation method and purposes are coupled the invention discloses a class compound anthracycline antibiotic.It is better on treatment eye disease that the anthracycline antibiotic is coupled medicine or medicament of the non-linear segmented copolymer than other forms.

Description

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

Technical field

Non-linear segmented copolymer is coupled the invention discloses the new compound anthracycline antibiotic of a class, and is prepared Method and purposes.

Background technology

Anthracycline antibiotic is the medicine of a kind of antimitotic and great cytotoxicity.Doxorubicin can successfully suppress Several Kinds of Malignancy, including acute leukemia, lymthoma, soft tissue and osteosarcoma, children malignant tumors and adult solid's knurl.Its The mechanism of action is that medicine can penetrate into cell, is combined with chromosome.In medicines structure planar rings insertion base-pair between from And combine to form compound, severe jamming DNA synthesis, DNA dependent rna synthesis and protein synthesis with DNA.But pass through the machine The drug concentration of tumor locus will height in Doxorubicin concentration ratio clinical treatment needed for system generation anti-proliferative effect.In addition anthracene nucleus Class antibiotic is also relevant with redox, may participate in reaction and obtains cytotoxic compound, such as peroxide, the hydrogen-oxygen of activity Base and hydrogen peroxide etc..The site of action of anthracycline antibiotic may be in cell membrane, and current anthracycline antibiotic is directed to various diseases Disease pharmacodynamic action do not reach common understanding in scientific circles also, for every kind of disease mechanism of action all do not have 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 in Point of View of Clinical, such as how soft ratio Star, common medicinal form is doxorubicin hydrochloride, i.e., need to become hydrochloride, because Doxorubicin solubility itself is very low, is not converted Into the form of salt, it is impossible to apply.And become after hydrochloride, the pharmacodynamics function of Doxorubicin declines to a great extent, therefore this is to clinic Using bringing very big difficulty.

Another of anthracycline antibiotic has the disadvantage due to strong CDCC itself, therefore the toxicity of itself is very big, Often there is obvious bone marrow suppression within 10 days or so after use, after one week, you can obviously intestines and stomach are not or not performance Good reaction and cardiac toxic, therefore must could be applied after accurate calculation during medication, and the half-life period of this medicine is very short, and same give should With bringing limitation.

When anthracene nucleus medicament is applied in eye, dosage is very sensitive with medicine efficacy relation, anthracycline antibiosis of the invention The non-linear segmented copolymer of element coupling, can be such that the new construction containing anthracycline antibiotic of preparation is dissolved in water, very well Control medicine delivery amount, and then reach treatment disease related with intraocular neovascularization or illness.

When end-product prepared by this patent is treating ocular angiogenesis with double (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 double (to carboxyphenoxy) hexane-polyethylene glycol, medication effect are non- Chang Hao.

The content of the invention

Present disclosure is as follows：

The invention discloses the non-linear segmented copolymer of the coupling of the anthracycline antibiotic shown in following formula I, its structure is such as Under：

Wherein R1, R2, R3, R4, R5 each stand alone as H, OH, CH₃、CH₂OH or OCH₃, R6 is H, OH, CH₃、CH₂OH、 OCH₃、COCH₃Or COCH₂OH, R7, R8 each 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 pyridine radicals, furyl, pyrrole radicals, thienyl or pyranose.R1, R2, R3, R4 preferably wherein Each stand alone as H, OH or OCH₃, R5 is OH, and R6 is H, OH, COCH₃Or COCH₂OH, R7, R8 each stand alone as OH, CH₃、 OCH₃、COCH₃, the integer between n=1-20.More preferably wherein R1 is H or OCH₃, R2 is H, and R3, R4 and R5 are OH, and R6 is COCH₃Or COCH₂OH, R7, R8 each 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.

Described polymer is respectively：

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

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

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

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

Most select structure as follows：

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

1) compound A is obtained with methoxy poly (ethylene glycol) amine and the reaction of hydroxyl glutaric acid；

2) double (to carboxyphenoxy) hexane reactions of compound A and 1,6- of acetylation obtain polymer B, wherein n=1- 200 integer, preferably 1-100；

3) polymer B obtains final product with anthracycline antibiotic C reactions；

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

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

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

2) methoxy poly (ethylene glycol) amine is mixed in a solvent with the dissolving of hydroxyl glutaric acid, and reaction at room temperature is stayed overnight, and is dried Obtain compound A；

3) double (to the carboxyphenoxy) hexanes of the 1 of acetylation, 6- are mixed with compound A, reacted at 100-200 DEG C, Reaction time 20min to 2h；After the cooling of question response mixture, 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 20 minutes, then homogenizer high-speed stirred 1-10 minutes, rotation volatilization obtained crude product, and rear centrifugation frozen dried 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 is obtained, it is easy to dissolved in water, and its half-life period More many than anthracycline antibiotic extension, such noval chemical compound solves the application limitation of anthracene nucleus medicament.

Disease or illness that end-product prepared by this patent can be treated or prevented, relate generally to retina and choroid disease Disease, it is characterised in that the disease or illness for the treatment of are in eye.Characterized in that, described retinal and choroidal disease is AMD disease, proliferative diabetic retinopathy, proliferative vitreoretinopathy, premature regards Retinopathy, pathological myopia, it is assumed that ocular histoplasmosis's syndrome, branch retinal vein occlusion remaining, retinal centre is quiet Arteries and veins blocks, Branch Retinal Artery obstruction, CRAO etc..

The polymer containing anthracycline antibiotic structure that the present invention is obtained, it is easy to dissolved in water, and its half-life period More many than anthracycline antibiotic extension, such noval chemical compound solves the application limitation of anthracene nucleus medicament.Prepared in this patent End-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.

Brief description of the drawings：

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

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

Embodiment

Specific embodiment is described in further detail to the present invention below, but the present invention not only limits to following examples.

Prepare embodiment as follows：

Embodiment 1

1) mixture backflows of double (to carboxyphenoxy) the hexane 25g of 1,6- in 500ml acetic anhydrides, to form acetylation Double (to the carboxyphenoxy) hexanes of 1,6-；

2) methoxy poly (ethylene glycol) amine 3.5g, hydroxyl glutaric acid 55mg, dicyclohexylcarbodiimide 165mg and pyridine 8mg Mixing, is stirred at room temperature overnight；Then washed, and be 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； Thing to be polymerized is cooled to room temperature chloroform and dissolved, and with petroleum ether and drying；

4) polymer of Doxorubicin and step 3 is put into 20ml dimethyl sulphoxide solutions 48 hours；Then baking is inserted 3 hours in case；In subzero 10-20 degree, ultrasound 15 minutes；Homogenizer high-speed stirred 1 minute, the cholic acid for being then put into 5% is molten 400 turns are stirred 2 hours in liquid；Freezed after being collected by centrifugation, produce end-product.

Embodiment 2

1) mixture backflows of double (to carboxyphenoxy) the hexane 30g of 1,6- in 300ml acetic anhydrides, to form acetylation Double (to the carboxyphenoxy) hexanes of 1,6-；

2) methoxy poly (ethylene glycol) amine 3g, hydroxyl glutaric acid 30mg, dicyclohexylcarbodiimide 130mg and pyridine 5mg are mixed Close, be stirred at room temperature overnight；Then washed, and be 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； Thing to be polymerized is cooled to room temperature chloroform and dissolved, and with petroleum ether and drying；

4) THP and the polymer is put into the solution mixed by 5ml ethanol and 5ml dichloromethane；Then insert 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% 400 turns are stirred 2 hours in cholic acid solution；Freezed after being collected by centrifugation, produce end-product.

Embodiment 3

1) mixture backflows of double (to carboxyphenoxy) the hexane 17g of 1,6- in 500ml acetic anhydrides, to form acetylation Double (to the carboxyphenoxy) hexanes of 1,6-；

2) methoxy poly (ethylene glycol) amine 2g, hydroxyl glutaric acid 28mg, dicyclohexylcarbodiimide 90mg and pyridine 4mg mixing 10ml chloroforms are added, are stirred at room temperature overnight；Then washed, and be 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； Thing to be polymerized is cooled to room temperature chloroform and dissolved, and with petroleum ether and drying；

4) by the polymer of Aclarubicin and step 3 be put into by 10ml dichloromethane and 6ml dimethyl sulfoxide (DMSO)s mix it is molten In liquid；Then insert in baking oven and dry 1 hour；In subzero 20-30 degree, ultrasound 1 minute, then 5 points of homogenizer high-speed stirred Clock, product is put into 2% cholic acid solution 400 turns and stirred 4 hours；Freezed after being collected by centrifugation, produce end-product.

Embodiment 4

1) mixture backflows of double (to carboxyphenoxy) the hexane 30g of 1,6- in 350ml acetic anhydrides, to form acetylation Double (to the carboxyphenoxy) hexanes of 1,6-；

2) methoxy poly (ethylene glycol) amine 4g, hydroxyl glutaric acid 50mg, dicyclohexylcarbodiimide 160mg, 4mg pyridine are mixed Close, add 20ml dichloromethane, be stirred at room temperature overnight；Then washed, and be dried under vacuum with ether, is polymerize Thing；

4) by daunorubicin and step 3) polymer be put into by 10ml dichloromethane and 10mlDMF mixing solution in； Then insert 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 800 turns to stir 2.5 hours；Freezed after being collected by centrifugation, produce end-product.

Effect experiment is as follows：

The embodiment 1-4 samples prepared and double (to the carboxyphenoxy) hexane-glycol copolymers of 1,6- are directly wrapped 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 CNV.

Stability test：

By double (to the carboxyphenoxy) hexanes of the sample and 1,6- of 1 group of preparation of embodiment-glycol copolymer parcel The nanoparticle pharmaceutical group of Doxorubicin, Doxorubicin ordinary preparation takes same amount (in terms of Doxorubicin) to determine absorbance respectively and put Enter in 20 degree of incubators 3 months, then take out and determine 480 nanometers of lower absorbances, as a result visible embodiment 1-4 groups and ordinary preparation Without change before and after the absorbance of the Doxorubicin of group, and the nanoparticle group absorbance wrapped up declines 36%.

Drug release in vitro is tested：

By 1 group of embodiment, the nanometer of the Doxorubicin of 1,6- double (to carboxyphenoxy) hexanes-glycol copolymers parcel Grain medicine group and Doxorubicin ordinary preparation component also known as take equivalent medicine (in terms of Doxorubicin, every group containing 10mg it is how soft Than star), then each group medicine after being soaked with PBS, is shaken, timing as in test tube in shaking table under 37 degrees Celsius After sampling, the content of medicine is determined under 480 nanometers of ultraviolet specrophotometer, and the medicament contg percentage of release is calculated after recording Than, do releasing curve diagram, abscissa be the time (my god), ordinate for release percentage.See Fig. 1, it is seen that embodiment release Medicine is more permanent, makes drug half-life longer.

Solubility test in water：

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

The solubility of table 1 compares

Inhibitory action of the medicine to choroidal neovascularization：

Male rat 35 is taken, 7 groups, i.e. control group, double (to the carboxyphenoxy) hexane-ethylene glycol of 1,6- are randomly divided into common The nanoparticle pharmaceutical group (chemical coupling reaction not occurring, Doxorubicin does not have structure change) of the Doxorubicin of polymers parcel, it is real Apply a 1-4 group, 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 μ g medicines of intraocular injection 10 of each group or the load medicine durative action preparation containing 10 μ g medicines, right in addition to control group According to group to isometric PBS solution.

With laser irradiation in rats eyeball, there is bubble to produce or (ring) mark with light sometimes with hyporrhea after light is solidifying and hit Broken Bruch films, are designated as available point.After laser photocoagulation, each group medicine 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 it is as follows：

The retina of table 2 and choroidal neovascularization Area comparison (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

The result of upper table shows that control group retina and choroid generation blood vessel hyperplasia area are larger, each therapeutic component Not different limits reduce blood vessel hyperplasia area.Experiment to rat ocular vascular proliferation area shows, each embodiment group It can reduce lesion retina and choroidal blood vessel hyperplasia area simultaneously, but effect difference, wherein the reality being coupled Apply a 1-4 group better.

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

Claims

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

Wherein R₁、R₂、R₃、R₄、R₅Each stand alone as H, OH, CH₃、CH₂OH or OCH₃, R₆For H, OH, CH₃、CH₂OH、OCH₃、 COCH₃Or COCH₂OH, R₇、R₈Each stand alone as H, OH, CH₃、OCH₃、COCH₃Or OR₉, the integer between n=1-200；Wherein R₉ For pyridine radicals, furyl, pyrrole radicals, thienyl or pyranose, PEG refers to polyethylene glycol, refers specifically to-C₂H₄(O C₂H₄)_W-, its In_WInteger between=1-500.

2. the copolymer of claim 1, wherein R₁、R₂、R₃、R₄Each stand alone as H, OH or OCH₃, R₅For OH, R₆For H, OH, COCH₃Or COCH₂OH, R₇、R₈Each 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 R₁For H or OCH₃, R₂For H, R₃、R₄And R₅It is OH, R₆For COCH₃Or COCH₂OH, R₇、R₈Each stand alone as OH, CH₃Or OR₉, the integer between n=1-200；Wherein R₉For pyranose.

4. the copolymer of claim 1, it is respectively：

1)R₁For OCH₃, R₂For H, R₃、R₄And R₅It is OH, R₆For COCH₂OH, R₇For CH₃, R₈It is whole between n=1-200 for OH Number；

2)R₁For OCH₃, R₂For H, R₃、R₄And R₅It is OH, R₆For COCH₂OH, R₇For CH₃, R₈For OR₉, R₉For pyranose, n=1- Integer between 200；

3)R₁For OCH₃, R₂For H, R₃、R₄And R₅It is OH, R₆For COCH₃, R₇For CH₃, R₈For OH, the integer between n=1-200；

4)R₁、R₂For H, R₃、R₄And R₅It is OH, R₆For COCH₃, R₇For CH₃, R₈For OH, the integer between n=1-200.

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

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

2) double (to carboxyphenoxy) hexane reactions of compound A and 1,6- of acetylation obtain polymer B, wherein n=1-200's Integer；

3) polymer B obtains final product with anthracycline antibiotic C reactions；

7. the preparation method described in claim 6, wherein anthracycline antibiotic C be selected from adriamycin, Epi-ADM, THP, Daunorubicin, idarubicin, aclacinomycin or carminomycin.

8. the preparation method of claim 6 or 7, wherein step 1) solvent is optionally employed, the solvent is selected from：Benzene, toluene, pyrrole One or more in pyridine, tetrahydrofuran, chloroform, carbon tetrachloride, dichloromethane, dicyclohexylcarbodiimide；Step 3) optionally Using solvent, the solvent is selected from methanol, ethanol, dichloromethane, chloroform, carbon tetrachloride, dimethyl sulfoxide (DMSO), N, N- dimethyl methyls One or more in acid amides.

9. the purposes of any one of claim 1-5 copolymer, purposes treats disease at eye choroid and retina to prepare Medicine.

10. the purposes of claim 9, wherein eye disease refer to AMD.