CN107929279A

CN107929279A - A kind of new flavones derived polymer Nano medication and its application in oncotherapy

Info

Publication number: CN107929279A
Application number: CN201711188421.1A
Authority: CN
Inventors: 姚静; 徐程; 于瑶
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2017-11-22
Filing date: 2017-11-22
Publication date: 2018-04-20

Abstract

The present invention relates to a kind of new flavones derived polymer Nano medication, the Nano medication is covalently attached by linking arm and heparin class polysaccharide macro-molecular by hydrophobicity flavonoid micromolecule and forms amphiphilic graft polymers, and self assembly obtains in an aqueous medium, level-one combination strategy of the Nano medication in oncotherapy is formed；The different hydrophobicity anti-tumor medicine of mechanism of action is loaded by the effect of π pi accumulations and hydrophobic driving effect equimolecular intermolecular forces, it is incorporated into a nanometer system, water solubility, stability and the targeting of hydrophobicity anti-tumor medicine in vivo are effectively improved, realizes its combination strategy of two level in oncotherapy；Loading the nanometer system of different mechanisms anti-tumor medicine can also be fitted to each other according to clinic needs, flexibly select, realize its combination strategy of three-level in oncotherapy, to realize tumor treatment model that a variety for the treatment of means develop simultaneously, maximize, optimize antitumous effect.

Description

A kind of new flavones derived polymer Nano medication and its application in oncotherapy

Technical field

The invention belongs to field of pharmaceutical preparations, be related to a kind of new flavones derived polymer Nano medication structure and its Application in oncotherapy.

Background technology

Multidrug resistance, heterogeneity and its complexity between microenvironment of tumour, which contact etc., to be faced in oncotherapy instantly Tough questions, it is alone a certain kind therapeutic modality may be difficult to achieve preferable antitumous effect.At present clinically, tumour is controlled The three big main means treated are surgery excision, chemotherapy and radiotherapy, and above means have played each in oncotherapy Advantage, but inevitably have the defects of certain at the same time, as being difficult to cut off minimal disease, easily recurrence during surgery excision, put Penetrate treatment cycle is long, easily damages immune function and normal cell, and toxic and side is big etc..In recent years, some new mechanisms, The discovery of novel targets allows more polynary combined therapy strategy to be possibly realized.The treatment means or anti-tumor medicine of different mechanisms " multi-pronged ", can effectively make up the limitation of monotherapy, improve tumor-killing effect, reduce drug resistance of tumor and recur several Rate.

Antiangiogenesis therapy is referred to as the available strategy of " the 4th kind " oncotherapy.Its pathophysiological basis is：When Growth of tumour cell is more than 1-3mm³When, blood vessel is supported if lacked, tumour cell will stop due to a lack of sufficient nutriment Only growth is even gradually dead.Promote blood vessel to obtain more nutriments, tumour cell meeting secretion of VEGF, bFGF etc. from body The factor is generated, inducing endothelial cell is migrated to tumor locus, forms capilary, and then support tumour cell.Blood vessel and tumour are thin Mutually promote between born of the same parents, produce positive feedback, this will accelerate tumor cell proliferation, the increase of knurl volume, and may increase tumour blood The probability of transfer.And antiangiogenesis therapy can effectively block tumour blood supply, and then suppress tumour rapid progress.Heparin class polysaccharide It is a kind of hydrophilic glycosaminoglycan macromolecular, there is good water solubility, biocompatibility and biodegradability.It can be combined The angiogenic factors such as VEGF, bFGF in microenvironment, block its acceptor corresponding on vascular endothelial cell to combine, in suppression Chrotoplast is to tumour chemotactic and into pipe；Meanwhile heparin class polysaccharide can also be combined with the heparin-binding site of tumor cell surface, and Enter tumour cell under the mediation in the site, lower and promote Angiogenesis correlation mRNA, suppressing tumour cell in transcriptional level closes Into with release angiogenic factors.Structurally, abundant carboxyl and hydroxyl makes it easier to chemical modification, to prepare nanometer Particle and medicine-carried system create good condition.

Flavonoid micromolecule compound is the natural products of nature rich reserves, its antitumor mechanism is various, such as fine grinding Silibin has antioxidation, suppresses the effect such as related enzyme activity, inducing cell cycle arrest；Chrysin can promote to press down cancer base Because expressing, intervening tumour cell signal transduction, reverse multidrug drug resistance, sensitive radiotherapy etc.；The antitumor activity of Quercetin presses down with it Activity, reduction tumor cell drug resistance, inducing apoptosis of tumour cell and the estrogen-like action of relevant enzyme processed etc. are related.But flavones Class compound is when being used alone, it will usually occur that hydrophobicity is strong, the shortcoming such as poor, dosage height of stability in body fluid, Toxic side effect even is brought when high dose is administered, it is weakened therewith as the preferable advantage of natural products security, is limited Its application clinically.

In traditional chemotherapy, cell toxicity medicament occupies significant proportion, these medicines are mainly swollen by damaging and being broken Oncocyte DNA skeleton structures, change DNA base methylate degree of modification, interference the mode such as DNA replication dna and transcription, direct killing Tumour cell suppresses tumor cell proliferation.Due to selectivity it is low, whole body major organs toxicity, immunotoxicity, genotoxicity compared with To be notable.Adriamycin such as one of anthracycline antibiotic can be inserted into DNA hydrophobic regions by hydrophobic driving effect, disturb DNA replication dna With transcription relevant enzyme system identification, thus play the role of suppress tumor proliferation, but during Clinical practice need closely monitoring Its extensive cardiac toxic and Toxicity of Kidney；The and for example Irinotecan of vinca, can be by suppressing topoisomerase I (TOPO- I), the DNA replication dna of injuring tumor cell, while HIF-1 α, VEGF and its mRNA expression can also be suppressed during daily low dosage administration Level, so produce angiogenesis suppression action, but Irinotecan may cause the hematotoxicities such as Neutrophilic granulocytopenia and Harmful effect is produced to cholinergic nerve function.

The high expression Cycloxygenase of tumour cell, thus high-level prostaglandin is produced in tumour cell and in mesenchyma stroma of tumors The class factor, the latter can stimulate vascular endothelial cell proliferation migration, suppress immunocyte to the killing activity of tumour and to micro-loop Macrophage, fibroblast produce Phenotypic Change effect in border.Non-steroidal anti-inflammatory drugs disturbs medicine as a major class lipid-metabolism Thing, can be by suppressing Cycloxygenase, and then it is the prostanoid factor to block arachidonic acid metabolic, plays antipyretic, analgesia, resists Inflammation effect, and this step is also their ability to one of important target spot of anti-tumor blood vessel.Research points out it by suppressing epoxidation Enzyme, and then block downstream mitogen-activated protein kinase approach (MAPK approach) and extracellular signal-regulated kinase (ERK approach) The tumor invasion of mediation, as celecoxib can reduce the risk that benign intestinal polyp is converted into pernicious intestinal cancer, its mechanism may be upper APC gene (APC) this tumor suppressor gene is adjusted, while suppresses proto-oncogene myc, and then suppresses cancerous lesion；Separately Outside, evidence suggests long-term low dose can effectively reduce using aspirin the probability of tumor in digestive tract, thus it can also make For a kind of tumor prevention medicine.However, such medicine is mostly aromatic acid structure, poorly water-soluble, limits it in Formulations for systemic administration Application；During such drug oral, due to playing suppression work to being distributed in intestines and stomach, protective effect cyclo-oxygenase With different degrees of gastrointestinal lesions will be produced；Which part medicine also has potential Cardiovascular Toxicity etc..

Photodynamic therapy is one kind based on being interacted between the oxygen three in exciting light, photosensitizer and microenvironment New disease treatment means, are the hot spots of therapeutic field of tumor in recent years.The radiation source of sensitiser absorption specific wavelength and by Excitation, the rear oxygen again passed to the luminous energy of absorption rapidly in microenvironment, make its be provoked into active oxygen (including superoxide ion, Hydrogen peroxide, singlet oxygen etc.), these active oxygens will destroy the integrality of DNA of tumor cell and cell membrane, produce potent killing Effect.In addition, photodynamic therapy can also kill tumor-microvessel, Chemotherapy medicine and activation body's immunity etc..By It is a kind of local therapeutic approaches in photodynamic therapy, i.e., target tissue, target cell, whole body is more specifically acted in light area Toxic side effect is small.But it is limited in that：Most of photosensitizer hydrophobicitys are very strong, easily quickly removed in vivo, and exist Reunite, wild effect, cause Formulations for systemic administration difficult；If using organic solvent solubilising or hydrotropy photosensitizer, solvent is considered again Toxicity；Photosensitizer is easily combined after entering in vivo with plasma protein, and high-bonding-ratio is allowed to most of and is retained in blood circulation, And only fraction is likely to enter tumor tissues；There is researcher to attempt to do photosensitizer molecule Hydrophilic modification, such as in aromatic ring The hydrophilic radicals such as outside modification glucose, lactose, although water solubility increased, but still cannot solve small molecule photosensitizer Whole body Dispersed precipitate, selectivity is low the defects of so that during using some photosensitizers, patient need to avoid direct sunlight without It can go out on daytime, bring serious body and mind to damage to patient.

For problem present in above-mentioned oncotherapy, present invention uses a kind of brand-new synthesis mode, is dredged to natural Water-based flavonoid micromolecule carries out aliphatic amines derivatization treatment, can be covalent by the carboxyl of heparin class large hydrophilic molecular Connection, obtains new heparin-flavonoids derived polymer, and then prepare new flavones derived polymer Nano medication.Its advantage It is：(1) the problem of phenolic hydroxyl group is reactive in flavones small molecule is effectively improved, solves flavonoid micromolecule well in body The problem of interior water solubility is low and stability is poor.(2) after the saccharoidal carboxyl of heparin is occupied by Hydrophobic small molecules, liver can effectively be reduced The saccharoidal anticoagulation of element, strengthens security during intravenously administrable.(3) the new heparin-flavonoids derived polymer is in work( Can on have flavone compound and the saccharoidal treatment advantage of heparin concurrently, that is, promote tumor death effect and blood vessel formation against function, Its own can realize that the level-one in oncotherapy is combined strategy with this separately as a kind of antitumor, anti-angiogenic medicaments. (4) polymer is amphiphilic macromolecular structure in structure, and a nanometer ruler can be self-assembly of in the aqueous environments such as body fluid The drug delivery system of degree, i.e., new flavones derived polymer Nano medication.(5) Nano medication by pi-pi accumulation effect and Acted between hydrophobic driving effect equimolecular, strongly load the hydrophobicity cancer therapeutics containing aromatic structure of other different mechanisms Thing, such as cell toxicity medicament, oncolipid metabolism chaff interferent and optical dynamic therapy medicine, by nanometer formulation targeting, efficiently Property etc. advantage, effectively improve the limitations brought such as the non-selection distribution of small molecule anti-tumor medicine poorly water-soluble, whole body, Reduce general toxicity and undershooting-effect；The anti-tumor medicine of different mechanisms is included in flavones derived polymer Nano medication, Realize that the two level in oncotherapy is combined strategy with this.(6) the new flavones derivative for loading different mechanisms anti-tumor medicine is poly- Compound Nano medication can also be needed to be combined with each other according to clinical treatment, flexibly selected, direct to tumour in conventional cell cytotoxic drug While killing, by suppressing tumor neogenetic blood vessels, or the pro-inflammatory cytokine of Lipid sources is reduced, and produce and kill using photosensitizer Wound property active oxygen, the unique advantage of a variety of therapeutic modalities is joined together by the nanometer system of two and the above, is realized Three-level combination strategy in oncotherapy.(7) the new flavones derived polymer Nano medication that the present invention obtains can be with other medicines Agent is subjected to supplementary product compatibility, is prepared into the formulation of the multipath administration such as injection, oral, has a good application prospect.

The content of the invention

An object of the present invention is to provide a kind of brand-new large hydrophilic molecular for hydrophobicity flavonoid micromolecule Method of modifying.Flavonoid micromolecule compound is using phenolic hydroxyl group and aromatic conjugated ring as main feature.The use of one end is bromine atoms Fatty amine linking arm does derivatization treatment to phenolic hydroxyl group, in combination with amido protecting and deprotection strategy, substantially increases reaction Selectivity and yield.The amino of high activity and the saccharoidal carboxyl covalent bond of heparin are obtained through heparin class polyose modification Hydrophobicity flavone derivative, i.e., new heparin-flavones derived polymer, its can self assembly in an aqueous medium, obtain new Type flavones derived polymer Nano medication.

Another object of the present invention is based on above-mentioned new flavones derived polymer Nano medication, there is provided Yi Taoquan The construction strategy of new anti-tumor medicine combination system.Multidrug resistance, heterogeneity and its answering between microenvironment of tumour Miscellaneous contact etc. is the Tough questions faced in current oncotherapy, and alone a certain kind therapeutic modality may be difficult to achieve preferable anti- Tumor effect.In the present invention, new flavones derived polymer Nano medication itself has anti-angiogenesis concurrently and promotees tumour cell Apoptotic effect, heparin class polysaccharide and flavonoid micromolecule become a system, it can be achieved that level-one is combined strategy by being covalently attached. In addition, the cytotoxic drug energy direct killing tumour cell or suppression tumour cell of interference DNA of tumor cell transmission and expression Propagation, it is thin that the lipid-metabolism interference medicament by reducing oncolipid correlation pro-inflammatory cytokine can reduce angiogenesis, suppression interstitial Born of the same parents' Phenotypic Change and then improvement microenvironment, the optical dynamic therapy medicine by producing active singlet oxygen causes tumour cell can not Inverse damage, new flavones derived polymer Nano medication nanometer system is included by the anti-tumor medicine of above-mentioned different mechanisms, can Realize two level combination strategy.In addition, the new flavones derived polymer Nano medication of different anti-tumor medicines is loaded, can also root Flexible combination is needed according to clinical treatment, is together administered, to realize that its three-level in oncotherapy is combined strategy.

A further object of the invention is based on above-mentioned new flavones derived polymer Nano medication, there is provided Yi Zhongzhen To the mode of the Synergy and attenuation of the hydrophobicity anti-tumor medicine containing aromatic conjugated structure.Hydrophobicity anti-tumor medicine is swollen Common problem present in knurl treatment is including stability is poor, is easy to settle aggregation, half-life short, whole body distribution, poor selectivity, poison Side effect is big etc..In the present invention, new flavones derived polymer Nano medication leads to using the hydrophobic conjugated backbone of flavones as kernel Cross and pi-pi accumulation effect, hydrophobic driving effect decile are produced between the hydrophobicity anti-tumor medicine containing aromatic conjugated structure Interact between son, realize the strength loading to these anti-tumor medicines, provided for these hydrophobicity anti-tumor medicines Its suitable scattered hydrophobic space, and then play effective solubilization；The hydrophilic outer shell that heparin class polysaccharide is formed can be protected effectively Inner hydrophobic anti-tumor medicine is protected in blood not by tachymetabolism, extends the half-life period of these medicines, and then played The stabilization of effect；Nano medication is a kind of drug delivery system of nanoscale, can pass through EPR effects and the length of hydrophilic outer shell Ringing, increases the targeting of tumor locus, medicine is enriched with tumor locus, strengthens therapeutic effect of the medicine to tumour, Whole body Dispersed precipitate is reduced at the same time, and then reduces toxic side effect.

Further object of the present invention is to provide the above-mentioned preparation method based on new flavones derived polymer Nano medication.

Final object of the present invention is to provide above-mentioned new flavones derived polymer Nano medication in oncotherapy Application.

In order to achieve the above object, the present invention provides a kind of new flavones derived polymer Nano medication.From both ends point Not Wei bromine atoms and amino, the different carbon numbers in interval linking arm set out, using necleophilic reaction principle and amido protecting with Deprotection strategy, does amino derivatization processing to the phenolic hydroxyl group of the hydrophobicity flavonoid micromolecule containing conjugated structure, makes flavones Micromolecular is coupled the aliphatic amines of high reaction activity, then passes through with the carboxyl of heparin class polysaccharide skeleton structure chemical even Connection, forms new heparin-flavones derived polymer, it self assembly can obtain new flavones derived polymer and receive in an aqueous medium Rice medicine；By simple physical mixed, cytotoxic drug, tumour cell lipid-metabolism chaff interferent and optical dynamic therapy are loaded This three major types anti-tumor medicine of medicine, forms multimachine and is formed on integral Nano medication；Load different mechanisms anti-tumor medicine Nanometer system each other can also flexible combination, be together administered；Combinations thereof mode can realize that kinds of tumors medicine exists Combination strategy step by step in oncotherapy.New flavones derived polymer Nano medication preparation process is simple, and yield is high, reappearance It is good；Combine closely, these hydrophobic anti-tumor medicines are played aobvious by different kinds of molecules intermolecular forces between medicine and carrier The solubilizing effect of work；And granularity is suitable, the EPR effects of nanometer system can be made full use of to accumulate in tumor tissues, tumour is improved and control Treat the targeting and therapeutic alliance effect of medicine.

New heparin-flavones the derived polymer, wherein hydrophobicity flavonoid micromolecule are selected from Chrysin, radix scutellariae Element, wogonin, Quercetin, glycyrrhizin, daidzein, Puerarin, apiolin, hesperetin；The new heparin-flavones derives Polymer, wherein heparin class polysaccharide include unfraction heparin, low molecular weight heparin and desulfated heparin.

The preparation method of the new flavones derived polymer Nano medication, wherein new heparin-flavones derives polymerization The preparation method of thing is as follows：

(1) the bromide linking arm containing free amine group is dissolved in appropriate organic solvent, adds appropriate amino protecting agent, with After add suitable activator and appropriate triethylamine, control reaction condition is to the reaction was complete, respectively with acid wash liquid, alkaline rinse Wash with saturated nacl aqueous solution or separated by silica gel column chromatography, be spin-dried for organic solvent, obtain thick liquid, be intermediate I；

(2) hydrophobic nature flavonoid micromolecule is dissolved in appropriate organic solvent, adds appropriate activator, in adding in right amount Mesosome I, control reaction condition is to the reaction was complete, and increasing amount water makes Precipitation, and ethyl acetate extraction is dry, is obtained after being spin-dried for Intermediate II；

(3) intermediate II is dissolved in appropriate organic solvent, adds appropriate amino deprotection agent, control reaction condition to reaction Completely, precipitating reagent is added, gained washing of precipitate, after being dried using appropriate drying means, obtain intermediate III；

(4) heparin class polysaccharide is dissolved in appropriate solvent, under inert gas shielding and condition of ice bath, adds appropriate carboxyl Activator；Appropriate intermediate III is added, adds appropriate triethylamine；Reaction condition is controlled to the reaction was complete, uses centrifugation or filter After film removes insoluble matter, precipitated with appropriate precipitating reagent；Precipitation is collected, adds water to redissolve, loads bag filter and is dialysed with single water that steams, dialysis After moisture removed using appropriate drying means, up to new heparin-flavones derived polymer.

In the preparation method, it is 2~6 that the bromide linking arm described in step (1), which includes internal carbon atoms number, Alkylidene bromo-amine and its hydrobromate or hydrochloride；The appropriate organic solvent is dichloromethane, chloroform, acetic acid second Ester；The amino protecting agent is selected from di-tert-butyl dicarbonate, benzyl chloroformate；The activator is selected from N, and N- dimethyl- 4- pyridines amine, 4- pyrollidinopyridines, I-hydroxybenzotriazole；The acid wash liquid is the dilute hydrochloric acid of 0.01~0.5mol/L Or dilute sulfuric acid；Alkaline rinse is saturated solution of sodium bicarbonate.

In the preparation method, the appropriate organic solvent described in step (2) is formamide, N, N- dimethyl formyls One or more of mixed system in amine, n,N-dimethylacetamide, dimethyl sulfoxide (DMSO)；The activator is potassium carbonate, carbon Sour sodium；

In the preparation method, appropriate organic solvent described in step (3) is dichloromethane, chloroform, methanol, Dioxane；The mixed system that the amino deprotection agent is trifluoroacetic acid, one or more of in hydrochloric acid, hydrobromic acid；It is described Precipitating reagent be ether or anhydrous ether；The appropriate drying means is to volatilize or be spin-dried for.

In the preparation method, the heparin class polysaccharide described in step (4) is selected from unfraction heparin, low molecular weight liver Element, desulfated heparin；The appropriate solvent is formamide, n,N-Dimethylformamide, n,N-dimethylacetamide, diformazan One or more of mixed system in base sulfoxide；The carboxyl activator is selected from 1- (3- dimethylamino-propyls) -3- ethyl carbon Diimmonium salt hydrochlorate and HOSu NHS or 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and 1- Hydroxybenzotriazole or dicyclohexylcarbodiimide and HOSu NHS；The appropriate precipitating reagent for ice acetone or Ice ethanol；The method of the collection precipitation filters for centrifugation or decompression；The appropriate drying means includes vacuum drying, spray Mist is dry or is freeze-dried.

The new flavones derived polymer Nano medication, can be separately as good antitumor, anti-angiogenic drugs Thing, since heparin class polysaccharide and flavonoid micromolecule are integrally formed by being covalently attached, realizes the level-one in oncotherapy whereby Federation policies；The Nano medication is alternatively arranged as nano-carrier, by mutual between pi-pi accumulation effect and hydrophobic driving effect equimolecular Effect physics contains the hydrophobicity anti-tumor medicine containing conjugated structure, and then by these antitumor mechanisms medicine with their own characteristics Thing includes a system jointly, and two level combination strategy is formed with this.The hydrophobicity cancer therapeutics containing conjugated structure Any hydrophobic drug in thing, including cytotoxic drug, tumour cell lipid-metabolism chaff interferent, optical dynamic therapy medicine.

The preparation method of the new flavones derived polymer Nano medication based on two level combination strategy is as follows：

Technique I：New heparin-flavones derived polymer is dissolved in suitable quantity of water；By a certain proportion of containing aromatic conjugated The hydrophobicity anti-tumor medicine of structure is dissolved with suitable organic solvents, is slowly dropped to new heparin-flavones derived polymer In solution.After stirring, ultrasound, small molecule and organic solvent are removed using dialysis or ultrafiltration, it is rear using vacuum drying, spraying Dry or freeze-drying removes moisture, can obtain the polymer nanocomposite pharmaceutical composition of uniform particle diameter.Obtained polymer nanocomposite Pharmaceutical composition can be applied directly, can also dry and solid pharmaceutical preparation is made.

Technique II：New heparin-flavones derived polymer is dissolved in suitable quantity of water；By a certain proportion of containing aromatic conjugated The hydrophobicity anti-tumor medicine of structure is dissolved with suitable organic solvents.Both are mixed, room temperature lower open mouth is stirred overnight or makes Organic solvent is removed with Rotary Evaporators, small molecule is removed using centrifugal process or post separation method, crosses 0.8 μm of filter membrane, it is rear using true Sky is dry, spray drying or freeze-drying remove moisture, can obtain the polymer nanocomposite pharmaceutical composition of uniform particle diameter.Obtain Polymer nanocomposite pharmaceutical composition can be applied directly, can also dry and solid pharmaceutical preparation is made.

Concrete scheme is as follows：

Hydrophily heparin class polysaccharide is introduced in the hydrophobic nature flavonoid micromolecule containing conjugated structure, is obtained new Heparin-flavones derived polymer, and then self assembly obtains new flavones derived polymer Nano medication in an aqueous medium, effectively Improve dissolubility and stability of the hydrophobicity flavonoid micromolecule in aqueous environments；Its hydrophilic outer shell is helped avoid by net Shape endothelial system swallows, and extends its circulation time in blood；Its nano-scale can make full use of EPR effects, coordinate length to follow Ring acts on the enrichment for realizing tumor locus, realizes that the level-one in oncotherapy is combined strategy with this；Slightly solubility cancer therapeutics Thing in the nanometer system, can be increased and tumor section is selectively delivered by physically encapsulation by nanometer system advantage, reduce Whole body toxic side effect, realizes that the two level in oncotherapy is combined strategy with this；Load receiving for different mechanisms anti-tumor medicine Rice medicine can each other continue to combine, three in oncotherapy realized with this according to the needs of clinical treatment, flexible selection percentage Level combination strategy.The above-mentioned equal size tunable of nanometer system, good evenness, drugloading rate is high, good quality after redissolution；It can also add corresponding Pharmaceutic adjuvant, be prepared into the corresponding preparation for being used to inject or be administered orally.

The preparation of the new flavones derived polymer Nano medication and its realization that strategy is combined in oncotherapy, Its specific embodiment is as follows：

First, the preparation of new heparin-flavones derived polymer

(1) the bromide linking arm containing free amine group is dissolved in appropriate organic solvent, adds appropriate amino protecting agent, with After add suitable activator and appropriate triethylamine, control reaction condition is to the reaction was complete, respectively with acid wash liquid, alkaline rinse Wash with saturated nacl aqueous solution or separated by silica gel column chromatography, be spin-dried for organic solvent, obtain thick liquid, be intermediate I。

Synthetic route is as follows：

(Boc-R₁：Di-tert-butyl dicarbonate, benzyl chloroformate；Br-R₂-NH₂：Internal carbon atoms number is the alkylene of 2~6 Bromide amine and its hydrobromate or hydrochloride；Activator：N, N- dimethyl -4- pyridines amine, 4- pyrollidinopyridines, 1- hydroxy benzenes And triazole)

(2) hydrophobic nature flavonoid micromolecule is dissolved in appropriate organic solvent, adds appropriate activator, in adding in right amount Mesosome I, control reaction condition is to the reaction was complete, and increasing amount water makes Precipitation, and ethyl acetate extraction is dry, is obtained after being spin-dried for Intermediate II.

Synthetic route is as follows：

(R₃-OH：Chrysin, baicalein, wogonin, Quercetin, glycyrrhizin, daidzein, Puerarin, apiolin, dried orange peel Element；Activator：Potassium carbonate, sodium carbonate)

(3) intermediate II is dissolved in appropriate organic solvent, adds appropriate amino deprotection agent, control reaction condition to reaction Completely, precipitating reagent is added, gained washing of precipitate, after being dried using appropriate drying means, obtain intermediate III.

Synthetic route is as follows：

(amino deprotection agent：One or more of mixed system in trifluoroacetic acid, hydrochloric acid, hydrobromic acid)

Synthetic route is as follows：

(R₄-COOH：Unfraction heparin, low molecular weight heparin, desulfated heparin；Carboxyl activator：1- (3- diformazan ammonia Base propyl group) -3- ethyl-carbodiimide hydrochlorides and HOSu NHS or 1- (3- dimethylamino-propyls) -3- ethyl carbon Diimmonium salt hydrochlorate and I-hydroxybenzotriazole or dicyclohexylcarbodiimide and HOSu NHS)

Bromide linking arm described in above-mentioned steps (1) include the alkylidene bromo-amine that internal carbon atoms number is 2~6 and Its hydrobromate or hydrochloride；The appropriate organic solvent is dichloromethane, chloroform, ethyl acetate；The amino Protective agent is selected from di-tert-butyl dicarbonate, benzyl chloroformate；The appropriate amino protecting agent refers to itself and bromide linking arm Molar ratio be 1~5: 1；The activator is selected from N, N- dimethyl -4- pyridines amine, 4- pyrollidinopyridines, 1- hydroxy benzenes And triazole；The appropriate activator refers to that the molar ratio of itself and bromide linking arm is 1~5: 5；The appropriate triethylamine The molar ratio for referring to itself and bromide linking arm is 1~5: 1；The acid wash liquid be 0.01~0.5mol/L dilute hydrochloric acid or Dilute sulfuric acid；Alkaline rinse is saturated solution of sodium bicarbonate；The reaction condition is 10~30 DEG C.

Appropriate organic solvent described in above-mentioned steps (2) is formamide, n,N-Dimethylformamide, N, N- dimethyl second One or more of mixed system in acid amides, dimethyl sulfoxide (DMSO)；The activator is potassium carbonate, sodium carbonate；Described is appropriate Activator refers to that the molar ratio of itself and hydrophobicity flavonoid micromolecule is 1~5: 1；The appropriate intermediate compound I refers to it with dredging The molar ratio of water-based flavonoid micromolecule is 1~5: 1；The reaction condition is 35~100 DEG C.

Appropriate organic solvent described in above-mentioned steps (3) is dichloromethane, chloroform, methanol, dioxane；It is described Amino deprotection agent be trifluoroacetic acid, hydrochloric acid, mixed systems one or more of in hydrobromic acid；The appropriate amino remove-insurance Shield agent refers to that the molar ratio of itself and intermediate II is 5~50: 1；The reaction condition is 10~30 DEG C；The precipitating reagent is Ether or anhydrous ether；The appropriate drying means is to volatilize or be spin-dried for.

Heparin class polysaccharide described in above-mentioned steps (4) is selected from unfraction heparin, low molecular weight heparin, desulfation liver Element；The appropriate solvent is formamide, a kind of in n,N-Dimethylformamide, n,N-dimethylacetamide, dimethyl sulfoxide (DMSO) Or several mixed system；The carboxyl activator is selected from 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides With HOSu NHS or 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and I-hydroxybenzotriazole, Or dicyclohexylcarbodiimide and HOSu NHS；The appropriate carboxyl activator refer to its with heparin class polysaccharide The molar ratio of carboxyl is 1~10: 1；The appropriate intermediate III refers to that the molar ratio of itself and carboxyl in heparin class polysaccharide is 1 ~10: 1；The appropriate triethylamine refers to that the molar ratio of itself and intermediate III is 2~20: 1；The reaction condition is ice Carboxyl activator is first added dropwise under the conditions of bath into heparin class polysaccharide, after keeping 0.5~4h of ice bath, adds intermediate III and three Ethamine, continues 6~72h of room temperature reaction；The appropriate precipitating reagent is ice acetone or ice ethanol；The method of the collection precipitation To centrifuge or depressurizing suction filtration；The appropriate drying means includes vacuum drying, spray drying or freeze-drying.

2nd, the preparation of the new flavones derived polymer Nano medication based on two level combination strategy

Technique I：New heparin-flavones derived polymer and water are mixed by weight the ratio of (mg/mg) 2~50: 1000 Solution is made in conjunction；A certain proportion of hydrophobicity anti-tumor medicine containing aromatic conjugated structure is molten with appropriate organic solvent Solution；Anti-tumor medicine solution is slowly dropped in new heparin-flavones derived polymer solution, 0.5~2h is stirred at room temperature Afterwards, 10~30min of Probe Ultrasonic Searching under ice bath；Small molecule and organic solvent are removed using dialysis or ultrafiltration, afterwards using vacuum Dry, spray drying or freeze-drying remove moisture, to obtain the final product；

Technique II：New heparin-flavones derived polymer and water are mixed by weight the ratio of (mg/mg) 2~50: 1000 Solution is made in conjunction；A certain proportion of hydrophobicity anti-tumor medicine containing aromatic conjugated structure is molten with appropriate organic solvent Solution；Both are mixed, 10~30min of Probe Ultrasonic Searching under ice bath, room temperature lower open mouth is stirred overnight or is removed using Rotary Evaporators Organic solvent, removes small molecule using centrifugal process or post separation method afterwards, crosses 0.8 μm of filter membrane, rear using vacuum drying, spray drying Or freeze-drying removes moisture, to obtain the final product.

Certain proportion described in technique I and technique II refers to that anti-tumor medicine derives with new heparin-flavones and polymerize The mass ratio (mg/mg) of thing is 1: 2~5；Organic solvent described in technique I is selected from n,N-Dimethylformamide, N, N- diformazans Yl acetamide, dimethyl sulfoxide (DMSO), ethanol, methanol, dichloromethane, pyridine, polyvinylpyrrolidone；It is appropriate described in technique I Organic solvent refers to that the volume ratio of itself and water is 1: 2~10；Organic solvent described in technique II be selected from dichloromethane, ether, Anhydrous ether；Appropriate organic solvent described in technique II refers to that the volume ratio of itself and water is 1: 2~10.

The hydrophobicity anti-tumor medicine containing aromatic conjugated structure includes：Cytotoxic drug, including Ah mould Element, Epi-ADM, mitoxantrone, Dacarbazine, harringtonine, homoharringtonine, umbelliferone, psoralen, Osthole, podophyllotoxin, camptothecine, hydroxycamptothecin, Etoposide, topotecan, Irinotecan and its active metabolite SN-38, taxol, docetaxel；Oncolipid is metabolized interference medicament, including aspirin, Diclofenac, Indomethacin, naphthalene General life, brufen, Meloxicam, celecoxib, rofecoxib；Photodynamic therapy medicines, including phthalocyanine, Phthalocyanine Zinc, CuPc, Nickel Phthalocyanine, silicon phthalocyanine, the derivative of phthalocyanine gallium and these phthalocyanine compounds.

The present invention is had the following advantages relative to the prior art and effect：

(1) present invention solves containing conjugated structure, hydrophobic nature flavonoid micromolecule phenolic hydroxyl group reactivity first The problem of low.The electron cloud of phenolic hydroxyl group oxygen atom is subject to the big π delocalizations of aromatic rings, reactivity ratio's aliphatic hydroxyl or amino Low, therefore, using the fatty amine linking arm attack phenolic hydroxyl group of one end bromine atoms, the amino for realizing phenolic hydroxyl group by necleophilic reaction spreads out Biochemistry, substantially increases the reactivity of flavonoid micromolecule, has expanded its chemical reaction scope that can be participated in.Additionally, it is contemplated that To the reactivity of amino and bromine atoms, the protection of amino is also introduced in synthesis and is deprotected strategy, simple and easy to do, purifying side Just, reaction selectivity and yield are higher.

(2) in the present invention, hydrophobicity flavonoid micromolecule utilizes the aliphatic amines derived and the saccharoidal carboxylic of heparin Base covalent bond, solving the problems, such as flavonoid micromolecule well, water-soluble low and stability is poor in vivo.Small point of flavonoids Son tends to coalescence sedimentation in aqueous environments, causes clinically intravascular administration difficulty.And it is heparin modified after flavones molecule It can also be subject to hydrophobic effect to drive in aqueous environments, produce aggregation tendency, but due to being connected with water-soluble macromolecule, the latter's structure Flavonoid micromolecule is wrapped in inside into hydrophilic outer layer, suppresses its coalescence and sedimentation, substantially increases flavonoid micromolecule Water-soluble and stability, plays a role easy to it in vascular drug delivery.

(3) in the present invention, it is connected using the amino that flavonoid micromolecule derives with the saccharoidal carboxyl of heparin, is improved Security of the heparin class polysaccharide in Formulations for systemic administration.The saccharoidal carboxyl of heparin is deemed likely to cause prolonged bleeding time, and The flavones of amino derivatization processing occupies the saccharoidal free carboxy of heparin by chemical reaction, and it is potential to effectively reduce heparin Anti-freezing, the side effect of bleeding, substantially increase security of the heparin polysaccharides in oncotherapy.

(4) the obtained new flavones derived polymer Nano medication of present invention appearance clarification, stable homogeneous in aqueous. Hydrophily shell of the heparin class polysaccharide as Nano medication, is fully extended, produces powerful steric hindrance, can effectively drop in water The low probability removed by netted phagocytosis system, realizes stability higher in blood circulation.Meanwhile the flavones of nano-scale derives Polymer nanocomposite medicine can make full use of the targeting that EPR effects are brought, and realize that tumor locus high concentration is enriched with, and play anti-swollen Knurl, anti-angiogenic effect, improve curative effect, reduce whole body toxic side effect.

(5) the new flavones derived polymer Nano medication itself that the present invention obtains can be used as anti-tumor medicine.Liver Plain class polysaccharide has a blood vessel formation against function, and flavonoid micromolecule has concurrently and promotees apoptosis of tumor cells and Antineoplastic angiogenesis Effect, covalent coupling make both be integrated, and are delivered to tumor locus jointly, realize collaboration oncotherapy effect, are formed with this Level-one combination strategy in oncotherapy.

(6) the problem of dissolubility of the invention for existing anti-tumor medicine generally existing is poor, draws into nanometer system Hydrophobic nature flavonoid micromolecule this structure is entered.The skeleton structure of flavonoid micromolecule not only has very strong hydrophobic Property, and aromatic ring structure therein has prominent conjugate character, thus pi-pi accumulation effect and hydrophobic interaction etc. can be passed through Intermolecular force, realizes the strength assembling to the hydrophobicity anti-tumor medicine containing aromatic conjugated structure, it is swollen to improve these The dissolubility and stability of knurl medicine in vivo, fully demonstrate the pervasive of the new flavones derived polymer Nano medication Property, this is also the basis of heretofore described two level combination strategy.

(7) the new flavones derived polymer Nano medication obtained in the present invention can effectively improve the target of anti-tumor medicine Tropism.For hydrophobicity anti-tumor medicine, powerful load medicine function makes it be efficiently coated on inside Nano medication, Physical stability and chemical stability in blood circulation are effectively ensured；Plasma protein, which combines, to be reduced, Half-life in vivo Extend, Internal pharmacokinetics behavior is obviously improved；Nanometer system give full play to nanometer delivering target, accurate advantage, improve cell The distribution of malicious class medicine, oncolipid metabolism interference medicament and optical dynamic therapy medicine in tumour and its microenvironment, further Realize while dosage is reduced, reach even preferably therapeutic effect identical with the anti-tumor medicine to dissociate.

(8) the new chromocor derivative polymer nanocomposite medicine obtained in the present invention can effectively reduce anti-tumor medicine Toxic side effect.Since medicine is wrapped in inside, its internal behavior depends on nanometer system, and long circulating action and EPR effects Nano medication is set to be significantly reduced in the distribution of non-target organ.The major organs toxicity of cytotoxic drug for example alopecia, cardiac toxic, Toxicity of Kidney, hematotoxicity, bone marrow toxicity etc. effectively reduce；Due to being loaded by nanometer system, oncolipid metabolism chaff interferent Whole body Dispersed precipitate effect mitigate, thus the lipid-metabolism of normal tissue is noiseless, reduces injury of gastrointestinal tract, angiocarpy The incidence of the adverse reactions such as risk；For optical dynamic therapy, hydrophobic photosensitizer obtained in nanometer system solubilising, Stablize, reduce its own tendency for being easy to assemble, settle, while the distribution to normal structure significantly reduces, and reduces body surface The phototoxicity of normal structure.

(9) the new flavones derived polymer Nano medication in the present invention can realize the antineoplastic Internet of Things of higher level With strategy.The flavones derived polymer Nano medication after medicine is carried except possessing the saccharoidal anti-angiogenic effect of heparin and flavonoids Outside antitumor, the anti-angiogenic effect of small molecule, cell toxicity medicament, tumour cell lipid-metabolism interference medicament or light are also combined and have moved Power medicine, the two level formed with this in oncotherapy are combined strategy；Load different anti-tumor medicines nanometer system it Between can also be needed according to clinic be combined with each other, flexibly select, when needing to receive kinds of tumors medicine such as patient, can at the same time group Close two and the above loads the nanometer system of different anti-tumor medicines, stablize them and coexist in same water-based system, at the same time Administration, avoids ordinary combination treatment from separating multiple injection problem, at the same can also mitigate patient because in the short time multiple dosing bring Pain, this is more extensive, higher levels of three-level combination strategy.

(10) the new flavones derived polymer Nano medication that the present invention obtains, has good biocompatibility and biology Degradability.

(11) the obtained new flavones derived polymer Nano medication of the present invention can be subjected to auxiliary material with other pharmacies and match somebody with somebody 5, the formulation of the multipath administration such as injection, oral is prepared into, is had a good application prospect.

Embodiment

The present invention is further illustrated with below by embodiment, but following embodiments are not intended to limit the right of this patent Scope.

Embodiment 1：The preparation of baicalein-desulfated heparin derived polymer

Weigh 2- bromine ethylamine hydrobromides to be placed in eggplant-shape bottle, add dichloromethane and make solvent, then add two carbonic acid two The tert-butyl ester.The molar ratio of di-tert-butyl dicarbonate and 2- bromine ethylamine hydrobromides is 1.05: 1.Separately weigh N, N- dimethyl -4- pyrroles Pyridine amine, draw triethylamine in 5mL EP pipes, and adding 2mL dichloromethane makes dissolving, is slowly dropped into above-mentioned eggplant-shape bottle.N, N- diformazan Base -4- pyridines amine and the molar ratio of 2- bromine ethylamine hydrobromides are 2: 5, and triethylamine and the molar ratio of 2- bromine ethylamine hydrobromides are 2∶1.After reacting at room temperature 40min, separatory funnel is transferred to, respectively with 0.1mol/L dilute sulfuric acids, saturated solution of sodium bicarbonate and full Washed respectively three times with sodium chloride solution.After washing, organic phase is transferred to conical flask, adds anhydrous sodium sulfate water suction 2h.Turn Enter sand core funnel suction filtration, filtrate revolving removes dichloromethane, obtains intermediate compound I.

The potassium carbonate for weighing 5 times of baicaleins of a certain amount of baicalein and mole loads eggplant-shape bottle, adds N, N- dimethyl Formamide, 50 DEG C are heated to reflux 60min.Intermediate compound I is dissolved in n,N-Dimethylformamide, is added dropwise above-mentioned eggplant-shape bottle, 50 DEG C It is heated to reflux down that the reaction was continued.The molar ratio of baicalein and intermediate compound I is 1: 1.05.After 5h, adding a large amount of water separates out product. Ethyl acetate repeatedly extracts, and merges organic phase, adds anhydrous sodium sulfate drying 2h, is transferred to sand core funnel suction filtration, is rotated on filtrate Ethyl acetate is removed, obtains intermediate II.

Weigh intermediate II and load eggplant-shape bottle, add dichloromethane and make solvent, in molar ratio trifluoroacetic acid：Intermediate II= Be added dropwise trifluoroacetic acid at 20: 1, and 40min is stirred at room temperature.After reaction, whole system is poured into ether, ice bath 30min Product is promoted to settle out.Centrifugation (4000rpm, 2min) must precipitate, and ether washs three times, and sediment is transferred to round-bottomed flask, revolving Residual ether is removed, obtains intermediate III.

Weigh appropriate desulfated heparin and be dissolved in formamide, the carboxyl of desulfated heparin and the molar ratio of intermediate III For 1: 2.After being heated to 60 DEG C of auxiliary dissolvings, 1- (3- dimethylamino-propyls) -3- ethyl carbodiimide salt is added under condition of ice bath Hydrochlorate and HOSu NHS, 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides, HOSu NHS with The molar ratio of the carboxyl of desulfated heparin is 4: 4: 1.After ice bath activation 2h, the dimethyl sulphoxide solution of intermediate III is added In the desulfated heparin solution for entering activation, the triethylamine of 10 times of intermediate IIIs of mole is added, reacts at room temperature 24h.Reaction After, insoluble matter is centrifuged off, clear liquid is poured into the ice acetone of 10 times of volumes, and gained precipitation is baicalein-desulfation liver Plain derived polymer.Distilled water redissolves, and is placed in bag filter the 48h that dialyses, and by 0.8 μm of miillpore filter, is freeze-dried, to obtain the final product.

Embodiment 2：The preparation of Chrysin-low molecular weight heparin derived polymer

Weigh 3- propantheline bromide hydrobromides to be placed in eggplant-shape bottle, add dichloromethane and make solvent, then add two carbonic acid two The tert-butyl ester.The molar ratio of di-tert-butyl dicarbonate and 3- propantheline bromide hydrobromides is 1.1: 1.Separately weigh N, N- dimethyl -4- pyrroles Pyridine amine, draw triethylamine in 5mL EP pipes, and adding 2mL dichloromethane makes dissolving, is slowly dropped into above-mentioned eggplant-shape bottle.N, N- diformazan Base -4- pyridines amine and the molar ratio of 3- propantheline bromide hydrobromides are 1: 5, and the molar ratio of triethylamine and 3- propantheline bromide hydrobromides is 1.05∶1.React at room temperature 40min after, be transferred to separatory funnel, respectively with 0.2mol/L dilute hydrochloric acid, saturated solution of sodium bicarbonate and Saturated nacl aqueous solution washs three times respectively.After washing, organic phase is transferred to conical flask, adds anhydrous sodium sulfate water suction 2h. Sand core funnel suction filtration is transferred to, filtrate revolving removes dichloromethane, obtains intermediate compound I.

The potassium carbonate for weighing 5 times of Chrysins of a certain amount of Chrysin and mole loads eggplant-shape bottle, adds N, N- dimethyl Formamide, 50 DEG C are heated to reflux 40min.Intermediate compound I is dissolved in n,N-Dimethylformamide, is added dropwise above-mentioned eggplant-shape bottle, 50 DEG C It is heated to reflux down that the reaction was continued.The molar ratio of Chrysin and intermediate compound I is 1: 1.05.After 5h, adding 10 times of amount water analyses product Go out.Extracted using a large amount of ethyl acetate, merge organic phase, added anhydrous sodium sulfate drying 2h, be transferred to sand core funnel suction filtration, filtrate Upper revolving removes ethyl acetate, obtains intermediate II.

Weigh intermediate II and load eggplant-shape bottle, add 2mL dichloromethane, in molar ratio trifluoroacetic acid：Intermediate II=10: 1 is added dropwise trifluoroacetic acid, and 60min is stirred at room temperature.After reaction, whole system is poured into anhydrous ether, ice bath 30min Product is promoted to settle out.Sand core funnel, which filters, to be precipitated, and anhydrous ether washs three times, and sediment is transferred to round-bottomed flask, and revolving is removed Residual anhydrous ether is removed, obtains intermediate III.

Weigh appropriate low molecular weight heparin and be dissolved in formamide, the carboxyl of low molecular weight heparin and the molar ratio of intermediate III For 1: 2.After being heated to 60 DEG C of auxiliary dissolvings, 1- (3- dimethylamino-propyls) -3- ethyl carbodiimide salt is added under condition of ice bath Hydrochlorate and HOSu NHS, 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides, HOSu NHS with The molar ratio of the carboxyl of low molecular weight heparin is 4: 4: 1.After ice bath activation 1h, the dimethyl sulphoxide solution of intermediate III is added In the low molecular weight heparin solution for entering activation, the triethylamine of 5 times of intermediate IIIs of mole is added, reacts at room temperature 24h.Reaction After, insoluble matter is centrifuged off, clear liquid is poured into the ice acetone of 10 times of volumes, and gained precipitation is Chrysin-low molecular weight liver Plain derived polymer.Distilled water redissolves, and is placed in bag filter the 48h that dialyses, and by 0.8 μm of miillpore filter, is freeze-dried, to obtain the final product.

Embodiment 3：The preparation of Quercetin-unfraction heparin derived polymer

Weigh 4- bromine 1- butylamine hydrobromates to be placed in eggplant-shape bottle, add dichloromethane and make solvent, then add chloro-carbonic acid Benzyl ester.The molar ratio of benzyl chloroformate and 4- bromine 1- butylamine hydrobromates is 3: 1.It is another to weigh 4- pyrollidinopyridines, draw three Ethamine is managed in 5mL EP, and adding 2mL dichloromethane makes dissolving, is slowly dropped into above-mentioned eggplant-shape bottle.4- pyrollidinopyridines and 4- The molar ratio of bromine 1- butylamine hydrobromates is 3: 5, and the molar ratio of triethylamine and 4- bromine 1- butylamine hydrobromates is 3: 1.Room temperature is anti- After answering 1h, separatory funnel is transferred to, respectively with 0.1mol/L dilute sulfuric acids, saturated solution of sodium bicarbonate and saturated nacl aqueous solution point Do not wash three times.After washing, organic phase is transferred to conical flask, adds anhydrous sodium sulfate water suction 2h.Sand core funnel suction filtration is transferred to, Filtrate revolving removes dichloromethane, obtains intermediate compound I.

The sodium carbonate for weighing 3 times of Quercetins of a certain amount of Quercetin and mole loads eggplant-shape bottle, adds N, N- dimethyl Acetamide, 50 DEG C are heated to reflux 60min.Intermediate compound I is dissolved in n,N-dimethylacetamide, is added dropwise above-mentioned eggplant-shape bottle, 50 DEG C It is heated to reflux down that the reaction was continued.The molar ratio of Quercetin and intermediate compound I is 1: 2.After 5h, adding a large amount of water separates out product.Second Acetoacetic ester repeatedly extracts, and merges organic phase, adds anhydrous sodium sulfate drying 2h, is transferred to sand core funnel suction filtration, rotates and remove on filtrate Ethyl acetate is removed, obtains intermediate II.

Weigh appropriate unfraction heparin and be dissolved in formamide, the carboxyl of unfraction heparin and the molar ratio of intermediate III are 1: 5.1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and I-hydroxybenzotriazole, 1- (3- are added under condition of ice bath Dimethylamino-propyl) molar ratio of carboxyl of -3- ethyl-carbodiimide hydrochlorides, I-hydroxybenzotriazole and unfraction heparin is 5∶5∶1.After ice bath activation 1h, the dimethyl sulphoxide solution of intermediate III is added in the unfraction heparin solution of activation, added The triethylamine of 10 times of intermediate IIIs of mole, reacts at room temperature 48h.After reaction, insoluble matter is centrifuged off, clear liquid is poured into The ice ethanol of 10 times of volumes, gained precipitation are Quercetin-unfraction heparin derived polymer.After distilled water redissolves, it is placed in Dialyse 48h in analysis bag, by 0.8 μm of miillpore filter, is dried in vacuo, to obtain the final product.

Embodiment 4：The preparation of Quercetin-low molecular weight heparin derived polymer

Weigh 2- bromine ethylamine hydrobromides to be placed in eggplant-shape bottle, add dichloromethane and make solvent, then add two carbonic acid two The tert-butyl ester.The molar ratio of di-tert-butyl dicarbonate and 2- bromine ethylamine hydrobromides is 1.03: 1.Separately weigh N, N- dimethyl -4- pyrroles Pyridine amine, draw triethylamine in 5mL EP pipes, and adding 2mL dichloromethane makes dissolving, is slowly dropped into above-mentioned eggplant-shape bottle.N, N- diformazan Base -4- pyridines amine and the molar ratio of 2- bromine ethylamine hydrobromides are 1: 5, and triethylamine and the molar ratio of 2- bromine ethylamine hydrobromides are 1.1∶1.React at room temperature 40min after, be transferred to separatory funnel, respectively with 0.2mol/L dilute hydrochloric acid, saturated solution of sodium bicarbonate and Saturated nacl aqueous solution washs three times respectively.After washing, organic phase is transferred to conical flask, adds anhydrous sodium sulfate water suction 2h. Sand core funnel suction filtration is transferred to, filtrate revolving removes dichloromethane, obtains intermediate compound I.

The potassium carbonate for weighing 5 times of Quercetins of a certain amount of Quercetin and mole loads eggplant-shape bottle, adds N, N- dimethyl Formamide, 60 DEG C are heated to reflux 60min.Intermediate compound I is dissolved in n,N-Dimethylformamide, is added dropwise above-mentioned eggplant-shape bottle, 60 DEG C It is heated to reflux down that the reaction was continued.The molar ratio of Quercetin and intermediate compound I is 1: 1.5.After 5h, adding a large amount of water separates out product. Ethyl acetate repeatedly extracts, and merges organic phase, adds anhydrous sodium sulfate drying 2h, is transferred to sand core funnel suction filtration, is rotated on filtrate Ethyl acetate is removed, obtains intermediate II.

Weigh intermediate II and load eggplant-shape bottle, add dichloromethane and make solvent, in molar ratio trifluoroacetic acid：Intermediate II= Be added dropwise trifluoroacetic acid at 15: 1, and 60min is stirred at room temperature.After reaction, whole system is poured into anhydrous ether, ice bath 30min promotes product to settle out.Sand core funnel, which filters, to be precipitated, and anhydrous ether washs three times, and sediment is transferred to round-bottomed flask, rotation Residual anhydrous ether is evaporated off, obtains intermediate III.

Weigh appropriate low molecular weight heparin and be dissolved in formamide, the carboxyl of low molecular weight heparin and the molar ratio of intermediate III For 1: 3.After being heated to 60 DEG C of auxiliary dissolvings, 1- (3- dimethylamino-propyls) -3- ethyl carbodiimide salt is added under condition of ice bath Hydrochlorate and HOSu NHS, 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides, HOSu NHS with The molar ratio of the carboxyl of low molecular weight heparin is 4: 4: 1.After ice bath activation 1.5h, by the dimethyl sulphoxide solution of intermediate III Add in the low molecular weight heparin solution of activation, add the triethylamine of 5 times of intermediate IIIs of mole, react at room temperature 24h.Instead After answering, insoluble matter is centrifuged off, clear liquid is poured into the ice acetone of 10 times of volumes, and gained precipitation is Quercetin-low molecular weight Heparin derivative polymer.Distilled water redissolves, and is placed in bag filter the 48h that dialyses, and by 0.8 μm of miillpore filter, is freeze-dried, i.e., .

Embodiment 5：The preparation of baicalein-low molecular weight heparin derived polymer

Weigh 4- bromine 1- butylamine hydrobromates to be placed in eggplant-shape bottle, add dichloromethane and make solvent, then add two carbonic acid Di tert butyl carbonate.The molar ratio of di-tert-butyl dicarbonate and 4- bromine 1- butylamine hydrobromates is 1.03: 1.Separately weigh 4- pyrrolidinyls Pyridine, draw triethylamine in 5mL EP pipes, and adding 2mL dichloromethane makes dissolving, is slowly dropped into above-mentioned eggplant-shape bottle.4- pyrrolidines The molar ratio of yl pyridines and 4- bromine 1- butylamine hydrobromates is 1: 5, and the molar ratio of triethylamine and 4- bromine 1- butylamine hydrobromates is 1.05∶1.After reacting at room temperature 1h, separatory funnel is transferred to, respectively with 0.2mol/L dilute sulfuric acids, saturated solution of sodium bicarbonate and full Washed respectively three times with sodium chloride solution.After washing, organic phase is transferred to conical flask, adds anhydrous sodium sulfate water suction 2h.Turn Enter sand core funnel suction filtration, filtrate revolving removes dichloromethane, obtains intermediate compound I.

The sodium carbonate for weighing 3 times of baicaleins of a certain amount of baicalein and mole loads eggplant-shape bottle, adds dimethyl sulfoxide (DMSO), 50 DEG C are heated to reflux 60min.Intermediate compound I is dissolved in dimethyl sulfoxide (DMSO), and above-mentioned eggplant-shape bottle is added dropwise, and 50 DEG C are heated to reflux lower continuation Reaction.The molar ratio of baicalein and intermediate compound I is 1: 3.After 5h, adding a large amount of water separates out product.Ethyl acetate repeatedly extracts, Merge organic phase, add anhydrous sodium sulfate drying 2h, be transferred to sand core funnel suction filtration, revolving removes ethyl acetate on filtrate, in obtaining Mesosome II.

Weigh intermediate II and load eggplant-shape bottle, add dichloromethane and make solvent, in molar ratio trifluoroacetic acid：Intermediate II= Be added dropwise trifluoroacetic acid at 20: 1, continues that 40min is stirred at room temperature.After reaction, whole system is poured into ether, ice bath 30min promotes product to settle out.Centrifugation (4000rpm, 2min) must precipitate, and ether washs three times, and sediment is transferred to round-bottomed flask, Revolving removes residual ether, obtains intermediate III.

Weigh appropriate low molecular weight heparin and be dissolved in formamide, the carboxyl of low molecular weight heparin and the molar ratio of intermediate III For 1: 4.1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and I-hydroxybenzotriazole are added under condition of ice bath, The carboxyl of 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides, I-hydroxybenzotriazole and low molecular weight heparin Molar ratio is 3: 3: 1.After ice bath activation 1h, the dimethyl sulphoxide solution of intermediate III is added to the low molecular weight heparin of activation In solution, the triethylamine of 5 times of intermediate IIIs of mole is added, reacts at room temperature 24h.After reaction, it is centrifuged off insoluble Thing, clear liquid are poured into the ice acetone of 10 times of volumes, and gained precipitation is baicalein-low molecular weight heparin derived polymer.Distilled water After redissolution, be placed in bag filter the 48h that dialyses, by 0.8 μm of miillpore filter, be dried in vacuo, to obtain the final product.

Embodiment 6：APTT methods detect the anticoagulating active of new heparin-flavones derived polymer

The anticoagulation that new heparin-flavones derived polymer is detected using activated partial thromboplastin time (APTT) method is lived Property.Rabbit auricular vein takes blood, is placed in the sodium citrate anti-freezing liquid (+9 parts of whole bloods of 1 part of anti-freezing liquid) containing 1/10 volume 0.109M In plastic tube, gently overturn and mix, 3000rpm centrifugation 15min, collect upper liquid (blood plasma).By 0.1mL sample solutions (20 μ g/ ML) add in 0.4mL citrated plasmas, add APTT the reagents 0.1mL, 37 DEG C of incubation 5min of 37 DEG C of pre-temperatures.While with Blank plasma is as control.Then, the 0.025mol/L calcium chloride solution 0.1mL of 37 DEG C of pre-temperatures are added, start stopwatch, record blood Setting time is starched, each sample does 3 multiple pipe measure, is averaged.It the results are shown in Table 1.The result shows that small point of hydrophobicity flavonoids After son is connected with heparin class polysaccharide covalent, the anticoagulating active of the latter can be substantially reduced, improves heparin class polysaccharide in vascular drug delivery In security.

The anticoagulating active of the new flavones derived polymer of table 1

Embodiment 7：It is anti-angiogenic with respect to the new heparin of content of hemoglobin (Rhb) evaluation of measuring-flavones derived polymer Generation activity

Matrigel is mixed from angiogenesis factor bFGF and different heparin-flavones derived polymer, it is subcutaneous respectively Male mice oxter is injected in, mouse is put to death after 10 days, isolates matrigel, is homogenized with hypotonic lysis liquid, centrifugation, takes supernatant Drabkin ' s reagents are added, absorbance (A is measured under 540nm_sample), calculate Rhb contents by formula (1).In this formula, The absorbance of negative control group is A_0%, the absorbance of positive controls is A_100%.The Rhb of each heparin-flavones derived polymer group Assay the results are shown in Table 2.The result shows that when being used alone compared to both, hydrophobicity flavonoid micromolecule and heparin class are more Sugar generates stronger anti-angiogenesis activity after being covalently attached.

Table 2 is with respect to content of hemoglobin (Rhb) evaluation of measuring heparin derivatives anti-angiogenesis activity

Embodiment 8：The shelf-stability of new heparin-flavones derived polymer solid state and solution state

A collection of Quercetin-desulfated heparin derived polymer is prepared, the closed placement of room temperature, took suitable respectively in 0 day, 20 days Measure it is soluble in water make into the Nano medication solution that concentration is 1mg/mL, measure particle diameter and polydispersity coefficient (PDI), evaluated newly with this Shelf-stability of the type flavones derived polymer Nano medication in solid state.From table 3 it can be seen that new flavones derives polymerization What thing Nano medication preserved at room temperature has good stability.

It is appropriate to weigh hesperetin-low molecular weight heparin derived polymer, is dissolved in appropriate distilled water, it is 1mg/ to be prepared into concentration The Nano medication solution of mL, room temperature place 48h, respectively in the particle diameter and polydispersity coefficient of 0h, 24h and 48h measure solution (PDI), the shelf-stability of new flavones derived polymer Nano medication solution is evaluated whereby.From table 4, it can be seen that new Huang Ketone derived polymer Nano medication has good stability in the solution.

The shelf-stability of 3 Quercetins of table-desulfated heparin derived polymer Nano medication (solid state)

The shelf-stability of 4 hesperetins of table-low molecular weight heparin derived polymer Nano medication (solution state)

Embodiment 9：Carry the preparation of Chrysin-unfraction heparin derived polymer Nano medication of Phthalocyanine Zinc

Weigh appropriate Phthalocyanine Zinc and be dissolved in appropriate polyvinylpyrrolidone, it is poly- to weigh appropriate Chrysin-unfraction heparin derivative Compound Nano medication is dissolved in distilled water, and after room temperature quickly stirs 30min, the polyvinylpyrrolidonesolution solution of Phthalocyanine Zinc is dripped dropwise Enter.The mass ratio of Phthalocyanine Zinc and Chrysin-unfraction heparin derived polymer Nano medication is 1: 3.Pop one's head in after mixing under ice bath Ultrasonic 30min, is transferred to super filter tube to remove small molecule and organic solvent by mixed system, vacuum drying, to obtain the final product.

Embodiment 10：Carry the preparation of Quercetin-desulfated heparin derived polymer Nano medication of celecoxib

Weigh appropriate celecoxib and be dissolved in dichloromethane, weigh appropriate Quercetin-desulfated heparin and be dissolved in distilled water.Plug The mass ratio for coming former times cloth and Quercetin-desulfated heparin derived polymer Nano medication is 1: 4.By both above-mentioned mixing, room Warm opening is stirred overnight to fling to dichloromethane.Remaining liq centrifuges 10min to remove insoluble small molecule, mistake in 3000rpm 0.8 μm of miillpore filter, freeze-drying, to obtain the final product.

Embodiment 11：Carry the preparation of baicalein-low molecular weight heparin derived polymer Nano medication of Irinotecan

Weigh appropriate Irinotecan and be dissolved in n,N-Dimethylformamide, weigh appropriate baicalein-low molecular weight heparin and derive Polymer nanocomposite medicine is dissolved in distilled water, after room temperature quickly stirs 30min, by the n,N-Dimethylformamide solution of Irinotecan Instill dropwise.The mass ratio of Irinotecan and baicalein-low molecular weight heparin derived polymer Nano medication is 1: 3.Speed is added dropwise Rate is per minute 2~4 drops.After being added dropwise, Probe Ultrasonic Searching 30min under condition of ice bath, is transferred to bag filter, and dialyse 24h, crosses 0.8 μ M miillpore filters, freeze-drying, to obtain the final product.

Embodiment 12：Load Epi-ADM, celecoxib, Chrysin-low molecular weight heparin derived polymer of Phthalocyanine Zinc The shelf-stability of Nano medication (solid state)

Prepare and load Epi-ADM, celecoxib, Chrysin-low molecular weight heparin derived polymer nanometer medicine of Phthalocyanine Zinc Thing, the closed placement of room temperature, being taken respectively in 0,20 day appropriate soluble in water makes into the Nano medication solution that concentration is 1mg/mL, measure The particle diameter and polydispersity coefficient (PDI) of derived polymer Nano medication solution, the flavones derived polymer after carrying medicine is evaluated with this The shelf-stability of Nano medication in the solid state.As can be seen from Table 5, the flavones derived polymer Nano medication after medicine is carried What is preserved at room temperature has good stability.

The shelf-stability of new flavones derived polymer Nano medication (solid state) after the load medicine of table 5

Embodiment 13：The substitution value and drugloading rate of new flavones derived polymer Nano medication

Substitution value detection method：Using UV-VIS spectrophotometry, to hydrophobicity flavonoid micromolecule maximum absorption wave Strong point measures the content of the Hydrophobic small molecules containing conjugated structure, and substitution value is calculated with formula (2).In this formula, m_sFor containing conjugation The content (g) of the Hydrophobic small molecules of structure, is calculated by standard curve；m_tFor the flavones derived polymer Nano medication weighed Quality (g), M_sFor the average molecular weight of the hydrophobicity flavonoid micromolecule containing conjugated structure, M_hepFor unfraction heparin, low point The average molecular weight of son amount heparin or desulfated heparin.It the results are shown in Table 6.

Drugloading rate detection method：Using high performance liquid chromatography, to the hydrophobicity anti-tumor medicine containing conjugated structure Standard curve of the peak area to concentration is drawn in its maximum absorption wave strong point.To the flavones derived polymer Nano medication after load medicine Peak area is measured in above-mentioned maximum absorption wave strong point, calculates concentration according still further to standard curve, and drugloading rate is calculated with formula (3). It the results are shown in Table 6.

The substitution value and drugloading rate of the new flavones of table 6-heparin derivative polymer nanocomposite medicine

Embodiment 14：The solubility of new flavones derived polymer Nano medication

The method for investigating solubility：The new heparin of 1g-flavones derived polymer is weighed respectively and is loaded with cancer therapeutics The new flavones derived polymer Nano medication of thing is dissolved in 1mL water.30s is vibrated every 5min at room temperature, is observed in 30min Whether can be completely dissolved.As shown in Table 7, after hydrophobicity flavonoid micromolecule being covalently attached to heparin class polysaccharide, flavonoids The dissolubility of small molecule in water significantly improves.Meanwhile the hydrophobicity anti-tumor medicine solubility containing aromatic conjugated structure Also greatly improve.

The solubility of the new flavones derived polymer Nano medication of table 7

Note："-" is almost insoluble, and "+" is solvable, and " ++ " is readily soluble.

Embodiment 15：Stability after the new flavones derived polymer Nano medication combination of two kinds of load medicines

Take the new flavones derived polymer Nano medication of two kinds of load medicines appropriate, after mixing plus water makes total concentration be 1mg/ ML, that is, obtain the new flavones derived polymer Nano medication solution based on three-level combination strategy.Room temperature places 48h, and observation is outer Whether sight has visible foreign matters or precipitation, and evaluation whereby carries the new mixed placement of flavones derived polymer Nano medication after medicine Stability.As can be seen that 48h after mixing, still has no visible foreign matters or precipitation, show compatible between each medicine-carried system in table 8 Property is preferable, without obvious incompatibility in terms of medicaments dispensing angle.

Table 8 carries the stability after the new flavones derived polymer Nano medication combination of medicine

Embodiment 16：Mtt assay detection carries inhibitory action of the new flavones derived polymer Nano medication to HepG2 cells

HepG2 cells are taken with 5 × 10³A/hole is inoculated in 96 orifice plates, and 37 DEG C of incubation 24h, suck nutrient solution, add respectively Enter the 200 μ L of new flavones derived polymer Nano medication culture medium solution of the anti-tumor medicine containing various concentrations, 37 DEG C incubate After educating 48h, 40 μ L Methyl thiazoly tetrazolium assays (MTT, 2.5mg/mL) are added, continue to be incubated 4h；Each hole culture medium is sucked, is added 150 μ L dimethyl sulfoxide (DMSO)s, shaking 10min make crystallization fully dissolve.The suction of each Nano medication is measured with microplate reader under 570nm Shading value (experimental group OD values).And blank group OD values and control group OD values, n=6 are measured in the same way.For being moved using light The experimental group of power medicine, adds the new flavones derived polymer nanometer medicine containing optical dynamic therapy medicine of various concentrations After 200 μ L of thing culture medium solution, using 10J/cm²Laser irradiates 10min, remaining is same as above.Subject cell is calculated by formula (4) to deposit Motility rate, and calculate half inhibiting rate IC of the new chromocor derivative Nano medication to HepG2 cells with survival results₅₀, IC₅₀ It the results are shown in Table 9.The result shows that the new flavones derived polymer Nano medication tumour for loading anti-tumor medicine has stronger swell Knurl inhibitory action；When two kinds of new flavones derived polymer Nano medication combinations, tumor inhibition effect is stronger.

Inhibitory activity of the new flavones derived polymer Nano medication of table 9 to HepG2 cells

Claims

1. a kind of new flavones derived polymer Nano medication, it is characterised in that using hydrophobic nature flavonoid micromolecule to set out Point, the amination derivative of flavonoid micromolecule is obtained by chemical synthesis, then carboxyl saccharoidal with heparin is covalently attached, and is obtained To new heparin-flavones derived polymer, and then prepare new flavones derived polymer Nano medication.

2. the new flavones derived polymer Nano medication according to claims 1, it is characterised in that based on the new Huang Ketone derived polymer Nano medication, can realize multi-cascade strategy in oncotherapy；The new heparin-flavones derives poly- The compound new flavones derived polymer Nano medication that self assembly obtains in an aqueous medium, itself can be used as antitumor, anti- Neonate tumour blood vessel medicine, forms a cascade based on the new flavones derived polymer Nano medication, in oncotherapy With strategy；The Nano medication is alternatively arranged as carrier, and by various intermolecular interactions, physical loading contains aromatic conjugated structure Hydrophobicity anti-tumor medicine, the anti-tumor medicine of a variety of mechanism of action is included a nanometer system, base formed with this In the new flavones derived polymer Nano medication, two level in oncotherapy be combined strategy；Load different oncotherapies The nanometer system of medicine can be needed to continue flexible combination by clinic, be formed with this based on the new flavones derived polymer nanometer medicine Thing, three-level in oncotherapy combination strategy.

3. the new flavones derived polymer Nano medication according to claims 1, it is characterised in that described is natural thin Water-based flavonoid micromolecule includes Chrysin, baicalein, wogonin, Quercetin, glycyrrhizin, daidzein, Puerarin, celery Element, hesperetin；The heparin class polysaccharide includes unfraction heparin, low molecular weight heparin and desulfated heparin.

4. the new flavones derived polymer Nano medication according to claims 1, it is characterised in that new heparin-flavones The preparation method of derived polymer includes the following steps：

(1) the bromide linking arm containing free amine group is dissolved in appropriate organic solvent, adds appropriate amino protecting agent, then added Enter appropriate activator and appropriate triethylamine, control reaction condition is to the reaction was complete, respectively with acid wash liquid, alkaline rinse and saturation Sodium chloride solution is washed or separated by silica gel column chromatography, is spin-dried for organic solvent, is obtained thick liquid, is intermediate compound I；

(2) hydrophobic nature flavonoid micromolecule is dissolved in appropriate organic solvent, adds appropriate activator, add appropriate intermediate I, control reaction condition is to the reaction was complete, and increasing amount water makes Precipitation, and ethyl acetate extraction is dry, and centre is obtained after being spin-dried for Body II；

(3) intermediate II is dissolved in appropriate organic solvent, adds appropriate amino deprotection agent, control reaction condition is to having reacted Entirely, precipitating reagent is added, gained washing of precipitate, after being dried using appropriate drying means, obtain intermediate III；

(4) heparin class polysaccharide is dissolved in appropriate solvent, under inert gas shielding and condition of ice bath, adds appropriate activated carboxylic Agent；Appropriate intermediate III is added, adds appropriate triethylamine；Reaction condition is controlled to be removed to the reaction was complete using centrifugation or filter membrane After removing insoluble matter, precipitated with appropriate precipitating reagent；Precipitation is collected, adds water to redissolve, loads bag filter and is dialysed with single water that steams, dialysis finishes Moisture is removed using appropriate drying means afterwards, up to new heparin-flavones derived polymer.

5. the preparation method of new heparin-flavones derived polymer according to claims 4, it is characterised in that：

Bromide linking arm described in step (1) includes the alkylidene bromo-amine and its hydrobromic acid that internal carbon atoms number is 2~6 Salt or hydrochloride；The appropriate organic solvent is dichloromethane, chloroform, ethyl acetate；The amino protecting agent choosing From di-tert-butyl dicarbonate, benzyl chloroformate；The appropriate amino protecting agent refers to its molar ratio with bromide linking arm For 1~5: 1；The activator is selected from N, N- dimethyl -4- pyridines amine, 4- pyrollidinopyridines, I-hydroxybenzotriazole；Institute The appropriate activator stated refers to that the molar ratio of itself and bromide linking arm is 1~5: 5；The appropriate triethylamine refers to itself and bromine The molar ratio of compound linking arm is 1~5: 1；The acid wash liquid is the dilute hydrochloric acid or dilute sulfuric acid of 0.01~0.5mol/L；Alkali Property washing lotion is saturated solution of sodium bicarbonate；The reaction condition is 10~30 DEG C；

Appropriate organic solvent described in step (2) is formamide, n,N-Dimethylformamide, n,N-dimethylacetamide, two One or more of mixed system in methyl sulfoxide；The activator is potassium carbonate, sodium carbonate；The appropriate activator is The molar ratio for referring to itself and hydrophobicity flavonoid micromolecule is 1~5: 1；The appropriate intermediate compound I refers to itself and hydrophobicity flavones The molar ratio of micromolecular is 1~5: 1；The reaction condition is 35~100 DEG C；

Appropriate organic solvent described in step (3) is dichloromethane, chloroform, methanol, dioxane；The amino takes off The mixed system that protective agent is trifluoroacetic acid, one or more of in hydrochloric acid, hydrobromic acid；The appropriate amino deprotection agent refers to The molar ratio of itself and intermediate II is 5~50: 1；The reaction condition is 10~30 DEG C；The precipitating reagent is ether or nothing Water ether；The appropriate drying means is to volatilize or be spin-dried for；

Heparin class polysaccharide described in step (4) is selected from unfraction heparin, low molecular weight heparin, desulfated heparin；Described Appropriate solvent is one or more of in formamide, n,N-Dimethylformamide, n,N-dimethylacetamide, dimethyl sulfoxide (DMSO) mixes Zoarium system；The carboxyl activator is selected from 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and hydroxysuccinimidyl Acid imide or 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and I-hydroxybenzotriazole or two hexamethylenes Base carbodiimide and HOSu NHS；The appropriate carboxyl activator refers to its mole with carboxyl in heparin class polysaccharide Than for 1~10: 1；The appropriate intermediate III refers to that the molar ratio of itself and carboxyl in heparin class polysaccharide is 1~10: 1；It is described Appropriate triethylamine refer to that the molar ratio of itself and intermediate III is 2~20: 1；The reaction condition be condition of ice bath under first to Carboxyl activator is added dropwise in heparin class polysaccharide, after keeping 0.5~4h of ice bath, adds intermediate III and triethylamine, continues room temperature React 6~72h；The appropriate precipitating reagent is ice acetone or ice ethanol；The method of the collection precipitation is centrifugation or decompression Filter；The appropriate drying means includes vacuum drying, spray drying or freeze-drying.

6. according to claims 2 it is based on the new flavones derived polymer Nano medication, in oncotherapy Two level combination strategy, it is characterised in that the hydrophobicity anti-tumor medicine containing aromatic conjugated structure includes：Cytotoxic drug, Including adriamycin, Epi-ADM, mitoxantrone, Dacarbazine, harringtonine, homoharringtonine, umbelliferone, benefit Bone fat element, Osthole, podophyllotoxin, camptothecine, hydroxycamptothecin, Etoposide, topotecan, Irinotecan and its activity Metabolin SN-38, taxol, docetaxel；Oncolipid is metabolized interference medicament, including aspirin, Diclofenac, indoles U.S. Pungent, naproxen, brufen, Meloxicam, celecoxib, rofecoxib；Photodynamic therapy medicines, including phthalocyanine, Phthalocyanine Zinc, phthalein Cyanines copper, Nickel Phthalocyanine, silicon phthalocyanine, the derivative of phthalocyanine gallium and these phthalocyanine compounds.

7. according to claims 2 it is based on the new flavones derived polymer Nano medication, in oncotherapy Two level combination strategy, it is characterised in that the implementation method of two level combination strategy includes following two techniques：

Technique I：New heparin-flavones derived polymer and water are mixed by weight the ratio of (mg/mg) 2~50: 1000 and made Into solution；A certain proportion of appropriate organic solvent of hydrophobicity anti-tumor medicine containing aromatic conjugated structure is dissolved；Will Anti-tumor medicine solution is slowly dropped in new heparin-flavones derived polymer solution, after 0.5~2h is stirred at room temperature, ice 10~30min of the lower Probe Ultrasonic Searching of bath；Using dialysis or ultrafiltration removing small molecule and organic solvent, rear use vacuum drying, Spray drying or freeze-drying remove moisture, to obtain the final product；

Technique II：New heparin-flavones derived polymer and water are mixed by weight the ratio of (mg/mg) 2~50: 1000 and made Into solution；A certain proportion of appropriate organic solvent of hydrophobicity anti-tumor medicine containing aromatic conjugated structure is dissolved；Will Both mix, and 10~30min of Probe Ultrasonic Searching under ice bath, room temperature lower open mouth is stirred overnight or is removed using Rotary Evaporators organic molten Agent, removes small molecule using centrifugal process or post separation method afterwards, crosses 0.8 μm of filter membrane, rear using vacuum drying, spray drying or freezing It is dry to remove moisture, to obtain the final product.

8. the implementation method of the two level combination strategy according to claims 7, it is characterised in that：

Certain proportion described in technique I and technique II refers to anti-tumor medicine and new heparin-flavones derived polymer Mass ratio (mg/mg) is 1: 2~5；Organic solvent described in technique I is selected from n,N-Dimethylformamide, N, N- dimethyl second Acid amides, dimethyl sulfoxide (DMSO), ethanol, methanol, dichloromethane, pyridine, polyvinylpyrrolidone；It is appropriate organic described in technique I Solvent refers to that the volume ratio of itself and water is 1: 2~10；Organic solvent described in technique II is selected from dichloromethane, ether, anhydrous Ether；Appropriate organic solvent described in technique II refers to that the volume ratio of itself and water is 1: 2~10.