CN102343099B

CN102343099B - Preparation method and application of folacin mediated tumor targeting adriamycin prodrugs

Info

Publication number: CN102343099B
Application number: CN2011102996511A
Authority: CN
Inventors: 周四元; 叶威良; 宦梦蕾; 滕增辉; 张邦乐; 梅其炳
Original assignee: Fourth Military Medical University FMMU
Current assignee: Fourth Military Medical University FMMU
Priority date: 2011-09-30
Filing date: 2011-09-30
Publication date: 2012-11-28
Anticipated expiration: 2031-09-30
Also published as: CN102343099A

Abstract

本发明提供两种阿霉素前体药物的制备方法以及应用，通过腙键的连接及叶酸的引入，得到两种兼具肿瘤主动靶向和酸敏突释特性的前药。这两种前体药物可能会增强阿霉素的抗肿瘤作用，降低药物毒副作用，提高靶向性，为抗肿瘤药物的开发提供新的思路。The invention provides the preparation method and application of two doxorubicin prodrugs. Through the connection of hydrazone bonds and the introduction of folic acid, two kinds of prodrugs with both active tumor targeting and acid-sensitive burst release characteristics are obtained. These two prodrugs may enhance the anti-tumor effect of doxorubicin, reduce drug side effects, improve targeting, and provide new ideas for the development of anti-tumor drugs.

Description

The preparation and the application thereof of folate-mediated cancer target adriablastina prodrug

Technical field

The present invention relates to the method for preparing and the application of two kinds of adriablastina prodrugs.

Background technology

Malignant tumor serious harm human health and existence are to cause one of main causes of death, and its sickness rate can be in any more always, have presented tangible rejuvenation trend at present.2000, World Health Organization's report, global cancer mortality example number surpasses 7,000,000, and wherein developed country accounts for 21%, and developing country accounts for 9%, and the death toll that tumor causes accounts for 12% of global death toll.Malignant tumor increases case about 2,000,000, dead 1,700,000 every year newly in the primary cause of death that China has become urban and rural residents.

Anthracycline antibiotics is one of clinical the most frequently used and effective antitumour medicine, and amycin is the representative medicine of this series antineoplastic medicament, early than from streptococcic metabolism of pigment product, obtaining the sixties in last century.Amycin is a kind of broad-spectrum chemotherapeutics, is usually used in treating breast carcinoma clinically, child's solid tumor, soft-tissue tumor, the malignant tumor such as lymphatic cancer of feeling sick.Clinical research is in recent years found, in the clinical practice of amycin, has occurred the drug resistance to tumor cell, and the cytotoxicity for normal structure has limited its further application simultaneously.The modal toxicity of this type of life-time service medicine is injury of myocardium, can bring out congestive heart failure.In view of above reason; People are continuing to the transformation and the modification of amycin always; Specialized transport carrier through will having tumor-targeting or can be combined with amycin by the molecule of tumor tissues specific recognition; Processing the adriablastina prodrug with tumor-targeting, is that present people are devoted to seek one of strategy that increases its drug effect and safety always.

At present, according to the design concept of prodrug, mainly concentrate on 13 carbonyl and 3 ' amino to the position of amycin structure of modification.Wherein, can 13 carbonyl be connected with the hydrazides group (modification group) of different molecular, the reaction of shrinking forms hydrazone key junctional complex, and the hydrazone key is more stable under neutral environment, and cleavable discharges the former medicine of amycin under the environment of meta-acid.Research shows, the growth of tumour cell metabolism is active, and pH value is starkly lower than normal structure on every side, so the formation of hydrazone key can bring into play acid-sensitive effect, has the effect of cancer target.

Different carrier molecules have different features, and itself and the bonded mode of former medicine or the characteristics that act on tumor tissues are difference to some extent.Polyethylene Glycol (PEG) can delay the speed that medicine is removed, and the half-life of prolong drug, the increase antitumor drug is accumulated tumor locus, and local concentration can reach 5-10 doubly, and then the effect of performance tumor tissues passive target.PEG can be connected through covalent bond with multiple medicine, thereby synthetic different macromolecule precursor medicament has been widely used in pharmaceutical preparation and the drug delivery system at present.

At present, the tumor cell folacin receptor receives extensive concern.Folacin receptor is the membrane glycoprotein that a kind of glycosylation phospholipid inositol connects, high expressed in many tumor cells, and in most of normal structures, express hardly.Therefore folic acid is connected with antitumor drug, can makes drug targeting to tumor cell, killing tumor cell optionally, and alleviate infringement to normal structure.

Summary of the invention

The purpose of this invention is to provide the method for preparing and the application of two kinds of adriablastina prodrugs; Amycin combined with Polyethylene Glycol, the folic acid modified respectively obtain two kinds and have acid-sensitive active adriablastina target precursor medicine; May strengthen the antitumor action of amycin; Reduce poisonous side effect of medicine, improve targeting property, for the exploitation of antitumor drug provides new thinking.

Precursor medicine of the present invention has two kinds: 1. amycin combines the amycin precursor medicine that obtains with Polyethylene Glycol, folic acid.

2. amycin is connected the amycin prodrug that obtains with folic acid through aminocaproic acid.

The structural formula of amycin is following:

The synthetic route of folic acid-aminocaproic acid-amycin:

Concrete synthetic method is following:

Synthesizing of 1 aminocaproic acid ethyl ester

Add dehydrated alcohol in the there-necked flask, the cryosel water-bath is cooled to below-10 ℃, drips the thionyl chloride that heavily steams, and maintains the temperature at below 0 ℃.Drip and finish, room temperature is stirring 10h down, adds aminocaproic acid temperature rising reflux reaction 7h, and decompression steams excessive thionyl chloride and ethanol, and residue solidifies, and uses methanol: (v: v=5: 1) recrystallization gets white solid to petroleum ether.

Synthesizing of 2 folic acid aminocaproic acid ethyl esters

Precision takes by weighing folic acid and adds anhydrous dimethyl sulphoxide (DMSO), and stirring and evenly mixing adds NHS more successively, DCC, triethylamine, room temperature nitrogen lucifuge activation 6h; Add the aminocaproic acid ethyl ester then, reaction 12h.Filtering reaction by-product DCU uses acetone-ether sedimentation at last, and ether washes repeatedly, gets bullion.Cross post separate pure article.

Synthesizing of 3 folic acid aminocaproic acid hydrazides

Precision takes by weighing folic acid aminocaproic acid ethyl ester, is dissolved among the anhydrous DMSO, adds the hydrazine hydrate of 4mL then, and 50 ℃ of lucifuge reaction 6h use ethanol precipitation at last, and ether washes repeatedly, gets bullion.Cross post separate pure article.

Synthesizing of 4 folic acid-aminocaproic acid-amycin

Precision takes by weighing folic acid aminocaproic acid hydrazides, is dissolved among the anhydrous DMSO.Fully the dissolving back adds the 75mg amycin, slowly drips the trifluoroacetic acid of 60 μ L, room temperature lucifuge reaction 12h; Use acetone-ether sedimentation at last, ether washes repeatedly, gets bullion.Cross post separate pure article.

The synthetic method of amycin-PEG-folic acid hydrazone key junctional complex is following:

1, HOOC-PEG-folic acid is synthetic

Folic acid is dissolved among the anhydrous DMSO, behind the preparatory activation 30min of nitrogen protection, adds HOOC-PEG-NH under the adding DCC/NHS room temperature ₂, system in the presence of triethylamine in room temperature reaction 2h.Add water after reaction finishes pH transferred to 7.5, the extraction that adds methylene chloride in the reactant liquor, add diethyl ether precipitate product.

2, amycin-PEG-folic acid hydrazone key junctional complex

HOOC-PEG-folic acid and tert-butyl group oxygen carbonyl hydrazine (BOC-hyd) react in DMSO under the catalysis of DCC/NHS and obtain FA-PEG-hyd-BOC, in 20% hydrochloric acid, slough the BOC blocking group and obtain FA-PEG-hyd.FA-PEG-hyd is dissolved among the DMSO, adds amycin, lucifuge reaction 48h gets amycin-PEG-folic acid hydrazone key junctional complex (DOX-hyd-PEG-FA) in the presence of trifluoroacetic acid.

Concrete synthetic route is following:

Description of drawings

Fig. 1 be folic acid-aminocaproic acid-amycin (FA-AMA-DOX) in 24h to KB cells in vitro anti-tumor activity curve chart.

Fig. 2 be folic acid-aminocaproic acid-amycin (FA-AMA-DOX) in 24h to A549 cells in vitro anti-tumor activity curve chart.

Amycin in Fig. 1 and Fig. 2 (DOX), aminocaproic acid-amycin (AMA-DOX), folic acid-aminocaproic acid-amycin (FA-AMA-DOX) in 24h to the cytotoxicity of KB cell (A) and A549 cell (B).

Fig. 3 is that amycin-PEG-folic acid hydrazone key junctional complex (DOX-hyd-PEG-FA) is hatched behind the 48h A549 cells in vitro anti-tumor activity figure.

Fig. 4 is that amycin-PEG-folic acid hydrazone key junctional complex (DOX-hyd-PEG-FA) is hatched behind the 48h KB cells in vitro anti-tumor activity figure.

Free amycin (DOX) in Fig. 3 and Fig. 4; Amycin-PEG amido link junctional complex (DOX-ami-PEG); Amycin-PEG-folic acid amido link junctional complex (DOX-ami-PEG-FA); Amycin-PEG hydrazone key junctional complex (DOX-hyd-PEG), amycin-PEG-folic acid hydrazone key junctional complex (DOX-hyd-PEG-FA) are hatched behind the 48h A549 cell (A), the cytotoxicity of KB cell (B).

The practical implementation method

The anti tumor activity in vitro research of adriablastina prodrug

1 purpose:

Through experiment in vitro, with the positive contrast of amycin, it is active to estimate the prodrug antineoplastic.

2 research methoies:

2.1 the prodrug vitro cytotoxicity is estimated

Various medicines to be measured are dissolved in (DMSO is lower than 0.1%) in the culture fluid that does not contain serum by setting concentration; Trypsinization with 0.25% grows to the negative A549 cell of expressing of KB cell, folic acid of the folic acid high expressed of exponential phase; Be inoculated in 96 orifice plates, density is 1 * 10 ⁴, add 0.001,0.01 respectively behind the cultivation 24h; 0.1, the DOX of 1,10 μ M (being the concentration of amycin), FA-AMA-DOX, DOX-hyd-PEG-FA (culture fluid that does not wherein contain serum is a blank); Adding 20 μ L MTT continue to hatch 4h after hatching 24h/48h, discard culture fluid, with 150 μ L DMSO dissolve purple crystallizations again; ELIASA reads the OD value under 570nm, draw the cell survival rate curve chart.

3 experimental results:

Experimental result is seen accompanying drawing.

Fig. 1 and Fig. 2 have shown folic acid-aminocaproic acid-amycin (FA-AMA-DOX) anti tumor activity in vitro.Compare with the junctional complex that is not connected folic acid, the anti tumor activity in vitro of FA-AMA-DOX demonstrates good antitumor activity on the KB of folacin receptor high expressed cell, and anti tumor activity in vitro is better than amycin; But for the negative A549 cell of cell surface folacin receptor, folate-mediated prodrug does not demonstrate ideal anti tumor activity in vitro.This result confirms that the tumor cell of FA-AMA-DOX pair cell surface folacin receptor high expressed has good in vitro and suppresses active.

Fig. 3 and Fig. 4 have shown the anti tumor activity in vitro of amycin-PEG-U folic acid hydrazone key junctional complex (DOX-hyd-PEG-FA).Similar with FA-AMA-DOX, for the KB cell of folacin receptor high expressed, the introducing of folic acid can increase the absorption of tumor cell to prodrug, thereby improves drug level in the cell, increases the anti-tumor activity of prodrug.Compare with amycin-PEG-folic acid amido link junctional complex (DOX-ami-PEG-FA) simultaneously; Hydrazone key junctional complex is because the introducing of the acid-sensitive active hydrazone key of tool; Realized prominent release of the former medicine of amycin in tumor cell; Thereby increased the concentration of free amycin in the tumor cell, demonstrated certain anti tumor activity in vitro.

4 conclusions:

Visible by above cell experiment result; Folate-mediated amycin-Polyethylene Glycol hydrazone key macromole junctional complex and amycin-aminocaproic acid-folic acid micromolecule junctional complex can be enriched in the abundant tumor cell of folacin receptor through folacin receptor mediated approach targeting; Reach targeting; Improve curative effect, reduce toxic and side effects, have good DEVELOPMENT PROSPECT.

Claims

1. The preparation method of folic acid-aminocaproic acid-doxorubicin prodrug is characterized in that aminocaproic acid is reacted with ethanol to obtain aminocaproic acid ethyl ester, and folic acid is connected with folic acid aminocaproic acid ethyl ester by DCC/NHS catalysis , carry out transesterification with hydrazine hydrate to obtain folic acid aminocaproic acid hydrazide; through the catalysis of trifluoroacetic acid, introduce doxorubicin to obtain the target compound folic acid-aminocaproic acid-doxorubicin.

2. The preparation method of doxorubicin-PEG-folate hydrazone linkage, which is characterized in that folic acid is catalyzed by DCC/NHS and HOOC-PEG-NH ₂ is connected to obtain HOOC-PEG-FA, HOOC-PEG-folic acid and tert-butyl Oxycarbonylhydrazine BOC-hyd reacts under the catalysis of DCC/NHS to obtain FA-PEG-hyd-BOC, removes the BOC protecting group to obtain FA-PEG-hyd; adds doxorubicin, and obtains in the presence of trifluoroacetic acid Doxorubicin-PEG-folate hydrazone linker DOX-hyd-PEG-FA.

3. The application of the folic acid-aminocaproic acid-doxorubicin prodrug according to claim 1 in the preparation of a medicament for treating tumors with high expression of folate receptors.

4. The application of the doxorubicin-PEG-folate hydrazone bond linker described in claim 2 in the preparation of a drug for treating tumors with high expression of folate receptors.