CN103130871B - Preparation method and application of prodrug of endopeptidase activated doxorubicin - Google Patents
Preparation method and application of prodrug of endopeptidase activated doxorubicin Download PDFInfo
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Abstract
本发明属于生物与医药技术领域，公开了内肽酶激活式多柔比星前药的制备方法，其特征在于包括以下步骤：（1）称取N-苄氧羰基-三肽，依次加入1-羟基苯并三唑，2-（7-偶氮苯并三氮唑）-四甲基脲六氟磷酸酯及N,N-二甲基甲酰胺，冰浴搅拌，加入N-甲基吗啡啉反应；（2）加入盐酸多柔比星，并补加N,N-二甲基甲酰胺，室温下反应，薄层层析监测产物点荧光强度至不再增加，停止反应；（3）采用柱层析法进行纯化。该法制备得到的内肽酶激活式多柔比星前药毒性低、纯度高，可应用于抗肿瘤领域。The invention belongs to the technical field of biology and medicine, and discloses a preparation method of endopeptidase-activated doxorubicin prodrug, which is characterized in that it comprises the following steps: (1) Weighing N-benzyloxycarbonyl-tripeptide, adding 1 -Hydroxybenzotriazole, 2-(7-azobenzotriazole)-tetramethyluronium hexafluorophosphate and N,N-dimethylformamide, stirring in ice bath, adding N-methylmorphine Phenol reaction; (2) Add doxorubicin hydrochloride, and add N,N-dimethylformamide, react at room temperature, monitor the fluorescence intensity of the product point by thin-layer chromatography until it no longer increases, and stop the reaction; (3) Purification was carried out by column chromatography. The endopeptidase-activated doxorubicin prodrug prepared by the method has low toxicity and high purity, and can be applied in the field of antitumor.
Description
Technical field
The invention belongs to biological and medical technical field, the particularly preparation method of endopeptidase activation type Dx prodrug, and this prodrug is in the application in antitumor field.
Background technology
Anthracene ring antitumor medicinal Dx (doxorubicin, Dox) be the wide spectrum powerful anticancer medicine that clinical First-line chemotherapy is conventional, effective to approximately 70% solid tumor, as mammary cancer, malignant lymphoma, lung cancer, acute leukemia etc., but there is the toxic side effect such as cardiac toxic and bone marrow depression.Therefore, reduce its toxicity and improve targeting, thus medicament curative effect enhancement, for chemotherapy medication with to improve the life quality of tumour patient significant.
For reducing or improve the toxic side effect and raising curative effect of Dx medicine; prodrug (prodrugs; abbreviation prodrug) chemistry that design theory is Dox changes structure provides theoretical foundation: in the structure of the former medicine Dox of small molecules, contain the free amine group that acylation reaction can occur, it is the crucial group that affects the chemistry and biology characteristic of medicine.When it, by other chemical group, modified or sealing, the external cytotoxicity of medicine that causes significantly reduces, and after entering in body, removes its modification group, and cytotoxicity can be recovered.Thereby neoplasm targeted therapy and prodrug design theory require the special molecular that a tumour is expressed with activation, to remove the object that modification group is realized activation prodrug as target spot conventionally.
The peptide quasi-molecule of some particular combination can be used as the modification group of former medicine and becomes the enzyme that tumour expresses and realize selectivity enzymolysis.Nature(Mackay J A, et al.Nat Mater, 2009,8 (12): 993-9.) reported that a kind of artificial synthetic polypeptide forms prodrug as the carrier of Dox, used the survival time of the tumor-bearing mice of this prodrug to use Dox to extend more than 3 times.Prodrug (Shahnaz G, et al.J Control Release, 2012,157 (3): 375-82.) that the pentapeptide of take in addition can extend plasma half-life and reduce plasma clearance function in carrier is carrier has synthesized a kind of new body.In addition, some specificity small peptide can be used as substrate and by the specific enzymes of tumor cells expression, is activated and degrade, and also can in the design process of prodrug, be utilized effectively.At present, the cancer therapy drug L-377202 that has entered the I phase clinical study stage is the prodrug of Dox (Dipaola R S, et al.J Clin Oncol, 2002,20 (7): 1874-9.), by Dox and peptide carrier N-glutaryl-[4-prolyl]-L-Ala-Serine-L-glutamic acid-Serine-leucine covalent attachment, formed, prodrug is non-activity in vitro, secreting under serine protease prostate specific antigen (PSA) enzymolysis, L-377202 activates as Dox form in cell.L-377202 to the anti-tumor activity of prostate cancer far away higher than Dox(Defeo-Jones D, et al.NatMed, 2000,6 (11): 1248-52.).
Lysosomal protein enzyme-l-asparagine endopeptidase (Asparaginyl endopeptidase, AEP claim again Legumain) of the kinds of tumor cells high expression level that research is found recently may become the novel targets for the treatment of.It expresses hardly (Chen J M, et al.Biochem J, 1998,335 (Pt1): 111-7.) except low expression in heart, kidney and placenta in other healthy tissuess.The activation of stress protein enzyme AEP and performance enzymolysis feature are to require the P1 site of substrate to have a l-asparagine (Dall E, Acta Crystallogr Sect F Struct Biol Cryst Commun, 2012,68 (Pt1): 24-31.).AEP expression amount in many mouse and people's tumor tissues significantly raises, and comprises people's multiple solid tumor, as mammary cancer, colorectal carcinoma, prostate cancer, tumor neovasculature endotheliocyte and tumor-associated macrophages etc.AEP has composing type and two kinds of phraseologies of secretor type at tumor microenvironment, and participates in Tumor-assaciated biology event (Briggs J J, et al.BMC Cancer, 2010,10:17; Murthy R V, et al.Clin Cancer Res, 2005,11 (6): 2293-9.).AEP can activate matrix metalloproteinase, promotes the newborn of tumor cell invasion, blood vessel and shifts (Liu C, et al.Cancer Res, 2003,63 (11): 2957-64.).The AEP of what is more important high expression level conventionally with the infiltration of tumour, transfer, terminal stage of a disease, poor prognosis, to resistance relevant (Gawenda J, et al.Breast Cancer Res Treat, 2007,102 (1): 1-6.) such as chemoradiotherapies.AEP is and the proteolytic enzyme of cancer therapy height correlation, can be used to its specificity enzymatic catalysis characteristics.Take AEP as action target, and the miniature vaccine target of design can act on the stroma cell of mammary cancer, with these inhibition tumor cell growth and vasculogenesis (Lewen S, et al.Cancer Immunol Immunother, 2008,57 (4): 507-15.).
So far, found to take that various former medicines directly act on AEP prodrug as basis has: carbobenzoxy-(Cbz)-Ala-Ala-Radix Asparagi quadrol Etoposide, former medicine Etoposide is brought into play antitumor action (Stern L by acting on DNA topoisomerase II, et al.Bioconjug Chem, 2009,20 (3): 500-10.).Take to micropipe aggregation have restraining effect anti-tumor activity pentapeptide Dolastatin-10 as the synthetic prodrug of former medicine can suppress tumour growth and transfer (Bajjuri K M, et al.ChemMedChem, 2011,6 (1): 54-9.).In addition a prodrug of the MMAE being activated by AEP, mouse breast cancer, lung cancer animal model show growth and transfer (Liu Y, et al.Mol Pharm, 2012,9 (1): 168-75.) that it has reduced tumour effectively.
But in existing research and development, the specificity of modified peptides and quantity, arrangement mode, antigenicity and validity are needed solution badly, the preparation method of the mixture that Dox covalent coupling polypeptide forms is mostly consuming time longer, and speed of reaction is not high, and the productive rate of product, stability and purity are lower.
Summary of the invention
In order to overcome the shortcoming and deficiency of prior art, primary and foremost purpose of the present invention is to provide a kind of preparation method of endopeptidase activation type Dx prodrug.
Another object of the present invention is to provide the endopeptidase activation type Dx being prepared by above-mentioned preparation method prodrug.
A further object of the present invention is to provide the application of above-mentioned endopeptidase activation type Dx prodrug in antitumor field.
Object of the present invention is achieved through the following technical solutions: the preparation method of endopeptidase activation type Dx prodrug, comprises the following steps:
(1) take N-carbobenzoxy-(Cbz)-tripeptides, add successively I-hydroxybenzotriazole (HOBT), 2-(7-azo benzotriazole)-tetramethyl-urea phosphofluoric acid ester (HBTU) and N, dinethylformamide (DMF), ice bath stirs, after system temperature declines, add N-methylmorpholine (NMM) reaction, obtain three peptide solutions of activation;
(2) add doxorubicin hydrochloride, and add DMF, under room temperature, react, thin-layer chromatography (TLC) monitoring product point fluorescence intensity is to no longer increase, stopped reaction;
(3) adopt column chromatography to carry out purifying, obtain endopeptidase activation type Dx prodrug.
Product is identified by TLC, and mass spectrum confirmation detects purity by high performance liquid chromatography (HPLC).
The described N-carbobenzoxy-(Cbz)-tripeptides of step (1) refers to N-carbobenzoxy-(Cbz)-alanyl-alanyl-l-asparagine (CBZ-Ala-Ala-Asn-OH, abbreviation CBZ-PAN-OH), N-carbobenzoxy-(Cbz)-prolyl-alanyl-l-asparagine (CBZ-Pro-Ala-Asn-OH, abbreviation CBZ-PAN-OH), N-carbobenzoxy-(Cbz)-threonyl-alanyl-l-asparagine (CBZ-Thr-Ala-Asn-OH, be called for short CBZ-TAN-OH) or N-carbobenzoxy-(Cbz)-prolyl-threonyl-l-asparagine (CBZ-Pro-Thr-Asn-OH is called for short CBZ-PTN-OH).
The mol ratio of N-carbobenzoxy-(Cbz)-tripeptides, HOBT, HBTU and NMM that step (1) is described is 1:1:1:1～5; The volume ratio of DMF and NMM is 20～100:1.
The time that the described ice bath of step (1) stirs is 15～60min.
The described system temperature of step (1) declines and refers to drop to-15～5 ℃.
The time of the described reaction of step (1) is 10～40min.
The mol ratio of the described doxorubicin hydrochloride of step (2) and the described N-carbobenzoxy-(Cbz)-tripeptides of step (1) is 1:1.0～1.5.
The described DMF volume of adding of step (2) is 5～10% of step (1) reaction system cumulative volume;
The chromatographic condition of the described column chromatography of step (3): conventional post (2.5 * 30cm), stationary phase is column chromatography silica gel, moving phase is ethyl acetate-methyl alcohol; Ethyl acetate used and the volume ratio of methyl alcohol are respectively 5:1 and 10:3.
The endopeptidase activation type Dx prodrug being prepared by aforesaid method.
Above-mentioned endopeptidase activation type Dx prodrug is in the application in antitumor field.
Mechanism of the present invention is: by small peptide covalent modification technology, the specific tripeptides that contains l-asparagine is connected on the amino of Dx by acylation reaction, forms new tripeptides covalent modification Dx prodrug the cytotoxicity of Dx is reduced greatly; Simultaneously, this prodrug express the tumor cell surface of endopeptidase AEP or by endocytosis after could discharge Dx, Dx can be enriched to around tumor tissues or in born of the same parents, nontoxic to health tissues or cell, thus improve Dx to the targeting of tumour and effect curative effect.
The present invention has following advantage and effect with respect to prior art:
(1) prodrug that preparation method provided by the invention prepares has following characteristic: 1), due to the sealing of Dx amino, the toxicity of the Dx prodrug of tripeptides covalent modification reduces greatly than its former medicine Dx; 2) tripeptides covalent modification Dx prodrug discharges Dx under the activation of tumour endopeptidase AEP, thereby realize Dx selectivity, discharges, and has reduced the toxicity of Dx normal tissue, improves the curative effect to tumor tissues chemotherapy;
(2) synthetic method of the present invention is simple, and building-up reactions takes short, and productive rate is high, the easy purifying of reaction after product.
Accompanying drawing explanation
Fig. 1 is the positive ion high resolution mass spectrum figure of the Dx prodrug CBZ-AAN-Dox for preparing of embodiment 1.
Fig. 2 is the positive and negative ion color atlas of the Dx prodrug CBZ-AAN-Dox for preparing of embodiment 1.
Fig. 3 is that after the administration of four kinds of Dx prodrugs, mouse moves mammary cancer and plants in knurl model and respectively organize relative tumor proliferation rate (%) (n=10).
Fig. 4 is the impact of four kinds of Dx prodrugs administration on body weight in mouse breast cancer transplanted tumor model.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
Embodiment 1:N-carbobenzoxy-(Cbz)-alanyl-alanyl-l-asparagine is modified the design of Dx prodrug (CBZ-AAN-Dox) with synthetic
(1) activation of N-carbobenzoxy-(Cbz)-alanyl-alanyl-l-asparagine (CBZ-AAN-OH):
Take 39.0mg(95 μ mol) the big bio tech ltd of Ztel, CBZ-AAN-OH(Guangzhou) be placed in 25mL round-bottomed flask, add 12.9mg(95 μ mol) HOBt and 54.0mg(95 μ mol) HBTU, add 1.0mL DMF, ice bath stirs 30min, treat that system temperature drops to-15～5 ℃, add 9.5 μ L(95 μ mol) NMM, reaction 15min.
(2) preparation of CBZ-AAN-Dox:
In step (1) system, add 50mg(86 μ mol) doxorubicin hydrochloride, and add 1.0mLDMF, under room temperature, react, to thin-layer chromatography (TLC), monitoring product point fluorescence intensity no longer increases, stopped reaction, ether extraction liquid makes the mixture of tripeptides covalent coupling Dx through rotary evaporation.
(3) purifying of CBZ-AAN-Dox:
Adopt column chromatography to carry out purifying to tripeptides covalent modification Dx.Chromatographic condition: conventional post (2.5 * 30cm).Stationary phase: column chromatography silica gel; Moving phase: ethyl acetate and methyl alcohol volume ratio are respectively 5:1 and 10:3.Obtain CBZ-AAN-Dox.Product identifies by TLC, mass spectrum confirmation (ES-MS:[M+Na]
+=956) (Fig. 1), by high performance liquid chromatography (HPLC), detect purity, purity reaches 93.86%(Fig. 2).
Embodiment 2:N-carbobenzoxy-(Cbz)-prolyl-alanyl-l-asparagine is modified the design of Dx prodrug (CBZ-PAN-Dox) with synthetic
(1) activation of N-carbobenzoxy-(Cbz)-prolyl-alanyl-l-asparagine (CBZ-PAN-OH):
Take 41.5mg(95 μ mol) the big bio tech ltd of Ztel, CBZ-PAN-OH(Guangzhou) be placed in 25mL round-bottomed flask, add 12.9mg(95 μ mol) HOBt and 54.0mg(95 μ mol) HBTU, add 1.0mL DMF, ice bath stirs, treat that system temperature drops to-15～5 ℃, add 20 μ L(200 μ mol) NMM, reaction 10min.
(2) preparation of CBZ-PAN-Dox:
In step (1) system, add 55mg(95 μ mol) doxorubicin hydrochloride, and add 1.0mL DMF, under room temperature, react, to thin-layer chromatography (TLC), monitoring product point fluorescence intensity no longer increases, stopped reaction, ether extraction liquid makes the mixture of tripeptides covalent coupling Dx through rotary evaporation.
(3) purifying of CBZ-PAN-Dox:
Adopt column chromatography to carry out purifying to tripeptides covalent modification Dx.Chromatographic condition: conventional post (2.5 * 30cm).Stationary phase: column chromatography silica gel; Moving phase: ethyl acetate and methyl alcohol volume ratio are respectively 5:1 and 10:3.Obtain N-carbobenzoxy-(Cbz)-prolyl-alanyl-l-asparagine and modify Dx prodrug.
Embodiment 3:N-carbobenzoxy-(Cbz)-threonyl-alanyl-l-asparagine is modified the design of Dx prodrug (CBZ-TAN-Dox) with synthetic
(1) activation of N-carbobenzoxy-(Cbz)-threonyl-alanyl-l-asparagine (CBZ-TAN-OH):
Take 41.8mg(95 μ mol) the big bio tech ltd of Ztel, CBZ-TAN-OH(Guangzhou) be placed in 25mL round-bottomed flask, add 12.9mg(95 μ mol) HOBt and 54.0mg(95 μ mol) HBTU, add 1.0mL DMF, ice bath stirs, treat that system temperature drops to-15～5 ℃, add 50 μ L(500 μ mol) NMM, reaction 8min.
(2) preparation of CBZ-TAN-Dox:
In step (1) system, add 37mg(63 μ mol) doxorubicin hydrochloride, and add 1.0mLDMF, under room temperature, react, to thin-layer chromatography (TLC), monitoring product point fluorescence intensity no longer increases, stopped reaction, ether extraction liquid makes the mixture of tripeptides covalent coupling Dx through rotary evaporation.
(3) purifying of CBZ-TAN-Dox:
Adopt column chromatography to carry out purifying to tripeptides covalent modification Dx.Chromatographic condition: conventional post (2.5 * 30cm).Stationary phase: column chromatography silica gel; Moving phase: ethyl acetate and methyl alcohol volume ratio are respectively 5:1 and 10:3.Obtain N-carbobenzoxy-(Cbz)-threonyl-alanyl-l-asparagine and modify Dx prodrug.
Embodiment 4:N-carbobenzoxy-(Cbz)-prolyl-threonyl-l-asparagine is modified the design of Dx prodrug (CBZ-PTN-Dox) with synthetic
(1) activation of N-carbobenzoxy-(Cbz)-prolyl-threonyl-l-asparagine (CBZ-PTN-OH):
Take 44.3mg(95 μ mol) the big bio tech ltd of Ztel, CBZ-PTN-OH(Guangzhou) be placed in 25mL round-bottomed flask, add 12.9mg(95 μ mol) HOBt and 54.0mg(95 μ mol) HBTU, add 1.0mL DMF, ice bath stirs, treat that system temperature drops to-15～5 ℃, add 20 μ L(200 μ mol) NMM, reaction 8min.
(2) preparation of CBZ-PTN-Dox:
In step (1) system, add 50mg(86 μ mol) doxorubicin hydrochloride, and add 1.0mLDMF, under room temperature, react, to thin-layer chromatography (TLC), monitoring product point fluorescence intensity no longer increases, stopped reaction, ether extraction liquid makes the mixture of tripeptides covalent coupling Dx through rotary evaporation.
(3) purifying of CBZ-PTN-Dox:
Adopt column chromatography to carry out purifying to tripeptides covalent modification Dx.Chromatographic condition: conventional post (2.5 * 30cm).Stationary phase: column chromatography silica gel; Moving phase: ethyl acetate and methyl alcohol volume ratio are respectively 5:1 and 10:3.Obtain N-carbobenzoxy-(Cbz)-prolyl-threonyl-l-asparagine and modify Dx prodrug.
Embodiment 5: the stability test of the synthetic prodrug of novel tripeptides covalent modification Dx
By the synthetic CBZ-AAN-Dox obtaining of embodiment 1, the synthetic CBZ-PAN-Dox obtaining of embodiment 2, the synthetic CBZ-TAN-Dox obtaining of embodiment 3 and the synthetic CBZ-PTN-Dox obtaining of embodiment 4 are placed in respectively hot conditions (60 ℃), (25 ℃ of super-humid conditions, relative humidity is 90% ± 5%), under high light condition (4500 ± 500LX), carry out study on the stability, then respectively equivalent compound is dissolved in to human normal plasma, mice plasma, in DMSO solution, in different time point 5, 10, 30, 60, 120min sampling, measure respectively wherein CBZ-AAN-Dox, CBZ-PAN-Dox, CBZ-TAN-Dox, four kinds of prodrugs such as CBZ-PTN-Dox content situation in time.Within the scope of experimental period, four kinds of prodrugs mass content in different solvents, all more than 98%, illustrates that the new synthetic prodrugs of the overwhelming majority can just degraded before arriving target organ when intravenously administrable.In addition, these four kinds of target compounds are all very stable in hot conditions (60 ℃), high light condition (4500 ± 500LX), but moisture absorption is serious when super-humid conditions (relative humidity is 92.5%), form liquid, point out the long-term preservation of such prodrug should be placed in dry airtight environment.
Single substrate specificity dynamical analysis of the release in vitro of the synthetic prodrug of 6: four kinds of novel tripeptides covalent modification Dxs of embodiment and specificity hydrolysis
Tripeptides covalent modification Dx prodrug is as the substrate of dynamic analysis, and CBZ-Phe-Tyr-Asn-Dox is substrate (CBZ-Phe-Tyr-Asn-Dox prepares according to embodiment 1 preparation method) as a control group.The determination of activity condition of enzyme: 25 ℃, containing 50mM sodium-acetate buffer, pH is 5.5,2mM DTT and 2mM EDTA.The final concentration scope of substrate is 0.006～0.4mM, and DMSO volumetric concentration is 3%.Preactivated AEP enzyme solution joins (every hole is 100 μ l altogether) in each hole.The excitation wavelength of selected fluorescence is 488nm, and emission wavelength is that 575nm measures; Application GraphPadPrism5.0 software analysis data, calculate enzyme kinetics parameter.The results are shown in Table 1, the tripeptides covalent modification Dx prodrug that the present invention prepares can specificity be AEP enzymolysis, and control group CBZ-Phe-Tyr-Asn-Dox cuts activity without enzyme.
Km and the Vmax of table 1 people endopeptidase AEP enzymolysis Dx prodrug substrate
| Dx prodrug | Km(ｕM) | Vmax?(ｕMs -1) |
| CBZ-AAN-Dox | 68±7 | 3.9±0.6 |
| CBZ-PAN-Dox | 85±9 | 4.4±0.3 |
| CBZ-TAN-Dox | 128±10 | 5.7±0.6 |
| CBZ-PTN-Dox | 122±13 | 6.9±0.6 |
| CBZ-FYN-Dox | - | - |
Note :-indicate without enzymolysis activity
The synthetic prodrug vitro cytotoxicity experiment of 7: four kinds of novel tripeptides covalent modification Dxs of embodiment
Adopt CCK-8Kit to detect leukemia cell line K562, the cell survival rate of breast cancer cell line mcf-7.Every hole adds 100 μ l, 5000 logarithmic phase cells.Respectively by 12.5,6.25,3.13,1.57,0.79,0.39, the CBZ-AAN-Dox(DMSO of 0.19,0.1 μ M is as solvent) join in cell.Every hole adds the CCK-8 solution of 10 μ l.And respective amount cell culture fluid, medicine and CCK-8 solution are set but do not add the hole of cell as blank.In cell culture incubator, continue to hatch, after 48h, by microplate reader, at 450nm, measure absorbancy respectively.Experimental group cell cultures adopts weary oxygen inducing culture method.Experimental result is in Table the IC of the more former medicine of cytotoxicity (to K562 and MCF-7) of 2, CBZ-AAN-Dox prodrug
50significantly raise, illustrate that its toxicity has reduced respectively 87.5% and 96.1% compared with the former medicine of Dox, CBZ-PAN-Dox, CBZ-TAN-Dox, other three kinds of more former medicines of prodrug such as CBZ-PTN-Dox have reduced respectively 92.9% and 97.3% to the toxicity of two kinds of cell strains, 94.1% and 96.9%, 96.1% and 97.3%, and reduced by 97.5% compared with human umbilical vein endothelial cell (HUVEC) contrast; Four kinds of prodrugs, after weary oxygen induction, can recover the cytotoxicity similar to former medicine.
Four kinds of novel prodrugs of table 2 and the IC of Dox effect after 48 hours
50relatively
In the synthetic prodrug body of 8: four kinds of novel tripeptides covalent modification Dxs of embodiment, anti-tumor activity detects
Every subcutaneous lipids pad inoculation 10 of Balb/C female mice
7individual MDA-MB-431 cell, tumour transplatation tumor-bearing mice is divided into physiological saline (NS) group at random, and Dox group and tripeptides are modified Dox group, and mouse gives respectively Dox(5mg/kg) and equimolar tripeptides modification Dox, within continuous three weeks, observe tumor size, calculate the relative tumor proliferation rate of respectively organizing.Result proves: while finishing to administration, the Dx prodrug after tripeptides is modified has the anti-tumor activity identical with former medicine Dx and curative effect (Fig. 3).
The synthetic prodrug toxicity in vivo experiment of 9: four kinds of novel tripeptides covalent modification Dxs of embodiment
According to the method for embodiment 5, take administration first day as zero beginning, in continuous three weeks, measure every three days Mouse Weight.Found that the body weight of the Dx prodrug group after tripeptides is modified is without considerable change, and Dx group reopens at the 8th day body the decline of beginning, and illustrates that the Dx prodrug after modifying reduces (Fig. 4) than former medicine Dx toxicity.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (7)
1. the preparation method of endopeptidase activation type Dx prodrug, is characterized in that comprising the following steps:
(1) take N-carbobenzoxy-(Cbz)-tripeptides, add successively I-hydroxybenzotriazole, 2-(7-azo benzotriazole)-tetramethyl-urea phosphofluoric acid ester and N, dinethylformamide, ice bath stirs, and after system temperature declines, adds the reaction of N-methylmorpholine;
(2) add doxorubicin hydrochloride, and add DMF, under room temperature, react, thin-layer chromatography monitoring product point fluorescence intensity is to no longer increase, stopped reaction;
(3) adopt column chromatography to carry out purifying, obtain endopeptidase activation type Dx prodrug;
The mol ratio of N-carbobenzoxy-(Cbz)-tripeptides, I-hydroxybenzotriazole, 2-(7-azo benzotriazole)-tetramethyl-urea phosphofluoric acid ester and N-methylmorpholine that step (1) is described is 1:1:1:1～5; The volume ratio of DMF and N-methylmorpholine is 500～750:1; Described N-carbobenzoxy-(Cbz)-tripeptides refers to N-carbobenzoxy-(Cbz)-alanyl-alanyl-l-asparagine, N-carbobenzoxy-(Cbz)-prolyl-alanyl-l-asparagine, N-carbobenzoxy-(Cbz)-threonyl-alanyl-l-asparagine or N-carbobenzoxy-(Cbz)-prolyl-threonyl-l-asparagine;
The chromatographic condition of the described column chromatography of step (3): conventional post 2.5 * 30cm, stationary phase is column chromatography silica gel, moving phase is ethyl acetate-methyl alcohol; Ethyl acetate used and the volume ratio of methyl alcohol are respectively 5:1 and 10:3.
2. the preparation method of endopeptidase activation type Dx prodrug according to claim 1, is characterized in that: the described system temperature of step (1) declines and refers to drop to-15～5 ℃.
3. the preparation method of endopeptidase activation type Dx prodrug according to claim 1, is characterized in that: the time of the described reaction of step (1) is 10～40min.
4. the preparation method of endopeptidase activation type Dx prodrug according to claim 1, is characterized in that: the doxorubicin hydrochloride that step (2) is described and the mol ratio of the described N-carbobenzoxy-(Cbz)-tripeptides of step (1) are 1:1.0～1.5.
5. the preparation method of endopeptidase activation type Dx prodrug according to claim 1, is characterized in that: described in step (2), adding DMF volume is 5～10% of step (1) reaction system cumulative volume.
6. the endopeptidase activation type Dx prodrug being prepared by preparation method described in claim 1～5 any one.
7. the application of endopeptidase activation type Dx prodrug according to claim 6 in preparing antitumor drug.
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| CN101374856A (en) * | 2005-11-29 | 2009-02-25 | 斯克里普斯研究学院 | Inhibit tumor cell invasion, metastasis and angiogenesis |
| CN102068703A (en) * | 2011-01-05 | 2011-05-25 | 武汉理工大学 | Doxorubicin multi-targeting drug delivery system and preparation method and application thereof |
| CN102225052A (en) * | 2011-06-09 | 2011-10-26 | 上海微纳科技有限公司 | pH-sensitive doxorubicin nano-liposome and preparation method thereof |
-
2013
- 2013-02-06 CN CN201310048238.7A patent/CN103130871B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101374856A (en) * | 2005-11-29 | 2009-02-25 | 斯克里普斯研究学院 | Inhibit tumor cell invasion, metastasis and angiogenesis |
| CN102068703A (en) * | 2011-01-05 | 2011-05-25 | 武汉理工大学 | Doxorubicin multi-targeting drug delivery system and preparation method and application thereof |
| CN102225052A (en) * | 2011-06-09 | 2011-10-26 | 上海微纳科技有限公司 | pH-sensitive doxorubicin nano-liposome and preparation method thereof |
Non-Patent Citations (2)
| Title |
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| A Novel Antitumor Prodrug Platform Designed to Be Cleaved by the Endoprotease Legumain;Liron Stern et al.;《Bioconjugate Chem》;20091231;第20卷;500-510 * |
| Liron Stern et al..A Novel Antitumor Prodrug Platform Designed to Be Cleaved by the Endoprotease Legumain.《Bioconjugate Chem》.2009,第20卷500-510. * |
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