CN101274100B - Method for preparing adriamycin-dipeptide complexes and applications - Google Patents
Method for preparing adriamycin-dipeptide complexes and applications Download PDFInfo
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- CN101274100B CN101274100B CN2008100263834A CN200810026383A CN101274100B CN 101274100 B CN101274100 B CN 101274100B CN 2008100263834 A CN2008100263834 A CN 2008100263834A CN 200810026383 A CN200810026383 A CN 200810026383A CN 101274100 B CN101274100 B CN 101274100B
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Abstract
The invention discloses a preparation method of an adriamycin-dipeptide compound. Dipeptide or polypeptide containing proline residue is connected to amino of the adriamycin by the acylation through the technique of peptide modification; the toxicity of the adriamycin can be reduced and at the same time the pro-drug can release the adriamycin under the action of tumor interstitial cells so as to ensure that the adriamycin can enrich around tumor tissue to improve the selectivity of the adriamycin to the tumor tissue. The preparation method of the invention has the advantages of fast reaction rate, short term, relatively high productive rate and purity.
Description
Technical field
The invention belongs to biological and medical technical field, be specifically related to a kind of preparation method and application with adriamycin-dipeptide complexes of active anticancer.
Background technology
Because it is antitumor action widely, amycin is usually used in the clinical treatment of hepatocarcinoma, gastric cancer, breast carcinoma, pulmonary carcinoma, ovarian cancer and multiple leukemia etc. as a line medicine of treatment cancer.Yet untoward reaction such as the bone marrow depression of amycin and cardiac toxicity often destroy patient's immunologic function, have had a strong impact on therapeutic effect.Therefore, seek and a kind ofly to reduce amycin toxicity, improve amycin the optionally method of modifying of tumor tissues is become one of emphasis of present research.
Contain the free amine group that acylation reaction can take place in the structure that a core of prodrug technology is many anti-tumor small molecular chemicalses; and these free amine groups often affect the chemistry and biology characteristic of medicine; in case modified or seal by other chemical groups; to cause the cytotoxicity of medicine significantly to reduce, and remove its cytotoxicity of modification group and then can recover.These medicines comprise: amycin (doxorubicin, Dox), daunomycin (daunomycin, DM) or the like.Baurain etc. have prepared the polypeptide derivative of a plurality of daunomycin, found that, the cytotoxicity of Ala-Leu-DM and Leu-Leu-DM drops to 1/100 and 1/250 of parent drug DM respectively.In case and polypeptide is removed in hydrolysis, the cytotoxicity of DM can recover [Baurain R, et al.J Med Chem.23 (11): 1171-4.1980].The Trouet report, the tetrapeptide derivative N-alanyl-leucyl-alanyl-leucyl-Dox of amycin only is the 1/9[Trouet A of amycin to the toxicity of normal mouse and tumor-bearing mice, etal.Cancer Res.61 (7): 2843-6.2001.]
Molecular targeted agents is according to the difference on the molecular biology between tumor cell and the normal cell, and selectively acting is in the gene of target cell, enzyme, signal transduction molecule etc., thereby reaches the purpose of treatment.Though the molecular targeted agents treatment is a kind of ideal therapeutic modality, single medicine result of use is not good, remains in many problems.Wherein owing to the unstability of tumor cell gene, tumor cell often passes through the modulation of target molecule or the attack that disappearance is escaped medicine, and causing molecular targeted agents to lose curative effect is exactly a problem that needs to be resolved hurrily.In recent years, researcher begins sight is turned to the stable mesenchyma stroma of tumors cell of genome.In mesenchyma stroma of tumors, fibroblast activates by interacting with tumor cell, this activation fibroblast is called as tumor associated fibroblast cell (Carcinoma-Associated Fibroblasts, CAF) [Bhowmick NA, et al.Nature.432 (7015): 332-7.2004.].A large amount of evidences show, CAF plays necessary support facilitation to the generation development of tumor, promote tumor-infiltrated and transfer [Tuxhorn JA, et al.ClinCancer Res.8 (9): 2912-23.2002.] such as the express cell factor, protease and adhesion molecule etc.In view of the importance of CAF for tumor development, selecting this group cell is target, cuts off its support facilitation to tumor cell, can yet be regarded as and improves the requisite measure of neoplasm targeted therapy effectiveness.Have now found that tumor associated fibroblast cell contains the protease of the peptide bond that second of multiple hydrolysis form by proline residue, such as becoming the fiber activator protein.This albuminoid enzyme is present in tumor tissues usually, the wound healing tissue, and embryo fibroblast [Niedermeyer J, et al.Int J CANCER.71:383-389.1997] makes the mesenchyma stroma of tumors cell have the characteristic different with normal surrounding tissue.
But in the prior art, the preparation method of adriamycin-dipeptide complexes is general consuming time longer, answers speed lower, and the productive rate of product and purity are also lower.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, a kind of preparation method of adriamycin-dipeptide complexes is provided, introducing can be modified amycin by the dipeptides of the proteolytic enzyme selective hydrolysis of mesenchyma stroma of tumors cellular expression.
Technical scheme of the present invention is:
A kind of preparation method of adriamycin-dipeptide complexes is provided, may further comprise the steps:
(1) take by weighing N-benzyloxycarbonyl group glycyl proline (dipeptides) and be dissolved in N, dinethylformamide (DMF) adds ethyl chloroformate, stirs under the room temperature, adds triethylamine, and stirring at room obtains the solution of activatory dipeptides;
(2) doxorubicin hydrochloride is dissolved in and contains the DMF that waits the Et3N of amount of substance with it, adds activatory two peptide solutions, stirring at room, and the extracted with diethyl ether product, extract makes adriamycin-dipeptide complexes through rotary evaporation;
(3) adopt column chromatography that adriamycin-dipeptide complexes is carried out purification.
Described ethyl chloroformate of step (1) and N-benzyloxycarbonyl group glycyl proline mol ratio are 1: 1.2.
Behind the described adding ethyl chloroformate of step (1) under the room temperature mixing time be 10 minutes; The stirring at room time is 40 minutes behind the described adding triethylamine.
The addition of described activatory two peptide solutions of step (2) is the mol ratios such as DMF solution with doxorubicin hydrochloride.
The described stirring at room time of step (2) is 5～6 hours.
The described ether consumption of step (2) be 5 times to the reactant liquor volume.
The chromatographic condition of the described column chromatography of step (3) is conventional post 2.5 * 30cm; Immobile phase is a column chromatography silica gel; Mobile phase is the ethyl acetate-methanol of 7.5: 1 and 5: 1.
Product detects purity with high performance liquid chromatography (HPLC) after identifying by thin layer chromatography.
The present invention is by the peptide modification technique; the dipeptides or the polypeptide that will contain proline residue are connected on the amino of amycin by acylation reaction; the toxicity of amycin is reduced; this prodrug can discharge amycin under the effect of mesenchyma stroma of tumors cell simultaneously; amycin can be enriched to around the tumor tissues, improve the selectivity of amycin tumor tissues.
The invention has the beneficial effects as follows:
(1) adriamycin-dipeptide complexes prepared of preparation method provided by the invention has the characteristics of two aspects: on the one hand because the sealing of amycin amino, adriamycin-dipeptide complexes is than the toxicity reduction of its female medicine amycin; On the other hand, adriamycin-dipeptide complexes discharges amycin under the effect of the proteolytic enzyme of mesenchyma stroma of tumors cell, thereby reach the enrichment function that amycin is discharged by selectivity, given adriamycin-dipeptide complexes to the normal structure low toxicity, tumor tissues has been had the therapeutic effect of lethal effect.
(2) method of the present invention is simple, the reaction rate height, and weak point consuming time, degree of purity of production and productivity ratio are higher.
Description of drawings
Fig. 1 plasma stability experimental result
Fig. 2 tumor growth curve
Laboratory animal body weight change curve in the experiment of Fig. 3 drug toxicity
The specific embodiment
Further specify the present invention below in conjunction with the drawings and specific embodiments.
Synthesizing of embodiment 1 benzyloxycarbonyl group glycyl prolyl amycin
(1) activation of N-benzyloxycarbonyl group glycyl proline:
The N-benzyloxycarbonyl group glycyl proline that takes by weighing 26.4mg (86.2 μ mol) is dissolved in the N of 1.5ml, dinethylformamide (DMF), add 9.8 μ l (86.2 * 1.2=103.4 μ mol) ethyl chloroformate, stirred 10 minutes under the room temperature, add 14.5 μ l (103.4 μ mol) triethylamine, stirring at room 40 minutes.
(2) preparation of N-benzyloxycarbonyl group glycyl prolyl amycin:
Take by weighing 40mg (86.2 μ mol) doxorubicin hydrochloride and be dissolved among the 1.5mL DMF, to wherein adding 12.5 μ l (86.2 μ mol) triethylamine and mixing.The doxorubicin hydrochloride solution that dissolving is good adds through above-mentioned activatory Z-GP solution, stirring at room 5～6 hours; Product extracts with the ether (5 times to the reactant liquor volume) of 30 ml, and decompress filter discards precipitation, and ether extraction liquid promptly gets crude product through rotary evaporation.
(3) purification of N-benzyloxycarbonyl group glycyl prolyl amycin:
Adopt column chromatography that thick product is carried out purification.Chromatographic condition: conventional post (2.5 * 30cm).Immobile phase: column chromatography silica gel; Mobile phase: ethyl acetate-methanol (7.5: 1,5: 1).Product identifies by thin layer chromatography, mass spectrum confirmation (ES-MS:[M+K]
+=870), detect purity with high performance liquid chromatography (HPLC), purity reaches 98%.
The experiment of embodiment 2 benzyloxycarbonyl group glycyl prolyl amycin (Z-GP-Dox) vitro cytotoxicities
With K562, the MOLT-4 cell inoculation is in 96 orifice plates, density is 10,000/ hole, will contain 0 μ M-2 μ M free amycin or etc. the Z-GP-Dox (solvent is DMSO) of molar concentration be added in the cell, perhaps with K562, the MOLT-4 cell culture medium replaces to the mEF cell culture medium of hatching 24 hours and 48 hours with the Z-GP-Dox of molar concentrations such as above-mentioned free amycin, after 24 hours and 48 hours, detect K562, the cell survival rate of MOLT-4 by mtt assay.Can get from the result, the Z-GP-Dox cytotoxicity reduces 4～7 times (K562 cells) and 9～24 times (MOLT-4 cell) respectively than the IC50 of Dox.And Z-GP-Dox through to can return to the cytotoxicity IC50 similar after the mEF cell is hatched to parent drug, see Table 1.
The cytotoxicity of Z-GP-Dox and Dox compared (IC50) before and after table 1mEF cell was hatched
| Cell strain | Dox (IC50) | Z-GP-Dox (the mEF cell is hatched preceding IC50) | Z-GP-Dox (the mEF cell is hatched back IC50) | |
| |
24 hours | 0.22394μM | 0.960753μM | 0.355691μM |
| 48 hours | 0.04469μM | 0.305835μM | 0.09653μM | |
| MOLT-4 | 24 hours | 0.04665μM | 0.460327μM | 0.05098μM |
| 48 hours | 0.01795μM | 0.440791μM | 0.03879μM | |
The experiment of embodiment 3 benzyloxycarbonyl group glycyl prolyl amycin (Z-GP-Dox) plasma stabilities
Contain 0.675 μ M Z-GP-Dox and containing 20% hepatoma carcinoma cell respectively, among patient with breast cancer's serum and the human normal plasma 6 hours, 12 hours, after 24 hours, to get supernatant and act on the K562 cell, mtt assay detects cell survival rate.The result shows that after 24 hours, cell survival rate still reaches more than 70%, and it is less to show that Z-GP-Dox is cracked into cell toxicant sexupara medicine amycin in serum, at liver cancer patient, is stable in patient with breast cancer and normal person's the serum.See shown in the accompanying drawing 1.
Embodiment 4 benzyloxycarbonyl group glycyl prolyl amycin (Z-GP-Dox) anti-tumor in vivo activity experiments
106 4T1 cells of every subcutaneous vaccination of Balb/C mice, the tumour transplatation tumor-bearing mice is divided into the normal saline group at random, Dox group and Z-GP-Dox group, mice is given and Dox (5mg/kg) and equimolar Z-GP-Dox (7.164mg/kg) respectively, observed the tumor size in continuous 12 days, it is little that the result proves that the amycin after dipeptides is modified is compared the anti-tumor activity variation with female medicine amycin, still to female medicine similar curative effect arranged, and sees tumor growth curve shown in the accompanying drawing 2.
The experiment of embodiment 5 benzyloxycarbonyl group glycyl prolyl amycin (Z-GP-Dox) toxicity in vivo
Press embodiment 4 methods, respectively 1,2,3,4,8,12,17,20 days measurement mice body weight.The body weight change that found that the amycin after dipeptides is modified is little, and amycin is reopened beginning decline at the 8th day body, illustrates that the amycin after modifying reduces than female medicine amycin toxicity.See in drug toxicity shown in the accompanying drawing 3 experiment change curve in the experimental animals.
In sum, the female medicine adriamycin of the adriamycin-dipeptide complexes that preparation method of the present invention prepares is compared, and toxicity reduces; Adriamycin-dipeptide complexes can be had the expressed enzymatic cracking of the MEC (Mouse EmbryoFibroblast, mEF) of similar characteristic with the mesenchyma stroma of tumors cell and be provided cytotoxic adriamycin; Adriamycin-dipeptide complexes is at liver cancer patient, and patient with breast cancer and normal human serum are stable; Transplant mouse model by 4T1 breast cancer and confirm to compare with female medicine adriamycin with the adriamycin-dipeptide complexes that preparation method of the present invention prepares, toxicity reduces, and antitumor curative effect is constant.
Claims (6)
1. the preparation method of an adriamycin-dipeptide complexes is characterized in that may further comprise the steps:
(1) take by weighing N-benzyloxycarbonyl group glycyl proline and be dissolved in N, dinethylformamide adds ethyl chloroformate, stirs under the room temperature, adds triethylamine, and stirring at room obtains the solution of activatory dipeptides; Described ethyl chloroformate and N-benzyloxycarbonyl group glycyl proline mol ratio are 1: 1.2;
(2) doxorubicin hydrochloride is dissolved in and contains the N that waits the triethylamine of amount of substance with it, and dinethylformamide adds activatory two peptide solutions, stirring at room, and the extracted with diethyl ether product, extract makes adriamycin-dipeptide complexes through rotary evaporation; The addition of described activatory two peptide solutions is the N with doxorubicin hydrochloride, mol ratios such as dinethylformamide solution;
(3) adopt column chromatography that adriamycin-dipeptide complexes is carried out purification.
2. according to the preparation method of the described adriamycin-dipeptide complexes of claim 1, it is characterized in that behind the described adding ethyl chloroformate of step (1) that mixing time is 10 minutes under the room temperature; The stirring at room time is 40 minutes behind the described adding triethylamine.
3. according to the preparation method of the described adriamycin-dipeptide complexes of claim 1, it is characterized in that the described stirring at room time of step (2) is 5～6 hours.
4. according to the preparation method of the described adriamycin-dipeptide complexes of claim 1, it is characterized in that the described ether consumption of step (2) be 5 times to the reactant liquor volume.
5. according to the preparation method of the described adriamycin-dipeptide complexes of claim 1, the chromatographic condition that it is characterized in that the described column chromatography of step (3) is conventional post 2.5 * 30cm; Immobile phase is a column chromatography silica gel; Mobile phase is the ethyl acetate-methanol of 7.5: 1 and 5: 1.
6. the application of adriamycin-dipeptide complexes in the preparation antitumor drug that the described preparation method of claim 1 prepares.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1606450A (en) * | 2001-12-21 | 2005-04-13 | 俄罗斯生物技术有限公司 | Polypeptide, the conjugate thereof with doxorubicin and a pharmaceutical composition based thereon |
| CN1973902A (en) * | 2006-12-12 | 2007-06-06 | 东北师范大学 | Anticancer medicine adriamycin composition with ginseng polyse as carrier and its prepn process |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1606450A (en) * | 2001-12-21 | 2005-04-13 | 俄罗斯生物技术有限公司 | Polypeptide, the conjugate thereof with doxorubicin and a pharmaceutical composition based thereon |
| CN1973902A (en) * | 2006-12-12 | 2007-06-06 | 东北师范大学 | Anticancer medicine adriamycin composition with ginseng polyse as carrier and its prepn process |
Non-Patent Citations (3)
| Title |
|---|
| André Trouet, et al..Extracellularly Tumor-activated Prodrugs for theSelectiveChemotherapy of Cancer: Application to DoxorubicinandPreliminary in Vitro and in Vivo Studies.CANCER RESEARCH61.2001,612843-2846. * |
| Gene M. Dubowchik, et al..Cathepsin B-labile Dipeptide Linkers for Lysosomal Release ofDoxorubicin from Internalizing Immunoconjugates: ModelStudies of Enzymatic Drug Release and Antigen-Specific InVitro Anticancer Activity.Bioconjugate Chem.13 4.2002,13(4),855-869. * |
| Scott C. Jeffrey, et al..Dipeptide-based highly potent doxorubicin antibodyconjugates.Bioorganic & Medicinal Chemistry Letters16.2005,16358-362. * |
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