CN103130854A - Vitamin E succinic acid esterification gemcitabine prodrug and application - Google Patents
Vitamin E succinic acid esterification gemcitabine prodrug and application Download PDFInfo
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- CN103130854A CN103130854A CN2013100368080A CN201310036808A CN103130854A CN 103130854 A CN103130854 A CN 103130854A CN 2013100368080 A CN2013100368080 A CN 2013100368080A CN 201310036808 A CN201310036808 A CN 201310036808A CN 103130854 A CN103130854 A CN 103130854A
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Abstract
The invention discloses a vitamin E succinic acid esterification gemcitabine prodrug which is characterized in that vitamin E succinic acid ester is connected with N4-amino of gemcitabine through an amido bond and the vitamin E succinic acid esterification gemcitabine prodrug has the following formula structure. By means of the vitamin E succinic acid esterification gemcitabine prodrug, the defect that gemcitabine (dFdC) is fast in metabolism is overcome, cycling time of the dFdC is a human body is prolonged, the mode in which the dFdC entering a cell is changed, namely the dFdC no longer enters into the cell relying on a nucleoside transport protein carrier, thus generation of drug resistance is avoided, meanwhile toxic and side effects of the dFdC are reduced furthest, curative effects and bioavailability are improved notably, and the bottleneck that the clinical effect of the dFdC on pancreatic is non-ideal at present is broken.
Description
Technical field
The present invention relates to antitumor drug research and development field, specifically a kind of VE-succinate gemcitabine (VES-dFdC) prodrug and application.
Background technology
Gemcitabine (Gemcitabine, dFdC), chemical name is (+) 2 ‵-deoxidation-2 ‵, 2 ‵-difluocytosine is a kind of novel pyrimidines deoxynucleoside analogue, can be inserted into the site of Deoxyribose cytidine in the DNA chain, and permission guanosine and its pairing, " sheltering " of guanosine makes its reparation that removes of avoiding exoribonuclease, and causes then that the DNA chain is synthetic to be stopped, and then DNA break, natural death of cerebral cells.It is wide that clinical practice proof dFdC has anticancer spectrum, can the characteristics such as synergy be arranged with multiple antitumor drug, is one of medicine commonly used in current combined chemotherapy, especially the malignant tumours such as treatment carcinoma of the pancreas, mammary cancer, nonsmall-cell lung cancer had curative effect preferably.Yet the amino on 4 ' of dFdC is very easily carbonyl and inactivation causes its Half-life in vivo short (only having 32～94 minutes) by the deoxycytidine deaminase metabolism.Therefore treat the needed optimum concn of tumour in order to reach, necessary persistent intravenous injection administration, and this persistence administration not only brings pain to sufferer, has also greatly increased toxic side effect simultaneously, has a strong impact on result for the treatment of.In addition, clinical practice shows: the tumours such as dFdC treatment carcinoma of the pancreas are easy to produce resistance, and the generation of resistance causes result for the treatment of undesirable usually, even treats unsuccessfully.The generation of dFdC resistance and its chemical structure and physico-chemical property are closely related.Water miscible dFdC must enter cell by specific nucleoside transporter carrier, and when this nucleoside transporter disappearance, tumour very easily produces resistance.Therefore, seek new and effectively, low toxic side effect has the dFdC prodrug of hypertonicity to become the effective measure that improve its therapeutic index to tumor tissues or cell.
For water-soluble anti-tumor medicine, it is carried out not only can improving drug molecule physico-chemical property in vivo after the lipid transformation, pharmacokinetics and pharmacodynamic property, and because most of film moiety in its structure and body is similar, make it have better membrane permeation ability; In addition, tumor growth needs a large amount of lipids as energy derive, and this just makes the prodrug after lipid can selective aggregation in tumor tissues, thereby produces result for the treatment of better, and reduces the toxic side effect of normal tissue.Patent (CN 102617679 A) is modified the amino of dFdC with conjugated linolic acid, discovery can by changing fat-soluble its cross-cell membrane transporting mechanism that affects of dFdC, overcome to reach the resistance that dFdC causes because of the nucleoside transporter disappearance.Zhengrong Cui etc. get stearic acidifying gemcitabine with the amino of dFdC with Stearate Modified, and further make solid lipid nanoparticle, find that stearic acidifying dFdC nanoparticle has good inhibition growth activity (Journal of Controlled Release to the lung cancer in nude mice tumour, 157 (2012), 132 – 140).
Vitamin-E is a kind of liposoluble vitamin, and its derivative VE-succinate (VES) has the effect of inducing the kinds of tumor cells apoptosis.It is worth mentioning that, VES has good antitumor selectivity, and in kill cancer cell, normal tissue can not produce toxic side effect.
Summary of the invention
The gemcitabine prodrug that the purpose of this invention is to provide a kind of lipid, namely for the deficiency of dFdC and existing dFdC prodrug, and take into full account the mechanism that it produces anti-tumor activity, design, synthesized VE-succinate gemcitabine amide (VES-dFdC) prodrug, and with the medicine of this prodrug as malignant tumours such as preparation treatment carcinoma of the pancreas.
The object of the present invention is achieved like this:
A kind of VE-succinate gemcitabine (VES-dFdC) prodrug, characteristics are: VE-succinate (VES) is connected by amido linkage with the N4 bit amino of gemcitabine (dFdC), and it has following structure:
The preparation method of a kind of VE-succinate gemcitabine (VES-dFdC) prodrug, the method comprises following concrete steps:
A) under the nitrogen protection, get vitamin-E amber ester, triethylamine is dissolved in anhydrous tetrahydro furan, isobutyl chlorocarbonate is dissolved in tetrahydrofuran (THF) is added dropwise to reaction system ,-15 ℃ are stirred 10～30 min and form the mixed acid anhydride solution; Wherein: the molar ratio of vitamin-E amber ester, isobutyl chlorocarbonate and triethylamine is 1:1:1.2, and the peak optimization reaction concentration of vitamin-E amber ester is 20-200 mM;
B) gemcitabine is dissolved in anhydrous DMF, is added dropwise to mixed acid anhydride solution, stirring at room 48～72 h under nitrogen protection in-15 ℃; Wherein: the molar ratio of gemcitabine and vitamin-E amber ester is 1:0.5～2, and the peak optimization reaction concentration of gemcitabine is 20-200 mM;
C) with step b) reaction solution concentrated, crude product is respectively with saturated sodium bicarbonate and pure water washing, and use ethyl acetate extraction, with the organic phase merging, adds anhydrous MgSO
4Fully dry, column chromatography is purified, and rotary evaporation is removed solvent, and vacuum-drying obtains described VE-succinate gemcitabine (VES-dFdC) prodrug; Wherein: the eluent that column chromatography uses is the mixed solvent of ethyl acetate and sherwood oil, and the volume ratio of ethyl acetate and sherwood oil is 1～3:1.
The application of a kind of VE-succinate gemcitabine (VES-dFdC) prodrug, characteristics are the application on preparation treatment carcinoma of the pancreas, mammary cancer, nonsmall-cell lung cancer, bladder cancer, leukemia medicament of this prodrug.
Prodrug of the present invention is dissolved in ethylene glycol ethyl ether according to prior art with the VES-dFdC prodrug, adds tensio-active agent, makes faint yellow settled solution; This solution is made injection with the glucose injection dilution of physiological saline or 5%.
VE-succinate gemcitabine of the present invention (VES-dFdC) prodrug, improved the fast defective of dFdC metabolism, extended its cycling time in vivo, change the pattern that it enters cell, no longer rely on the nucleoside transporter carrier and enter cell, therefore overcome the generation of resistance, reduce to greatest extent its toxic side effect simultaneously, significantly improve curative effect and bioavailability, broken through the undesirable bottleneck of present dFdC treatment carcinoma of the pancreas clinical effectiveness.
Description of drawings
Fig. 1 is the chemical structural drawing of prodrug of the present invention;
Fig. 2 is the nuclear magnetic spectrogram of prodrug of the present invention;
Fig. 3 is the infrared spectrum of prodrug of the present invention;
Fig. 4 is the cytotoxicity figure of prodrug VES-dFdC of the present invention;
Fig. 5 is that prodrug injection of the present invention is to the influence curve figure of transplanted tumor volume; Wherein: curve 1 is the physiological saline group; Curve 2 is commercially available GEMCITABINE HYDROCHLORIDE group; Curve 3 is experiment synthesizing gemcitabine hydrochloride group; Curve 4 is VES-dFdC group of the present invention;
Fig. 6 is that prodrug injection of the present invention is to the influence curve figure of the relative knurl volume of transplanted tumor; Wherein: curve 1 is the physiological saline group; Curve 2 is commercially available GEMCITABINE HYDROCHLORIDE group; Curve 3 is experiment synthesizing gemcitabine hydrochloride group; Curve is VES-dFdC group of the present invention;
Fig. 7 is that prodrug injection of the present invention is to the experimental therapy action effect figure of transplanted tumor; Wherein: control group is the physiological saline group; The G positive is commercially available GEMCITABINE HYDROCHLORIDE group; G is tested for testing synthesizing gemcitabine hydrochloride group; VE-G is VES-dFdC group of the present invention.
Embodiment
The below further illustrates the present invention with embodiment, but the present invention is not limited to this.
Synthesizing of embodiment 1, VES-dFdC prodrug
Get VES(530 mg, 1mmol), triethylamine (0.17 mL, 1.2 mmol) is dissolved in 15 mL anhydrous tetrahydro furans (THF), is cooled to-15 ℃.After homo(io)thermism, 0.13 mL isobutyl chlorocarbonate is dissolved in 5 mL THF is added dropwise in reaction system ,-15 ℃ of reaction 30 min under nitrogen protection.Get dFdC(263mg, 1mmol) be dissolved in 15 mL DMFs (DMF), dropwise add reaction system.After dropwising, stirring at room 72 h.Reaction solution is concentrated, obtain the pale yellow oily liquid body, successively wash for several times ethyl acetate extraction, combined ethyl acetate phase, anhydrous MgSO with saturated sodium bicarbonate solution and pure water
4Fully dry, filter, concentrated, obtain white sticky solid.Take ethyl acetate and sherwood oil as eluent, column chromatography separates that to obtain clean product be the white solid powder, productive rate: 53%, and liquid chromatography purity is 99.4%; Structure and structural characterization are seen Fig. 1, Fig. 2 and Fig. 3.
To be in logarithmic phase BxPC-3 cell suspension in the fresh RPMI1640 substratum that contains 1% pair of anti-and 10% calf serum, and be inoculated in 96 orifice plates (5 * 10
3Individual cells/well), in 37 ℃ with contain the CO of 5% concentration
2Cultivate 24 h in cell culture incubator, this nutrient solution of reject, every hole add the dFdC that is dissolved with different concns or the fresh culture 100 μ L of VES-dFdC, after continuing to cultivate 24 h, every hole adds the phosphate buffer solution 10 μ L that are dissolved with 5 mg/mL tetrazolium bromides again, at 37 oC and 5% CO
2Continue to cultivate 4 h under the environment of concentration, the sucking-off nutrient solution, the methyl-sulphoxide Rong Xie formazan that adds 100 μ L in every hole adopts microplate reader (wavelength 570 nm place) to measure the absorbancy (OD value) of Gai formazan solution, and uses the relative survival rate of following formula calculating BxPC-3 cell.
Cell survival rate (%)=(OD
Experimental group/ OD
Control group) * 100%
It the results are shown in Figure 4.
24 uniform nude mices of tumor growth are divided into 4 groups at random: (1) Control group, 6, weekly tail vein injection gives 0.9% NaCl solution 0.1 mL/10g; (2) the positive group of gemcitabine (listing medicine) (50 mg/kg), 6, weekly tail vein injection gives Jixitabin solution 0.1 mL/10g of 5.0 mg/mL; (3) tested group of gemcitabine (50 mg/kg), 6, weekly tail vein injection gives tested solution 0.1 mL/10g of gemcitabine of 5 mg/ml; (4) VES-dFdC injection group (50 mg/kg), 6, weekly tail vein injection gives VES-dFdC injection 0.1 mL/10g of 5 mg/mL.Measure weekly gross tumor volume and body weight twice, weighed in the 15th day, measure gross tumor volume, get knurl piece weighing knurl after the execution nude mouse and weigh, calculate relative tumour volume (RTV), relative tumour appreciation rate (T/C) and tumor suppression percentage, do statistical analysis.Be calculated as follows gross tumor volume V:
V?=?3.14?(a?×?b
2)?/?6
A in formula, b are respectively the minimum and maximum size of tumour, the results are shown in Figure 5, Fig. 6 and Fig. 7.
Claims (3)
1. a VE-succinate gemcitabine prodrug, is characterized in that VE-succinate is connected by amido linkage with the N4 bit amino of gemcitabine, and it has following structure:
2. the preparation method of the described prodrug of claim 1 is characterized in that the method comprises following concrete steps:
A) under the nitrogen protection, get vitamin-E amber ester, triethylamine is dissolved in anhydrous tetrahydro furan, isobutyl chlorocarbonate is dissolved in anhydrous tetrahydro furan is added dropwise to reaction system ,-15 ℃ are stirred 10～30 min and form the mixed acid anhydride solution; Wherein: the molar ratio of vitamin-E amber ester, isobutyl chlorocarbonate and triethylamine is 1:1:1.2, and the reaction density of vitamin-E amber ester is 20～200 mM;
B) gemcitabine is dissolved in anhydrous DMF, is added dropwise to mixed acid anhydride solution, stirring at room 48～72 h under nitrogen protection in-15 ℃; Wherein: the molar ratio of gemcitabine and vitamin-E amber ester is 1:0.5～2, and the reaction density of gemcitabine is 20～500 mM;
C) with step b) reaction solution concentrated, crude product is respectively with saturated sodium bicarbonate and pure water washing, and use ethyl acetate extraction, with the organic phase merging, adds anhydrous MgSO
4Fully dry, column chromatography is purified, and rotary evaporation is removed solvent, and vacuum-drying obtains described VE-succinate gemcitabine prodrug; Wherein: the eluent that column chromatography uses is the mixed solvent of ethyl acetate and sherwood oil, and the volume ratio of ethyl acetate and sherwood oil is 1～3:1.
3. the application of the described VE-succinate gemcitabine of claim 1 prodrug is characterized in that the application of this prodrug on preparation treatment carcinoma of the pancreas, mammary cancer, nonsmall-cell lung cancer, bladder cancer and leukemia medicament.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8956613B2 (en) | 2012-11-13 | 2015-02-17 | BoYen Therapeutics, Inc. | Gemcitabine prodrugs and uses thereof |
| CN105001291A (en) * | 2014-04-15 | 2015-10-28 | 上海知萌生物医药科技有限公司 | Gemcitabine chemical transfer prodrug, preparation method and applications thereof |
| EP3204004A4 (en) * | 2014-10-08 | 2018-05-30 | Epigenetics Pharma LLC | Vitamin e-nucleoside prodrugs |
| CN108341798A (en) * | 2017-01-23 | 2018-07-31 | 沈阳药科大学 | Rotigotine derivative and its preparation and application |
| CN109152791A (en) * | 2016-02-26 | 2019-01-04 | 表观遗传学药业有限责任公司 | The method of -2 ' of the fluoro- 5- azepine of 2 ', 2 '-two-deoxycytidine or its prodrug treatment TP53 wild type tumor |
| CN113307838A (en) * | 2021-05-11 | 2021-08-27 | 中国医学科学院医药生物技术研究所 | Hybrid molecular compound and application thereof in treating tumors |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8956613B2 (en) | 2012-11-13 | 2015-02-17 | BoYen Therapeutics, Inc. | Gemcitabine prodrugs and uses thereof |
| US9540410B2 (en) | 2012-11-13 | 2017-01-10 | BoYen Therapeutics, Inc. | Gemcitabine prodrugs and uses thereof |
| US9890189B2 (en) | 2012-11-13 | 2018-02-13 | BoYen Therapeutics, Inc. | Gemcitabine prodrugs and uses thereof |
| CN105001291A (en) * | 2014-04-15 | 2015-10-28 | 上海知萌生物医药科技有限公司 | Gemcitabine chemical transfer prodrug, preparation method and applications thereof |
| CN105001291B (en) * | 2014-04-15 | 2018-12-04 | 上海知萌生物医药科技有限公司 | Gemcitabine chemistry transmits prodrug and its preparation method and application |
| EP3204004A4 (en) * | 2014-10-08 | 2018-05-30 | Epigenetics Pharma LLC | Vitamin e-nucleoside prodrugs |
| US10071112B2 (en) | 2014-10-08 | 2018-09-11 | Epigenetics Pharma Llc | Vitamin E-nucleoside prodrugs |
| CN109152791A (en) * | 2016-02-26 | 2019-01-04 | 表观遗传学药业有限责任公司 | The method of -2 ' of the fluoro- 5- azepine of 2 ', 2 '-two-deoxycytidine or its prodrug treatment TP53 wild type tumor |
| EP3419631A4 (en) * | 2016-02-26 | 2019-12-04 | Epigenetics Pharma, LLC | Method of treatment of tp53 wild-type tumors with 2',2'-difluoro-5-aza-2'-deoxycytidine or prodrugs thereof |
| CN108341798A (en) * | 2017-01-23 | 2018-07-31 | 沈阳药科大学 | Rotigotine derivative and its preparation and application |
| CN108341798B (en) * | 2017-01-23 | 2021-05-25 | 沈阳药科大学 | Rotigotine derivative and preparation and application thereof |
| CN113307838A (en) * | 2021-05-11 | 2021-08-27 | 中国医学科学院医药生物技术研究所 | Hybrid molecular compound and application thereof in treating tumors |
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