Preparation method and application of connected conjugated linoleic acid and gemcitabine prodrug

A technology of conjugated linoleic acid and gemcitabine, which is applied in the field of preparation of the prodrug, can solve the problems of easy drug resistance of tumors, increase of toxic and side effects, and influence of clinical curative effect, and achieve excellent anti-tumor therapeutic effect, reduce Effects of Toxicity and Resistance, Strong Antitumor Efficacy

Active Publication Date: 2012-08-01
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the presence of deoxycytidine deaminase in the blood will deactivate the 4'-amino group of gemcitabine, so that the half-life of gemcitabine in vivo is very short (8-17min), and continuous intravenous administration is necessary to maintain its toxicity to cancer cells , this dose-limiting toxicity affects clinical efficacy and greatly increases toxic side effects[1]
At the same time, water-soluble gemcitabine can only enter tumor cells through specific nucleic acid transporters. When the nucleic acid transporter is lacking, the tumor is prone to drug resistance[2]

Method used

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  • Preparation method and application of connected conjugated linoleic acid and gemcitabine prodrug
  • Preparation method and application of connected conjugated linoleic acid and gemcitabine prodrug
  • Preparation method and application of connected conjugated linoleic acid and gemcitabine prodrug

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Conjugated linoleic acid and gemcitabine-linked prodrug (CLA-GEM conjugate) Conjugated linoleic acid (0.50g, 1.78mmol) and triethylamine (0.20g, 2.0mmol) were dissolved in 3ml In tetrahydrofuran, keeping the temperature at -10°C, ethyl chloroformate (0.19 g, 1.78 mmol) was added dropwise with stirring, and stirred at -15°C for 30 minutes after the dropwise addition. Gemcitabine hydrochloride (0.53g, 1.78mmol) and triethylamine (0.20g, 2.0mmol) were then dissolved in 5ml of N,N-dimethylformamide, and added dropwise to the above-mentioned conjugated linoleic acid solution at -15°C , the reaction solution was stirred at room temperature for 72 hours under nitrogen protection, and dried in vacuo. Take the material obtained from the above reaction, add 50ml of saturated aqueous sodium bicarbonate solution and ethyl acetate solution to wash with water, collect the ethyl acetate layer, dry it in vacuum, separate it with a silica gel column, and use gradient elution. ...

Embodiment 2

[0036] Example 2 Confirmation of the structure of the prodrug (CLA-GEM conjugate) linked to conjugated linoleic acid and gemcitabine

[0037] The structure of the prepared prodrug (CLA-GEM conjugate) was verified by 400MHz 1H-NMR (Bruker AVANCEIII 400, Germany).

[0038] 1H NMR (400MHz, d-DMSO, δppm)

[0039] δ10.97 (1H, s, NHCO), 8.24 (1H, d, J=7.6Hz, 6-CH), 7.29 (1H, d, J=7.6Hz, 5-CH), 6.30-6.31 (1H, m , CH=CH), 6.28 (1H, m, CH=CH), 6.15 (1H, t, J=7.6Hz, 1'-CH), 5.89 (1H, m, CH=CH), 5.65 (1H, m , CH=CH), 4.18 (1H, m, 3'-CH) 3.63-3.90 (3H, m, 4'-CH and 5'-CH2), 2.398 (2H, t, J=7.6, CO-CH2) , 2.1 (4H, m, 2CH2), 1.2-1.5 (18H, m, CH2), 0.88 (3H, t, CH3)

Embodiment 3

[0040] Example 3 Evaluation of prodrug (CLA-GEM conjugate) in vitro cytotoxicity and cell membrane transport mechanism

[0041] Using ammonium thiocyanate B (SulforhodamineB) (SRB) determination method. MCF-7 human breast cancer cells were inoculated in a 96-well plate (2500 cells / well), and after incubation for 24 hours, the gemcitabine hydrochloride aqueous solution and the DMSO solution of the CLA-GEM conjugate were diluted into drug solutions of different concentrations, and added Into the tumor cells, 6 wells per sample, continue to incubate for 24 hours, discard the medium, fix the cells with 10% trichloroacetic acid at 4°C for 1 hour, rinse with purified water, dry and rinse with 0.4% SRB at 4°C After staining for 30 minutes, take it out and wash it with 0.1% acetic acid, add 10uM Tris solution after drying, shake it on a shaker for 30 minutes, and measure the absorbance at 540nm with a microplate reader.

[0042] The research results showed that after 24 hours, 5uM CL...

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PUM

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Abstract

The invention relates to a preparation method and application of a connected conjugated linoleic acid and gemcitabine prodrug. The conjugated linoleic acid is connected with the gemcitabine via an amide bond. The preparation method comprises the following steps that: under the protection of nitrogen, the conjugated linoleic acid is dissolved in a solvent, and an activator is added at a low temperature of -15 DEG C under stirring to activate the conjugated linoleic acid to form a highly reactive mixed acid anhydride; the gemcitabine is dissolved in a solvent to be added dropwise to the mixed acid anhydride, and the reaction is undergone under stirring at room temperature; the solvent refers to one or a mixed solution of triethylamine, tetrahydrofuran, N,N-dimethyl formamide, and the activator is ethyl chloroformate; the reaction time is 2-72 hours, and the feed molar ratio of the gemcitabine to the conjugated linoleic acid is 1:(0.5-5); and the molar ratio of the activator to the conjugated linoleic acid is 1:(0.5-5). The object of the invention is to increase the anti-tumor efficacy of the gemcitabine.

Description

technical field [0001] The invention relates to a prodrug in which conjugated linoleic acid is linked to the amino group at the N4 position of gemcitabine through an amide bond. Compared with gemcitabine, the prodrug of the invention has better antitumor activity. The present invention also relates to the preparation method of said prodrug. Background of the invention [0002] Gemcitabine (gemcitabine, GEM) is a new type of pyrimidine deoxynucleoside analogs, its hydrochloride is commonly used, its chemical name is (+) 2'-deoxy-2'2'-difluorocytosine hydrochloride, through The anti-metabolism effect exerts the anti-tumor effect, mainly acts on the DNA synthesis phase, and can prevent cells from progressing from the G1 phase to the S phase under certain conditions. Clinically, it is often used in combination with other drugs or administered alone as the first-line drug for breast cancer, non-small cell lung cancer, bladder cancer, and pancreatic cancer. However, the presence...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H19/073C07H1/00A61K31/7068A61P35/00
Inventor 王坚成张强陶小妹张烜
Owner PEKING UNIV
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