CN103130854A - Vitamin E succinic acid esterification gemcitabine prodrug and application - Google Patents

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

Prodrug of vitamin E succinate gemcitabine and its application

technical field

The invention relates to the field of research and development of antitumor drugs, in particular to a prodrug of vitamin E succinate gemcitabine (VES-dFdC) and its application.

Background technique

Gemcitabine (Gemcitabine, dFdC), the chemical name is (+) 2‵-deoxy-2‵, 2‵-difluorocytosine, is a new type of pyrimidine deoxynucleoside analogs, which can be inserted into the DNA chain deoxycytidine The site of guanosine and allows guanosine to pair with it, and the "masking" of guanosine prevents it from being removed and repaired by exoribonuclease, which then leads to the cessation of DNA chain synthesis, resulting in DNA fragmentation and cell apoptosis. Clinical practice has proved that dFdC has a broad anti-cancer spectrum and can have synergistic effects with a variety of anti-tumor drugs. It is one of the commonly used drugs in combination chemotherapy, especially for the treatment of malignant tumors such as pancreatic cancer, breast cancer, and non-small cell lung cancer. Have better curative effect. However, the amino group at the 4' position of dFdC is easily metabolized to a carbonyl group by deoxycytidine deaminase and inactivated, resulting in a short half-life in vivo (only 32-94 minutes). Therefore, in order to achieve the optimal concentration required for the treatment of tumors, continuous intravenous administration is necessary, and this continuous administration not only brings pain to patients, but also greatly increases toxic and side effects, seriously affecting the therapeutic effect. In addition, clinical practice has shown that: dFdC treatment of pancreatic cancer and other tumors is prone to drug resistance, and the generation of drug resistance usually leads to unsatisfactory treatment effects or even treatment failure. The generation of dFdC drug resistance is closely related to its chemical structure and physical and chemical properties. Water-soluble dFdC must enter cells through a specific nucleoside transporter carrier. When this nucleoside transporter is missing, tumors are prone to drug resistance. Therefore, finding new dFdC prodrugs with high efficiency, low toxicity and high permeability to tumor tissues or cells has become an effective measure to improve their therapeutic index.

For water-soluble antineoplastic drugs, lipid modification can not only improve the physical and chemical properties, pharmacokinetics and pharmacodynamic properties of drug molecules in vivo, but also because of their structure and most of the membrane components in the body Similarly, it has better membrane penetration; in addition, tumor growth requires a large amount of lipids as an energy source, which allows lipidated prodrugs to selectively aggregate in tumor tissues, resulting in better therapeutic effect, and reduce the toxic side effects on normal tissues. The patent (CN 102617679 A) uses conjugated linoleic acid to modify the amino group of dFdC, and found that it can change the lipid solubility of dFdC to affect its transmembrane transport mechanism, so as to overcome the drug resistance of dFdC caused by the lack of nucleoside transporter. Zhengrong Cui et al. modified the amino group of dFdC with stearic acid to obtain stearated gemcitabine, and further made solid lipid nanoparticles, and found that stearated dFdC nanoparticles had good growth inhibitory activity on lung cancer tumors in nude mice (Journal of Controlled Release, 157 (2012), 132–140).

Vitamin E is a fat-soluble vitamin, and its derivative vitamin E succinate (VES) can induce apoptosis of various tumor cells. It is worth mentioning that VES has good anti-tumor selectivity, while killing cancer cells, it will not produce toxic side effects on normal tissues.

Contents of the invention

The purpose of the present invention is to provide a lipidated gemcitabine prodrug, which aims at dFdC and the deficiencies of existing dFdC prodrugs, and fully considers the mechanism of its anti-tumor activity, designs and synthesizes vitamin E succinate The gemcitabine amide (VES-dFdC) prodrug is used as a drug for the preparation of malignant tumors such as pancreatic cancer.

The purpose of the present invention is achieved like this:

A prodrug of vitamin E succinate gemcitabine (VES-dFdC), which is characterized in that vitamin E succinate (VES) is connected to the N4 amino group of gemcitabine (dFdC) through an amide bond, and has the following structure:

 

 

A preparation method of vitamin E succinate gemcitabine (VES-dFdC) prodrug, the method comprising the following specific steps:

a) Under nitrogen protection, dissolve vitamin E succinate and triethylamine in anhydrous tetrahydrofuran, dissolve isobutyl chloroformate in tetrahydrofuran and add dropwise to the reaction system, and stir at -15°C for 10 to 30 minutes to form a mixed anhydride solution; : The molar ratio of vitamin E succinate, isobutyl chloroformate and triethylamine is 1:1:1.2, and the optimal reaction concentration of vitamin E succinate is 20-200 mM;

b) Dissolve gemcitabine in anhydrous N,N-dimethylformamide, add the mixed anhydride solution dropwise at -15°C, and stir at room temperature for 48-72 h under nitrogen protection; wherein: the molar ratio of gemcitabine to vitamin E succinate The optimal reaction concentration of gemcitabine is 20-200 mM;

c) Concentrate the reaction solution of step b), wash the crude product with saturated sodium bicarbonate and pure water respectively, and extract with ethyl acetate, combine the organic phases, add anhydrous _MgSO4 fully dry, purify by column chromatography, spin The solvent was evaporated and dried in vacuo to obtain the vitamin E succinate-esterified gemcitabine (VES-dFdC) prodrug; wherein: the eluent used in column chromatography was a mixed solvent of ethyl acetate and petroleum ether, ethyl acetate The volume ratio with petroleum ether is 1-3:1.

The application of a prodrug of vitamin E succinate gemcitabine (VES-dFdC) is characterized in that the prodrug is used in the preparation of drugs for treating pancreatic cancer, breast cancer, non-small cell lung cancer, bladder cancer and leukemia.

According to the prior art, the prodrug of the present invention dissolves the VES-dFdC prodrug in ethylene glycol ether, adds a surfactant, and makes a light yellow clear solution; the solution is diluted with normal saline or 5% glucose injection to prepare Injection.

The vitamin E succinate gemcitabine (VES-dFdC) prodrug of the present invention improves the defect of fast metabolism of dFdC, prolongs its circulation time in the body, changes its mode of entering cells, and no longer relies on nucleoside transporter carriers Entering cells, thus overcoming the generation of drug resistance, while minimizing its toxic and side effects, significantly improving the curative effect and bioavailability, breaking through the bottleneck of the current unsatisfactory clinical effect of dFdC in the treatment of pancreatic cancer.

Description of drawings

Fig. 1 is the chemical structure figure of prodrug of the present invention;

Fig. 2 is the NMR spectrum of prodrug of the present invention;

Fig. 3 is the infrared spectrogram of prodrug of the present invention;

Fig. 4 is the cytotoxicity figure of prodrug VES-dFdC of the present invention;

Fig. 5 is the influence curve figure of prodrug injection of the present invention on transplanted tumor volume; Wherein: Curve 1 is normal saline group; Curve 2 is commercially available gemcitabine hydrochloride group; Curve 3 is experimentally synthesized gemcitabine hydrochloride group; Curve 4 is the VES-dFdC group of the present invention;

Fig. 6 is a graph showing the influence of the prodrug injection of the present invention on the relative tumor volume of transplanted tumors; wherein: curve 1 is the normal saline group; curve 2 is the commercially available gemcitabine hydrochloride group; curve 3 is the experimentally synthesized gemcitabine hydrochloride group ; The curve is the VES-dFdC group of the present invention;

Fig. 7 is the effect diagram of the experimental therapeutic effect of the prodrug injection of the present invention on transplanted tumors; wherein: the control group is the normal saline group; G positive is the commercially available gemcitabine hydrochloride group; G is tested as the experimentally synthesized gemcitabine hydrochloride group; VE-G is the VES-dFdC group of the present invention.

Detailed ways

Further illustrate the present invention with embodiment below, but the present invention is not limited thereto.

Embodiment 1, the synthesis of VES-dFdC prodrug

Dissolve VES (530 mg, 1 mmol) and triethylamine (0.17 mL, 1.2 mmol) in 15 mL of anhydrous tetrahydrofuran (THF) and cool to -15°C. After the temperature was constant, 0.13 mL of isobutyl chloroformate dissolved in 5 mL of THF was added dropwise into the reaction system, and reacted at -15°C for 30 min under nitrogen protection. Dissolve dFdC (263 mg, 1 mmol) in 15 mL N, N-dimethylformamide (DMF), and add it to the reaction system dropwise. After the dropwise addition, it was stirred at room temperature for 72 h. Concentrate the reaction solution to obtain a light yellow oily liquid, wash it several times with saturated sodium bicarbonate solution and pure water, extract with ethyl acetate, combine the ethyl acetate phases, fully dry with anhydrous MgSO ₄ , filter, and concentrate to obtain a white viscous Thick solid. Using ethyl acetate and petroleum ether as eluents, the pure product was separated by column chromatography as a white solid powder, with a yield of 53% and a liquid chromatography purity of 99.4%; the structure and structural characterization are shown in Figure 1, Figure 2 and image 3.

Example 2, VES-dFdC cytotoxicity

BxPC-3 cells in the logarithmic growth phase were suspended in fresh RPMI1640 medium containing 1% double antibody and 10% calf serum, and seeded in 96-well plates (5×10 ³ cells/well), Cultivate at 37°C for 24 h in a CO ₂ cell incubator containing 5% concentration, discard the culture medium, add 100 μL of fresh medium dissolved in different concentrations of dFdC or VES-dFdC to each well, and continue to culture for 24 h Add 10 μL of phosphate buffer solution containing 5 mg/mL thiazolyl blue to each well, continue to incubate for 4 h at 37 oC and 5% CO2 concentration, suck out the _culture medium, and add 100 μL of dimethylformazol to each well Dissolve formazan in sulfoxide, measure the absorbance (OD value) of the formazan solution with a microplate reader (at a wavelength of 570 nm), and use the following formula to calculate the relative survival rate of BxPC-3 cells.

Cell survival rate (%) = (OD _{experimental group} /OD _{control group} ) × 100%

The results are shown in Figure 4.

Example 3, Evaluation of VES-dFdC Injection Inhibiting Pancreatic Cancer Growth Activity

24 nude mice with uniform tumor growth were randomly divided into 4 groups: (1) Control group, 6 mice were given 0.9% NaCl solution 0.1 mL/10g by tail vein injection once a week; (2) Gemcitabine (marketed drug) positive group (50 mg/kg), 6 rats, administered 5.0 mg/mL gemcitabine solution 0.1 mL/10g by tail vein injection once a week; (3) Gemcitabine test group (50 mg/kg), 6 rats, once a week Intravenous injection of 5 mg/ml gemcitabine test solution 0.1 mL/10g; (4) VES-dFdC injection group (50 mg/kg), 6 rats, 5 mg/mL VES-dFdC by tail vein injection once a week Injection 0.1 mL/10g. The tumor volume and body weight were measured twice a week, and the body weight was weighed on the 15th day to measure the tumor volume. After the nude mice were sacrificed, the tumor block was taken to weigh the tumor weight, and the relative tumor volume (RTV) and relative tumor growth rate (T/ C) and tumor inhibition percentage, for statistical analysis. The tumor volume V was calculated according to the following formula:

V = 3.14 (a × b ² ) / 6

In the formula, a and b are the largest and smallest size of the tumor, respectively, and the results are shown in Fig. 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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