新的核苷类化合物β-L-D4A的化学合成及体外抗HBV作用

Synthesis of a novel L-nucleoside , β-L-D4A and its inhibition on the replication of hepatitis B virus in vitro

目的以D型谷氨酸为原料,通过一系列化学转化,合成了新的核苷类化合物βLD4A,并初步探索其体外抗HBV作用。方法合成βLD4A,用红外光谱、核磁共振氢谱和质谱确证目标化合物的结构,以2.2.15细胞(HepG2细胞进行HBV基因组转染后所得)培养为基础,Southern印迹法检测不同浓度化合物体外抑制HNVDNA复制作用,并求出50%抑制的药物浓度,即EC50。以四噻唑蓝(MTT)比色分析法检测不同浓度药物的细胞毒性,求出IC50。结果化合物βLD4A经红外光谱、核磁共振氢谱和质谱确证;2.2.15细胞培养上清液病毒DNA的Southern印迹、自显影结果显示病毒的抑制呈明显的浓度依赖性,计算出EC50为0.2μmol·L-1,胞内DNA的Southern印迹、自显影显示类似的结果;细胞毒性实验显示IC50为200μmol·L-1。结论体外实验显示βLD4A具有明显的抑制病毒DNA复制作用,且无明显的细胞毒性,TI值为1000,高于临床用Lamivudine(750),有望开发为临床抗HBV用药。

Aim Nucleoside analogues have become the most promising candidates of anti-HBV drugs. In this study, β-L-D4A was synthesized and explored its inhibitiory action against hepatitis B virus （HBV） in 2.2.15 cells derived from HepG2 cells transfected with HBV genome. Methods β-L-D4A was stereo-controlled synthesized from D-glutamic acid, and the structure was identified by IR, ^1H NMR and MS. 2.2.15 Cells were placed at a density of 5 × 10^4 per well in 12-well tissue culture plates, and treated with various concentrations of β-L-D4A for 6 days. At the end, medium was processed to obtain virions by a polyethlene glycol precipitation method. At the same time, intracellular DNA was also extracted and digested with Hind III. Both of the above DNA were subjected to Southern blot, hybridized with a ^32P-labeled HBV probe and autoradiographed. The intensity of the autoradiographic bands was quantitated by densitometric scans of computer and EC50 was calculated. 2.2.15 cells were also seeded in 24-well tissue culture plates, and cytotoxicity with different concentrations was examined by MTT method. IC50 was calculated. Results The synthesized compound structure conformed with β-L-D4A; Autoradiographic bands showed similar for supernatant and intracellular HBV DNA. Episomal HBV DNA wasinhibited in a dose-dependent manner. EC50 0. 2μmol · L^-1 The experiment of cytotoxicity gained IC50 200μmol · L^-1 Conclusion β-L-D4A has been synthesized successfully. β-L-D4A possessed potent inhibitory effect on replication of HBV in vitro with low cytotoxicity, TI value was 1 000. It is expected to be developed clinically into a new anti-HBV drug.