CBZ-AAN-Dox新前药的计算机辅助设计及合成与活性检测

Studies on a novel doxorubicin prodrug with computer-aided design, synthesis and activity detection

目的探究一种以多柔比星为母药的低毒新型抗肿瘤前药。方法采用Sybyl 8.0的Sketch模块进行前药的计算机辅助设计,通过多种短肽共价偶联多柔比星形成前药库。虚拟筛选后用化学方法合成前药,通过硅胶柱和凝胶柱进行纯化,HPLC法检测与鉴定,体外特异性酶解试验及细胞毒性试验进行活性检测。结果计算机辅助药物设计所得到的新型前药N-苄氧羰基-丙氨酰-丙氨酰-天冬酰胺-多柔比星物理及化学参数较好,其化学合成产物纯度为94%,其天冬酰胺内肽酶酶解Km和Vmax分别为(68±7)μmol/L,(3.9±0.6)μmol/(L·s),对2种体外培养肿瘤细胞株的毒性作用约降低了约90%。结论计算机辅助药物设计在指导多柔比星前药合成过程中具有参考意义;前药合成与分离纯化方法简单可靠,分离效果好;新合成前药细胞毒性降低,可被天冬酰胺内肽酶酶解。

Objective To explore a novel prodrug of short peptides covalently bound doxorubicin with reduced toxicity. Methods Sybyl 8.0 Sketch module was used for the targeting prodrug design, it was synthesized and purified by silica gel column. HPLC was used for the purification detection and identification. Specific enzyme activity detection tests and in vitro cytotoxicity were determined. Results A novel prodrug of N-benzyloxycarbonyl-alanyl- alanyl-Asn-doxorubicin with better physical and chemical parameters was obtained by the computer-aided drug design and then chemically synthesized. The purity was 94%. The prodrug asparagine endopeptidase digestion K and Vm~ were （68＋7）gmol/L, （3.9＋0.6）~unol/（L＇s） respectively. The toxicity of two kinds of tumor cell lines K562 and MCF- 7 in vitro were reduced by approximately 90%. Conclusion Computer-aided drug design is helpful to doxorubicin prodrug in guiding the synthesis process with a reference value. The prodrug synthesis and purification method is simple and reliable. The novel prodrug has lower cytotoxic and can be digested by asparagine endopeptidase enzyme.