莽草酸衍生物的合成及其对紫杉醇耐药人乳腺癌细胞的逆转作用研究

Study on the Synthesis of Shikimic Acid Derivatives and Their Reversal Effects on Paclitaxel-resistant Human Breast Cancer Cells

目的:对莽草酸进行结构修饰,并探讨其衍生物对紫杉醇耐药人乳腺癌细胞MCF-7/PTX的逆转作用。方法:以莽草酸为先导结构,通过酯化、酰胺化、氢化、还原等方法对其1位羧基进行结构修饰,合成一系列莽草酸衍生物;以非耐药细胞MCF-7为参照,采用MTT法筛选具有抑制活性的衍生物,并同法考察活性衍生物对MCF-7/PTX细胞的半数抑制浓度(IC50)和逆转倍数(RI);以耐药相关蛋白转胶蛋白2为靶点,采用Glide 1.0计算机辅助设计软件将活性衍生物与转胶蛋白2进行分子对接。结果:共合成得到15个衍生物(T1~T15),其中T4~T15为首次合成所得。MTT试验结果显示,(3R,4S,5R)-N-苄基-3,4,5-三羟基-1-环己烯-1-甲酰胺(T7)、(3R,4S,5R)-N-(3,4,5-三羟基-1-环己烯甲基)-苄胺(T14)、(3R,4S,5R)-3,4-O-异亚丙基-5-O-乙酰基-1-环己烯-1-甲酸甲酯(T15)对MCF-7和MCF-7/PTX细胞均具有一定的抑制作用,且各剂量T7、T14、T15+紫杉醇联用组对MCF-7/PTX细胞的IC50值均显著低于阴性对照(紫杉醇单用)组(P<0.05);T14、T15的RI较高,其最高剂量的RI已达8.8、9.3,与阳性对照药物维拉帕米(10.8)相当。分子对接结果显示,T7 3、4位上的羟基可与转胶蛋白2催化区域的Arg625、Asp627形成多个氢键;T14除上述氢键外,其1位上的二级胺侧链还可与转胶蛋白2的Glu657形成氢键;T15母核上的羟基被供电基团衍生化后,其与转胶蛋白2的结合更为紧密,抑制作用有所增强。结论:衍生物T7、T14、T15对紫杉醇耐药人乳腺癌细胞均具有一定的逆转活性。母核上的多羟基结构是莽草酸及其衍生物与转胶蛋白2形成氢键的主要结构区域,对其进行供电基团衍生化或在莽草酸1位羧基引入二级胺、疏水基团等均可有利于增强衍生物的耐药逆转活性。

OBJECTIVE:To structurally modify shikimic acid,and to investigate the reversal effects of its derivatives on paclitaxel-resistant human breast cancer cells MCF-7/PTX. METHODS:Using shikimic acid as the lead structure,1-position carboxyl group was structurally modified to synthesize a series of shikimic acid derivatives through esterification,amidation,hydrogenation and reduction,etc. Using non-drug resistant cells MCF-7 as reference,MTT assay was used to screen derivatives with inhibitory activity as well as half-inhibitory concentration(IC50)and reversal index(RI)of derivatives to MCF-7/PTX. With the drug resistance-related transgelin 2 as the target,the molecular docking of the active derivatives with the drug resistance-related protein was carried out by using Glide 1.0 computer-aided design software. RESULTS:Totally 15 derivatives were obtained(T1-T15),of which T4-T15 were obtained for the first time. MTT assay showed that(3 R, 4 S, 5 R)-N-benzyl-3, 4,5-trihydroxy-1-cyclohexene-1-formamide(T7),(3 R,4 S,5 R)-N-(3,4,5-trihydroxy-1-cyclohexenylmethyl)-benzylamine(T14),(3 R,4 S,5 R)-3,4-O-isopropyl-5-O-acetyl-1-cyclohexene-1-methyl formate(T15) inhibited MCF-7 and MCF-7/PTX cells to a certain extent;IC50 values of T7,T14 and T15 combined with pacliaxel to MCF-7/PTX cells were significantly lower than that in negative control(Paclitaxel alone)group(P<0.05). RIs of T14 and T15 were higher,and RIs of the highest dose were 8.8 and 9.3,which were equivalent to positive control verapamil(10.8). The results of molecular docking showed that the hydroxyl groups at positions 3,4 of T7 could form multiple hydrogen bonds with Arg625 and Asp627 in the catalytic region of transgelin 2. In addition to the hydrogen bond mentioned above at T7,the secondary amine side chain at position 1 of T14 could also form hydrogen bond with Glu657 of transgelin 2. When the hydroxyl group on the T15 mother nucleus was derived from the donor group,the binding of the hydroxyl group to transgelin 2 was closer and the inhibition was enhanced. CONCLUSIONS:The derivatives T7,T14 and T15 have certain reverse activity to paclitaxel-resistant human breast cancer cells. The polyhydroxy structure of the mother nucleus is the main structural region of the hydrogen bond between shikimic acid and its derivatives and transgelin 2. The derivation of its power supply group or the introduction of secondary amines and hydrophobic groups into the1-carboxyl group of shikimic acid is benifit for enhancing the drug resistance reversal effect of derivative.