抗肿瘤番荔枝内酯维生素偶联物的合成及其受体介导靶向输送作用

Annonaceous acetogenins (ACGs) are a class of polyketides only occurring in some Annonaceae plants. Many members of ACGs have been found to exhibit strong antitumor activities in vitro and in vivo and ever been considered to be the best hope for new antitumor natural drugs. However, all antitumor ACGs have been found to be also cytotoxic toward normal cells and have a narrow therapeutic index due to their poor selectivity to tumor cells. This is the main reason that none of ACGs or their derivatives has ever entered the stage of clinical trials so far. Therefore, the key problem that needs to be solved in research and development of ACGs as antitumor drug candidates is how to increase their selectivity for tumors. Based on the vitamin-mediated tumor-targeting delivery mechanism, we have chosen biotin and folic acid as the tumor-targeting molecules and ACGs as the antitumor agents to construct tumor-targeting delivery drug conjugates. Recently we have synthesized a series of ACG-biotin conjugates starting from highly potent ACGs and observed that several conjugates showed an increased selectivity for the tumor cell lines which over-express biotin receptor, suggesting that ACG-vitamin conjugates are potential as tumor-targeting drug candidates. This prompts us to propose this project in order to continue our investigation. In this proposed project, (1) more ACG-vitamin conjugates including additional ACG-biotin conjugates and a series of ACG-folate conjugates will be designed and synthesized; (2) their selectivity for tumor cells over-expressing vitamin receptors and their tumor-targeting delivery properties will be evaluated to discover conjugates with both potent antitumor activity and good tumor-targeting delivery property; (3) regio-selective synthesis methods for the tumor-targeting antitumor ACG-vitamin conjugates will be established; and (4) in vivo antitumor activity and acute toxicity of the tumor-targeting antitumor ACG-vitamin conjugates will be evaluated. It is predictable that this project can achieve a significant breakthrough in research and development of ACGs as antitumor drug leads and discover novel tumor-targeting antitumor drug candidates.

番荔枝内酯是一类来自番荔枝科植物的聚酮化合物，由于其极强的抗肿瘤活性而曾受到国内外的高度重视。但由于其对肿瘤细胞的选择性较差，经近30年的研究一直难以在有效性与安全性间取得理想平衡而未能成药。为了提高对肿瘤的选择性，基于抗肿瘤药物-维生素偶联物的受体介导靶向输送原理，我们在前期合成了一系列抗癌活性番荔枝内酯与生物素的偶联物，并发现部分偶联物对生物素受体高表达肿瘤细胞的选择性有显著提高。本项目将在此前期工作基础上继续设计、合成高活性番荔枝内酯与生物素、叶酸等维生素的偶联物；测定偶联物对维生素受体高表达肿瘤细胞的选择性和受体介导性能；研究有靶向输送作用的高活性偶联物的选择性合成方法并测试其体内抗瘤作用和初步安全性，获得对肿瘤选择性高、活性强的番荔枝内酯-维生素偶联物。该项目将能在番荔枝内酯抗肿瘤药物研究上取得突破、获得新的靶向输送抗肿瘤药物候选物。项目还能为番荔枝植物资源的利用提供新的途径。

前期研究发现部分抗瘤活性番荔枝内酯(ACG)的生物素偶联物对生物素受体高表达肿瘤细胞有更高的选择性。在此基础上，本项目进行了下列研究：(1)以抗瘤活性ACG成分Squamocin (SQ)和Bullatacin为母体化合物合成了31个ACG与生物素和叶酸的新偶联物。测定了生物素偶联物对生物素受体高表达细胞(4T1、P815)和非高表达细胞(L1210)的活性，结果显示偶联物15-O-(4-oxo-4-(6-biotinylaminohexyl)amino)butyrylsquamocin对4T1细胞的活性和选择性相比SQ分别提高了约10倍和8倍；而其它偶联物对4T1细胞的活性和选择性均低于前期发现的偶联物15,28-Di-O-(6-biotinylamidohexanoyl)squamocin 和28-O-Biotinylsquamocin (SB)。通过合成方法学研究，建立了SB的选择性合成方法，并以较高的产率合成获得了该偶联物。最后，对SB和SQ进行了小鼠急性毒性和体内抗瘤试验，结果显示SB对小鼠腹腔注射的急性毒性(LD50=0.175 mg/kg)强于SQ (LD50=0.2025 mg/kg)，SB和SQ在中、高剂量下可降低S180荷瘤鼠的肿瘤增殖率，但与模型组相比无显著性差别，而且SB和SQ二者的抑瘤率无明显差别。此研究结果表明偶联生物素不一定能增强ACG的体内抗瘤活性，但可能增加其体内毒性。(2)合成了9个ACG与葡萄糖和半乳糖的偶联物，测定了这些偶联物对A549、HeLa、HepG2细胞的活性，结果表明糖基化修饰能保持ACG的活性、提高抗瘤活性ACG对不同肿瘤细胞的选择性。(3)合成了11个ACG的甘脯二肽偶联物，测定了其对4T1细胞的毒性，结果显示在抗瘤活性ACG五元不饱和内酯环和邻双THF环二个关键药效团邻近位置引入甘脯二肽可显著降低其细胞毒性，表明ACG-甘脯二肽偶联物在FAPa酶激活抗瘤前药研发上有潜力。(4)对二个抗肿瘤活性C-烷基黄酮成分淫羊藿素(ICT)和2′,4′-二羟基-6′-甲氧基-3′,5′-二甲基查尔酮(DMC)进行了结构修饰，获得了二个对雌激素受体高表达乳腺癌细胞有显著活性和良好选择性的ICT新衍生物、二个与紫杉醇有显著协同作用的DMC新衍生物。研究结果为ICT和DMC的结构优化提供了新路径。