【作者】 赵磊；

【导师】 田瑄；

【作者基本信息】 兰州大学 ， 有机化学， 2009， 博士

【摘要】 鬼臼毒素（Podophyllotoxin）又叫鬼臼素、鬼臼毒、鬼臼脂素,由于其用药历史悠久,且具有多种显著的生物活性而闻名于世,特别是其糖苷类衍生物如etoposide（VP-16）,teniposide（VM-26,）,和etopofos已广泛用于癌症的临床治疗。大量的构效关系（SARs）的研究显示,鬼臼毒素及其衍生物的生物活性有明显的立体化学特异性,在研究鬼臼的结构改造和生物活性的评价的过程中,我们发现急需要一种快速鉴别和检测鬼臼及其衍生物立体异构体的方法,本论文以鬼臼毒素类系列化合物的2位、4位立体异构体为研究对象,发展并建立了HPLC-DAD、HPLC-ESI/MSⁿ方法,可成功地分离和快速鉴别混合物中的鬼臼毒素类化合物的2位、4位立体异构体。具体内容包括以下几个部分:第一部分首先,应用HPLC-ESI/MS/MS快速分离和鉴定了8对鬼臼及其酯类化合物2位异构体,所采用的方法,首次运用梯度洗脱成功地分离了这8对异构体,有趣的是,这些化合物的色谱保留行为是有规律的,即:在相同的色谱条件下,2α-H的化合物（苦鬼臼类化合物）总是比相应的2β-H（鬼臼类化合物）的异构体先流出来,高效液相色谱分离的8对鬼臼及其酯类化合物2位异构体的流分在线的一级正离子模式质谱中主要是形成了铵离子加合离子[M+NH₄]⁺和碎片[M-ROH+H]⁺,可以明确地确定每个化合物的分子量;接着以[M-ROH+H]⁺（ion397）为前体离子,在串联质谱中碰撞诱导解离（CID）,产生了一系列[M-ROH+H]⁺裂解的碎片,[A+H]⁺（ion 313）,[M-ROH-ArH+H]⁺（ion 229）特征性的离子,且[M-ROH+H]⁺（ion 397）与[M+NH₄]⁺的离子丰度比以及在二级质谱中的[A+H]⁺（ion 313）与[M-ROH+H]⁺（ion 397）的丰度比,[M-ROH-ArH+H]⁺（ion 229）与[M-ROH+H]⁺（ion 397）的离子丰度比具有立体化学的特异性,可以用来快速鉴别每一对2位异构体。其次,在以上研究的基础上,采用HPLC-DAD、HPLC-ESI/MS/MS快速分离和鉴定了4对氮氧自由基自旋标记的鬼臼类化合物的2位立体异构体,并研究了其色谱和质谱特征,并进一步证实了以上结果,这对不能用核磁共振方法鉴别立体异构体的自旋标记的鬼臼类化合物的分离和鉴别是非常有用的;同时,运用HPLC-ESI/MS,ESI-MS/MS方法分离和鉴定了7对表鬼臼及其酯类化合物2位异构体,并对其色谱及质谱特征进行了研究并给出了合理的解释;另采用HPLC-DAD、CD方法研究了6对天然产物中鬼臼类化合物的2位异构体。第二部分采用HPLC-ESI/MS快速分离和鉴定了5对氮氧自由基自旋标记的鬼臼类化合物的4位立体异构体,首先运用简单的三相体系,分离了这5对4位异构体,实验发现在反相色谱中,这5对化合物出峰是有规律的,总是4β-H取代的异构体（氮氧自由基自旋标记的鬼臼类化合物）总是比4α-H取代的相应的异构体（氮氧自由基自旋标记的表鬼臼类化合物）先出峰,高效液相色谱分离的这5对自旋标记的鬼臼类化合物4位异构体的流分在线的一级正离子模式质谱中,主要是形成了质子加合离子[M+H]⁺,且在离线的一级质谱中,[M-ROH+Na]⁺和[M+Na]⁺离子离子的相对丰度比都显示出了一定的区别,具有立体化学的特异性,对不易用核磁共振（NMR）方法监测的自旋标记的化合物4位异构体来说,无疑是一种新的分离和鉴定方法。第三部分采用HPLC-ESI/MS方法,在正离子模式下,比较了不同提取方法对桃儿七的活性成分的影响,通过对照品对照、准分子离子峰及查阅文献确定十八个化合物,主要为活性成分木脂素类化合物,为抗癌药材桃儿七的质量控制提供了实验依据。更多还原

【Abstract】 Podophyllotoxin is a well-known natural product due to its long history of use in folk medicine and the biological activity of its many derivatives. In particular, its glucopyranodide derivatives such as etoposide （VP-16）, teniposide （VM-26）, and etopofos have been widely used in the chemotherapy of cancers. Research on structure-activity relationships （SARs） indicated that the biological activity and toxicity of podophyllotoxin and its derivatives are stereochemically specific. In this dissertation, novel HPLC-DAD and HPLC-ESI/MSⁿ methods were developed and successfully applied in rapid separation, determination and identification of the diastereoisomers of podophyllotoxin and its analogues at 2 and 4 position. The method could be used in rapidly identifying the purity and monitoring of the epimerization of 2-H, 4-H of podophyllotoxin and its analogues from natural products, chemical reactions, and pharmaceutical metabolism.I A rapid and stable high-performance liquid chromatography-diode array detection （HPLC-DAD） and a high-performance liquid chromatography-electrospray ionization tandem mass spectrometry （HPLC-ESI/MS/MS） method were developed and validated for the separation, determination, and identification of eight pairs of diastereoisomers of podophyllotoxin and its esters at C-2 position. Interestingly, every 2α-H compound migrated before its corresponding 2β-H epimer under optimum conditions. In the HPLC-ESI/MS spectra, each pair of diastereoisomers of the derivatives in the mixture was directly confirmed and identified by [M+NH₄]⁺ ions and ion ratios of relative abundance of [M-ROH+H]⁺ （ion 397） to [M+NH₄]⁺. When the [M-ROH+H]⁺ ions （at m/z 397） were selected as the precursor ions to perform the MS/MS product ion scan. The characteristic elimination from the precursor protonated ions and the product ions at m/z 313, 282, and 229 were the common diagnostic masses. The ion ratios of relative abundance [M-ROH+H]⁺ （ion 397） to [M+NH₄]⁺, [A+H]⁺ （ion 313） to [M-ROH+H]⁺, and [M-ROH-ArH+H]⁺ （ion 229） to [M-ROH+H]⁺ in the ESI/MS/MS spectra of each pair of diastereoisomers of the lignans specifically exhibited a stereochemical effect. The method could be rapidly identified the each pair of diastereoisomer of the lignans.On the basis of the above study, a high-performance liquid chromatography-diode array detection （HPLC-DAD） and a high-performance liquid chromatography-electrospray ionization tandem mass spectrometry （HPLC-ESI/MS/MS） method were developed and validated for the separation, identification of four pairs of diastereoisomers of spin-labeled derivatives of podophyllotoxin at C-2 position. The results support the research of the above study, revealing that provided a basis for identifying spin-labeled diastereoisomers of podophyllotoxin and its analogues at C-2 position in the mixture. Meanwhile, using HPLC-DAD, HPLC-ESI/MS, ESI/MS/MS, seven pairs of epipodophyllotoxin and its esters were separated and identified. The characteristics of the chromatography and mass spectrometry were studied and given a reasonable explaination. Moreover, six pairs of natural podophyllotoxin were separated and identified by HPLC-DAD and CD.II Spin-labeled nitroxide derivatives of podophyllotoxin had better antitumor activity and less toxicity than that of the parent compounds. However, configurations of these spin-labeled derivatives cannot be determined by Nuclear Magnetic Resonance （NMR） methods. HPLC-ESI/MS and ESI/MS methods were developed and validated for the separation, identification of five pairs of diastereoisomers of spin-labeled derivatives of podophyllotoxin at C-4 position. It was found that the migration behaviors of the compounds with different types were regular, that is, every 4β-H compound migrated before its corresponding 4α-H epimer under optimum conditions. In the HPLC-ESI/MS spectra, each pair of diastereoisomers of the lignans in the mixture was directly confirmed and identified by [M+H]⁺ ions and EIC. Off-line ESI-MS were performed to study the relationship between the structural characteristics and ESI/MS fragmentation pathways of the diastereoisomers at C-4 of the lignans. The ion ratios of relative abundance [M-ROH+Na]⁺ to [M+Na]⁺ in the ESI/MS spectra of each pair of diastereoisomers of the lignans specifically exhibited a stereochemical effect. Undoubtedly, for the Spin-labeled nitroxide derivatives of podophyllotoxin, this is a novel separation and identification method.III Podophyllum emodi Wall var. Chinesis. which is existed in the western China. Plant containing podophyllotoxin analogues have been used as folk remedies in traditional medicinal of many diverse cultures. Compounds extracted from P. Wall var. Chinesis. in three extraction methods were analyed by HPLC-DAD, HPLC-ESI/MS. Eighteen components were unambiguously identified or tentatively characterized based on their retention times, UV spectra and mass spectra in comparison with the data from the reference standards. The results may be useful for the quality control of P. Wall var. Chinesis.and its related preparations.更多还原