Synthesis and insecticidal activities of pyridine ring derivatives of podophyllotoxin

In an attempt to find the biorational pesticides, we synthesized 12 pyridinyl derivatives of podophyllotoxin (PPT) and 4′-demthylepipodophylltoxin (4′-DMEP) in this study. Their structures and the α/β substitution at C-4 were confirmed by ¹H NMR, IR, MS spectral analyses and elemental analysis. The insecticidal activities were tested against fifth-instar larvae of Pieris rapae and the third-instar larvae of Cullex pipiens pallens at concentrations of 250 and 10 μg ml⁻¹. Four derivatives of PPT, 4.1, 4.2, 4.3 and 4.5, showed higher insecticidal activities against P. rapae than PPT, while three derivatives of PPT, 4.4, 4.5 and 4.6, displayed higher mosquito larvicidal activity than PPT, with LC₅₀ values of 1.66, 3.96 and 1.54 mg l⁻¹, respectively. Interestingly, we also found that the pyridine ring derivatives of PPT showed delayed insecticidal activity, which is different from traditional neurotic insecticides. The results suggest that 4′-OCH₃ in the PPT derivatives is essential to keep the insecticidal activity and the insecticidal activities of pyridine ring derivatives of PPT are higher than that of the derivatives of 4′-DMEP, supporting PPT has the potential to be a lead structure of semi-synthetic insecticides.     

Evaluation of insecticidal activity of podophyllotoxin derivatives against Brontispa longissima

Twenty podophyllotoxin analogues were first tested for their insecticidal activity against the fifth-instar larvae of Brontispa longissima in vivo. Among them, compounds 6-9 and 19 showed more promising and pronounced insecticidal activity than toosendanin, a commercial insecticide derived from Melia azedarach. The different insecticidal activity ranges of compounds 1-20 indicated that variation of chemical structures in the podophyllotoxin skeleton markedly affected the activity profiles of this compound class, and some important SAR information has been revealed from it. The results obtained from SAR analysis show good correlation with the docking models as well as with QSAR studies, which allows for the rational design of more potent podophyllotoxin derivatives in the development of potential new insecticides.     

天然抗肿瘤药物鬼臼毒素的生物转化研究

[目的]筛选能够转化鬼臼毒素的菌株,得到其转化产物并鉴定结构,以期为高抗肿瘤活性天然药物的获取和研究奠定基础。[方法]采用微生物分批转化法对鬼臼毒素进行结构修饰,利用薄层色谱法和高效液相色谱法对鬼臼毒素及其转化产物进行检测,筛选出能够转化鬼臼毒素的菌株,将其中一株能够转化鬼臼毒素的菌株进行扩大培养,对其发酵液进行乙酸乙酯萃取、常规硅胶柱层析分离提取转化产物,应用现代波谱技术鉴定其转化产物结构。[结果]通过转化鬼臼毒素的研究,筛选出能转化鬼臼毒素的菌株为BS.Str-1、BS.Str-2、BS.Str-3、BS.Str-7、BS.Str-18、BS.Str-21和BS.Str-29;对BS.Str-21扩大培养,提取分离得到唯一的转化产物,经鉴定为苦鬼臼毒素。[结论]该研究表明通过生物转化的方法可以获得鬼臼毒素衍生物,该结果为高抗肿瘤活性天然药物的获取奠定了基础。     

Podophyllum lignans array of Podophyllum hexandrum Royle populations from semi-desert alpine region of Zanskar valley in Himalayas

Podophyllum hexandrum Royle (Syn. P. emodi Wale) a perennial rhizomatous herb found in alpine region distributed in the entire range of Himalayas from Ladakh to Sikkim at an altitude of 3000-4200 m asl is an preferred commercial source of Podophyllum lignans. It contains three times more Podophyllotoxin than the American species, Podophyllum peltatum. The present study was aimed to investigate variation of Podophyllum lignans contents based on six marker compounds viz. Podophyllotoxin; Deoxypodophyllotoxin; Picropodophyllotoxin; Podophyllotoxin β-d-glucopyanoside; Isopicropodophyllone; 4′-Demethyldeoxypodophyllotoxin, β-d-lucopyanoside, in P. hexandrum population growing at three locations. Further, ontogenetic and morphogenetic variations of Podophyllum lignan contents were studied to investigate dynamics of accumulation of these compounds. Representative collections from three locations viz., Panikhar, Padam and Tangoli located in Trans Himalayan semi-desert region of Zanskar valley were harvested at three stages (dormancy, active growth and maturity). Plants were dissected into root, rhizome and rhizome-buds, dried separately and assayed for Podophyllum lignan contents by high performance liquid chromatography.     

Molecular characterization and marker based chemotaxonomic studies of Podophyllum hexandrum Royle

Detailed chemical studies and RAPD analysis were done in different populations of Podophyllum hexandrum collected from high altitude regions of North Western Himalayas. Random amplified polymorphic DNA (RAPD) analysis revealed a high degree of genetic diversity among the 12 collected accessions, attributed to their geographical and climatic conditions. HPLC analysis also revealed variation in the concentration of two major marker compounds which lead to the identification of a chemotype. The study demonstrated that RAPD and chemical markers are very useful tools to compare the genetic relationship and pattern of variation among such prioritized and endangered medicinal plants.     

鬼臼毒素在两种溶剂中核磁共振谱图数据的比较

比较了微生物源的鬼臼毒素在氘代氯仿(CDCl3)和二甲亚砜(DMSO)2种溶剂中的核磁共振数据,结果表明:在溶剂DMSO中,核磁共振能提供更多可靠的信号;据解析,确定了在2种溶剂中所有13C NMR、1HNMR谱的信号归属。     

鬼臼毒素和脱氧鬼臼毒素对粘虫生物活性初步研究

采用小叶碟添加法测定了鬼臼毒素 ( Podophyllotoxin)和脱氧鬼臼毒素 ( Deoxypodophyllotoxin)对粘虫 ( Mythimna separata W.)的生物活性。结果表明 ,二者对粘虫 4龄幼虫均有很强的拒食作用 ,2 4h、48h和72 h的 AFC50 值分别为 1 .40 86、1 2 .45 1 4、0 .8881 mg/m L和 0 .4686、1 .1 0 66、0 .2 5 99mg/m L;二者对粘虫的生长发育抑制作用也较强 ,致使粘虫生长发育各历期均延迟 ,化蛹率、羽化率、产卵量等均明显降低     

Synthesis and insecticidal activities of novel derivatives of podophyllotoxin: Part XII

In order to find the biorational pesticides, eight novel 4β-substituted phenoxyaniline derivatives of podophyllotoxin have been synthesized with significant stereoselectivity and improved yields by employing the BF₃·Et₂O/NaI reagent system and evaluated for their antifeedant effect against 5th instar larvae of Pieris rapae and effect on the development of 5th instar larvae of P. rapae as well as for insecticidal activity against P. rapae. The antifeedant activities showed that these compounds exhibited less potent than podophyllotoxin. Though these derivatives showed less potent antifeedant activities than podophyllotoxin, they acted as growth development inhibitor to 5th instar larvae of P. rapae, which were found that the animals treated with podophyllotoxin and its derivatives showed moulting disturbances and/or deformities. Also, the insecticidal activity results show that all of these derivatives of PPT showed delayed insecticidal activity, which differs from traditional neurotoxic insecticides. Among them, compounds possessing a 4β-phenoxyaniline moiety substituted (CO₂C₂H₅, Cl and OH) at para position exhibited greater insecticidal activity against P. rapae than podophyllotoxin.     

酶联免疫吸附法分析鬼臼毒素在水体中的降解

利用固相间接竞争酶联免疫吸附法（Indirect competitive enzyme—linked immunosorbent assay，IC—ELISA）对鬼臼毒素在不同pH值水体中的稳定性进行了研究，并以高效液相色谱法（HPLC）进行验证。结果表明：鬼臼毒素在酸性条件下稳定，半衰期约为145d；在中性条件下，半衰期约为10d；在碱性条件下降解速度快，半衰期仅为5．78h。IC—ELISA法和HPLC法测得鬼臼毒素在pH为5．0、7．0水体中的水解半衰期结果相似，但IC—ELISA法检测灵敏度更高，且具有处理量大，方便快速等优点。因此，在一定条件下IC—ELISA法可用于水体中鬼臼毒素含量的测定。根据鬼臼毒素的水溶性、Koc系数及半衰期等判定，鬼臼毒素是不会给水体带来严重污染的。     

Assessment of diversity in Podophyllum hexandrum by genetic and phytochemical markers

For successful conservation and domestication of a species, evaluation of its genetic diversity by different markers is important. Morphological characteristics, phytochemical variation and random amplified polymorphic DNA (RAPD) profiles were generated in different accessions of Podophyllum hexandrum in order to determine the genetic diversity. Random amplified polymorphic DNA (RAPD) analysis revealed a high degree of genetic diversity among the accessions used in the study. There was also high diversity in the concentration of marker compounds in the collected samples as revealed by HPLC analysis. It is shown that the approaches used in the work successfully discriminate between the accessions of this species and thus they constitute interesting tools to analyze molecular, biochemical and phenotypic diversity within this species. Similarity measurement using UPGMA followed by cluster analysis resulted in formation of many groups based on geographical distribution that generally reflected expected trends between the genotypes. There were also some important exceptions like PW-S, an accession from Wastoorwan, Khrew showing close resemblance to PG-S and PG-B collected from Gulmarg but grown at two different gene banks at Srinagar and Bonera. Further an accession PSH-B from Keller was significantly diverse from the rest of the native genotypes phytochemically, morphologically and at molecular level. RAPD data analysis was found to be significant predictor of phytochemical markers in cultivated P. hexandrum germplasm. Twelve accessions grown in gene bank repository were subjected to RAPD analysis and were assessed for content of podophyllotoxin and podophyllotoxin β-d-glycoside by HPLC. Individual regressions of podophyllotoxin and podophyllotoxin β-d-glycoside by RAPD analysis against HPLC has been found to determine linear values. Strong correlation and a strong association of values of the phytochemical variables and the DNA polymorphism data has been recorded.