Study on synthesis and inhibitory activities of NR2B ca 2 flux of isoindoline derivatives and study on chemical constituents of marsdenia tenacissima and calotropis gigantea

学校代码：10251 学 号：010110066 博士学位论文 题 目： 异吲哚啉衍生物的合成及其抑制 NR2B Ca2+ 流量研究和通关藤和牛角瓜的化学成分研究 专 业： 研究方向： 有机化学 天然药物化学 姓 名： 范文康 (PHAM VAN KHANG) 导 师： 胡立宏 教授 定稿时间： 2014 年 01 月 15 日 分类号： O62 密级： U D C： 华 东 理 工 大 学 学 位 论 文 异吲哚啉衍生物的合成及其抑制 NR2B Ca2+流量 研究和通关藤和牛角瓜的化学成分研究 范文康 (PHAM VAN KHANG) 指导教师姓名： 胡立宏 教授, 华东理工大学药学院 马磊 副教授, 华东理工大学药学院 申请学位级别： 博士 专 论文定稿日期： 2014-01-15 业 名 称： 论文答辩日期： 学位授予单位： 有机化学 2014-04-28 华东理工大学 学位授予日期： 答辩委员会主席： 沈旭 教授 评 李剑 教授 柳红 教授 刘璇 教授 沈旭 教授 段文虎 教授 阅 人： 作者说明 我郑重声明: 本人悟守学术道德, 崇尚严谨学风。所呈交的学位论文, 是本人在导师的指 导下, 独立进行研究工作所取得的结果。除文中明确注明和引用的内容外, 本论文不包 含任何他人已经发表或撰写过的内容。论文为本人亲自撰写, 并对所写内容负责。 论文作者签名： 年 月 日 华东理工大学博士学位论文 第I页 Study on synthesis and inhibitory activities of NR2B Ca2+-flux of isoindoline derivatives, and study on chemical constituents of Marsdenia tenacissima and Calotropis gigantea Abstract The whole thesis consists of four parts: (1) Study on synthesis of isoindolin-1-imine derivatives via one-pot reaction and their inhibitory activities of NR2B Ca2+-flux; (2) Study on synthesis of 2-substituted-3-(2-oxoalkyl)isoindolin-1-one derivatives via one-pot reaction; (3) Study on C21 steroidal glycosides from the Stems of Marsdenia tenacissima; (4) Study on the isolation, structural determination and cytotoxicities of cardenolides from the bark of Calotropis gigantea Isoindolin-1-imine derivatives are an important class of isoindole scaffold, which exhibit typical pharmacological activities as NR2B-selective NMDA (N-Methyl-D-aspartate) receptor antagonists, and the thrombin receptor (PAR-1) inhibitors In an effort to explore new method for synthesis of isoindolin-1-imine derivatives, a novel one-pot method for the synthesis of isoindolin-1-imine derivatives has been developed via a simple three-component condensation of 2-cyanobenzaldhyde, ammonium acetate, and 4-hyroxycoumarin derivatives or 1,3dicarbonyl compounds, or 4-hydroxyquinolin-2(1H)-one in ethanol under reflux condition for 20-60 with excellent yields Moreover, a new, simple, efficient procedure for the preparation of 3-(2-substituted-3-iminoisoindolin-1-yl)-2-hydroxy-4H-chromen-4-one analogs is decribed in this thesis via a three-component condensation of 2-cyanobenzaldehyde, primary amine, and 4-hydroxycoumarin derivatives in dry dichloromethane without catalyst at room temperature The condensation reactions are proceeded smoothly and quickly to afford products in excellent yields The inhibitory activities of NR2B Ca2+-flux of synthesized isoindolin-1-imine analogs have been evaluated via the fluorescence measurement of free concentrations of intracellular calcium of L(tk-) cells expressing NR1a/NR2B receptors The results showed that all tested isoindolin-1-imine derivatives exhibited potent inhibitory activity of Ca2+flux in cells An efficient one-pot procedure for the synthesis of 2-substituted-3-(2oxoalkyl)isoindolin-1-one analogs has been developed from phthalaldehydic acid, primary amine, and ketone in aqueous solution under reflux condition in the presence of VB1 Various substrates can be applied to this procedure with operational simplicity, good yields, short reaction time, and environmental friendly conditions Marsdenia tenacissima (Roxb.) Wight et Arn., is known as a famous traditional Chinese medicine, which is widely used in the treatment of cancer, and other diseases From ethanolic extract of stem of Marsdenia tenacissima, 20 compounds have been isolated, including new compounds and 17 known ones, and their structures have been elucidated via NMR spectroscopic identification and LC-MS analysis Calotropis species are known as a source of biological active substances, in particular it 第 II 页 华东理工大学博士学位论文 is one of a good source of cardenolides From the 90% ethanolic extract of the bark of Calotropis gigantea (C gigantea), three new cardenolides and eleven known ones have been isolated, and their structures have been elucidated via NMR spectroscopic identification and LC-MS analysis The inhibitory activities of all isolated compounds have been evaluated against non-small cell lung carcinoma (A549) and human cervix epithelial adenocarcinoma cell line (HeLa), and several cardenolides exhibit strong potent cytotoxicities Keywords: Isoindolin-1-imine; gigantea; cardenolides isoindolin-1-one; Marsdenia tenacissima; Calotropis 华东理工大学博士学位论文 第 III 页 异吲哚啉衍生物的合成及其抑制 NR2B Ca2+流量研究和 通关藤和牛角瓜的化学成分研究 摘要 本论文内容包括四个部分：（1）异吲哚啉-1-亚胺衍生物的多组分合成及其抑制 NR2B Ca2+流量抑制研究；（2）异吲哚啉-1-酮衍生物的多组分合成方法学研究；（3）通 关藤茎中 C21 甾体皂苷的化学成分研究；（4）牛角瓜化学成分及其细胞毒活性研究。 异吲哚啉-1-亚胺衍似物是一类非常重要的异吲哚类化合物，它们具有多种生物活 性，如拮抗 N-甲基-D-天冬氨酸（NMDA）受体, 抑制凝血酶受体(PAR-1)和抗增值等作 用。为了开发异吲哚啉-1-亚胺衍似物的新合成方法，通过探索研究，我们发现了以邻 醛基氰基取代的苯环与相应的亲核试剂发生缩合反应，可高效, 简便, 绿色构建异吲哚 啉-1-亚胺衍生物类骨架。首先探索了 2-氰基苯甲醛, 乙酸铵，与 4-香豆素, 或 1,3-二羰 基化合物, 或 2,4-二羟基喹啉等亲核试剂构建异吲哚啉-1-亚胺骨架的多组分合成反应的 最佳反应条件和底物适用性。其次，发现以烷基伯胺替代乙酸铵，该反应在二氯甲烷, 室温条件下以高产率得到 2-取代-3-烷氧基异吲哚啉-1-亚胺-香豆素类衍生物。最后，对 所合成的 2-（4-羟基香豆素取代）异吲哚啉-1-亚胺衍似物进行了抑制 NR2B Ca2+流量 活性评价。结果显示大多数 2-（4-羟基香豆素取代）异吲哚啉-1-亚胺衍似物是对细胞 钙外流的都具有强的抑制作用。 此外，我们还开发了一种简单，有效的三组分法反应，三组分苯醛酸, 伯胺, 酮， 在 VB1 催化下，在水中回流以高产率构建 2-取代-3-(2-氧代烷基) 异吲哚啉-1-酮衍生物 类似物。该反应底物适应性广, 操作简单, 产率高, 反应时间短, 绿色环保。 通关藤是一种常用的抗肿瘤中药，广泛生长在中国的西南部以及热带地区。我们 从通关藤的茎中分离鉴定了 20 个化合物，其中包括 个新化合物。 牛角瓜是一种萝藦科植物，文献报道其主要化学成分是强心苷类。我们从牛角瓜 皮的 90%乙醇提取物中分离得到 个新的强心苷，11 个已知强心苷。 化合物的结构通 过 NMR 及 LC-MS 得到鉴定。部分化合物显示强的细胞毒活性。 关键词 异吲哚林-1-亚胺； 异吲哚林；通关藤；牛角瓜；强心苷 第 IV 页 华东理工大学博士学位论文 Table of contents Chapter Study on synthesis of isoindolin-1-imine derivatives via one-pot reaction and their inhibitory activities of NR2B Ca2+-flux 1.1 Introduction 1.2 Design for synthesis of isoindolin-1-imine derivatives 1.3 Experiment 1.3.1 Synthesis of 3-substituted isoindolin-1-imine derivatives 1.3.1.1 Synthesis of 2-hydroxy-3-(3-iminoisoindolin-1-yl)-4H-chromen-4-one (4a) 1.3.1.2 The optimization of reaction conditions 1.3.1.3 The scope and limitations of reaction substrates 1.3.1.4 The structural determination 1.3.1.5 The plausible mechanism for synthesis of isoindolin-1-imine 10 1.3.2 Synthesis of 2,3-disubstituted isoindolin-1-imine derivatives 10 1.3.2.1 The optimization of reaction conditions 11 1.3.2.2 The scopes and limitations of reaction substrates 12 1.3.2.3 The plausible mechanism for synthesis of isoindolin-1-imines 11 13 1.3.3 Activity of isoindolin-1-imine derivatives 14 1.3.3.1 Introduction 14 1.3.3.2 Effect of isoindolin-1-imine derivatives as NR2B Ca2+ flux inhibitor 14 1.3.3.2.1 Effect of products 14 1.3.3.2.2 Effect of products 11 17 1.3.4 Preparation section 18 1.3.4.1 Preparation of isoindolin-1-imines 4, 8, and 10 18 1.3.4.1.1 Preparation of products 18 1.3.4.1.2 Preparation of products 22 1.3.4.1.3 Preparation of products 10 22 1.3.4.2 Preparation of products 11 23 1.3.4.3 Preparation of Calcium flux functional assay 28 1.4 Conclusions 29 Chapter Study on synthesis of 2-substituted-3-(2-oxoalkyl)isoindolin-1-one derivatives via one-pot reaction 30 2.1 Introduction 30 2.2 Results and Discussion 32 2.2.1 The optimization of reaction catalyst 32 2.2.2 The optimization of reaction solvent 33 2.2.3 The scope and limitations of reaction substrates 34 2.3 The plausible mechanism 35 2.4 Conclusions 35 2.5 Experiment section 36 华东理工大学博士学位论文 第V页 Chapter Study on C21 steroidal glycosides from the stems of Marsdenia tenacissima 38 3.1 Introduction 38 3.1.1 Chemical compositions of M tenacissima 38 3.1.2 Biological activities of of M tenacissima 43 3.1.2.1 Biological activities of the total extract of M tenacissima 43 3.1.2.2 Biological activities of M tenacissima steroids 45 3.2 Results and discussion 46 3.2.1 New isolated compounds 47 3.2.1.1 Compound 10 47 3.2.1.2 Compound 13 48 3.2.1.3 Compound 14 50 3.2.2 Known compounds 52 3.3 Conclusions 58 3.4 Experimental section 58 3.4.1 General experimental procedures 58 3.4.2 Extraction and isolation 58 3.4.3 Hydrolysis of compounds 59 Chapter Study on the isolation, structural determination, and cytotoxicities of cardenolides from the bark of Calotropis gigantea (Linb.) 60 4.1 Introduction 60 4.2 Chemical components 61 4.3 Pharmacological activities of extracts and isolated components 63 4.4 Aims of study 66 4.5 Results and discussion 67 4.5.1 Structural determination of isolated compounds 67 4.5.1.1 New compounds 68 4.5.1.1.1 Compound 68 4.5.1.1.2 Compounds 11 and 12 69 4.5.1.2 Known compounds 70 4.5.2 Inhibitory activity against cancer cell lines 73 4.6 Conclusions 74 4.7 Experimental 74 4.7.1 General experimental procedures 74 4.7.2 Plant material 75 4.7.3 Extraction and isolation 75 4.7.4 Hydrolysis of compounds 11 and 12 76 4.7.5 Cytotoxicity assays 76 Chapter Summary 77 References 79 Appendix 91 第 VI 页 华东理工大学博士学位论文 Appendix 94 Appendix 104 Publications 106 致谢 107 华东理工大学博士学位论文 No 11 12 13 14 15 16 17 18 19 20 m.p H 13 第 93 页 C MS HRMS [α] Ref 云南植物研究 1999, 21(3), 369-377 Hel Chim Acta 2005, 88, 2675–82 New compound New compound Hel Chim Acta 2005, 88, 2675–82 Hel Chim Acta 2006, 89, 2738–44 J Asian Nat Prod Res 2011, 13(6), 477-85 Phytochemistry 1996, 41, 1385–1388 CN101463060 A Phytochemistry 1996, 41, 1385–1388 Chapter 4 10 11 12 13 14 AA New compound Chem Pharm Bull 1998, 46, 528–530 Magn Reson Chem 2002, 40(9), 599-602 Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry (1972-1999) 1989, 9, 1563-70 Phytochemistry 1994, 37(1), 217-26 Phytochemistry 1994, 37(1), 217-26 Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry (1972-1999) 1989, 9, 1563-70 Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry (1972-1999) 1989, 9, 1563-70 Arthropod-Plant Interactions 2011, 5, 219-225 Arthropod-Plant Interactions 2011, 5, 219-225 New compound New compound Tetrahedron Lett 1963, 33, 63-67 J Nat Prod 2005, 68(10), 1494-9 Tetrahedron Lett 1981, 22, 2405–2408 华东理工大学博士学位论文 第 94 页 Appendix Copy NMR of some compounds 4b Fig P1 1H, 13C-NMR of 2-hydroxy-3-(3-iminoisoindolin-1-yl)-6-methyl-4H-chromen-4-one (4b) 3' 3a O HN 7a Fig P3 1H NMR of isoindolin-1-one 4a 1.05 4' 1.03 H 4a' 3.54 3.53 3.49 3.48 3.20 3.18 3.16 3.14 H 2' 4.58 4.54 5' 1' 1.00 6' O 1.04 7' 8a' 1.02 8' 1.08 1.98 1.26 1.98 3.16 5.26 9' 7.94 7.93 7.92 7.82 7.80 7.60 7.58 7.56 7.50 7.50 7.49 7.46 7.44 7.42 7.40 7.28 7.27 7.26 7.26 7.24 7.22 7.20 7.19 5.28 5.27 5.25 5.11 5.07 华东理工大学博士学位论文 第 95 页 OH HN Fig P2 HMBC correlations for compound 4b O N O 第 96 页 华东理工大学博士学位论文 Fig P4 1H and 13C NMR of 10 华东理工大学博士学位论文 Fig P5 HMBC correlations for 10 Fig P6 1H NMR spectrum of 13 第 97 页 第 98 页 华东理工大学博士学位论文 Fig P7 13C NMR spectrum of 13 Fig P8 HMBC correlations for 13 华东理工大学博士学位论文 第 99 页 Fig P9 1H NMR spectrum of 14 Fig P10 13C NMR spectrum of 14 第 100 页 华东理工大学博士学位论文 Fig P11 HMBC correlations for 14 华东理工大学博士学位论文 Fig P12 HMQC and HMBC correlations for 第 101 页 第 102 页 华东理工大学博士学位论文 Fig P13 HMQC and HMBC correlations for 11 华东理工大学博士学位论文 Fig P14 HMQC and HMBC correlations for 12 第 103 页 华东理工大学博士学位论文 第 104 页 Appendix Abbreviations Ac ALP ATPase Bn BSA cacld DBU DCE DCM DMF DMSO Dox DPP EAC ESI Et EtOAc FBS fluo-3 AM h HEPES HIV-1 PR HMBC HPLC HRMS HSQC IC50 IR KDR K526 LP MDM2 Me Acetone Alkaline phosphatase Adenosinetriphosphatase Benzyl Bovine serum albumin Calculated 1,8-Diazabicyclo[5.4.0]undec-7-ene 1,2-Dichloroethane Dichloromethane N,N-dimethylformamide Dimethyl sulfoxide Doxorubicin 2,6-Dimethyldifuro-8-pyrone Endocrine active compound Electrospray ionization Ethyl Ethyl acetate Fetal bovine serum 4-(6-Acetoxymethoxy-2,7-dichloro-3-oxo-9-xanthenyl)-4′-methyl2,2′(ethylenedioxy)dianiline-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl) ester Hour(s) 4-(2-Hydroxyethyl)-1-piperazineethanesuffonic acid Human immunodeficiency virus type protease Heteronuclear multiple bond correlation High performance liquid chromatography High-resolution mass spectrum Heteronuclear single quantum coherence Half maximal (50%) inhibitory concentration of a substance Infrared spectroscopy Kinase insert domain receptor Erythromyeloblastoid leukemia cell line Lipoprotein Murine double minute Methyl 华东理工大学博士学位论文 MEM mg mL μL μM N NMR nM PE Pr r.t SDS SFDA SGOT SGPT Tf THF TMS 5-HT Minimum essential medium 1×10-3 g Minute(s) 1×10-3 L 1×10-6 L 1×10-6 mol/L mol/L Nuclear magnetic resonance 1×10-9 mol/L Petroleum ether Proply Room temperature Sodium dodecyl sulfate State food and drug administration Serum glutamic oxaloacetic transaminase Serum glutamic pyruvate transaminase Trifluoromethanesulfonic Tetrahydrofuran Trimethylsilane 5-Hydroxytryptamine 第 105 页 华东理工大学博士学位论文 第 106 页 Publications Khangvan Pham, Zhongguo Zhang, Sida Shen, Lei Ma, Lihong Hu A one-pot synthesis of isoindolin-1-imine derivatives Tetrahedron 2013, 69, 10933-10939 Pham Van Khang, Zhong Guo Zhang, Yu Hui Meng, De An Guo, Xuan Liu, and LiHong Hu, Lei Ma Cardenolides from the bark of Calotropis gigantea Natural Product Research 2014 Accepted Sida Shen, Pham Van Khang, Yijia Chen, Min Lei and Lihong Hu Catalyst-free one-pot synthesis of isoindolin-1-imine derivatives via three-component reaction Arkivoc 2013, 3, 413-423 Lei Ma, Wenjuan Xiang, Pham Van Khang, Yang Liang, Yaping Xiao, Lihong Hu New dammarane-type glycosides from the roots of Gynostemma pentaphyllum Planta Medica 2012, 78, 597-605 华东理工大学博士学位论文 第 107 页 致谢 时光荏苒，岁月如梭，3 年的博学习生涯即将结束，在毕业论文临近完成之际，我 首先要衷心地感谢我的导师胡立宏教授, 马磊副教授这 年来对我的关心和帮助，我的 每一点进步都与他们的悉心指导和亲切关怀息息相关。胡老师学识渊博,治学严谨,思维 广阔,勇于创新同时又十分平易近人，所有这些品质都是值得我学习的。感谢胡老师给 我提供了严谨积极不断突破的科研氛围和完备优越的实验条件，在这样的环境中使我 学习到丰富的知识以及做人做事的基本准则，这一切都会让我受益终生。在此谨向胡 老师表达我最诚挚的谢意和崇高的敬意！ 特别感谢上海药物所果德安研究员，刘璇研究员以及孟宇辉同学，陈梅元(越南河 内医药大学)他们承担了本论文中活性的测试工作。本论文能够顺利完成与这些优秀合 作伙伴的辛勤努力和付出是密不可分的，在此表示衷心的感谢！ 由衷感谢本课题组的每一位成员，我很幸运能够在这样一个关系和睦融洽、相互 勉励支持、共同努力前行的课题组氛围中度过我 年的博士研究生涯，感谢每一位课 题组成员给予我的帮助和鼓励。他们包括：雷敏,沈思达，向文娟,陈旭冰,刘军华,马彪, 赵坤,陈方磊,尤晗昀,刘鹏,陈虎,颜建明,徐星宇,胡领军,陈轶嘉,王琪琪,张忠国， 徐婷，陈 刘欢。其中，特别感谢沈思达，张忠国，徐婷本论文能够顺利完成与你们尽心尽力的 帮助和付出是分不开的。 感谢中国政府，越南政府对于我的支持。 感谢华东理工大学，药学院，国际教育学院各位领导，各位老师和同学们在我学 习和生活上的关心和帮助。 感谢越南教育部，越南太原大学， 越南太原师范大学对于我的支持。 最后，衷心的感谢我的父母与家人。感谢他们多年来对我学业和生活上的理解和 支持。 ... 华东理工大学博士学位论文 第I页 Study on synthesis and inhibitory activities of NR2B Ca2 + -flux of isoindoline derivatives, and study on chemical constituents of Marsdenia tenacissima and Calotropis gigantea Abstract... 129 .1 (2C), 128 .9, 128 .2 (2C), 128 .1, 127 .8, 126 .6, 123 .6, 123 .3, 122 .8, 122 .6, 122 .1, 122 .0, 121 .7, 118.4, 117.8, 88 .2, 64.0, 45.6; MS (ESI): m/z 433.45 [M+H]+; HRMS (ESI) Calcd for C28H21N2O3... 130.9, 129 .2, 127 .7, 125 .6, 123 .5, 122 .6, 122 .5, 122 .0, 116 .2, 90.1, 62. 7, 55.4, 29 .8, 29 .5, 29 .3, 25 .9, 25 .2; MS (ESI): m/z 373.45 [M-H]-; HRMS (ESI): m/z [M-H]- Calcd for C23H21N2O3: 373.15 42; Found: