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研究生：	蔡志偉 Tsai, Chih-Wei
論文名稱：	C6胺甲基isofagomine分子群之合成及其生物活性之探討 Synthesis and biological evaluation of C6 aminomethyl isofagomine-based molecules
指導教授：	鄭偉杰 Cheng, Wei-Chieh
共同指導教授:	黃福永 Huang, Fu-Yung
學位類別：	碩士 Master
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2011
畢業學年度：	100
語文別：	英文
論文頁數：	136
中文關鍵詞：	亞胺醣、抑制劑、助疊小分子、高雪氏症
外文關鍵詞：	iminosugar, inhibitor, chaperone, Gaucher disease
相關次數：	點閱：83 下載：4
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亞胺醣如isofagomine和1-deoxynujirimycin (DNJ)等，是非常具有藥效潛力的分子，例如治療代謝疾病、抑制腫瘤生長和抑制病毒和細菌活性等。由於這些分子具有許多生物活性效果，其製備方法顯得非常重要。從化學結構的角度來看，亞胺醣被認為是模仿糖苷酶水解時的過渡態oxocarbonium ion。在本論文中，我們設計了一個新的合成路徑，使用普遍且容易改質的方法來製備新的骨架稱為 C6-aminomethyl hydroxymethyl piperidine diol（AHPD）。我們將AHPD library的分子進行葡萄糖苷水解酶抑制活性測試，發現亞胺醣衍生物A17與A76為非常好的抑制劑(at nanomolar level)。此外，合成出的新穎分子也應用在助疊小分子以治療高雪氏症(Gaucher disease)。經由細胞檢測(N370S)發現，亞胺醣衍生物B117約增加突變酵素活性2.5倍為相當具有潛力的小分子。

Iminosugars such as isofagomine and 1-deoxynujirimycin (DNJ) have significant therapeutic potential for the treatment of metabolic diseases, inhibition of tumor metastasis and control infections of fungi and viruses. Due to their versatile biological activities, the preparation of them becomes very attractive and important. From the structural point of view, iminosugars might be thought as the transition state mimic of oxocarbonium-ion during the enzymatic hydrolysis of glycosidase. In this project, we have developed a new synthetic strategy to prepare new scaffolds called C6-aminomethyl hydroxymethyl piperidine diol (AHPD) with specific configuration via our general and flexible synthetic approach. These AHPD-based libraries were investigated for the inhibition of glycosidases, such as beta-glucocerebrosidase and several potent inhibitors were found at namomolar level. Besides, we also applied several AHPD-based molecules to treat N370S in fibroblasts (Gaucher disease) and these chemical chaperones increase the cellular activity around 2.5 fold.

Abstract I
Acknowledgement III 
Table of contents V
Index of Figures VII
Index of Tables IX
Index of Schemes X
Abbreviations XII

Chapter 1. Introduction 1
1-1 Introduction of Iminosugars 1
1-1.1 Mechanism for retaining and inverting glycosidases 2
1-1.2 Transition state mimics of substrate 4
1-2 Synthesis and biological applications of Isofagomine 4
1-2.1 Gaucher disease 9
1-2.2 Treatment of Gaucher disease 10
1-3 Motivation 12

Chapter 2. Results and Discussion 13
2-1 General strategy of design new cores 13
2-1.1 Retrosynthesis analysis 14
2-1.2 Method A 14
2-1.2.1 The preparation of alpha,beta-unsaturated nitroalkene 10 14
2-1.3 Method B 18
2-1.4 Synthesis of cis-AHPD and trans- AHPD 20
2-1.4.1 Proposed mechanism for nucleophile attacking cyclic nitrone 26
2-1.4.2 Preparation of libraries 28
2-2 Inhibition assays against beta-glucocerebrosidase and chaperone effects 30
2-2.1 Computation 36
2-3 Conclusions 39

Chapter 3 Experimental Section 40
3-1 Biological assays 40
3-2 General experimental procedure 43
3-2.1 General procedure A for preparation of amide products (library A) 43
3-2.2 General procedure B for preparation of amide products (library B) 43
3-3 Procedures and experimental data 44

References 64

Appendix 70

Index of Figures
Figure 1.1. Structures of NJ, DNJ, swainsonine, castanosperine, fagomine, Miglitol, Zevasca, and isofagomine 1
Figure 1.2. Common structural classes of iminosugars 2
Figure 1.3. Mechanism of retaining and inverting enzymatic hydrolysis 3
Figure 1.4. Transition state mimics of sugar-processing substrates 4
Figure 1.5. Schematic representation of the interactions 5
Figure 1.6. Various starting materials of isofagomine 5
Figure 1.7. Disease-related applications of Isofagomine-based molecules 9
Figure 2.1. (a) 1H-1H COSY of 3; (b) 1H-1H NOESY of 3 21
Figure 2.2. (a) 1H-1H COSY of 4; (b) 1H-1H NOESY of 4 23
Figure 2.3. (a) 1H-1H COSY of 26; (b) 1H-1H NOESY of 26 26
Figure 2.4. Inhibition percentages of A1~A80 against beta-glucosidase at 100 nM 30
Figure 2.5. Inhibition percentages of A81~A134 against beta-glucosidase at 100 nM 30
Figure 2.6. Structures of potent inhibitors with inhibition percentages (%) in A(1~134) at 100 nM 31
Figure 2.7. Structures of resynthesis inhibitors with their IC50 32
Figure 2.8. Chaperone effects of B1~B80 on cellular N370S GC activity at 1 M 33
Figure 2.9. Chaperone effects of B81~B134 on cellular N370S GC activity at 1 M 33
Figure 2.10. Chaperone effects of B81~B134 on cellular N370S GC activity at 1 M 33
Figure 2.11. Structures of A76, A81, A17 and A112 with their IC50 34
Figure 2.12. Structures of A76, A81, A17 and A112 with their IC50 34
Figure 2.13. The Lineweaver–Burk plots of (a)A17, (b)A76 and (c)A112 for the inhibition of human GCase. The increasing concentrations of substrate were used to determine the Ki values and the data were plotted as 1/v versus 1/[S] 35
Figure 2.14. Surface representation of GCase near the active site. (a) IFG-bound inh. (b) inh-like configuration in neutral. (c) Glucosylceramide-docked inh. Glucosylceramide has been modified by truncating its alkyl chains. (d) Schematic diagram of hydrogen bonding interactions involved in stabilizing IFG in the active site of GCase 36
Figure 2.15. Docking for (a) A76;(b) A81;(c) A17;(d) A112 37

Index of Tables
Table 2.1. Preparation of 13 via Michael addition 16
Table 2.2. Preparation of alcohol 17 18

Index of Schemes
Scheme 1.1. Synthesis of isofagomine 8 from Levoglucosan 6

Scheme 1.2. Synthesis of isofagomine 8 from butadiene monoxide 7

Scheme 1.3. Synthesis of isofagomine 8 from L-xylose 8

Scheme 2.1. General strategy of design new cores 13

Scheme 2.2. Retrosynthetic analysis of AHPD cores 14

Scheme 2.3 The preparation of alpha,beta-unsaturated nitroalkene 10 15

Scheme 2.4. Michael addition of 10 with Cuprate reagent 15

Scheme 2.5. Model of Michael addition of 10 with Cuprate reagent 16

Scheme 2.6. Preparation of piperidine 2 from 11 17

Scheme 2.7. Preparation of amine 16 from 11 18

Scheme 2.8. Mechanism of Nef reaction 19

Scheme 2.9. Preparation of 21 from 11 20

Scheme 2.10. Preparation of cis-AHPD 3 20

Scheme 2.11. The preparation of trans-AHPD 4 23

Scheme 2.12. The preparation of 5,6-trans piperidine 26 25

Scheme 2.13. Proposed mechanism for nucleophilic addition with TMSCN 27

Scheme 2.14. Proposed mechanism for nucleophilic addition with Grignard reagent 28

Scheme 2.15. Preparation of libraries 30

Scheme 3.1. Inhibition assay against beta-glucosidase 40 

                                    

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