PART I. SYNTHESIS AND BIOLOGICAL EVALUATION OF PHOSPHOGLYCOLIPID PGL1 ANALOGUES. PART II. SYNTHESIS AND BIOLOGICAL EVALUATION OF ANDROGRAPHOLIDE ANALOGUES. HADHI WIJAYA NATIONAL UNIVERSITY OF SINGAPORE 2014 PART I. SYNTHESIS AND BIOLOGICAL EVALUATION OF PHOSPHOGLYCOLIPID PGL1 ANALOGUES. PART II. SYNTHESIS AND BIOLOGICAL EVALUATION OF ANDROGRAPHOLIDE ANALOGUES. HADHI WIJAYA (BSc. (Hons.) NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2014 ACKNOWLEDGEMENTS First and foremost I would like to express my sincere gratitude to my supervisor, A/P Lam Yulin, who has given me the opportunity to join her research lab as a graduate student. I would like to thank her for her guidance provided to me throughout my PhD studies. She has always been patient and understanding by giving me sufficient time and advice to overcome difficulties that I encountered over the course of my research. I would also like to thank our collaborator Prof. Wu Shih-Hsiung, Prof. Hua Kuo-Feng, Dr. Yang Feng-Ling, Prof. Yao Shao Qin, Dr. Li Lin, Ms. Sun Chenyang, Prof. Wong Wai-Hsiu and Ms. Loh Xinyi. They have provided valuable ideas and are responsible for the biological studies discussed in this thesis. I would like to express my sincere appreciation to Mdm. Han Yanhui and Dr. Wu Ji’En from NMR lab, Mdm. Wong Lai Kwai, Mdm. Lai Hui Ngee and Dr. Liu Qiping from Mass spec lab as well as Ms. Tan Geok Kheng and Ms. Hong Yimian from X-ray lab for their assistance in the compounds characterization. To all past and present members of A/P Lam lab, Dr. Kong Kah Hoe, Dr. Fang Zhanxiong, Dr. Che Jun, Dr. Wong Lingkai, Dr. Samanta Sanjay, Dr. Woen Susanto, Lin Xijie, Alan Sim, Cliff Anderson, Ang Wei Jie, Poh Zhong Wei, Ng Cheng Yang, Ran Jiangkun, Niu Zilu, Gan Chin Heng, Linus Lim Wei Jie and Chng Yong Sheng, I would like to say thank you for your advice and help. ii I would like to thank my family for their continuous support. Without them, I would not have the opportunity to further my study in Singapore. Last but not least, I woud like to express my gratitude to my partner Ms. Pulvy Iskandar. I would like to thank her for her encouragement, motivation, patience and understanding. For the past fifteen years, she has always been there for me, giving me the strength and courage to face all the challenges and obstacles that I encountered in my life. iii TABLE OF CONTENTS DECLARATION i ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iv SUMMARY vii LIST OF TABLES ix LIST OF FIGURES xi LIST OF SCHEMES xv LIST OF ABBREVIATIONS xvii CHAPTER 1. INTRODUCTION 1.1 Overview 1.2 Definition, Classification and sources of Natural Products 1.3 Natural Products with Anti-inflammatory Activity 1.4 Synthesis of Natural Product and Analogues 12 1.5 Purpose of Research Work in this Thesis 14 1.6 References 15 CHAPTER 2. SYNTHESIS AND BIOLOGICAL 19 EVALUATION OF PHOSPHOGLYCOLIPID PGL1 ANALOGUES 2.1 Introduction 19 2.2 Results and Discussions 24 2.2.1 Retrosynthesis 24 2.2.2 Synthesis of glycosyl donor 2-3 25 2.2.3 Synthesis of glycerate 2-4 28 2.2.4 Synthesis of H-phosphonate 2-2 30 iv 2.2.5 Synthesis of glycolipid 2-29 and 2-35 38 2.2.6 Synthesis of glycosyl donor 2-41 42 2.2.7 Synthesis of PGL1 analogues 43 2.3 Biological Results 50 2.4 Conclusion 54 2.5 References 55 CHAPTER 3. SYNTHESIS OF PROBES FOR ACTIVITY 58 BASED PROTEIN PROFILING OF POTENTIAL CELLULAR TARGETS OF ANDROGRAPHOLIDE 3.1 Introduction 58 3.2 Results and Discussions 63 3.2.1 Design of probes 63 3.2.2 Synthesis of probes 67 3.3. Biological Results 73 3.3.1 Cell Proliferation Assay and Western Blott Analysis 73 of STAT Phosphorylation in HepG2 cell line 3.3.2 In situ protein profiling in HepG2 cell line with AP1 75 3.3.3 In situ protein profiling of all probes in HepG2 cell lines 76 3.3.4 In situ protein profiling by AP1 in different cell lines 77 3.3.5 Pull down and Target Validation 78 3.3.6 Fluorescence enzymatic assay 80 3.3.7 Cellular imaging with APNP, AP1NP and AP2NP 82 3.4 Conclusion 84 3.5 References 86 v CHAPTER 4. SYNTHESIS AND BIOLOGICAL EVALUATION 90 OF ANDROGRAPHOLIDE ANALOGUES AS POTENTIAL INHIBITORS OF NF-B. 4.1 Introduction 90 4.2 Results and Discussion 93 4.2.1 Synthesis of Analogues 93 4.2.2 Biological Results 98 4.3 Conclusion 103 4.4 References 104 APPENDICES Appendix A. Experimental Procedures and Characterization 106 Data for Compounds Appendix B. Experimental Procedures and Characterization 210 Data for Compounds Appendix C. Experimental Procedures and Characterization 234 Data for Compounds Appendix D. NMR Spectra of Some Intermediates and Analogues vi 251 SUMMARY Natural products play an important role in drug discovery. Numerous life saving medicine and medical breakthrough have been associated and intrinsically linked to natural products. Today, half of the drugs approved for marketing are of natural origin or derived from natural products. Currently, less than 10% of world biodiversity has been explored for medicinal purposes. Many more biologically active compounds from nature have yet to be discovered and developed into useful drugs for the treatment of various diseases. In this thesis, the synthesis and biological evaluation of analogues of two natural products are presented. In Chapter 2, the synthesis and biological evaluation of phosphoglycolipid PGL1 analogues are described. The synthetic route towards PGL1 was successfully established and a library consisting of 21 analogues was prepared. The analogues were initially evaluated for their immunostimulation activity. However, no positive results were obtained. This could be due to various factors such as incorrect chiral centre of the glycerol moiety or incorrect fatty acid chains (length and branching). The analogues were then evaluated for their inhibition activity agains TNF- and IL-6 instead. Two of the analogues, PGL1j and PGL1s showed significant inhibition against IL-6. In Chapter 3, the synthesis of andrographolide probes as well as Activity Based Protein Profiling (ABPP) of potential andrographolide targets are described. The probes were designed based on previous structure-activity vii relationship study and anti-inflammatory mechanism of andrographolide. Protein profiling and subsequent target validation confirmed p50 as the protein target in HepG2 cell. Pull down/LC-MS/MS analysis identified NAMPT as one of the potential targets in A549 cell. The fluorophore containing probes were successfully utilized in live cell imaging which produced fluorescent signal upon reacting with the targets. The imaging result were also consistent with the pull down and Western blot analysis. Finally, the synthesis and biological evaluation of andrographolide analogues for inhibition of NF-B are described in Chapter 4. The synthesis involved modification of the lactone ring of andrographolide. Biological evaluation revealed that one of the analogues, 4-7c showed a concentrationdependent inhibition of NF-B in A549 cell. viii O H O O O H O 4-4e O H O O O H O 4-4e 299 O O O HO H HO 4-5a O O O HO H HO 4-5a 300 O O HO H HO 4-5b O O HO H HO 4-5b 301 O O O HO H HO 4-5c O O O HO H HO 4-5c 302 O O HO H HO 4-5d O O HO H HO 4-5d 303 O H O O HO H HO 4-5e O H O O HO H HO 4-5e 304 S HN O S O O H O 4-6a S HN O S O O H O 4-6a 305 HN O O S O O H O 4-6b HN O O S O O H O 4-6b 306 NH O O O H O 4-6c NH O O O H O 4-6c 307 S HN S O O HO H HO 4-7a S HN O S O HO H HO 4-7a 308 O HN S O O HO H HO 4-7b HN O O S O HO H HO 4-7b 309 NH O O HO H HO 4-7c NH O O HO H HO 4-7c 310 O N SiPh2 Me 4-9 O N SiPh2 Me 4-9 311 N O O O H O 4-10 N O O O H O 4-10 312 N O O HO H HO 4-11 N O O HO H HO 4-11 313 O O AcO H AcO 4-12 O O AcO H AcO 4-12 314 [...]... cell Figure 3.10 In situ protein profiling in HepG2 cell with AP1 xii 75 Figure 3.11 In situ protein profiling of all probes and 76 andrographolide in HepG2 cell Figure 3.12 In situ protein profiling of AP1 in different cell lines 77 Figure 3.13 In-gel fluorescence scanning and Western blot analysis 78 (anti p50) of HepG2 cell Figure 3.14 In-gel fluorescence scanning and Western blot analysis 79 (anti-p50,... exerts its antiinflammatory activity by inhibiting NF-B DNA-binding activity.44 Andalusol and parthenolide are natural products belonging to the terpenoid class which have also been reported to inhibit NF-B.45,46 The ability of parthenolide in inhibiting NF-B proved to be useful in increasing the sensitivity of cancer cells towards chemotherapeutic drugs This is evidenced by its ability to decrease... or Physiology, which was awarded to Sir Alexander Fleming in 1945 More 5 importantly, many lives have been saved as a result of using penicillin to treat bacterial infections, which caused very serious diseases decades ago Penicillin exerts its antibiotic activity by specifically inhibiting transpeptidase, the enzyme which catalyzes the final steps in cell wall biosynthesis of bacteria.16,17 This results... the migration of leukocytes (neutrophils) into the tissue The neutrophils attempt to eliminate the offending agents by releasing reactive oxygen species (ROS), reactive nitrogen species and elastase.32 After the elimination of the inflammatory inducer, a resolution of inflammation and tissue repair take place Unsuccessful elimination of inducers often results in chronic inflammation 9 The inflammation... 97 xvi LIST OF ABBREVIATIONS ABPP activity based protein profiling Ac2O acetic anhydride ACN acetonitrile AcOH acetic acid AcSH thioacetic acid aq aqueous BAIB bis(acetoxy)iodobenzene BF3.OEt2 boron trifluoride diethyl etherate Bn benzyl BnBr benzyl bromide CCl3CN trichloroacetonitrile CDI 1,1’-carbonyldiimidazole DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene DCC N,N’-dicyclohexylcarbodiimide DCM dichloromethane... heteronuclear single quantum coherence IL-1 interleukin 1 IL-6 interleukin 6 K2CO3 potassium carbonate LCMS-IT-TOF liquid chromatography mass spectrometer-ion traptime of flight LDA lithium diisopropylamine LDL low-density lipoprotein LPSs lipopolysaccharides Me methyl M.W microwave irradiation NaOMe sodium methoxide NF-B nuclear factor kappa-light-chain-enhancer of activated B cells NIS N-iodosuccinimide NMR... antimalarial drug The endo-peroxide functionality of this compound was reported to be essential for its biological activity.13 The antimalarial effect of artemisinin originates from its ability to modify protein synthesis within the malaria parasite, Plasmodium falciparum.14 O O O NH O OH O H O OH O OH O O O O Paclitaxel 1-1 Figure 1.3 Structure of paclitaxel 1-1 H H 3C CH 3 OO O HO H CH3 O Artemisinin... 2.14 Synthesis of glycosyl donor 2-41 43 Scheme 2.15 Synthesis of PGL1 45 Scheme 2.16 Solvent participation in glycosylations 45 Scheme 3.1 Mechanism of inhibition of NF-B by andrographolide 64 xv Scheme 3.2 Proposed mechanism of self-reporting property of 66 probe APNP Scheme 3.3 Synthesis of AP1 and AP2 68 Scheme 3.4 Synthesis of AP3 69 Scheme 3.5 Synthesis of NC1 70 Scheme 3.6 Synthesis of APCM and. .. microtubules.11 This binding process results in the inhibition of cell division and eventually cell death, which serves to kill the rapidly dividing tumor cells Another natural product derived from plant origin is artemisinin 1-2 (Figure 1.4) This compound was isolated from the leaves of Artemesia annua Artemesia has been used as traditional Chinese medicine for over 2000 4 years.12 Today, it is used primarily as... using bio-orthogonal reactions 60 Figure 3.4 ABPP of parthenolide using biotinylated derivative 61 Figure 3.5 Structural features of andrographolide 62 Figure 3.6 Structures of andrographolide probes 64 Figure 3.7 Structure of fluorophore-containing andrographolide 65 probes Figure 3.8 Cell proliferation assay of andrographolide and probes 74 Figure 3.9 Western blot analysis of STAT3 phosphorylation in . NATIONAL UNIVERSITY OF SINGAPORE 2014 PART I. SYNTHESIS AND BIOLOGICAL EVALUATION OF PHOSPHOGLYCOLIPID PGL1 ANALOGUES. PART II. SYNTHESIS AND BIOLOGICAL EVALUATION OF ANDROGRAPHOLIDE ANALOGUES. . PART I. SYNTHESIS AND BIOLOGICAL EVALUATION OF PHOSPHOGLYCOLIPID PGL1 ANALOGUES. PART II. SYNTHESIS AND BIOLOGICAL EVALUATION OF ANDROGRAPHOLIDE ANALOGUES. HADHI WIJAYA. protein profiling in HepG2 cell with AP1 75 xiii Figure 3.11. In situ protein profiling of all probes and 76 andrographolide in HepG2 cell Figure 3.12. In situ protein profiling of AP1 in different