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.
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.
iii

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. iv

TABLE OF CONTENTS
DECLARATION i

ACKNOWLEDGEMENTS ii

TABLE OF CONTENTS iv
SUMMARY vii

LIST OF TABLES ix

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 3 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 vi

CHAPTER 4. SYNTHESIS AND BIOLOGICAL EVALUATION 90
OF ANDROGRAPHOLIDE ANALOGUES AS POTENTIAL
INHIBITORS OF NF-B.
4.1 Introduction 90

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
viii

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 concentration-
dependent inhibition of NF-B in A549 cell.
ix

LIST OF TABLES

Figure 1.2. Classification of Natural Products 4
Figure 1.3. Structure of paclitaxel 1-1 5
Figure 1.4. Structure of artemisinin 1-2 5
Figure 1.5. Structure of penicillin 1-3 6
Figure 1.6. Structure of bryostatin 1-4 7
Figure 1.7. Structure of captopril 1-5 7
Figure 1.8. Structure of teprotide 1-6 8
Figure 1.9. Examples of Natural products with anti-inflammatory 11
activity
Figure 1.10. Diverted Total Synthesis of Migrastatin Ether 13
Figure 1.11. Semi-synthesis of Paclitaxel 14
Figure 2.1. General structure of Gram-negative LPS 19
Figure 2.2. Structure of Lipid A of E. coli 21
Figure 2.3. Glycosphingolipid from sphingomonas 21
Figure 2.4. Structures of PGL1 and PGL2 22
Figure 2.5. HMBC correlation of monoester 2-26 (proton H
a
) 36
Figure 2.6. Overlapped HSQC/HMBC Spectrum (zoom in) 37
of compound 2-26
xii

Figure 2.7. Overlapped HSQC/HMBC Spectrum of compound 2-26 37
Figure 2.8. HMBC correlation of compound 2-42 (proton H
d
) 48
Figure 2.9. HSQC spectrum of 2-42 48
Figure 2.10. Dihedral angle between proton at C1 and proton at 48
C2 for  and  glycosides
Figure 2.11. HMBC spectrum of 2-42; Correlation between H

of APCM
Figure 3.16. Fluorescence activated cell sorting of live HepG2 cell 81
incubated with APCM and APNP
Figure 3.17. One-photon excited fluorescence images of HepG2 (a) 83
and A549 (b) cells upon treatment with APNP, AP1NP
and AP2NP (10.0 µM)
Figure 4.1. Activation pathway (canonical) of NF-B. 91
Figure 4.2. Different natural products that inhibit NF-κB 92
Figure 4.3. X-ray crystal structure of 4-10 97
Figure 4.4. SEAP reporter assay (inhibition of NF-B) for 99
4-5b, 4-5d, 4-5e, 4-7b
Figure 4.5. SEAP reporter assay (inhibition of NF-B) for 100
4-5a, 4-5c, 4-7a, 4-7c and 4-11
xiv

Figure 4.6. SEAP reporter assay (inhibition of NF-B) for 100
andrographolide
Figure 4.7. SEAP reporter assay (inhibition of NF-B) for 4-12 101
Figure 4.8. Flow cytometric analysis of cytotoxicity of 102
andrographolide, 4-7c and 4-12 against A549 cell

xv

LIST OF SCHEMES
Scheme 2.1. Retrosynthesis of PGL1 24
Scheme 2.2. Synthesis of glycosyl donor 2-3a 26
Scheme 2.3. Synthesis of glycosyl donor 2-3b 27
Scheme 2.4. Synthesis of amine 2-7a and 2-7b 28
Scheme 2.5. Synthesis of amine 2-7d 28
Scheme 2.6. Synthesis of glycerate 2-4 29

xvii

LIST OF ABBREVIATIONS
ABPP activity based protein profiling
Ac
2
O acetic anhydride
ACN acetonitrile
AcOH acetic acid
AcSH thioacetic acid
aq aqueous
BAIB bis(acetoxy)iodobenzene
BF
3
.OEt
2
boron trifluoride diethyl etherate
Bn benzyl
BnBr benzyl bromide
CCl
3
CN trichloroacetonitrile
CDI 1,1’-carbonyldiimidazole
DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene
DCC N,N’-dicyclohexylcarbodiimide
DCM dichloromethane
DHP dihydropyran
DIAD diisopropyl azodicarboxylate
DMAP 4-Dimethylaminopyridine
DMF N-N-dimethylformamide

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 nuclear magnetic resonance
PAGE polyacrylamide gel electrophoresis
PAMPs pathogen-associated molecular patterns
xix

PDC pyridinium dichromate
PGLs phosphoglycolipids
Ph phenyl
PhSH thiophenol
PMA phosphomolybdic acid
PPh
3
triphenylphosphine
PRRs pattern recognition receptors
r.t. room temperature
SDS sodium dodecyl sulfate
TBAF tetrabutylammonium fluoride
TBDMS tert-butyldimethyslsilyl ethers
TEMPO (2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl
TFA trifluoroacetic acid
THF tetrahydrofuran
TLC thin-layer chromatography
TMSOTf trimethylsilyl trifluoromethanesulfonate

4
Natural products continue to inspire the development of therapeutic
treatment today. This is demonstrated by number of approved anti-cancer
drugs (Figure 1.1),
5
From 1981 to 2010, up to 50% of the approved drugs were
natural products or of natural product origin.

Despite their abundance in nature, currently only less than 10% of the
world’s biodiversity has been explored for their potential therapeutic
applications.
6
The nature pool thus presents many exciting opportunities for
new compounds to be discovered and applied for various medicinal purposes.

2 Figure 1.1. Approved Anticancer Drugs, 1981 – 2010.
5
(“B”: biological,
usually large protein, “N”: natural product, “NB”: natural product “Botanical”,
“ND”: derived from Natural product, usually semi-synthetic, “S”: totally
synthetic drug, “S*”: made by total synthesis, natural product pharmacophore,
“NM”: natural product mimic, “v”: vaccine.

1.2 Definition, Classification and Sources of Natural Products
There are different ways in which a natural product can be defined. The
general definition of a natural product is a substance isolated from a living
organism in nature. In the organic chemistry context, natural products are

tetracyclic carbon skeleton

Non
ribosomal
polypeptide
peptide-like compounds
synthesized by nonribosomal
peptide synthetases without direct
RNA transcription

Alkaloids
a class of natural product, which
contains basic nitrogen atoms.
Enzyme
cofactor
non-protein component of
enzymes which is usually
inorganic ions or small organic
molecules Fatty Acids
carboxylic acids that contain long
hydrocarbon chain (saturated or

N
HN
O
H
N
O
O
O
N
H
O
N
O
N
O
Actinomycin D
N
O
O
OH
O
H
H
Cryptocin
NHHN
S
O
H H
COOH
Biotin


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