Semi-synthesis and structure-activity relationship studies of Stemona alkaloids and related

274 Pages·2011·10.53 MB·English

University of Wollongong
Research Online
University of Wollongong Thesis Collection University of Wollongong Thesis Collections
2011
Semi-synthesis and structure-activity relationship
studies of Stemona alkaloids and related analogues
Kwankamol Sastraruji
University of Wollongong, [email protected]
Recommended Citation
Sastraruji, Kwankamol, Semi-synthesis and structure-activity relationship studies of Stemona alkaloids and related analogues, Doctor
of Philosophy thesis, , University of Wollongong, 2011. http://ro.uow.edu.au/theses/3345
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Semi-Synthesis and Structure-Activity Relationship
Studies of Stemona Alkaloids and Related Analogues

A thesis submitted in fulfilment of the requirements
for the award of the degree of

Doctor of Philosophy
from
University of Wollongong

Kwankamol Sastraruji
B.Sc. Chemistry (Hons.)

Supervisors: Professor Stephen G. Pyne and
Associate Professor Alison T. Ung

School of Chemistry
July, 2011

DECLARATION

I, Kwankamol Sastraruji, declare that this thesis, submitted in fulfilment of
the requirements for the award of Doctor of Philosophy, in the School of Chemistry,
University of Wollongong, is wholly my own work unless reference is provided.
This document has not been submitted for qualifications at any other academic
institution.

Kwankamol Sastraruji
July, 2011
i
ACKNOWLEDGEMENTS
Firstly, I would like to express my gratitude and appreciation to both of my
excellent supervisors, Professor Stephen G. Pyne and Associate Professor Alison T.
Ung (UTS), who have allowed a great opportunity to further my knowledge at
University of Wollongong. Without their invaluable supervision, guidance, patience
and both physically and mentally supports throughout the course of my Ph.D. study,
this degree would never have been completed.
I would like to thank University of Wollongong for financial support in the
form of an International Student Tuition Scholarship and a University Postgraduate
Award (UPA) Scholarship.
Secondly, I am grateful to Associate Professor Renate Griffith (UNSW) for
her kindly supervision on my computational studies. A thank to Dr. Thanapat
Sastraruji for his help on performing the AChE assays and the HPLC analysis. I am
thankful for the research collaborators from Chiang Mai University, Thailand,
Associate Professor Araya Jatisatienr’s and Associate Professor Pornngarm
Limtrakul’s research groups, who have also partially contributed in this project. A
thank to Dr. Anthony Willis (ANU) for the X-ray crystallography analysis.
I would like to thank all the technical staffs at School of Chemistry,
University of Wollongong, especially for Dr. Wilford Lie for his expertise support on
the NMR experiments, Dr. John Korth, Dr. Thitima Urathamakul, Ben Cummings
and Roger Kanitz from the mass spectrometry group and Roza Dimeska for her
kindly assistance on the IR operation. To present and past members of the Pyne
group, I appreciate all the experiences that we have had together.
Special thanks to Barbara Pyne, Dr. Yoke Berry and people who I have not
named in here for their love and care during the hardest time of my life.
Finally, I would like to thank my parents for their unconditional love and all
their best efforts in shaping me into a person as I am today. Thanks again to my
brother for being my life assistance. Thanks to all members of my family for their
good wills, prayers and encouragements. For myself, thank you that I has stood up
again and finally made the right decision.

ii
ABSTRACT
The Stemona species are monocotyledonous plants that belong to the
Stemonaceae family. These plants are widely spread throughout South East Asia and
northern Australia. The crude extracts from the roots of Stemona sp. have been used
in China and South East Asia for agricultural and medicinal purposes. Stemona
alkaloids have been reported for their insecticidal activities which may be associated
with their ability to inhibit the enzyme acetylcholinesterase (AChE). Inhibitors of
this enzyme are currently used to treat patients with Alzheimer’s disease and
therefore the discovery of new AChE inhibitors is of medical importance. During the
course of this study stemofoline was reported to increase sensitivity of anticancer
drugs in treatment of multidrug resistance (MDR) cervical cancer cells.
The aims of the project were to use the known stemofoline-type alkaloid,
11(Z)-1',2'-didehydrostemofoline, as a template to prepare rare Stemona alkloids and
their analogues for testing as AChE inhibitors and structure-activity relationship
(SAR) studies and to test their abilities to increase the sensitivity of anticancer drugs
to MDR cancer cells.
11(Z)-1',2'-Didehydrostemofoline was isolated in grams quantities from the
root extracts of unknown Stemona sp. which had been collected in Amphur Mae
Moh, Lampang, Thailand.
From this starting material, four Stemona alkaloids, included stemoburkilline,
oxystemofoline, methoxystemofoline and (1'R)-hydroxystemofoline, were prepared
along with many analogues that incorporated hydroxyl and amino groups at the C-3
side chain position on the stemofoline back bone. The semi-synthesis of
stemoburkilline was reported in Chapter 3. Using 11(Z)-1',2'-didehydrostemofoline
as the starting material, stemoburkilline was prepared in four steps, these included
the hydrogenation of the side chain alkene to produce stemofoline. Hydrogenation of
the C-11-C-12 alkene moiety of stemofoline followed by a base catalysed ring-
opening reaction of 11,12-dihydrostemofoline in the presence of TMSCl to give the
TMS protected version of stemoburkilline. Mild TMS deprotection then gave
stemoburkilline. The ring-opening process was proposed to occur through an E1cB
mechanism. The NMR spectroscopic data of the synthesised stemoburkilline, which
indicated the formation of the Z-isomer, was identical to those of the natural product.
iii
This study led to the revision of the stemoburkilline structure from an E-isomer to a
Z-isomer.
Chapter 4 of this thesis reports the preparation of the key aldehyde
intermediate which was used to prepare several stemofoline derivatives that
contained a hydroxyl group in the C-3 butyl side chain. These included the natural
products, oxystemofoline, methoxystemofoline and (1'R)-hydroxystemofoline. The
key aldehyde was prepared in two steps from 11(Z)-1',2'-didehydrostemofoline via
an asymmetric dihydroxylation reaction of the C-3 1-butenyl side chain and then
oxidative cleavage of the subsequence diol. A modified Julia olefination reaction on
the aldehyde was employed as a key step in the preparation of oxystemofoline and
methoxystemofoline, using sulfone reagents which had 4-hydroxybutyl and 4-
methoxybutyl side chains, respectively. The synthesis of oxystemofoline and
methoxystemofoline allowed reassignment of the 13C NMR signals for C-6 and C-1′
from those reported for the natural products. Allylation of the aldehyde under
indium-mediated conditions or using chiral allylborane reagents provided (1′R) and
(1′S)-homoallylic alcohol products that lead to (1′R)-hydroxystemofoline and its (S)-
epimer, respectively. Surprisingly, the synthesised (1′R)-hydroxystemofoline proved
to be identical to a natural product that was later isolated from the root extracts of
Stemona aphylla. The Wittig reaction of the aldehyde with
(triphenylphosphoranylidene)acetaldehyde provided a mixture of three aldehyde
products formed from consecutive Wittig reactions. These aldehyde products were
later reduced to three different alcohols, included the enol, the dienol and the trienol.
The A, B, C ring core structure of stemofoline was also provided in two steps via an
Upjohn dihydroxylation reaction of stemofoline followed by an oxidative cleavage of
the corresponding C-11, C-12 diol. The oxidation reaction of 11(Z)-1',2'-
didehydrostemofoline using Na WO ·2H O as a catalyst provided its N-oxide
2 4 2
product in a shorter reaction time than the formerly reported method.
In Chapter 5 of this thesis, the reductive amination reactions of the aldehyde
were reported to prepare 17 secondary amine derivatives in yields ranging from 25-
93%. Some secondary amines were used in further methylation and carbamylation
reactions to prepare tertiary amine and carbamate derivatives, respectively. A
guanidine derivative was also prepared as its HCl salt.
iv
In Chapter 6 of this thesis, an examination of CuI-catalysed click reactions
was studied around a C-3 ethyne stemofoline analogue. The alkyne was prepared in a
one step reaction from the key aldehyde and the Bestmann-Ohira reagent. Four
triazoles were prepared using two different methods, a classical method using azides
and a microwave-assisted method in which the azides were generated in situ. While
two isoxazoles were prepared using chlorooximes as precursors to the corresponding
nitrile oxides. The Sonogashira coupling of the alkyne gave the phenylalkyne
product and the alkyne dimer which was more efficiently and directly prepared under
Eglington coupling conditions. A hydrogenation reaction of the phenylalkyne
product provided a compound with a flexible C-3 2-phenylethyl side chain.
In Chapter 7 the AChE inhibitory activities of the synthesised stemofoline
alkaloids and analogues are reported using a TLC bioautographic method which
measured the activity as a minimum inhibitory requirement (MIR) in ng or nmol
against electric eel AChE (eeAChE). Galanthamine was used as a positive control
having a MIR of 1 ng (0.003 nmol). The four Stemona alkaloids synthesised from
this study, stemoburkilline, oxystemofoline, methoxystemofoline and (1′R)-
hydroxystemofoline, showed MIRs of 50, 50, 50 and 5 ng, respectively. (1′R)-
Hydroxystemofoline also showed the highest activity among the alcohol derivatives.
Compared to 11(Z)-1',2'-didehydrostemofoline (MIR = 5 ng), its N-oxide had similar
activity (MIR = 5 ng) while that of stemofoline, which lacked the side chain alkene
functionality, showed less activity with a MIR of 10 ng. Lacking the lactone ring of
the stemofoline, the A, B, C ring core structure derivative was 10 times less active
than stemofoline (MIR = 100 ng). The cyclopentyl amino carbamate, the
dimethylamine and the alkyne derivatives had the highest activities in the group with
MIR values of 1 ng. In general it was found that most of the amine derivatives were
more active than the alcohol derivatives. While the click products showed moderate
activities in the range of 50-100 ng except for the benzyl triazole derivative which
had a higher activity with a MIR value of 5 ng. Compared to the alkyne (MIR = 1
ng), the phenylalkyne and the alkyne dimer were less active (MIRs = 50 and 100 ng,
respectively). However, the compound with a flexible 2-phenylethyl side chain was
10 times more active than the phenylalkyne with a MIR of 5 ng. Some compounds
were tested for their IC values against eeAChE and human AChE (hAChE) using a
50
colorimetric assay (known as Ellman’s method). 11(Z)-1',2'-Didehydrostemofoline
v
and an isopropylamine analogue showed good activities against eeAChE (IC values
50
= 19.2 and 12.9 M, respectively) and hAChE (IC values = 25.0 and 19.9 M,
50
respectively) but were not as potent as galanthamine (IC values = 0.9 and 0.6 M
50
for eeAChE and hAChE, respectively). While other Stemona derivatives showed
lower activities against eeAChE and hAChE with IC values in the range of 52.5-
50
302.3 M and 28.7-52.4 M, respectively. The MDR-reversing properties of some
Stemona compounds were performed using the colorimetric MTT assay. Among the
tested compounds, stemofoline showed the highest modulating effect on resistant
KB-V1 cells by decreasing the IC of paclitaxel from 10.06  1.56 M to 1.4  0.45
50
M and that of vinblastine from 0.61  0.05 M to 0.09  0.01 M. Stemofoline had
the highest modulating effect on the resistant KB-V1 cells.
In Chapter 8 of this thesis, SAR studies are described using pharmacophore
generation and molecular docking. The best seven different pharmacophore models
were generated in order to search for the binding mode of the Stemona compounds.
Unfortunately, based on the results from pharmacophore mapping alone we could not
confirm the exact binding site. Thus, protein-ligand docking was performed using
three different AChEs. The results from molecular docking suggested that the
Stemona compounds were more likely to fit vertically in the active-site gorge of
AChEs and bind between the active site and the PAS of AChEs. These
computational studies showed that Stemona compounds may inhibit AChEs by
allosterically binding at the PAS and blocking acetylcholine from reaching the active
site.
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