hplc-based activity profiling for herg channel inhibitors from galenia africana and gnidia ...

209 Pages·2015·12.29 MB·English

HPLC-BASED ACTIVITY PROFILING FOR HERG CHANNEL INHIBITORS FROM
GALENIA AFRICANA AND GNIDIA POLYCEPHALA, AND COUNTER-CURRENT
CHROMATOGRAPHIC ISOLATION OF ANTIMICROBIALS FROM
COLOPHOSPERMUM MOPANE

KUN DU
HPLC-BASED ACTIVITY PROFILING FOR HERG CHANNEL INHIBITORS FROM
GALENIA AFRICANA AND GNIDIA POLYCEPHALA, AND COUNTER-CURRENT
CHROMATOGRAPHIC ISOLATION OF ANTIMICROBIALS FROM
COLOPHOSPERMUM MOPANE

Thesis submitted in fulfillment of the requirements for the degree

PHILOSOPHIAE DOCTOR

in the

Department of Chemistry
Faculty of Natural and Agricultural Sciences

at the

University of the Free State
by

Kun Du

Supervisors

Professor Jan Van der Westhuizen
(University of the Free State, South Africa)
Professor Matthias Hamburger
(University of Basel, Switzerland)

January 2015
Bloemfontein
DECLARATION

I, Kun Du, hereby declare that this thesis is submitted by me for the degree of Philosophiae Doctor
in Chemistry, at the University of the Free State. To the best of my knowledge, this is my own
original work with the exception of such references used. This thesis has not been previously
published or submitted to any university for a degree. I further cede copyright of the thesis in favor
of the University of the Free State.
This thesis is dedicated to my MSc/PhD supervisor the Late Professor Andrew Marston (November
16, 1953 - March 26, 2013).
Kun Du
January 2015
ACKNOWLEDGMENTS
At the end of this road, I would like to express my sincere gratitude and appreciation to the
following people for their contribution towards this study:
 My late MSc/PhD supervisor Prof. Andrew Marston for believing in me, for introducing me
into phytochemistry, and for his supervision, support, encouragement, persistent, guidance
and friendship. I miss the years that we worked together on the same bench often until the
late night. You were a true inspiration. You may be gone but you will never be forgotten. I
keep you in my heart forever.
 Prof. Jan van der Westhuizen and Prof. Matthias Hamburger for their supervision, assistance,
guidance, patience, invaluable advice, and correcting and giving suggestions to this thesis.
 Dr. Maria De Mieri for her guidance, assistance, patience, and contribution to the projects of
this thesis.
 I would particularly like to thank Prof. M. Hamburger for kindly accommodating me in his
group at the University of Basel (Switzerland) and introducing me the state-of-the-art
techniques and research strategies, and Dr. M. De Mieri from the same group for her kind
help and support all the time in the laboratory.
 Prof. Sandy van Vuuren for running the antimicrobial assay, Prof. Steffen Hering and
co-workers (Priyanka, Khanya and Elmarie) for running the hERG assay, Dr. Markus
Neuburger for X-ray crystallography and Dr. Pieter Zietsman for collection and
identification of the plant materials.
 My group colleagues in University of the Free State and University of Basel for the help and
the good times we shared and most of all the friendly environment conductive for this study.
 The South African National Research Foundation (NRF), and European Union Framework 7
(NO. PIRSES-GA-2011-295174) are gratefully thanked for financial support.
 Finally, this work would not have been possible without the support of my family. They
have always been the source of my motivation for work and study.
Mr. Kun Du
Bloemfontein
January 2015
LIST OF ABBREVIATIONS

AC Absolute configuration
APCI Atmospheric pressure chemical ionization
API Atmospheric pressure ionization
CAM Complementary and alternative medicine
CCC counter-current chromatography
CD Circular dichroism
CDA Chiral derivatizing agent
COSY Correlation spectroscopy
DAD Diode array detector
DMSO Dimethyl sulfoxide
ECD Electronic circular dichroism
ECG Electrocardiogram
ELSD Evaporative light scattering detection
ESI Electrospray ionization
FDA Food and Drug Administration
GABA receptor Gamma aminobutyric acid type A receptor
A
HDES Hydrodynamic equilibrium system
hERG Human ether-a-go-go related gene
HM Herbal medicine
HMBC Heteronuclear multiple bond correlation
HPLC High performance liquid chromatography
HSCCC High-speed counter-current chromatography
HSES Hydrostatic equilibrium system
HSQC Heteronuclear single quantum correlation
HTS High-throughput screening
IC Half inhibitory concentration
50
LQT A long time interval from the point Q to point T in an electrocardiogram
MDR Multidrug resistant
MIC Minimal Inhibitory Concentration
MPA α-methoxyphenylacetic acid
MS Mass spectrometry
NMR Nuclear magnetic resonance spectroscopy
NOESY Nuclear Overhauser enhancement spectroscopy
NP Natural product
OR Optical rotation
ORD Optical rotatory dispersion
OTC Over the counter
PDA Photo-diode array
QT The time from the point Q to point T in an electrocardiogram
ROESY Rotating frame Overhauser effect spectroscopy
TCM Traditional Chinese Medicine
TD DFT Time-dependent density function theory
TdP arrhythmia Torsadesde pointes arrhythmia
TEVC Two-microelectrode voltage clamp
TLC Thin-layer chromatography
TM Traditional medicine
TOCSY Total correlation spectroscopy
TOF Time-of-flight
UV Ultraviolet
VCD Vibrational circular dichroism
Vis Visable
WHO World Health Organization
XRD X-ray diffraction
KEYWORDS

Natural products, drug discovery, hERG channel inhibitors, antimicrobials, HPLC-based activity
profiling, high-speed counter-current chromatography, medicinal plants, Galenia africana
(Aizoaceae), Gnidia polycephala (Thymelaeaceae), Colophospermum mopane (Fabaceae).
ABSTRACT

The human ether-a-go-go-related gene (hERG) channel plays a critical role in cardiac action
potential repolarization. Blocking of the channel by drugs may lead to a fatal type of arrhythmia,
named “torsades de pointes” (TdP). The hERG channel is thus considered as a primary antitarget in
safety pharmacology. Several drugs have been withdrawn from the market or received severe
restrictions on use for this reason, and numerous compounds have been blocked from progressing
further into phases of clinical development because of hERG channel inhibition. In contrast to the
routine screening in industry for potential hERG liabilities of drug leads, comparably little is known
about hERG inhibitors in medicinal plants (viz. phytomedicine). These are widely used as
complementary medicines and continue to increase in popularity. There is an urgent need to
critically assess the potential risks of these botanical products.
A library of 700 extracts from different parts of 142 South African medicinal plants has been
screened by our research group for the potential of hERG channel inhibition with a Xenopus laevis
oocytes based bioassay. The DCM extracts from stems and leaves of Galenia africana (Aizoaceae)
and from roots of Gnidia polycephala (Thymelaeaceae) showed the strongest inhibition [50.3 ± 5.4%
(n = 3) and 58.8 ± 13.4% (n = 3) respectively at a concentration of 100 g/mL]. The molecules
responsible for the blocking activity were investigated with the HPLC-based activity profiling
approach. Compounds in the active time window were isolated for further pharmacological testing.
We also isolated structurally related compounds in the inactive fractions in view of deriving some
structure-activity related information. Structures were elucidated by a combination of advanced
off-line analytical methods including MS and highly sensitive microprobe NMR. The absolute
configurations were determined by single-crystal X-ray diffraction with Cu Kα radiation or by
comparison of their experimental and calculated ECD spectra.
HPLC-based activity profiling of Galenia africana enabled the identification of nine flavonoids in
the active time windows. However, the hERG-channel inhibition of isolated compounds was less
pronounced than that of extract and active microfractions (hERG inhibition between 10.1 ± 5% and
14.1 ± 1.6% at 100 M). The two major constituents, 7,8-methylenedioxyflavone and
7,8-dimethoxyflavone were quantified (4.3% and 9.4%, respectively) in the extract. Further hERG
inhibition tests for 7,8-methylenedioxyflavone and 7,8-dimethoxyflavone at 300 M showed a
concentration-dependent inhibitory activity (33.2 ± 12.4% and 30.0 ± 7.4%, respectively). In a
detailed phytochemical profiling of the active extract, a total of 20 phenolic compounds, including
six new ones were isolated and characterized.

HPLC-based activity profiling of Gnidia polycephala enabled the identification of three
daphnane-type diterpenoid orthoesters (DDOs) as the hERG channel inhibitors with inhibition of
55.4 ± 7.0% (n = 4); 42.5 ± 16.0% (n = 3) and 51.3 ± 9.4% (n = 4) respectively at 100 M. DDOs
have demonstrated remarkable bioactivities. This is the first report of DDOs as hERG channel
inhibitors and they represent a new scaffold for hERG channel inhibition. In a detailed
phytochemical profiling of the active extract, a total of sixteen compounds, including two new
DDOs, two new guaiane sesquiterpenoids, polycephalone A and B with an unprecedented carbon
skeleton and ten known compounds were isolated and characterized.

New antimicrobials need to be discovered and developed urgently due to the constant evolution of
resistant microorganism phenotypes, the emergence of new diseases, and toxicity associated with
current drugs. A preliminary screen of the lipophilic extracts (DCM) of seeds, leaves and hulls of
Colophospermum mopane (Fabaceae) had shown positive antimicrobial activities. Our
antimicrobial bioassay requires relatively a larger quantity of sample (2 to 5 mg for pure
compounds), thus an efficient preparative isolation of the secondary metabolites in the mixture was
needed. High-speed counter-current chromatography (HSCCC) an all-liquid technique with an
unique separation mechanism was employed. Three new and two known labdane diterpenoids, one
new isolabdane, three new and two known clerodane diterpenoids, were isolated from the seeds,
husks and leaves of Colophospermum mopane. The structures of the isolates were elucidated with
spectroscopic (1D and 2D NMR) and spectrometric methods (MS). The absolute configurations of
compounds with a crystalline form were determined by single-crystal X-ray diffraction with Cu Kα
radiation. The absolute configuration of a labdane diterpenoid and an isolabdane diterpenoid
without a crystalline form was established by modified the Mosher’s method, and corroborated by
comparison of experimental and calculated ECD spectra of their 3-p-bromobenzoate derivatives.
The compounds were evaluated for antimicrobial activities. A clerodane diterpenoid was the most
active with MIC values as low as 51.3 µM, 51.3 µM and 102.9 µM against Escherichia coli,
Staphylococcus aureus and Enterococcus faecalis, respectively.