NATURAL PRODUCTS: STRATEGIES AND IMPORTANCE IN DRUG DISCOVERY, AND THE MALAYSIAN ASPIRATIONS

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NATURAL PRODUCTS STRATEGIES AND IMPORTANCE IN
DRUG DISCOVERY, AND THE MALAYSIAN ASPIRATIONS
Prof. Dr. Khalijah Awang

• Chemistry Department
• Faculty of Science
• University of Malaya
• Malaysia

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WHAT ARE NATURAL PRODUCTS?

• Is everything found naturally (from nature ) a
  natural product???
• Natural products are secondary metabolites
  produced by an organism, small molecules (mol wt
  lt 1500 amu approx.) But are not strictly
  necessary for the survival of the organism.

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EXAMPLES OF NATURAL PRODUCTS
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NATURAL PRODUCTS AND DRUG DISCOVERY

• Plants as a form of medication
• 1803-Derosne extracted a mixture of narcotine and
  morphine
• 1806-Serturner recognised the alkaline nature of
  the principle content of opium
• However the structures of these complex molecules
  were made known only in the 2nd half of the 20th
  century
• NMR X-ray- the tedious task has become much
  easier.

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NATURAL PRODUCTS AND DRUG DISCOVERY contd

• Many drugs were discovered from natural products
  and their complex structures have intrigued many
  chemists and due to this, synthesis of them were
  of great challenge.
• 1952-morphine was synthesised.
• 1950s reserpine was synthesized.

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NATURAL PRODUCTS AND DRUG DISCOVERY contd

• Molecules structures morphine, reserpine,
  vinblastine-vincristine, taxol.
• Drug synthesis expensive so extraction and
  hemisynthesis.

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NATURAL PRODUCTS AND DRUG DISCOVERY -contd

• Drug development cost has increased tremendously
  800 million USD
• 1990s combinatorial technology
• Synthesis high throughput screening to cut
  cost
• For about one decade natural products research
  was dampened
• But now industrialists realised the importance of
  natural products diversity and biocompatibility

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NATURAL PRODUCTS AND DRUG DISCOVERY contd

• TO FIND NEW LEADS
• Natural products high throughput screening
• Natural product research is revived and
  revitalized
• Preliminary biological screening of samples from
  living organisms plants and others
• Bioassay guided studies of the bioactive extracts
• Structural elucidation
• Biological testing of the isolated compounds
• Sar studies

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PRELIMINARY BIOLOGICAL SCREENING OF SAMPLES

• Plant collection
• Identify the botanical name
• Prepare a herbarium sample
• Dry and grind sample
• Extract the samples with various solvents
• Send extracts for biological screening

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HERBARIUM SAMPLE

• Herbarium KL 4937
• Name Fissistigma manubriatum (HK. F. et Th.)
  Merr.
• Family Annonaceae
• Locality and habitat 57 km from Mersing towards
  Kluang, Johore.
• Field description
• A woody climber, main stem 5-7 cm diam, bark
  brown to darkish brown young parts rusty
  tomentous leaves thinly coriaceous, from oblong
  to oblanceolate
• Duplicate Kew, Leiden, Paris, K.L.
• Date 20 Apr. 2000
• Ident L. E. Teo Din

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BIOASSAY GUIDED STUDIES OF THE BIOACTIVE EXTRACTS
HUNTING FOR THE ACTIVE MOLECULE/S

• Fractionation of the active extracts
• Send fractions for biological screening
• Isolate compounds from active fractions
• Structural elucidation of isolated compounds
• Send pure compounds for biological screening
• Hopefully active molecule/s is/are found

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BIOASSAY GUIDED STUDIES OF THE BIOACTIVE EXTRACTS
contd

• Fractionation and isolation
• The right chromatography techniques CC, TLC,
  VC, MPLC, HPLC solvent systems and stationary
  phase critical in hunting down the bioactive
  compound/s.

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MOST COMMON CHROMATOGRAPHIC TECHNIQUES

• CHROMATOGRAPHIC TECHNIQUES
• Column chromatography
• Thin layer chromatography
• MPLC
• HPLC

• STATIONARY PHASE
• Silica-nonpolar
• Bonded silica-C18, C-8, CN etc
• Polyacrylamide
• Polysaccharides sephadex

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STRUCTURAL ELUCIDATION

• Spectroscopic methods
• Infrared (IR)
• - indicates presence of functional groups
• CO 1670 1750 cm-1
• amide, ketone, ester
• OH, NH/NH2 3100 cm-1 to 3500 cm-1
• Limitation non polar and semi polar
• compounds only.

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STRUCTURAL ELUCIDATION contd

• Mass Spectrometry
• Enables the determination of molecular weight.
• Aids structural elucidation fragmentation
  peaks loss of CO (M- 28), loss of H2O (M- 18).
  
• Enables identification of mixtures MS-MS.
• Various ionisation techniques to accommodate
  different compounds polar, ionic, nonpolar,
  macromolecules.
• Various Analysers usage (MS-MS, HRMS), cost.

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COMMON MODES OF MS IONIZATION

• Electron Impact (EI)
• Chemical Ionization (CI)
• Fast Atom Bombardment (FAB)
• Thermospray Ionization
• Electrospray Ionization (ES)
• Atmospheric Pressure Chemical Ionization (APCI)

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COMMON MASS ANALYZERS

• Magnetic Deflection Mass Analyzer
• Double Focusing Sector Spectrometer
• Quadrapole Mass Analyzer
• Triple Quadrapole Analyzer
• Ion Trap
• Time of Flight Spectrometer

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MASS FRAGMENTATIONS OF ERYTHROCARPINE A
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NUCLEAR MAGNETIC RESONANCE

• Nuclear Magnetic resonance
• Permits the establishment of the structural
  skeleton of the compound investigated.
• 1HNMR showed resonances of protons while 13C NMR
  showed the C resonances.
• Able to establish the connectivity between
  carbons and protons.
• One dimensional and two dimensional techniques
  available
• COSY, HMQC, HMBC, NOESY etc.
• For 1HNMR 1-5 mg (pure) sufficient
• For 13C NMR 20 mg sufficient.

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lysicamine
norboldine
pallidinine
tetrahydropronuciferine
reticuline
O-methyldauricine
phoebescortechiniine
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2-OMe
1-OMe
5
11
10
8
4
9
lysicamine
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10-OMe
1-OMe
8
3
11
norboldine
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7-OMe
2-NMe
12-OMe
7-OMe
6-OMe
2-NMe
6-OMe
8 8
5 5
11 13
13 14
10 14
10
11
O-methyldauricine
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6-OMe
3-OMe
-NMe
4
1
5
16
10ax
9
15
8
10ef
pallidinine
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4-OMe
-NMe
2-OMe
1-OMe
2
5
2
3
1-NH
phoebescortechiniine
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reticuline
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-NMe
2-OMe
1-OMe
3
7
6a
12
tetrahydropronuciferine
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INACTIVE COMPOUNDS IMPORTANT?

• Today inactive but 10 years later ???
• Taxol (anticancer drug)
• Structure identified in 1971, mechanism of action
  1979
• Rhazinilam (anti tubulin agent)
• Structure identified in 1972, anti-tubuline
  (anticancer) activity discovered in 1990.
• Biogenesis relationship between the compounds.
• Chemical relationship between the active compound
  and major compound hemisynthesis
• 10 deacetylbaccatine III ? taxol and taxotere.

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HEMI-SYNTHESIS OF TAXOL
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NEW NATURAL PRODUCTS DRUGS
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NATURAL PRODUCTS ON CLINICAL TRIAL
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THE MALAYAN BORNEO FLORA

• One of the oldest and richest in the world
• More than 200 million years of age
• About 15,000-18,000 flowering species (Malayan
  Borneo)

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FIELD TEAM (2000)
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HERBARIUM
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ANNONACEAE
Monocarpia
Fissistigma
Artabotrys
Cyathostemma
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LAURACEAE
Phoebe grandis
Phoebe lanceolata
Kryptocarya kurzii
Dehaasia sp.
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MELIACEAE
Aglaia malaccensis
Chisocheton tomentosus
Aglaia argentia
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APOCYNACEAE
Alstonia angustifolia
Kopsia pauciflora
Kopsia teoi
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HERBARIUM SAMPLE

• Herbarium KL 4937
• Name Fissistigma manubriatum (HK. F. et Th.)
  Merr.
• Family Annonaceae
• Locality and habitat 57 km from Mersing towards
  Kluang, Johore.
• Field description
• A woody climber, main stem 5-7 cm diam, bark
  brown to darkish brown young parts rusty
  tomentous leaves thinly coriaceous, from oblong
  to oblanceolate
• Duplicate Kew, Leiden, Paris, K.L.
• Date 20 Apr. 2000
• Ident L. E. Teo Din

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BIOACTIVE COMPOUNDS FROM THE MALAYAN FLORA
Argentinic acid A methyl ester
Forbesine
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rhazinilam
2R kurziflavolactone A 2S kurziflavolactone B
kurzilactone
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Pedicin
(-)- sebiferine
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(-)-sebiferine
Erythrocarpine D
(-)-0,0-dimethylgrisabine
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PRESENT WORK

• Huncaniterine A
• A new bisindole alkaloid from Hunteria
  zeylanica Kg. Padang, Kuala Lipis, Pahang
• Exhibited vasorelaxation effects on isolated rat
  aorta
• Showed responses against norepinephrine (3x10-7
  M)
• Bisbenzyl and secobisbenzyl isoquinoline
  alkaloids
• Phaeanthus crassipetalus Dungun, Terengganu
• Used traditionally in Borneo, especially Sabah,
  for the treatment of wounds and high blood
  pressure
• 3 bisbenzyls and 3 secobisbenzyls that possess
  antihypertensive activity

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PRESENT WORK-Contd

• Labdane diterpenes
• Andrographis paniculata
• 6 knowns labdane diterpenes were isolated and
  have shown coronary vasodilation activity.
• Limonoids
• Chisocheton erythrocarpus Hutan Simpan Terenas,
  Kedah
• Dichloromethane extract showed cytotoxic
  activity against P388 murine leukaemia cells
  (IC50 18mg/ml)
• Six new and cytotoxic limonoids were isolated
  and identified

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Hunteria zeylanica
Huncaniterine A (Hunteria zeylanica) Heterocycles
Vol. 74 (2007)
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ANTI-HYPERTENSIVE ALKALOIDS FROM PHAENTUS
CRASSIPETALUS
Phaeanthus crassipetalus Becc.

• Phaeanthus crassipetalus Dungun, Terengganu
• Only 2 species in Malayan Forest P.
  crassipetalus P. ophthalamicus
• Used traditionally in Borneo, especially Sabah,
  for the treatment of wounds and high blood
  pressure
• 3 bisbenzyls and 3 secobisbenzyls that possess
  antihypertensive activity

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3 BISBENZYLS
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3 SECOBISBENZYLS
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BIOASSAY GUIDED STUDIES ON ANDROGRAPHIS PANICULATA

• Family Acantaceae
• Local name
• Hempedu bumi
• King of bitter
• Hypertension, diabetes, malaria and fever

Andrographis paniculata
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BIOASSAY contd
The effects of hexane (HE), dichloromethane (DE)
and water (WE) extracts of Andrographis
paniculata, and sodium nitroprusside (positive
control) on coronary perfusion pressure in
isolated rat hearts. denotes p lt 0.05, paired
t-test extract versus its vehicle control). The
methanol extract did not show significant effects
on coronary perfusion pressure, data not presented
Group Basal coronary perfusion pressure (mmHg) Change in coronary perfusion pressure (mmHg) (mean S.E.M) Change in coronary perfusion pressure (mmHg) (mean S.E.M) Change in coronary perfusion pressure (mmHg) (mean S.E.M) Change in coronary perfusion pressure (mmHg) (mean S.E.M) Change in coronary perfusion pressure (mmHg) (mean S.E.M) Change in coronary perfusion pressure (mmHg) (mean S.E.M)
Group Basal coronary perfusion pressure (mmHg) Extract (mg) Extract (mg) Extract (mg) Extract (mg) Extract (mg) Sodium nitroprusside 1000nmol
Group Basal coronary perfusion pressure (mmHg) 0.001 0.01 0.1 1 3 Sodium nitroprusside 1000nmol
HE (n4) Vehicle 94.0 9.9 2.5 3.2 3.8 3.8 -5.0 2.9 -2.5 1.4 -17.5 5.2 3.8 5.9 -47.5 14.8 12.5 6.3 - - -43.8 14.1
DE (n5) Vehicle 96.4 12.7 -2.4 2.4 -2.6 2.6 -8.6 2.7 10.0 6.3 -14.0 4.8 -4.6 3.0 -29.4 8.5 -4.0 12.6 -24.5 3.0 12.5 7.9
WE (n4) Vehicle 85.0 20.2 0 0 0 0 -12.0 6.0 0 -27.3 11.8 0 -36.3 18.0 0
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BIOASSAY contd

• The results showed that hexane, CH2Cl2 and water
  extract of the AP extracts tested were able to
  modulate rat cardiovascular function directly.
• Except for the methanol extract, all other
  extracts caused coronary vasodilation, as shown
  by reductions in coronary perfusion pressure.
• The hexane extract appeared to be the most active
  in this effect. In addition, the water extract
  was able to increase cardiac contractility, as
  observed from the increase in developed tension.

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BIOASSAY contd
andrographolide
14-deoxy-11,12-dihydroandrographolide
neoandrographolide
14-deoxy-12-hydroandrographolide
14-deoxydihydroandrographolide
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BIOASSAY contd
The effects of diterpenoids isolated from the
dichloromethane extract of Andrographis
paniculata, sodium nitoprusside (SNP) and 40
EtOH on coronary perfusion pressure of isolated
rat hearts. The results are expressed as mean
s.e.m., plt0.05, students paired T-test,
compounds versus vehicle (40 ethanol). Basal
coronary perfusion pressure 72.15.6 mmHg.
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BIOASSAY contd

• All of these diterpenoids reduced coronary
  perfusion pressure (CPP) significantly but
  compound 4 reduced CPP most (-19.5 7.0 mmHg).
  
• This value was comparable to sodium nitroprusside
  (positive control), which reduced CPP by 22.0
  5.4mmHg. The combination of all diterpenoids of
  the same amount reduced CPP by 10.8 3.7 mmHg.
• None of the five diterpenoids showed any
  significant effect on heart rate even though the
  positive control, isoprenaline, was effective.

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BIOASSAY GUIDED STUDY OF CHISOCHETON
ERYTHROCARPUS AGAINST P388 MURINE LEUKAEMIA CELLS

• Collected from Hutan Simpan Terenas, Kedah.
• 26/51 Chisocheton species in Peninsular Malaysia.
• The very first Malaysian species to be studied
  chemically.
• Dichloromethane extract IC50 18mg/ml

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CYTOTOXIC ACTIVITY AGAINST P388 MURINE LEUKAEMIA
CELLS
Compounds IC50 (mg/ml)
Erythrocarpine A (1) 2.0
Erythrocarpine B (2) 6.0
Erythrocarpine C (3) 6.0
Erythrocarpine D (4) 9.9
Erythrocarpine E (5) 10.0
Erythrocarpine F (6) 16.0
MMC (mitomycin C) 0.15
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MALAYSIAN ASPIRATIONS

• Malaysia rich in both natural resources and
  cultural heritage
• Natural Resources megabiodiversity with our
  Malayan and forests
• Cultural heritage traditional medicines
• Malaysia is represented by three major ethnic
  groups Malay, Chinese and Indian
• Three different type of traditional medicine
  practiced by all communities
• Jamu (Malay)
• Sensei (Chinese)
• Ayurvedic (India)

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MALAYSIAN ASPIRATIONS Contd

• Malaysian aspiration in the natural products
  research
• To execute a thorough biological and chemical
  studies on the Malayan and Borneo rain forests
  (less than 5 of plant species have been
  studied)
• Chemotaxonomy
• Bioassay guided studies Lead discoveries
• Help to develop drug discovery program (with
  foreign collaboration/pharmaceutical industries)-
  very high cost
• To study thoroughly the known medicinal
  plants/herbs for the development of
  nutraceuticals and public knowledge.
• To upgrade the present traditional medicines/
  herbal food supplements in the market
  bioactivities, bio-assay guided,
  standardization, toxicology and clinical studies.
  
• Help modernize the herbal industry to be
  accepted globally
• The population will benefit quality herbal and
  pharmaceutical
• products at affordable price.

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PROBLEMS OF NATURAL PRODUCT RESEARCH

• Lack of co-ordination and monitoring
• Projects should be well managed and monitored
• Better / stronger cooperation among researchers
  and institutions
• Documentation (Burkill)
• Industries (linkage)
• Lack of funding
• Human resource (expertise) / training
• Equipments / services
• Lack of focus
• Researchers-too many administrative
  responsibilities
• Training for staffs and students
• Regulations
• National Bereau of Pharmacy

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CHALLENGES

• Capitalise on our assets
• Overcome our problems professionally
• Think global not local
• Governmentresearchers industries better
  communication and trust

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INSYAALLAH

• Malaysia will be a global force in natural
  products research and pharmaceutical
  /nutraceutical industries.

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TERIMA KASIH THANK YOU