Fighting Malaria:
From Artemisinin to ACT’s.
Ana Francis Carballo-Arce
May 5th, 2011
www.hopeforallministries.org .(accessed January, 2011)
Outline
Malaria facts
First antimalarial treatments
Artemisia annua and artemisinin discovery
Bioactivation of artemisinin and mode of action
Molecular targets of artemisinin
Dosage and sources for artemisinin
2
Malaria facts
500 million people are
diagnosed with malaria
in the world annually,
and more than 1 million
die (mainly children).
• Plasmodium resistance to
antimalarial medicines is
one of the major problems.
The use of mono
therapy leads to
resistance.
• WHO has recommended
artemisinin based
combination therapies
(ACTs) as first-line treatment
for uncomplicated malaria
since 2005
http://www.nature.com/news/specials/malaria/index.html (accessed January, 2011)
3
Global map for Malaria
www.rollbackmalaria.org/worldmalariaday (accessed, April 28th)
4
Principal available antimalarial
drugs
http://www.viablehealth.com/library3/herbs/herbs_cinchona.htm (accessed February 2011)
5
Malaria over the past 50 years
Engineering microorganisms
Artemisinin analogues
(ACT)
First total synthesis
2012
2004
Discovery and isolation
of artemisinin
Malaria resistance
1983
1970-1975
1960
6
Malaria
cycle
in the
body
Wellems, Thomas E.; et Al. J Clin. Invest. 2009;119(9):2496–2505
http://www.youtube.com/watch?v=wbe4LTb8Dt4 .(accessed march 2011)
7
1960 – Malaria Resistance
•DDT was removed from
the market because of the
environmental damage
•Mosquito developed
resistance to DDT
•The parasite developed
resistance to available
drugs
e.g. chloroquine
Brown, Geoffrey D. Molecules 2010, 15, 7603-7698
Shel, Ellen Ruppel l. Atlantic Magazine.1997. Available at:
http://www.theatlantic.com/magazine/archive/1997/08/malaria-resurgence-of-a-deadly-disease/5755/
8
Search for a new treatment

Late 1960’s and 1970’s, Tu You-You,
head of the antimalarial research
group at the China Academy of
Traditional Chinese Medicine,
discovered the activity of the plant.
◦ “Sweet Annie”

The activity of the plant was first
reported during the Jin dynasty
◦ 284-346AD
Liao; Fulong. Molecules . 2009, 14, 5362-5366
http://www.chinavitae.com/biography/Tu_Youyou (picture)
9
Isolation of active compound
200 Chinese herbs were
tested against Malaria
Artemisa annua L.
(Sweet Annie) extract
shows activity, but high
toxicity.
Basic extract
retains activity
Artemisia annua
Acid extract did
not show any
activity
•1972, a colourless crystalline substance is isolated.
•The structure and stereochemistry were not determined until
1975 and published in 1977.
Liao; Fulong. Molecules . 2009, 14, 5362-5366
10
Artemisinin

Representing a new class of antimalarial agents,
artemisinin is a sesquiterpene trioxane lactone.
Arsenault, Patric R. et Al. Current medicinal chemistry, 2008, 15, 2886-2896
Covello, Patric S. Phytochemistry. 2008, 69, 2881-2885
11
Red blood stage in malaria
cycle
O’Neill, Paul M.; Barton, Victoria E.; Ward, Stephen A. Molecules 2010, 15, 1705-1721
12
Mode of action of artemisinin
Meshnick 1991:
• the bioactivation of 1,2,4 trioxanes is triggered by
iron (II) to generate reactive oxygen species.
• the selectivity of artemisinin towards parasiteinfected erythrocytes was then explained by the
iron dependent bioactivation of the endoperoxide
bridge.
O’Neill, Paul M.; Barton, Victoria E.; Ward, Stephen A. Molecules. 2010, 15, 1705-1721
Meshnick, Steven R. et Al. Mol.Biochem. Parasitol. 1991. 49. 181-189
13
O’Neill, Paul M.; Barton, Victoria E.; Ward, Stephen A. Molecules 2010, 15, 1705-1721
14
O’Neill, Paul M. et Al. Acc. Chem. Res. 2004, 37, 397-404
15
O’Neill, Paul M. et Al. Acc. Chem. Res. 2004, 37, 397-404
16
Molecular targets of artemisinin
I.Serca
Sarco-Endoplasmic Reticulum
Ca2+-ATPase
pfATP6ase
Jonathan Gershenzon & Natalia Dudareva. Nature Chemical Biology . 2007. 408 - 414 .
17
Molecular targets of artemisinin
II. Alkylation of heme groups
Artemisinin
O’Neill, Paul M.; Posner, Gary H. Journal of Medicinal Chemistry, 2004, 47, 12, 2945- 2964
18
Molecular targets of artemisinin
III. ROS generation
O’Neill, Paul M.; Posner, Gary H. Journal of Medicinal Chemistry, 2004, 47, 12, 2945- 2964
19
Dosage of Artemisinin

Monotherapy: 20 mg/kg in a divided loading dose
on the first day, followed by 10 mg/kg once a day
for 6 days.

Combination therapy: 20 mg/kg in a divided
loading dose on the first day, followed by 10 mg/kg
once a day for two more days plus mefloquine
http://www.rollbackmalaria.org/cmc_upload/0/000/014/923/am2_1-8.htm (accessed, may 2010)
20
Sources of Artemisinin
Plant MaterialBiosynthesis
Organic synthesis
•Analogs
•Trioxalanes
Synthetic Biology –
Heterologous expression
21
Plant Source



The content in the plant is low
between 0.01-1.5%.
It is the commercial source.
It is found in the glandular trichomes.
22
Biosynthesis
Brown, Geoffrey. Molecules. 2010, 15, 7603-7698
23
Carbon skeleton assembly
Brown, Geoffrey. Molecules. 2010, 15, 7603-7698
24
Oxidations
CYP 71AV1
Brown, Geoffrey. Molecules. 2010, 15, 7603-7698
25
Introduction of Oxygen
Brown, Geoffrey. Molecules. 2010, 15, 7603-7698
Brown, Geoffrey .Tetrahedron. 2002, 58, 897-908
26
Artemisinin ring closing
L.-K. Sy, G. D. Brown .Tetrahedron.2002, 58 897-908
27
Total syntheses

G. Schmid, W .
Hofheinz in 13 steps,
1983

Yadav in 9 steps, 2010
28
Synthesis by G. Schmid, W .
Hofheinz
G. Schmid, W. Hofheinz, J. Am. Chem. Soc. 1983, 105 , 624
29
Synthesis by G. Schmid, W .
Hofheinz
G. Schmid, W. Hofheinz, J. Am. Chem. Soc. 1983, 105 , 624
30
Silicon induced chemistry
G. Schmid, W. Hofheinz, J. Am. Chem. Soc. 1983, 105 , 624
31
Synthesis by G. Schmid, W .
Hofheinz
Overall yield 2.1%
G. Schmid, W. Hofheinz, J. Am. Chem. Soc. 1983, 105 , 624
32
Yadav total synthesis
J.S. Yadav , B. Thirupathaiah, P. Srihari, Tetrahedron. 2010.66 .2005–2009
33
Yadav total synthesis
Overall yield from citronellal to the photoxidation: 13%
J.S. Yadav , B. Thirupathaiah, P. Srihari, Tetrahedron. 2010.66 .2005–2009
34
Analogues: First generation
Dejan M.; Bogdan A. Šolaja. J. Serb. Chem. Soc. 2009. 74 (11) 1155–1193
SD90: required dose for 90% suppression of
parasitemia.
35
Second generation
Dejan M.; Bogdan A. Šolaja. J. Serb. Chem. Soc. 2009. 74 (11) 1155–1193
36
1,2,4 trioxalanes
Dong; Y. et Al. J. Med. Chem. 2005, 48, 4953-4961
37
Spiroadamantane ring
IC50 > 100 ng/mL
IC50 : 44 ng/mL
IC50 : 2.2 ng/mL
Dong; Y. et Al. J. Med. Chem. 2005, 48, 4953-4961
38
Key figures for analogs
O’Neil, Paul. Nature. 2004. 430, 838-839
39
OZ439
Roche-Medicines and Malaria Venture
-Single dose
-Low cost of goods
-High oral bioavailability
-phase III
Charman, Susan A. et Al. PNAS. 2011.108, 11, 4400-4005
40
Synthesis of trioxolanes
The compounds are synthesized using the Griesbaumco-ozonolysis
Tan, Yuanqing. et Al. J. Org. Chem. 2004. 69. 6470-6473
41
Cost of making artemisinin
•Artemisia annua cycle between 12 -18 months
•The yield from the plant extract is low; 17,000 ha of
the plant are required to produce 100 million adult
treatments per year.
•In 2007, the demand of treatments was 400 million
doses
•Synthesis of artemisinin is not cost effective, in
2006 was estimated in a range between $900$1600/kg
Sambo L.G. Am. J. Trop. Med. Hyg., 2007, 77(Suppl 6), pp. 198–202
42
Synthetic biology Artemisinin
◦ Kiesling and his group, have engineered two
organisms, to produce artimisinic acid, at
levels up to 25 g/L.
 Saccharomyces cerivisiae
 E. coli
Sambo L.G. Am. J. Trop. Med. Hyg., 2007, 77(Suppl 6), pp. 198–202
43
Kiesling and Ro; engineered
yeast
Sambo L.G. Am. J. Trop. Med. Hyg., 2007, 77(Suppl 6), pp. 198–202
Ro; Dae-Kyun et al .Nature . 2006, 440, 940-943
http://chemistry.umeche.maine.edu/CHY431/Synthetic2.html image (accessed February 2011)
44
Making Artemisinin commercially
available
Sambo L.G. Am. J. Trop. Med. Hyg., 2007, 77(Suppl 6), pp. 198–202
45
Patrick Covello
Jay Keasling
“This new development in the production of a malaria treatment
represents a major development in the fight against the disease. It will
strengthen Canada’s position as a world leader in health research and
provide a reliable and affordable solution.”
Saskatoon StarPhoenix
• Canada’s federal government has spent $869,000 over eight years.
•Sanofi-aventis will begin commercial-scale production in 2012.
•The Bill & Melinda Gates Foundation, has contributed with $42.6
million.
http://www.vancouversun.com/business/Canadians+make+malaria+breakthrough/4471342/story.html (accessed march 2011)
46
Summary
Nature is a good source for new molecules for
drug developing process.
Synthesis has not provide a cost efficient route
for artemisinin.
The last step in the production of artemisinin
still has a low yield.
Synthetic trioxanes, inspired by the artemisinin
structure, with an adamantyl substituent show
promising antimalarial activity.
Shel, Ellen Ruppel l. Atlantic Magazine.1997. Available at:
http://www.theatlantic.com/magazine/archive/1997/08/malaria-resurgence-of-a-deadly-disease/5755
47
Acknowledgements
Dr. Arnason group
Dr. Durst group
48
Other uses for Artemisinin
O’Neill; Paul M.; Barton, Victoria E. ; Ward, Stephen A. Molecules. 2010, 15(3), 1705-1721
49
Potential non-malarial uses of
artemisinin and derivatives
DIBAL
Artemisinin
Dihydroartemisinin
Ovarian Cancer, Oral
Cancer
Artesunate: Leukemia
Melanoma, Prostate
Cancer.
Dimers have enhanced activity
against cancer, but not good
activity against malaria
Patrick R. Arsenault, Kristin K. Wobbe and Pamela J. Weathers.Current Medicinal Chemistry, 2008, 15, 2886-2896
50
Plasmodium falciparum
http://sandwalk.blogspot.com/2007/07/plasmodium-falciparum-causes-malaria.html. accessed feb 2011
51
Biosynthesis of IPP and
DMAPP via the
mevalonate pathway (A)
and the mevalonateindependent (DXP)
pathway (B).
Mahmoud S S , Croteau R B PNAS 2001;98:8915-8920
©2001 by The National Academy of Sciences
52
Biosynthesis of GPP
prenyl transf erase
OPP
OPP
HR
DMAPP
HS
HS
HR
O PP
O PP
geranyl PP
(GPP)
53
Biosynthesis of FPP
geranyl PP
(GPP)
OPP
HR
HS
OPP
HS
HR
OPP
Farnesyl PP
(FPP)
54
H
H
O
OO
O
O
OO
Lewis Acid
H
O
O
H
H2O O
OH
H
OH
OO
+ H2O
O
H
- H2O
H
OH
OO
O
O
H
OH
OO
H
Fenton
ChemistryO
H
O
O
Fe(II)
OH
O
H
HO
O
HO
O
H
H
OO
O
O
H
H
O
Secondary carbon
centred radical
HO
H
OO
O
H
O
4-hydroxy
deoxyartemisinin
O’Neill, Paul M.; Barton,Victoria E.; Ward, Stephen A. Molecules 2010, 15, 1705-1721
55
http://pmn.plantcyc.org/PLANT/NEW-IMAGE?type=PATHWAY&object=PWY-5195&detail-level=3
56
Basic extraction
Artemisinin
57
Groves, John T. PNAS . 2003 .100 .7 3569–3574
Romeo, John T. Evolution of metabolic pathways. Ed. Pergamon.2000.
151-189
58
Hydroxylations on an sp3 C-H
bond
Groves, John T. PNAS . 2003 .100 .7 3569–3574
59
Carbonyl formation
oxidations
Dewick, M. Paul. Medicinal Natural Products: a biosynthetic approach. Wiley. 2nd ed. p24
60
Organocatalysis
J.S. Yadav , B. Thirupathaiah, P. Srihari, Tetrahedron. 2010.66 .2005–2009
61
Removal of methyl silyl (Me3Si) group
TBAF
G. Schmid, W. Hofheinz, J. Am. Chem. Soc. 1983, 105 , 624
62