2-Hexadecynoic acid inhibits plasmodial FAS-II enzymes and arrests erythrocytic and liver stage Plasmodium infections

Article abstract of DOI:10.1016/j.bmc.2010.08.055

Acetylenic fatty acids are known to display several biological activities, but their antimalarial activity has remained unexplored. In this study, we synthesized the 2-, 5-, 6-, and 9-hexadecynoic acids (HDAs) and evaluated their in vitro activity against erythrocytic (blood) stages of Plasmodium falciparum and liver stages of Plasmodium yoelii infections. Since the type II fatty acid biosynthesis pathway (PfFAS-II) has recently been shown to be indispensable for liver stage malaria parasites, the inhibitory potential of the HDAs against multiple P. falciparum FAS-II (PfFAS-II) elongation enzymes was also evaluated. The highest antiplasmodial activity against blood stages of P. falciparum was displayed by 5-HDA (IC₅₀ value 6.6 μg/ml), whereas the 2-HDA was the only acid arresting the growth of liver stage P. yoelii infection, in both flow cytometric assay (IC₅₀ value 2-HDA 15.3 μg/ml, control drug atovaquone 2.5 ng/ml) and immunofluorescence analysis (IC₅₀ 2-HDA 4.88 μg/ml, control drug atovaquone 0.37 ng/ml). 2-HDA showed the best inhibitory activity against the PfFAS-II enzymes PfFabI and PfFabZ with IC ₅₀ values of 0.38 and 0.58 μg/ml (IC₅₀ control drugs 14 and 30 ng/ml), respectively. Enzyme kinetics and molecular modeling studies revealed valuable insights into the binding mechanism of 2-HDA on the target enzymes. All HDAs showed in vitro activity against Trypanosoma brucei rhodesiense (IC₅₀ values 3.7-31.7 μg/ml), Trypanosoma cruzi (only 2-HDA, IC₅₀ 20.2 μg/ml), and Leishmania donovani (IC₅₀ values 4.1-13.4 μg/ml) with generally low or no significant toxicity on mammalian cells. This is the first study to indicate therapeutic potential of HDAs against various parasitic protozoa. It also points out that the malarial liver stage growth inhibitory effect of the 2-HDA may be promoted via PfFAS-II enzymes. The lack of cytotoxicity, lipophilic nature, and calculated pharmacokinetic properties suggests that 2-HDA could be a useful compound to study the interaction of fatty acids with these key P. falciparum enzymes.

Full text of DOI:10.1016/j.bmc.2010.08.055

Bioorganic & Medicinal Chemistry 18 (2010) 7475–7485
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
2-Hexadecynoic acid inhibits plasmodial FAS-II enzymes and arrests
erythrocytic and liver stage Plasmodium infections
Deniz Tasdemir a, , David Sanabria b, Ina L. Lauinger a, Alice Tarun c,d, Rob Herman c, Remo Perozzo e,
⇑
Mire Zloh a, Stefan H. Kappe c, Reto Brun f, Néstor M. Carballeira b,
⇑
a Department of Pharmaceutical and Biological Chemistry, School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, United Kingdom
b Department of Chemistry, University of Puerto Rico, PO Box 70377, San Juan, PR 00936-8377, USA
c Seattle Biomedical Research Institute, 307 Westlake Avenue N, Suite 500, Seattle, WA 98109-5219, USA
d Department of Infectious Diseases and Microbiology, University of Pittsburgh, 130 Desoto St, Pittsburgh, PA 15261, USA
e School of Pharmaceutical Sciences, University of Geneva, Quai Ernest-Ansermet 30, 1211 Geneva 4, Switzerland
f Department of Medical Parasitology and Infection Biology, Swiss Tropical Institute, Socinstr. 57, Basel 4002, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 June 2010
Revised 14 July 2010
Accepted 29 August 2010
Available online 18 September 2010
Acetylenic fatty acids are known to display several biological activities, but their antimalarial activity has
remained unexplored. In this study, we synthesized the 2-, 5-, 6-, and 9-hexadecynoic acids (HDAs) and
evaluated their in vitro activity against erythrocytic (blood) stages of Plasmodium falciparum and liver
stages of Plasmodium yoelii infections. Since the type II fatty acid biosynthesis pathway (PfFAS-II) has
recently been shown to be indispensable for liver stage malaria parasites, the inhibitory potential of
the HDAs against multiple P. falciparum FAS-II (PfFAS-II) elongation enzymes was also evaluated. The
highest antiplasmodial activity against blood stages of P. falciparum was displayed by 5-HDA (IC50 value
Keywords:
Hexadecynoic acid
Malaria
Liver stage
Blood stage
6.6
both flow cytometric assay (IC50 value 2-HDA 15.3
nofluorescence analysis (IC50 2-HDA 4.88 g/ml, control drug atovaquone 0.37 ng/ml). 2-HDA showed the
best inhibitory activity against the PfFAS-II enzymes PfFabI and PfFabZ with IC50 values of 0.38 and
0.58 g/ml (IC50 control drugs 14 and 30 ng/ml), respectively. Enzyme kinetics and molecular modeling
studies revealed valuable insights into the binding mechanism of 2-HDA on the target enzymes. All HDAs
showed in vitro activity against Trypanosoma brucei rhodesiense (IC50 values 3.7–31.7 g/ml), Trypano-
soma cruzi (only 2-HDA, IC50 20.2 g/ml), and Leishmania donovani (IC50 values 4.1–13.4 g/ml) with gen-
l
g/ml), whereas the 2-HDA was the only acid arresting the growth of liver stage P. yoelii infection, in
l
g/ml, control drug atovaquone 2.5 ng/ml) and immu-
l
Protozoa
Type II fatty acid biosynthesis
l
l
l
l
erally low or no significant toxicity on mammalian cells. This is the first study to indicate therapeutic
potential of HDAs against various parasitic protozoa. It also points out that the malarial liver stage growth
inhibitory effect of the 2-HDA may be promoted via PfFAS-II enzymes. The lack of cytotoxicity, lipophilic
nature, and calculated pharmacokinetic properties suggests that 2-HDA could be a useful compound to
study the interaction of fatty acids with these key P. falciparum enzymes.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
hepatocytes and begin to develop into merozoites. This so-called li-
ver stage (LS) or exoerythrocytic forms takes 2–16 days, depending
Malaria continues to be a cause of concern in underdeveloped
areas of the world. The World Health Organization (WHO) reports
around 300 million new cases, and over one million deaths due to
malaria each year.1 Malaria parasite Plasmodium has a complex life
cycle involving two hosts. The infection is initiated when Plasmo-
dium sporozoites enter the human (host 1) through the bite of an
infected Anopheles mosquito (host 2). The sporozoites inoculated
under the skin of the host migrate to the liver, where they infect
on the Plasmodium species, then thousands of LS merozoites are re-
leased into the bloodstream, where they invade red blood cells and
start multiple rounds of the asexual blood stages (BS). The entire
asexual BS cycle is completed within 1–2 days, again depending
on the Plasmodium species, producing large numbers of infected
erythrocytes (>1012 per host).2 During the BS, some merozoites
transform into the sexual stages, the male and female gametocytes,
which can be taken up by mosquitoes during blood meals. Game-
tocytes undergo fertilization in the mosquito midgut, producing
oocyst sporozoites that migrate to the salivary glands, ready to ini-
tiate a new round of infection.
⇑
Corresponding authors. Tel.: +44 20 77535845; fax: +44 20 77535909 (D.T.);
tel.: +1 787 764 0000x4791; fax: +1 787 756 8242 (N.M.C.).
E-mail addresses: deniz.tasdemir@pharmacy.ac.uk (D. Tasdemir), nmcarballeir-
a@uprrp.edu (N.M. Carballeira).
Past and current malaria drug discovery has been primarily di-
rected against the easy-to-grow asexual BS, which is responsible
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.055

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D. Tasdemir et al. / Bioorg. Med. Chem. 18 (2010) 7475–7485
for the clinical symptoms as well as mortality and morbidity of the
disease. Mainly due to technical challenges and high costs, LS has
been little exploited, despite its longer life span (6–7 days in Plas-
modium falciparum) than the BS (2 days in P. falciparum) and the
generation of much smaller number of parasites. Recently, a seri-
ous consideration has been given to asymptomatic LS parasites
as its full inhibition provides true causal malaria prophylaxis, that
is, prevents the blood stage infection and its clinical manifesta-
tions. The development of molecules inhibiting the growth of Plas-
modium hepatic forms could be useful in malaria prevention for
people living in malaria endemic areas, as well as for refugees
and travelers who are exposed to malaria risk for a limited time.
Inhibition of LS also reduces the risk of transmission because the
generation of the gametocytes will be interrupted.3 Furthermore,
the low parasitic load with limited multiplication substantially re-
duces the likelihood for drug-resistant forms to emerge. Hepatic
stage parasites represent further complication for Plasmodium vi-
vax and Plasmodiun ovale infections, as some of the parasites in
the hepatocytes transform into hypnozoites, which can stay dor-
mant up to several years and cause relapse.4 A few drugs, for exam-
ple, atovaquone, 8-aminoquinolines primaquine and tafenoquine
are effective against LS, but the primaquine is the only FDA li-
censed drug. However, its use is restricted, particularly in Africa
because of the frequency of genetic glucose-6-phosphate 1-dehy-
drogenase (G6PD) deficiency. Primaquine is also toxic and has a
very short half-life.4 Many other non-8-aminoquinolines lack oral
bioavailability, and a few natural products with anti-LS activity
have low selectivity.5,6 Hence, the search for new natural or syn-
thetic drugs targeting the LS of the malaria parasite is timely and
necessary.
2-HDA could target the PfFabI (and potentially other elongation en-
zymes from PfFAS-II system) and lead to death of LS malaria para-
sites. Herein we report (a) the synthesis of four HDAs, (b) their
in vitro growth inhibitory potential against BS and LS malaria par-
asites, (c) inhibitory activity against multiple PfFAS-II elongation
enzymes, PfFabI, PfFabZ, and PfFabG (b-ketoacyl-ACP reductase),
(d) the enzyme kinetics, docking studies, and calculated pharmaco-
kinetic properties, (e) in vitro activity against other parasitic proto-
zoa, that is, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and
Leishmania donovani and (f) Selective toxicity toward primary
mammalian and hepatoma cells. Palmitic acid (PA), the parent
compound lacking the triple bond was also tested in each assay
for comparison.
2. Results
2.1. Synthesis of hexadecynoic acid derivatives
We developed a short and versatile procedure to prepare the 5-,
6-, and 9-hexadecynoic acids as outlined in Figure 1. In general, the
appropriate bromo alcohol was protected with tert-butyldimethyl-
silyl chloride (TBSCl) yielding the TBS-protected bromo alcohols in
80–99% yields. Alkyne coupling with the required chain length of
the 1-alkynes in the presence of n-BuLi in tetrahydrofuran–hexam-
ethylphosphoramide (THF-HMPA) produced the desired TBS-pro-
tected alkynols in 23–32% yields. Deprotection of the TBS group
with tetrabutylammonium fluoride (TBAF) in THF followed by oxi-
dation of the resulting alcohols with pyridinium chloride (PDC) in
dimethylformamide (DMF) afforded the desired hexadecynoic
acids in 40–70% yields. The synthesis of the 2-HDA followed an al-
ready published procedure18 wherein commercially available 1-
pentadecyne reacted with n-BuLi in THF followed by quenching
with CO2 and final protonation with NH4Cl (Fig. 1). The purity of
the synthesized compounds was determined to be >95% by capil-
lary GC–MS and 13C NMR. The spectral data of the synthesized
HDAs (Sections 5.2 and 5.3) were in agreement with those
published.13,20,21
Due to inherent technical difficulties in studying the LS Plasmo-
dium parasites, little progress has been made in the identification
of new LS biological targets for drug discovery and design. Very re-
cent studies7,8 indicate that LS malaria parasites exhibit an abso-
lute requirement for de novo type II fatty acid biosynthesis (FAS-
II), which was previously thought to operate in blood stage.9 The
FAS-II pathway appears to be essential only for late hepatic stages
and deletion of critical elongation enzymes such as FabB/F (b-
ketoacyl-ACP synthase) and FabZ (b-hydroxyacyl-ACP dehydra-
tase) in Plasmodium yoelii cause a failure to generate exoerythro-
2.2. In vitro antimalarial activity toward P. falciparum blood
stages
cytic merozoites, that is, inability to cause
a
BS infection.7
The in vitro antiplasmodial activity of the HDAs against multi-
drug-resistant P. falciparum K1 strain was determined by using
the established 3H-hypoxanthine method. As shown in Table 1,
5-HDA showed the highest antiplasmodial activity among the alky-
Similarly, FabI (enoyl-ACP reductase)-deficient Plasmodium berghei
sporozoites were much less infective in mice and failed to com-
plete liver stage development.8 These data render the plasmodial
FAS-II pathway an attractive target for malaria prophylaxis.
Fatty acids have shown antimalarial activity10–12 but the litera-
ture reports have been scarce and there is not a consensus as to
what structural characteristics (i.e., unsaturation level, position
or chain length) favor the best antimalarial fatty acids. We believed
that a systematic study of the antimalarial activity of a series of
isomeric C16 acetylenic fatty acids could shed light on the struc-
tural properties required for antimalarial activity, in particular
how the antimalarial activity depends on the position of the triple
bond in a C16 acyl chain. For this purpose, we chose an isomeric
series of hexadecynoic acids (HDA), that is, the 2-, 5-, 6-, and 9-
HDAs, some of which were shown to be antibacterial, antifungal,
and antimycobacterial,13–16 but never investigated for antimalarial
potential, and synthesized them. Another reason for choosing C16
acetylenic acids, and not longer or shorter fatty acids (FAs), was be-
cause earlier studies indicated that 2-HDA inhibited fatty acid
elongation.17,18 The 2-HDA has recently been shown to inhibit
InhA, the enoyl-ACP reductase (FabI) analogue enzyme found in
the FAS-II pathway of the tubercle bacillus, Mycobacterium tubercu-
losis.16 In a recent publication, we reported unsaturated fatty acids
inhibiting enoyl-ACP reductases of P. falciparum (Pf) and M. tuber-
culosis.19 These facts suggested that C-16 alkynoic acids, especially
noic acids with an IC50 value of 6.6
tent 2-HDA and 9-HDAs (IC50s 10.4 and 10.3
and finally 6-HDA (IC50 22.5 g/ml). Interestingly, the parent com-
pound PA was more active (IC50 3.8 g/ml) than all acetylenic fatty
l
g/ml, followed by the equipo-
lg/ml, respectively)
l
l
acids, indicating that a triple bond is not favored for antimalarial
activity toward blood stage parasites.
2.3. In vitro activity toward P. yoelii liver stages
Next, we wanted to assess life stage selectivity of the HDAs and
investigated their growth inhibitory activity against LS malaria
parasites. Hepatic stage P. falciparum infection in humans is exper-
imentally intractable for practical purposes, hence has remained
poorly understood. In contrast, the LS of the disease in the rodent
malaria model is amenable to direct experimental interrogation.2
Thus, we have employed a medium throughput LS growth inhibi-
tion assay using rodent P. yoelii parasites, which have conserved
metabolic and antigenic profile as P. falciparum,22 to infect
HepG2:CD81 cell line. Infection was assayed by both flow cytomet-
ric (FC) and immunofluorescence analysis (IFA) methods, using
atovaquone as reference (Table 1). In the FC assay, the

D. Tasdemir et al. / Bioorg. Med. Chem. 18 (2010) 7475–7485
7477
n-BuLi, THF, -78ºC
TBSCl, imidazole
HMPA, 24h
TBSO
Br
HO
Br
n
n
CH2Cl2, rt, 1h
(80-99%)
H
m
(23-32%)
O
1) TBAF, THF, 0ºC, 24 h
2) PDC, DMF, rt
(40-70%)
TBSO
HO
m
m
n
n
5-HDA n = 2, m = 7
6-HDA n = 3, m = 6
9-HDA n = 6, m = 3
1. n-BuLi, THF, -78ºC, CO2
CO2H
H
2. NH4Cl
(10%)
10
10
2-HDA
Figure 1. Synthesis of the hexadecynoic acids.
Table 1
Inhibitory activities of the HDAs and palmitic acid (PA) against P. falciparum blood stage (BS) and P. yoelii liver stage (LS) parasites and PfFAS-II enzymes (FC, flow cytometry; IFA,
immunofluorescence analysis)
Compound
Standard
P. falciparum BS
P. yoelii LS FC
P. yoelii LS IFA
PfFabI
PfFabZ
PfFabG
0.056a
0.0025b
0.00037b
0.014c
0.03d
0.32d
2-HDA
5-HDA
6-HDA
9-HDA
PA
10.4
6.6
22.5
10.3
3.8
15.3
>25
>25
>25
>25
4.88
>25
>25
>25
>25
0.38 0.03
3.1 0.57
3.6 0.14
2.8 1.13
>100
0.58 0.11
14.0 1.41
12.0 2.83
12.5 2.12
>100
3.50 0.35
>100
>100
>100
>100
IC50 values are in
(À)-epigallocatechin gallate.
l
g/ml and represent the average of at least two independent assays performed in duplicates. Reference compounds a chloroquine; b atovaquone; c triclosan;
d
HepG2:CD81 cells are infected with transgenic P. yoelii sporozoites
that express green fluorescent protein (PyGFP), exposed to com-
pound, and allowed to develop for 43 h to measure infection rates.
To verify the results of the FC-based assay and to determine the ef-
fects of the drugs on the morphology and development of the LS
parasites, IFAs of P. yoelii-infected HepG2:CD81 cells were done
after drug treatment. In this assay, the LS parasites were detected
using a mouse monoclonal antibody against P. berghei HSP70 that
cross-reacts with P. yoelii HSP7023 and polyclonal antibody against
theP. yoelii UIS4 protein.24 The results of both FC and IFA assays are
shown in Table 1. In the FC assay, only 2-HDA appeared to be active
2.4. PfFAS-II enzyme inhibition studies
In an attempt to understand the mechanism of action of the
HDAs against LS parasites, we determined their inhibitory effect
against three key enzymes of the fatty acid biosynthesis of
P. falciparum, that is, PfFabI, PfFabZ, and PfFabG, all of which were
recombinantly prepared and assayed following an established pro-
cedure.26 Essentially, the HDAs were inhibitory toward the studied
PfFab enzymes following the order PfFabI > PfFabZ > PfFabG (Table
1). From the data obtained, it became clear that among the studied
HDAs, the 2-HDA displayed the best overall inhibitory activity
with an IC50 value of 15.3
l
g/ml. Atovaquone, a potent inhibitor of
against PfFabI (IC50 0.38 0.03
0.58 0.11 g/ml), and it was the only inhibitory acetylenic acid
against PfFabG (IC50 3.5 0.35 g/ml). The 5-, 6-, and 9-HDAs were
not inhibitory toward PfFabG (IC50 >100 g/ml), and their inhibi-
tory potential against two other target enzymes were 10-fold
(PfFabI) to 50-fold (PfFabZ) less. Notably, the parent compound
PA was inactive against all enzymes tested even at the highest con-
lg/ml) and PfFabZ (IC50
LS development, had an IC50 value of 2.5 ng/ml. In the IFA, atovaqu-
one was observed to potently inhibit LS development resulting in
much smaller parasites with an IC50 value of 0.37 ng/ml (Table
1), similar to what has been described before.25 Smaller LS para-
sites compared to the control were observed in the IFA after incu-
l
l
l
bation with 2-HDA with an IC50 value of 4.88 lg/ml, whereas other
alkynoic and palmitic acids had no effect, even at the highest test
concentrations. Figure 2 (20Â objective magnification) shows an
IFA of the late liver stage-infected HepG2:CD81 cells (43 h post-
infection) treated with DMSO, PA, and HDAs. The DAPI staining
of infected cells (Fig. 2) indicated none of the HDAs to exert toxicity
toward the liver cells. Figure 3 displays the close-up fluorescence
microscopy images (100Â objective magnification) of P. yoelii liver
stage schizonts detected by antibody against HSP70 protein at 43 h
post-infection treated with different concentrations of 2-HDA, as
well as DMSO and atovaquone. 2-HDA was non-toxic against
hepatoma cells in FC or IFA (see Section 5.6) or against primary
mammalian cells (see Sections 2.6. and 5.12) indicating its selec-
tive toxicity against the LS parasites.
centrations (IC50 >100 lg/ml). These data indicate that the hepatic
stage growth inhibitory activity of 2-HDA may be due to the inhi-
bition of the PfFAS-II enzymes.
2.5. Enzyme kinetics, docking studies, and pharmacokinetic
calculations
In order to shed more light into the inhibitory mechanism of 2-
HDA, in vitro enzyme kinetic studies as well as molecular modeling
(docking) studies were performed. Kinetic studies indicated that 2-
HDA inhibits the PfFabI enzyme in a non-competitive manner with
respect to crotonyl-CoA and the cofactor NADH (Fig. 4). This im-
plies that 2-HDA binds to the PfFabI enzyme at a site distinct of

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D. Tasdemir et al. / Bioorg. Med. Chem. 18 (2010) 7475–7485
Figure 2. Immunofluorescence microscopy images of P. yoelii-infected HepG2:CD81cells at 43 h post-infection treated with 1% DMSO or various compounds taken at 20Â
objective magnification. The top images for each set of pictures show the liver stage parasites detected by antibody against HSP70 protein (FITC, green) while the bottom
images show the merged images of the same section stained with the nuclear stain DAPI (blue) and the liver stage parasites that were doubly stained with antibodies against
HSP70 (FITC, green) and UIS4 protein (Texas Red).
Figure 3. Detailed fluorescence microscopy images of P. yoelii liver stage schizonts detected by antibody against HSP70 protein at 43 h post-infection treated with different
concentrations of 2-HDA, as well as DMSO and atovaquone (100Â objective magnification).
the substrate or the cofactor site. The calculated Ki values are
0.73 g/ml (=2.90 M, substrate varied) and 0.65 g/ml
(=2.58 M, cofactor varied). The inhibition type against the PfFabZ,
a dehydratase, which does not require a cofactor, is competitive (Ki
1.2 g/ml = 4.8 M) with respect to the substrate (Fig. 5). As shown
in Figure 6, 2-HDA inhibits the PfFabG enzyme in a competitive
manner when the substrate acetoacetyl-CoA is varied (Ki 3.6 g/
ml = 14.3
NADPH is varied (Ki 1.5
l
M) and in a non-competitive manner when the cofactor
g/ml = 6 M).
l
l
l
l
l
l
Molecular modeling studies supported the predicted binding
mode of the acetylenic acids determined by the kinetic studies
and revealed insights for understanding the superior enzyme
inhibitory activity of 2-HDA over the other HDAs. The molecular
docking of these ligands into the binding site of FabZ27,28 was
l
l
l

D. Tasdemir et al. / Bioorg. Med. Chem. 18 (2010) 7475–7485
7479
a
90
80
70
60
50
40
30
20
10
0
b
90
80
70
60
50
40
30
20
10
0
1 µg/ml
1 µg/ml
0.5 µg/ml
0 µg/ml
0.5 µg/ml
0 µg/ml
-0.06 -0.04 -0.02
0
0.02
0.04
0.06
0.08
0.1
0.12
-0.08
-0.03
0.02
0.07
0.12
1/crotonoyl - CoA (µM)-1
1/NADH (µM)-1
Figure 4. Kinetic plots of the binding of 2-HDA to PfFabI showing a non-competitive inhibition type when the substrate crotonyl-CoA (a) and the cofactor NADH (b) was
varied.
300
250
200
150
100
difference between activities of 2-HDA and other ligands. The
interaction of ligands and the X-ray structure of PfFabI complexed
with the NADH (pdb entry 1v35) was used to investigate the non-
competitive inhibition of PfFabI activity. The molecular interaction
fields were generated using GRID 22A software (Molecular Discov-
ery) and the energy of binding of ligands to the protein complex
was estimated using GLUE module of this software.29,30 The whole
surface of the protein was target for the interaction and all ligands
exhibited multiple binding positions on the surface (Fig. 7b), with
similar preferences. Importantly, ligands appear to protrude into
the protein surface and to make close contacts with the protein
substrate. These interactions could possibly affect the protein con-
formation and therefore inhibiting the protein activity. The 2-HDA
protrudes the most of all ligands due to the extended structure
around the carboxylic group and possibly has the strongest impact
on protein conformation and flexibility.
1 µg/ml
0.5 µg/ml
0 µg/ml
50
0
-0.02
0
0.02
0.04
0.06
0.08
0.1
0.12
1/crotonoyl-CoA (µM)-1
Figure 5. Kinetic plots of the binding of 2-HDA to PfFabZ showing a competitive
inhibition type with respect to the substrate crotonyl-CoA.
Molecular properties of HDAs and PA were calculated using
ChemSilico and Vega ZZ, which indicated the compliance of 2-
HDA with Lipinski’s rules.31 As shown in Table 3, the predicted
log P values by CS and Vega ZZ of 2-HDA are 5.6 and 5.47, respec-
tively, while the predicted log P values of PA are 5.4 and 6.04. The
predicted PSA/SA (polar surface areas/surface areas) values and hu-
man intestinal absorptions (HIA) of 2-HDA are very similar to those
of PA. Since the PA readily crosses the blood–brain barrier (BBB),32
and the PSA correlates with the ability of molecules to cross the
BBB,33 it is likely that the 2-HDA has a good penetration ability
through cell membranes, and will even diffuse through the BBB.
carried out using the crystal structure given in pdb entry 1z6b. The
best fit of ligands and their binding energies predicted by Autodock
indicate that all 4 ligands interact favorably with the protein, how-
ever the 2-HDA binds most strongly (Fig. 7a). Although there is no
direct correlation between activity and binding energy, a trend was
observed for energy of interaction (E2-HDA < E9-HAD < E5-HDA < E6-HDA),
which corresponds to the trend of activity. Docking studies indi-
cate that only 2-HDA form a hydrogen bond with the backbone
NH atoms of Gly142 and Val143 and the electron density of the tri-
ple bond of 2-HDA interacts with the active site residue Glu147.
These differences most likely explain why the 2-HDA is the most
active molecule against PfFabZ.
2.6. Inhibitory activity against other parasitic protozoa and
primary L6 cells
The docking of ligands into PfFabG site has indicated that all
ligands bind closely to the cofactor site, and again the 2-HDA binds
with most favorable energy of interactions. The orientation of
ligands are different too (results not shown), but further studies
would be needed to understand the reasons for such significant
Finally, the species-specific antiprotozoal activity of the HDAs
against Trypanosoma brucei rhodesiense (bloodstream forms),
90
140
3 µg/ml
a
b
3 µg/ml
80
70
120
100
80
60
40
20
0
60
2.5 µg/ml
0 µg/ml
2.5 µg/ml
50
40
0 µg/ml
30
20
10
0
-0.02
0
0.02
0.04
0.06
0.08
0.1
0.12
-0.06 -0.04 -0.02 0.00
0.02
0.04
0.06
0.08
0.10
0.12
1/acetoacetyl-CoA (µM)-1
1/NADPH (µM)-1
Figure 6. Kinetic plots of the binding of 2-HDA to PfFabG when the substrate acetoacetyl-CoA (a) and the cofactor NADPH (b) was varied.

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D. Tasdemir et al. / Bioorg. Med. Chem. 18 (2010) 7475–7485
Table 3
Calculated pharmacokinetic properties of HDAs and PA
Fatty acid
log Pa
log Pb
HIAa
PSAb
SAb
PSA/SAb (%)
2-HDA
5-HDA
6-HDA
9-HDA
PA
5.60
5.40
5.38
5.36
5.40
5.47
5.47
5.47
5.47
6.04
89.1
89.1
89.1
89.1
88.8
101.9
92.6
89.9
88.6
91.3
599.9
635.7
640.3
632.2
615.2
17.0
14.6
14.0
14.0
14.8
a
Calculated by ChemSilico (CS) software.
Calculated by bVega ZZ software log P: octanol/water partition coefficient; HIA:
b
Human intestinal absorption; PSA: polar surface area; SA: surface area.
T. cruzi (intracellular amastigotes in L6 rat skeletal myoblasts), and
L. donovani (axenic amastigotes) was determined. As shown in
Table 2, the studied HDAs displayed the best overall antiprotozoal
activities against L. donovani, with 2-HDA and 9-HDA being equally
active (IC50s 4.5 and 4.1
activity (IC50 9.8 g/ml), whereas 5-HDA and PA were the least ac-
tive ones (IC50 13.4 and 14.5 g/ml). The 9-HDA displayed the best
activity toward T. b. rhodesiense with an IC50 value of 3.7 g/ml,
lg/ml). 6-HDA displayed a twofold less
l
l
l
while the remaining compounds were 6–10-fold less active. The
2-HDA was the only fatty acid with moderate T. cruzi activity
(IC50 20.2 lg/ml). Overall, the 9-HDA showed the broadest spec-
trum antiprotozoal activity. An early study34 has pointed out the
cidal effect of palmitic (hexadecanoic) acid against the promasti-
gote stages of L. donovani, L. tropica and the epimastigote and tryp-
omastigote stages of T. cruzi. To our knowledge, this is the first
study assessing trypanocidal and leishmanicidal effects of the four
HDAs.
Selective toxicity of all fatty acids was determined against L6
cells, a primary cell line derived from rat skeletal myoblasts. As dis-
played in Table 2, the 2-HDA shows negligible toxicity and 5-HDA
is completely non-toxic at the highest test concentrations (90 lg/
ml). Only 6-HDA and PA bear some moderate cytotoxic effects
against L6 cells.
3. Discussion
In contrast to easily cultivable BS, working with LS malaria par-
asites is difficult and costly since primary hepatocytes or hepatoma
cells need to be infected with live sporozoites from Plasmodium-in-
fected mosquitoes. This requires continuous supply of blood con-
taining infectious gametocytes and a mosquito breeding facility
to produce viable, uncontaminated, and infective sporozoites. LS
infection has long been unamenable to standard biochemical as-
says because of generally low infection rates of hepatocytes and
hepatoma cells.35 However, technological developments in the
field of microscopy and the generation of parasites with specific
cell types expressing fluorescent proteins permitted fascinating
live imaging of LS infection. Nowadays, automated microscopy,
infrared fluorescence scanning, flow cytometry, and immunofluo-
rescence techniques enable medium to high throughput screening
in LS parasites. The current study is the first to investigate the LS
malaria parasite growth inhibitory effect of 2-, 5-, 6-, and 9-HDAs,
by using rodent P. yoelii parasites to infect HepG2:CD81cell line.
Infection was assayed by two medium throughput LS growth inhi-
bition assays, FC and IFA. In the FC assay, the HepG2:CD81 cells
that are infected with GFP expressing P. yoelii sporozoites are ex-
posed to compound for 43 h. The number of GFP-positive hepa-
toma cells is determined by FC and is used to measure infection
rates. One disadvantage of the FC assay is that it only provides
information on the LS infection rates after 43 h of post-infection.
In order to determine the effects of the drugs on the morphology
and development of the LS parasites, we undertook IFAs of
Figure 7. Interaction of acetylenic ligands with PfFabZ and PfFabI predicted by
molecular docking: (a) interaction of ligands within the binding site of PfFabZ
represented as surface (2-HDA represented as thick sticks inside the surface, green
lines depict hydrogen bonds), (b) surface map of electrostatic potentials of the
PfFabI with ligands bound to the multiple binding sites (2-HDA—green; 5-HDA—
orange; 6-HDA—cyan) and (c) detailed view of the ligands bound to the protein in
respect to the substrate at the site encircled in b).
Table 2
Trypanocidal and leishmanicidal activities of the HDAs and PA
Compound T. brucei
rhodesiense
Trypanosoma Leishmania
L6 cell
cytotoxicity
cruzi
0.489b
20.2
>30
>30
>30
>30
donovani
Standard
0.003a
0.26c
0.004d
2-HDA
5-HDA
6-HDA
9-HDA
PA
21.1
25.5
31.7
3.7
4.5
13.4
9.8
85.9
>90
30.3
80.8
52.7
4.1
14.5
30.4
IC50 values are in
lg/ml and represent the average of at least two independent
a
b
assays performed in duplicates. Reference compounds melarsoprol; benznidaz-
c
d
ole; miltefosine; podophyllotoxin.

D. Tasdemir et al. / Bioorg. Med. Chem. 18 (2010) 7475–7485
7481
the calculated pharmacokinetic properties of the remaining HDAs
were very similar to those of 2-HDA and PA (Table 3), ruling out
the permeability as a problem. The good drug-like properties, espe-
cially their ability to cross membranes, including the BBB, of HDAs
could make them very useful for the treatment of advanced phases
of cerebral malaria and sleeping sickness caused by Trypanosoma
brucei rhodesiense.
P. yoelii-infected HepG2:CD81 cells after drug treatment. IFA does
not only provide data on morphology and the size of LS parasites,
but also gives a visual indication of host cytotoxicity. The LS para-
sites were detected using a mouse monoclonal antibody against
P. berghei HSP70 that cross-reacts with P. yoelii HSP70 and poly-
clonal antibody against the P. yoelii UIS4 protein. While infection
rates are difficult to assess from the IFA (because host cell numbers
were not counted), the reduction in LS sizes is a more sensitive
indicator for the inhibitory effect of a drug on LS development
compared to the FC-based assay. As shown in Figures 2 and 3,
the 2-HDA was the only acid inhibiting the growth of P. yoelii LS
parasites in both assays. The IC50 values of 2-HDA and the stan-
dard, atovaquone, are much smaller in the IFA, showing the sensi-
tivity of the assay.
Most of the current antimalarial agents target the parasite’s
blood stages. Only a few compounds, such as primaquine, tafenoq-
uine, and atovaquone have activity against the LS parasites, how-
ever their use is limited due to hematological toxicity in
individuals with G6PD-deficiency and high costs (atovaquone).
Although discussed controversially, it is logical and desirable to
have one drug with a dual effect on both LS and BS parasites, that
is, both prophylactic and therapeutic. Quinine, chloroquine, meflo-
quine, and artemisinins have little or no efficacy against the hepa-
tic parasites, contributing to the relapse of the disease, particularly
in P. vivax, which develops liver hypnozoites. An earlier study20 re-
ported 2-HDA to inhibit the growth of hepatoma cells, however, we
observed no toxicity even at the highest test concentrations to-
ward HepG2:CD81 cells by any HDA compounds in any of the assay
employed. The HDAs generally lacked toxicity against mammalian
L6 cells, indicating a reasonable therapeutic index. Hence, 2-HDA
appears to be a promising small molecule for its inhibitory activity
on both LS and BS malaria parasites, with good cell viability on he-
patic cells. The inhibition of LS by a compound, such as 2-HDA, will
also reduce the risk of transmission by interrupting the formation
of the gametocytes in late BS. The next logical step would be to test
HDAs on P. vivax and P. ovale hypnozoites. However, none of the ro-
dent Plasmodium species including P. yoelii produces dormant he-
patic stages in laboratory animals3 hence this could not be
performed in our laboratories.
It has been reported that 2-HDA competitively inhibits the
mycobacterial InhA (MtFabI) enzyme16 with a similar potency
(Ki value 2.1 0.6 lM) to that obtained with PfFabI. Although
the exact binding mode of 2-HDA on PfFabI is not completely
clear, it is obvious that the significant enzyme inhibitory activity
of 2-HDA is not a simple, non-specific detergent effect. Again,
similar to its antimycobacterial (and probably antifungal) activ-
ity, the hepatic stage parasite activity of 2-HDA does not seem
to be due to non-specific fatty acid-mediated lysis. On the other
hand, 2-HDA acts as a prodrug and requires metabolization in
Mycobacterium species.16 Whether this same phenomenon occurs
in Plasmodium species remains unclear and warrants further
studies.
Our findings concerning LS development were generated with
the rodent malaria parasite P. yoelii, which has a much shorter
LS cycle (50–60 h), but the high conservation of FAS-II among Plas-
modium species22 may suggest similar results on P. falciparum and
P. vivax hepatic stage development. In a recent paper, Yu et al.
(2008)8 compared FA profiles of wild-type and FabI-mutant para-
sites in the blood stage of P. falciparum and P. berghei. The spec-
trum of synthesized FA’s was different in P. falciparum where
C-16 and C-18 FAs were detected while in P. berghei C-12 to
C-24 FAs were predominant.8 The 14C-acetate (radiolabeled FA
precursor) isotope-incorporation studies showed no difference in
the FA profiles in the FabI mutant and wild-type blood stages in
both P. falciparum and P. berghei indicating clearly that FAS-II is
not involved in the synthesis of these FA species. Thus, the differ-
ent FAs in the blood stage might be formed by fatty acid elongases
and the differences in FA synthesis in the two Plasmodium species
might be due to the number of involved elongases detected in
their genome (three for P. falciparum, four for P. berghei).8 A recent
paper by Déchamps et al. (2010)38 showed that rodent and mam-
malian Plasmodium blood stage parasites differ in their phospho-
lipid pathways. Hence it is possible that lipid metabolic
pathways are different in the rodent and human Plasmodium and
yet the parasite FAS-II pathway will be essentially similar. Since
the FAS-II pathway does not appear to be operational in blood
stages7,8 one cannot say that the observed effects by the HDA
and/or palmitic acid in the blood and liver stages would be
through the same mechanisms.
Due to the inherent difficulty in studying the LS parasites, only
little progress has been made in the identification of new LS drug
targets for rational drug design. Recent studies show the impor-
tance of type II FAS in the LS, particularly in the transition point
of parasites from liver to blood. As FAS-II deficiency leads to loss
of infective capability of red blood cells in vitro and in vivo,7,8
the plasmodial FAS-II system appears to be a promising drug target
for the clinically silent LS infection. So far, only hexachlorophene, a
PfFabG inhibitor, was shown to inhibit the LS development in vitro
in
(=1.95
a
dose-dependent manner with an IC50 value of 4.8
l
lM
4. Conclusions
g/ml).36 Recently, the potent PfFabI inhibitor triclosan,
which was previously reported to inhibit the blood stage P. falcipa-
In this study, we report the rapid synthesis and broad antipro-
tozoal activity of four HDAs. Of the four HDAs studied herein, only
the 6-HDA is a natural compound, recently isolated from the plant
Sommera sabiceoides.15 Many unsaturated fatty acids, including
the natural product scleropyric acid, a C-17 fatty acid with a dou-
ble and triple bond have been shown to have antiplasmodial
activity,12 but to our knowledge, this is the first report of antima-
larial activity of the 2-, 5-, 6-, and 9-HDAs against the BS of P. fal-
ciparum parasites. The data presented in this manuscript suggest
that the presence and the position of the triple bond in the acyl
chain to be of paramount importance for determining the antipro-
tozoal activity of acetylenic C16 fatty acids. Considering that the
PA was the most active compound against BS P. falciparum para-
sites, it seems that the absence or a more distal position (C-5)
of a triple bond is favored for BS activity. However, the presence
rum infection, has been reported to be cidal at the late liver phases
of P. berghei with an IC50 value of 39.4 lM (=11.4 l
g/ml).37 Thus, 2-
HDA appears to be the third PfFAS-II inhibitor with activity against
LS malaria parasites. The inhibition of multiple PfFAS-II enzymes
by 2-HDA is tantalizing, as it bears lower risk to develop resistance.
The remaining fatty acids also inhibit PfFabI and PfFabZ moderately
and in the FC assay, they cause a 10% decrease in infection (data
not shown) at the highest test concentration, 25 lg/ml (data not
shown). This might imply that they possibly have some activity
at higher concentrations, however, due to toxicity of DMSO against
hepatic cells, we were unable to test higher doses. On the other
hand, since PfFabZ and PfFabI are inhibited by all compounds,
one can suggest that the lack of LS activity of the other HDAs stems
from their low penetration into the infected hepatocytes. However,

7482
D. Tasdemir et al. / Bioorg. Med. Chem. 18 (2010) 7475–7485
of an acetylenic function at C-2 appears to be a critical require-
ment for LS parasite growth arrest as PA and all other HDAs are
inactive. The presence of a triple bond next to the carbonyl group,
as found in 2-HDA, seems to increase the conformational flexibil-
ity and dipole moment of 2-HDA, thus enabling it to interact with
PfFAS-II target enzymes more efficiently. The presence of a single
acetylenic function at C-9 however, increases the trypanocidal and
leishmanicidal activity of the C16 acids significantly. At this point,
a relevant question emerges concerning the impact of the chain
length, as well the number of triple bonds within the acetylenic
acid on their biological activity. We performed a docking study
calculating the binding energies of the 2-tetradecynoic acid (2-
TDA, C14), the 2-octadecynoic acid (2-ODA, C-18), and the 2,5-
hexadecadiynoic acid (2,5-HDDA). This study showed that 2-
HDA was still the best binder toward the two target PfFAS-II en-
zymes, PfFabG and PfFabZ (data not shown). The strength or mode
of binding of 2-TDA and the 2-ODA against PfFabI was different,
which may indicate a weaker or the absence of activity. The 2,5-
HDDA had close binding energies to 2-HDA toward PfFabG and
PfFabZ, but better binding energy toward PfFabI. However, the ori-
entation of the 2,5-HDDA molecule in the binding region is differ-
ent due to steric constraints of triple bond combination, thus it
might not have the same inhibitory activity as the 2-HDA. These
calculations confirm that the C-16 HDAs were a good choice for
this study and 2-HDA had better activity against a wider range
of target enzymes. The in vitro activity of 2-TDA, 2-ODA, and
2,5-HDDA, however, needs to be assessed in whole cell parasite
assays, following their successful synthesis.
5.2. Synthesis and structure elucidation of the 5-, 6-, and
9-HDAs
5.2.1. Alkyne couplings with the bromoalkyloxy-tert-
butyldimethylsilanes
To a stirred solution of the alkyne (3.74–4.36 mmol), prepared
from the bromoalcohol and tert-butyldimethylsilylchloride, in dry
THF (5.0–15.0 ml), n-Buli (2.5 M, 10.90–11.22 mmol) in dry hexane
was added dropwise while keeping the temperature at À60 °C.
After 45 min, HMPA (1.12–10.0 ml) and the bromoalkyloxy-tert-
butyldimethylsilane (3.74–4.36 mmol) were added dropwise to
the reaction mixture at À60 °C. After 24 h, the reaction mixture
was worked up by pouring into a large volume of water and
extracting with diethyl ether (2 Â 15 ml). The organic layer was
washed with brine (1 Â 15 ml) before drying (MgSO4). Filtration,
rotoevaporation of the solvent, and fractional distillation afforded
the tert-butyldimethylsilyloxy-alkynes in 23–32% yields after puri-
fication by Kugelrohr distillation (130–137 °C/3 mmHg) of the
impurities.
5.2.2. Oxidation to the carboxylic acids
To a stirred solution of the alkynol (0.2–1.3 mmol), after depro-
tection with tert-butylammonium fluoride (TBAF), in 3.0–7.0 ml of
DMF was slowly added pyridinium dichromate (0.8–7.9 mmol) at
rt. After 24–48 h, the reaction mixture was worked up by pouring
10 ml of water and extracting with hexane (3 Â 12 ml). Once the
solvent was evaporated and dried in vacuo, the acetylenic fatty
acids were obtained in 40–70% yields.
In conclusion, the current study has identified acetylenic C16
acids as a novel class of antiprotozoal agents. The discovery of
these easily synthesizable fatty acids with promising antimalarial
and malaria prophylactic effects may represent an opportunity
for the development of new agents, particularly those derived from
2-HDA, for the control and potentially eradication of malaria. Tak-
ing into account their synthetic accessibilities, antiprotozoal
potencies, no apparent toxicity on hepatocytes at active concentra-
tions, ability to affect both BS and LS malaria parasites, as well as
good pharmaceutical (lipophilic) and pharmacokinetic properties,
the acetylenic acids, in particular 2-HDA, might be useful tools to
understand the potential of fatty acids as drug candidates. Our fu-
ture work will involve the synthesis, in vitro and in vivo evaluation
of more potent acetylenic fatty acids with multiple triple and dou-
ble bonds.
5.2.2.1. 5-Hexadecynoic acid (5-HDA).
5-HDA was obtained
in a 75% yield (249 mg) from the reaction of 313 mg (1.3 mmol)
of 5-hexadecyn-1-ol with 3.0 g (7.9 mmol) of pyridinium dichro-
mate. IR (neat) mmax 3105–2900, 2926, 2854, 2206, 1711, 1465,
1261, 1207, 1090, 920, 803, 722 cmÀ1 1H NMR (500 MHz, CDCl3)
.
dH 2.48 (2H, t, J = 7.5 Hz, H-2), 2.12 (4H, m, H-4, H-7), 1.80 (2H,
quintet, J = 7.2 Hz, H-3), 1.46 (2H, quintet, J = 7.3 Hz, H-8), 1.35–
1.25 (14H, m, –CH2–), 0.88 (3H, t, J = 6.9 Hz, H-16). 13C NMR
(125 MHz, CDCl3) dC 178.5 (s, C-1), 81.5 (s), 78.5 (s), 32.6 (t,
C-2), 31.9 (t, C-14), 29.6 (t), 29.5 (t), 29.3 (t), 29.1 (t), 29.0
(t), 28.9 (t), 24.0 (t), 22.7 (t), 18.7 (t, C-7), 18.3 (t, C-4), 14.1 (q,
C-16).
5.2.2.2. 6-Hexadecynoic acid (6-HDA).
6-HDA was obtained
in a 42% yield (87 mg) from the reaction of 195 mg (0.8 mmol) of
6-hexadecyn-1-ol with 1.2 g (3.3 mmol) of pyridinium dichromate.
IR (neat) mmax 3105–2900, 2927, 2851, 1709, 1469, 1260, 1207,
5. Experimental
5.1. General
1077, 899, 797, 718 cmÀ1 1H NMR (300 MHz, CDCl3) dH 2.37 (2H,
.
t, J = 7.4 Hz, H-2), 2.15 (4H, m, H-5, H-8), 1.78–1.26 (19H, m, –
CH2–), 0.87 (3H, t, J = 6.7 Hz, H-16). 13C NMR (75 MHz, CDCl3) dC
179.9 (s, C-1), 80.8 (s), 79.3 (s), 33.4 (t, C-2), 31.9 (t), 29.5 (t),
29.3 (t), 29.15 (t), 29.10 (t), 28.9 (t), 28.4 (t), 23.8 (t), 22.7 (t),
18.7 (t), 18.4 (t), 14.1 (q, C-16).
(Trimethylsilyl)-acetylene and other reagents used were pur-
chased from Aldrich, TCI America, and Alfa Aesar. All synthetic
products were analyzed by 1H and 13C Nuclear Magnetic Reso-
nance (NMR) using a Bruker Avance DRX-500 or a Bruker Avance
DPX-300. The samples were dissolved in chloroform-d (CDCl3)
and the solvent signals at 7.26 and 77.0 ppm were used as internal
standard for proton and carbon, respectively. Mass spectral data
were acquired on a GC–MS (Hewlett–Packard 5972A MS Chem Sta-
tion) instrument at 70 eV, equipped with a 30 m  0.25 mm special
performance capillary column (HP-5MS) of polymethylsiloxane
cross-linked with 5% phenyl methylpolysiloxane. The GC–MS anal-
ysis conditions were identical to those previously described.39
Infrared (IR) spectra were recorded on a Nicolet 600 FTIR spectro-
photometer. Melting points (°C, uncorrected) were determined in a
MEL-TEMP capillary melting point apparatus. Thin layer chroma-
tography was carried out on aluminium TLC plates pre-coated with
Silica Gel 60 F254 (Merck, 0.25 mm).
5.2.2.3. 9-Hexadecynoic acid (9-HDA).
9-HDA was obtained
in an 80% yield (60 mg) from the reaction of 70 mg (0.25 mmol)
of 9-hexadecynal with a NaClO2–NaH2PO4 buffer solution. IR
(neat) mmax 3313–2900, 2954, 2929, 2852, 1690, 1465, 1441,
1414, 1335, 1277, 1263, 1094, 1015, 918, 726 cmÀ1 1H NMR
.
(300 MHz, CDCl3) dH 2.34 (2H, t, J = 7.5 Hz, H-2), 2.13 (4H, m, H-
8, H-11), 1.63 (2H, quintet, J = 7.0 Hz, H-3), 1.46 (4H, quintet,
J = 6.4 Hz, H-7, H-12), 1.30 (12 H, m, –CH2–), 0.87 (3H, t,
J = 6.5 Hz, H-16). 13C NMR (75 MHz, CDCl3) dC 179.9 (s, C-1), 80.4
(s), 80.0 (s), 34.0 (t, C-2), 31.5 (t), 29.7 (t), 29.1 (t), 29.0 (t), 28.9
(t), 28.8 (t), 28.6 (t), 28.5 (t), 24.6 (t), 22.6 (t), 18.75 (t), 18.71 (t),
14.1 (q, C-16).

D. Tasdemir et al. / Bioorg. Med. Chem. 18 (2010) 7475–7485
7483
5.3. 2-Hexadecynoic acid (2-HDA)
as 10 mM solutions in DMSO and tested at concentrations of 10–
100 lM in complete media. Atovaquone, a drug with known LS
2-HDA was obtained as a white solid in a 10% yield from the
reaction of 1-pentadecyne (1.00 g, 4.8 mmol) and excess dry CO2.
Mp 49–51 °C. IR (neat) mmax 3400–2900 (br), 2914, 2848, 2235,
inhibitory activity,25 was used as a positive control.
To verify the results of the FC assay and to determine the effect
of the compounds on the morphology and development of the LS
parasites, immunofluorescence analysis (IFAs) of P. yoelii-infected
HepG2:CD81 cells after compound treatment was also carried
out. Subconfluent HepG2:CD81 cells that were seeded in 8-well
chambered slides (Nunc) and maintained at 37 °C in 5% CO2 and
subsequently treated with the compounds. After 43 h of incuba-
tion, the infected cells were fixed with 10% neutral buffered forma-
lin. IFA was carried out using a mouse monoclonal antibody against
the Plasmodium HSP70 protein together with a rabbit polyclonal
antibody raised against either the P. yoelii UIS4 or FabI proteins.
The second antibody was used to confirm the presence of the liver
stage parasite. The slides were scanned with a fluorescence micro-
scope and images were captured using a 20Â objective. The result-
ing images were analyzed with the METAMORPH program (Molecular
Devices) which allows for automated measurements of liver stage
parasites stained with the HSP70 antibody (Fig. 2). Approximately
20–25 liver stage parasites were measured for each compound
treatment and the average sizes were compared with DMSO-trea-
ted controls. The normalized reduction in liver stages from infec-
tions treated with the compounds compared with DMSO-treated
controls was calculated and used for IC50 estimation using the ICES-
TIMATOR program (http://bichat.inserm.fr/equipes/Emi0357/Palu/in-
dex.htm). Compounds were dissolved in DMSO and tested at
1679, 1467, 1409, 1277, 1077 cmÀ1 1H NMR (500 MHz, CDCl3) dH
.
2.35 (2H, t, J = 7.2 Hz, H-4), 1.59 (2H, quintet, J = 7.4 Hz, H-5),
1.41–1.22 (20H, m, –CH2–), 0.88 (3H, t, J = 7.0 Hz, H-16). 13C NMR
(125 MHz, CDCl3) dC 157.2 (s, C-1), 92.6 (s, C-3), 72.5 (s, C-2),
31.9 (t, C-14), 29.65 (t), 29.63 (t), 29.6 (t), 29.4 (t), 29.3 (t), 29.0
(t), 28.8 (t), 27.4 (t), 22.7 (t, C-15), 18.8 (t, C-14), 14.1 (q, C-16).
5.4. Antimalarial activity against blood stage (BS) P. falciparum
In vitro activity against erythrocytic stages of P. falciparum was
determined by a modified [3H]-hypoxanthine incorporation as-
say,40 using the chloroquine- and pyrimethamine-resistant K1
strain and the standard drug chloroquine. Briefly, parasite cultures
incubated in RPMI 1640 medium with 5% Albumax (without hypo-
xanthine) were exposed to serial drug dilutions in microtiter
plates. After 48 h of incubation at 37 °C in a reduced oxygen atmo-
sphere, 0.5 l
Ci 3H-hypoxanthine was added to each well. Cultures
were incubated for a further 24 h before they were harvested onto
glass-fiber filters and washed with distilled water. The radioactiv-
ity was counted using a Betaplate™ liquid scintillation counter
(Wallac, Zurich, Switzerland). The results were recorded as counts
per minute (CPM) per well at each drug concentration and ex-
pressed as percentage of the untreated controls. IC50 values were
calculated from graphically plotted dose–response curves.
concentrations up to 25 lg/ml in complete media. Atovaquone
was used as a positive control. An 100Â oil immersion objective
was used for the close-up images (Fig. 3).
5.5. Antimalarial activity against liver stage (LS) P. yoelii
5.6. Toxicity assessments toward HepG2:CD81 cells
To assay for inhibitory activity against Plasmodium liver stages,
two assays based on in vitro infections of P. yoelii in the hepatoma
cell line HepG2:CD81 were developed. HepG2:CD81 cells stably
express the CD81 protein that is necessary and sufficient to give
high infection rates with P. yoelii sporozoites in vitro.41 P. yoelii
wild-type and transgenic P. yoelii sporozoites that express green
fluorescent protein (PyGFP)42 parasites were cycled between
Anopheles stephensi mosquitoes and swiss-webster mice (Harlan).
Infected mosquitoes were maintained at 24 °C and 70% humidity
on sugar water (dextrose). The first assay is a flow cytometry-
based analysis using the BD-LSRII (BD Biosciences) flow cytometer.
Briefly, 100,000 HepG2:CD81 cells grown in complete media (ad-
vanced DMEM/F12 supplemented with 10 FBS, 2% penicillin/strep-
tomycin, 2% glutamine and 1% amphotericin) were seeded in 48
Toxicity of the compounds to HepG2:CD81 cells was deter-
mined by using the non-membrane permeant nuclear stain
7-aminoactinomycin D (7-AAD). This stain is generally excluded
from live cells and thus generally stains only dead cells. The pro-
portion of 7-AAD positive cells detected in the flow assay is used
an indicator for dead cells in the HepG2:CD81 cell culture and
thus when compared to %7-AAD-positive cells in DMSO-treated
cells can be used as an indirect measure for toxicity of the com-
pounds in the treated cultures. In the IFA, toxicity of the com-
pounds was assessed by comparing the DAPI staining of infected
HepG2:CD81 cells treated with DMSO alone or with various con-
centrations of the compounds. Toxic concentrations of drugs such
as high concentrations of atovaquone result in cell death and
detachment from the culture slides which result in less DAPI-
stained cells.
well plates previously coated with 20 lg/ml ECL attachment ma-
trix (Upstate Labs). The day after the cells were seeded, they were
infected with 50,000 PyGFP sporozoites (in RPMI media at a vol-
ume of 100
l
l/well) that were isolated from manually dissected
5.7. Plasmodial FAS-II enzyme inhibition assays and enzyme
kinetics
PyGFP-infected mosquitoes 14 days post-blood meal. Two hours
after the sporozoites were added, media in each well were re-
moved and replaced with fresh complete media containing various
concentrations of compounds. The HepG2:CD81 infected cells were
continuously exposed to the compounds with a media change 24 h
after infection and allowed to develop for a further 43 h at 37 °C in
a CO2 incubator. The cells were trypsinized to prepare a single cell
Expression and purification of the PfFab enzymes as well as the
assays were performed as described.26 Briefly, assays were moni-
tored using an Uvikon 941 Plus spectrophotometer (Kontron
Instruments) in 1.0 ml of 20 mM HEPES pH 7.4, and 150 mM NaCl.
Compounds were dissolved in DMSO (max. final concentration is
suspension, centrifuged, and resuspended in 100
l
l complete med-
1%). For PfFabI 100
lM NADH (cofactor, Sigma) was added to
ium containing 0.1% 7-amino-actinomycin D (7-AAD, Invitrogen),
an impermeant nucleic acid dye that could be used to stain mem-
brane-compromised cells (i.e., dead cells). After transfer to 96-well
v-bottom plates, the number of GFP-positive hepatoma cells is
determined by flow cytometry using the BD-LSRII HTS system. In
most cases the BD-LSRII can count 50,000–100,000 events from
each well with 1.5–2.0% GFP-positive cells in wells treated with
0.5% or 1.0% DMSO (untreated control). Compounds were prepared
1
l
g enzyme and the reaction was started by addition of 50 lM
crotonyl-CoA (substrate, Sigma). The change of absorbance of the
mixture was recorded spectrophotometrically at 340 nm during
1 min. Triclosan was used as a positive control and was analyzed
the same way. For PfFabG different cofactor (NADPH, Fluka) and
substrate (acetoacetyl-CoA, Sigma) were used. PfFabZ was
measured at 263 nm for 2 min in the presence of 25
noyl-CoA and 0.5 g enzyme. Reference compound for the latter
lM croto-
l

7484
D. Tasdemir et al. / Bioorg. Med. Chem. 18 (2010) 7475–7485
two enzymes was (À)-epigallocatechin gallate (Sigma). IC50 values
were estimated from graphically plotted dose–response curves.26
The inhibition mechanism for 2-HDA was determined under
Michaelis–Menten steady-state conditions. For PfFabI, with respect
were read in a Spectramax Gemini XS microplate fluorometer
(Molecular Devices Cooperation, Sunnyvale, CA, USA) using an
excitation wavelength of 536 nm and an emission wavelength of
588 nm. Data were analyzed using the microplate reader software
Softmax Pro (Molecular Devices Cooperation, CA, USA).
to the substrate, 1
concentration (100
(0–1 g/ml). The reaction was initiated by the addition of croto-
nyl-CoA (10–50 M). The inhibition mechanism with respect to
NADH was determined in a similar way. For this, PfFabI was incu-
bated with varying NADH concentrations (10–50 M) and different
inhibitor concentrations (0–1 g/ml). The reaction was initiated by
the addition of 50 M crotonyl-CoA. Kinetic studies for PfFabG
were performed in a similar way, using NADPH as cofactor and
acetoacetyl-CoA as substrate. For PfFabZ, 0.5 g enzyme was incu-
bated with increasing inhibitor concentrations (0–1 g/ml) and the
reaction was started by the addition of crotonyl-CoA (10–50 M).
l
g enzyme was incubated with a fixed NADH
l
M) and increasing inhibitor concentrations
l
5.10. Trypanocidal activity against T. cruzi
l
Rat skeletal myoblasts (L6 cells) were seeded in 96-well micro-
l
titre plates at 2000 cells/well in 100
10% FBS and 2 mM l-glutamine. After 24 h the medium was
removed and replaced by 100 l per well containing 5000 trypo-
lL RPMI 1640 medium with
l
l
l
mastigote forms of T. cruzi Tulahuen strain C2C4 containing the
l
b-galactosidase (Lac Z) gene.46 After 48 h, the medium was re-
l
moved from the wells and replaced by 100
or without a serial drug dilution of seven threefold dilution steps
covering a range from 90 to 0.123 g/ml. After 96 h of incubation
ll fresh medium with
l
Ki values were obtained from Dixon and secondary plots. The re-
l
ported values represent means of two independent experiments.
the plates were inspected under an inverted microscope to assure
growth of the controls and sterility. Then the substrate CPRG/Non-
5.8. Molecular modeling and pharmacokinetic calculations
idet (50 ll) was added to all wells. A color reaction developed
within 2–6 h and could be read photometrically at 540 nm. Data
were transferred into the graphic programme Softmax Pro (Molec-
ular Devices), which calculated IC50 values. Benznidazole was the
standard drug used.
The 3D structure of ligands used for the docking were built by
Maestro GUI v8.5 (Schrodinger LLC) and initial conformations with
the correct protonation states were generated using LigPrep
(Schrodinger LLC) and OPLS-2005 force field. The protein struc-
tures FabZ (id:1z6b), FabI (id:1v35), and FabG (id:2c07) were
downloaded from the RCSB Protein Data Bank (PDB) and prepared
for docking by Rebol script ‘Receptor.r’ implemented in VegaZZ.43
The calculations of ligands docking to the binding site of PfFabZ
and PfFabG were carried out using Autodock and Gridock and Ve-
gaZZ as graphic user interface. Binding site was centered on the
residues of the active site a cube with an edge length of 32 Å was
defined as a bounding box in the protein. The best pose generated
for each ligand was selected based on lowest energy value of the
Autodock scoring function. The docking calculations of ligands into
the PfFabI were carried out using GLUE program (GRID package from
MolDiscovery Ltd). The surface of the whole protein was selected
to generate GRID maps for the probes that best simulate ligands.
The docking was carried out taking into account electrostatic con-
tributions and flexibility of ligands at default values of GLUE set-
tings. Interaction energies scores (kcal/mol) were used to rank
the interaction between ligand and a protein, but all solutions were
considered in the analysis of the results. The pharmacokinetics and
estimated molecular properties of ligands were estimated using
Vega ZZ software and ChemSilico online server (http://www.
chemsilico.com).
5.11. Leishmanicidal activity against L. donovani
Amastigotes of L. donovani strain MHOM/ET/67/L82 were grown
in axenic culture at 37 °C in SM medium at pH 5.4 supplemented
with 10% heat-inactivated fetal bovine serum under an atmosphere
of 5% CO2 in air. One hundred microliters of culture medium with
105 amastigotes from axenic culture with or without a serial drug
dilution were seeded in 96-well microtitre plates. Serial drug dilu-
tions covering a range from 90 to 0.123 lg/ml were prepared. After
72 h of incubation the plates were inspected under an inverted
microscope to assure growth of the controls and sterile conditions.
Ten microliters of a resazurin solution (12.5 mg resazurin dissolved
in 100 ml double-distilled water)47 was then added to each well
and the plates were incubated for another 2 h. Then the plates
were read in a Spectramax Gemini XS microplate fluorometer
using an excitation wavelength of 536 nm and an emission wave-
length of 588 nm. Data were analyzed using the software Softmax
Pro. Decrease of fluorescence (=inhibition) was expressed as per-
centage of the fluorescence of control cultures and plotted against
the drug concentrations. From the sigmoidal inhibition curves the
IC50 values were calculated. Miltefosine was used as a reference
drug.
5.9. Trypanocidal activity against Trypanosoma brucei
rhodesiense
5.12. Cytotoxicity against L6 cells
STIB 900 strain of T. b. rhodesiense and the standard drug melar-
soprol were used for the assay. This stock was isolated in 1982
from a human patient in Tanzania and after several mouse pas-
sages cloned and adapted to axenic culture conditions.44 Minimum
Assays were performed in 96-well microtiter plates, each well
containing 100 ll of RPMI 1640 medium supplemented with 1%
L
-glutamine (200 mM) and 10% fetal bovine serum, and 4 Â 104
L6 cells (a primary cell line derived from rat skeletal myoblasts).
Serial drug dilutions of seven threefold dilution steps covering a
Essential Medium (50 ll) supplemented with 25 mM HEPES, 1 g/l
additional glucose, 1% MEM non-essential amino acids (100Â),
0.2 mM 2-mercaptoethanol, 1 mM Na-pyruvate, and 15% heat inac-
tivated horse serum was added to each well of a 96-well microtiter
plate.45 Serial drug dilutions of seven threefold dilution steps cov-
range from 90 to 0.123
tion the plates were inspected under an inverted microscope to as-
sure growth of the controls and sterile conditions. 10 l of a
resazurin solution (12.5 mg resazurin dissolved in 100 ml distilled
water) was then added to each well and the plates were incubated
for another 2 h. Then the plates were read with a Spectramax Gem-
ini XS microplate fluorometer using an excitation wavelength of
536 nm and an emission wavelength of 588 nm. Data were ana-
lysed using the microplate reader software Softmax Pro. Podophyl-
lotoxin was the standard drug used.
lg/ml were prepared. After 72 h of incuba-
l
ering a range from 90 to 0.123
bloodstream forms of T. b. rhodesiense STIB 900 in 50
to each well and the plate incubated at 37 °C under a 5% CO2 atmo-
sphere for 72 h. 10 l of a resazurin solution (12.5 mg resazurin
l
g/ml were prepared. Then 104
l was added
l
l
dissolved in 100 ml double-distilled water) was then added to each
well and incubation continued for a further 2–4 h. Then the plates

D. Tasdemir et al. / Bioorg. Med. Chem. 18 (2010) 7475–7485
7485
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Acknowledgments
Part of the project described herein was supported by Award
Number SC1GM084708 from the National Institute of General
Medical Sciences (N.M.C) and partly by grants from the Bill and
Melinda Gates Foundation and Medicines for Malaria Ventures
(S.H.K.). The content is solely the responsibility of the authors
and does not necessarily represent the official views of the Na-
tional Institute of General Medical Sciences or the National Insti-
tutes of Health. Ina L. Lauinger thanks the School of Pharmacy for
the PhD scholarship.
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