Design, synthesis, and biological and crystallographic evaluation of novel inhibitors of plasmodium falciparum enoyl-ACP-reductase (pf fabi)

Article abstract of DOI:10.1021/jm400637m

Malaria, a disease of worldwide significance, is responsible for over one million deaths annually. The liver-stage of Plasmodium's life cycle is the first, obligatory, but clinically silent step in malaria infection. The P. falciparum type II fatty acid biosynthesis pathway (PfFAS-II) has been found to be essential for complete liver-stage development and has been regarded as a potential antimalarial target for the development of drugs for malaria prophylaxis and liver-stage eradication. In this paper, new coumarin-based triclosan analogues are reported and their biological profile is explored in terms of inhibitory potency against enzymes of the PfFAS-II pathway. Among the tested compounds, 7 and 8 showed the highest inhibitory potency against Pf enoyl-ACP-reductase (PfFabI), followed by 15 and 3. Finally, we determined the crystal structures of compounds 7 and 11 in complex with PfFabI to identify their mode of binding and to confirm outcomes of docking simulations.

Full text of DOI:10.1021/jm400637m

Article
pubs.acs.org/jmc
Design, Synthesis, and Biological and Crystallographic Evaluation
of Novel Inhibitors of Plasmodium falciparum Enoyl-ACP-reductase
(Pf FabI)
Federica Belluti,*,†,∞ Remo Perozzo,*,‡,∞ Leonardo Lauciello,‡ Francesco Colizzi,§ Dirk Kostrewa,∥
Alessandra Bisi,† Silvia Gobbi,† Angela Rampa,† Maria Laura Bolognesi,† Maurizio Recanatini,†
Reto Brun,⊥ Leonardo Scapozza,‡ and Andrea Cavalli†,#
†Department of Pharmacy and Biotechnology, University of Bologna, Via Belmeloro 6, I-40126 Bologna, Italy
‡Pharmaceutical Biochemistry Group, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne,
Quai Ernest-Ansermet 30, CH-1211 Geneva 4, Switzerland
§International School for Advanced Studies, Via Bonomea, 265, I-34136 Trieste, Italy
∥Department of Biochemistry, Gene Center Munich, Ludwig-Maximilians-University Munich, Munich 81377, Germany
⊥Swiss Tropical & Health Institute, Socinstrasse 57, CH-4002 Basel, Switzerland
#Department of Drug Discovery and Development, Istituto Italiano di Tecnologia, Via Morego 30, I-16163 Genova, Italy
S
* Supporting Information
ABSTRACT: Malaria, a disease of worldwide significance, is responsible
for over one million deaths annually. The liver-stage of Plasmodium’s life
cycle is the first, obligatory, but clinically silent step in malaria infection.
The P. falciparum type II fatty acid biosynthesis pathway (PfFAS-II) has
been found to be essential for complete liver-stage development and has
been regarded as a potential antimalarial target for the development of
drugs for malaria prophylaxis and liver-stage eradication. In this paper, new
coumarin-based triclosan analogues are reported and their biological
profile is explored in terms of inhibitory potency against enzymes of the
Pf FAS-II pathway. Among the tested compounds, 7 and 8 showed the
highest inhibitory potency against Pf enoyl-ACP-reductase (PfFabI),
followed by 15 and 3. Finally, we determined the crystal structures of
compounds 7 and 11 in complex with Pf FabI to identify their mode of
binding and to confirm outcomes of docking simulations.
meal. After migration to the liver, the sporozoites invade and
infect hepatocytes leading to the liver stage (LS) of the parasite
life cycle.4−6 During this stage, which is clinically silent, the
sporozoites grow and undergo replication, culminating in the
release of tens of thousands of merozoites into the bloodstream
to invade erythrocytes for subsequent asexual propagation. This
so-called blood stage (BS) is responsible for most of the malaria
symptoms. In P. vivax and P. ovale, a number of LS parasites
remain in the hepatocytes as a dormant form (hypnozoite),
which is difficult to eradicate and can lead to recurrent infections
even after months or years.7
With the exception of primaquine, the currently available
antimalarial drugs are blood schizontocidals targeting the
pathogenic, asexual BS parasites.8 However, full inhibition of
the parasite development during the obligatory LS would lead to
true causal prophylaxis, thus preventing pathology or at least
decreasing the severity of the disease. Furthermore, the low
INTRODUCTION
■
Malaria, caused by protozoan parasites of the genus Plasmodium,
is one of the most widespread and devastating infectious tropical
diseases, killing more than one million people each year and
continuously undermining the economic development oppor-
tunities of countries in the tropical and subtropical zones of the
world.1 The current emergence and spread of chloroquine-
resistant Plasmodium falciparum (Pf) strains represent major
obstacles for the control of the disease, impairing both
prophylaxis and chemotherapy. Even worse, the first signs of
resistance to artemisinin, the newest and most effective
antimalarial drug currently available, were identified in Cambodia
in 2006, and the resistance has now spread along the border with
Thailand.2 Although attempts to develop a vaccine for malaria are
ongoing, drugs continue to be the only treatment option.3
Consequently, there is an urgent need to develop potent and safe
antimalarial drugs.
The Plasmodium life cycle in the mammalian host begins with
sporozoites in the saliva of a female Anopheles mosquito, which
are injected into the dermis of the human host during the blood
Received: January 6, 2013
© XXXX American Chemical Society
A
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number of hepatic forms substantially reduces the likelihood of
selecting for drug-resistant parasites. Transmission would also be
interrupted because it depends on gametocytes that mature in
red blood cells.9 Thus, agents directed to the LS constitute a
promising strategy for malaria prophylaxis and eradication and
are likely to be beneficial for high-risk groups (e.g., children and
pregnant women) living in malaria endemic areas or travelers
exposed to the disease while visiting those areas. To achieve the
goal of malaria eradication, drug discovery efforts must continue
to produce new drugs, targeted at unconventional vital parasite
functions, such as new metabolic pathways.
Recent transcriptomic and proteomic findings on Plasmodium
parasites in isolated LS hepatocytes revealed protein classes and
metabolic pathways that have the potential to serve as drug
targets for malaria prophylaxis.10 Among others, several enzymes
involved in fatty acid synthesis, including those of the Pf type II
fatty acid biosynthesis (Pf FAS-II) pathway, are overrepresented
in the LS parasites.10 This is well in line with the recent discovery
that the PfFAS-II pathway is essential for correct LS develop-
ment,11,12 although it was previously considered to function only
in the BS parasite.13,14 The inherent differences between the fatty
acid biosynthesis pathway of the parasite (FAS-II) and that of the
human host (FAS-I) offer a great opportunity to develop highly
specific antimalarial agents.11,12
the 2-hydroxy-4-chlorophenyl fragment of 1 (called ring A) plays a
fundamental role in binding affinity, since both the aryl moiety and
its functional groups, together with the cofactor NADH, are
involved in several noncovalent interactions with the side chains of
amino acid residues. In particular, the hydroxyl group and the ether
oxygen linking the two aromatic rings are critical for binding.19
When the 2′,4′-dichlorophenyl moiety (called ring B) of 1 was
replaced with an extended naphthalene function bearing a
hydroxyl group (2, Figure 1), three new hydrogen bonds could
be established via the additional hydroxyl moiety.19 On the basis of
these findings, drug discovery projects have largely focused on
analogues of 1, affording a large collection of diaryl ethers with
strong activity toward the parasite or the target enzyme.19,26−28
The present study reports on the design and synthesis of novel
analogues of 1 obtained by replacing its B ring with a coumarin
moiety, which represents a naturally occurring scaffold found in
several marketed drugs. The molecules were then tested using
biochemical and cell-based assays to assess their antiplasmodial
profile. Docking simulations supported the design strategy, and
the X-ray cocrystal structures of compounds 7 and 11 in complex
with PfFabI were solved to disclose the binding mode. The
promising results point to these new chemical entities as potential
hit compounds for discovering novel antimalarial compounds to
be utilized as effective agents for malaria prophylaxis.
Fatty acids are produced via enzymatic steps consisting of
repeated cycles of elongation reactions, each including
condensation, dehydration, and reduction. The growing acyl
substrate is covalently bound to the acyl carrier protein (ACP),
which shuttles and delivers the substrate from one enzyme to the
other.15 The FAS-II pathway is composed of a series of distinct
and highly conserved enzymes, among which are β-ketoacyl-
ACP-reductase (FabG), β-hydroxacyl-ACP-dehydratase (FabZ),
and enoyl-ACP-reductase (FabI, also known as ENR). FabI,
responsible for the concluding reduction step of each elongation
cycle, represents a key physiological regulator of fatty acid
biosynthesis and has been validated as an important drug target
for the development of antibacterials16 and antimalarials.17,18
FabI is a key enzyme that is responsible for the NADH-
dependent reduction of trans-2-enoyl-ACP to saturated acyl-
ACP, and its three-dimensional structure in complex with several
inhibitors has been characterized by X-ray crystallography.19
Among the inhibitors reported in the literature, triclosan,
5-chloro-2-(2,4-dichlorophenoxy)phenol (compound 1, Figure 1),
RESULTS AND DISCUSSION
■
Design Strategy. Coumarins, a class of compounds found
widely in nature, show a broad spectrum of activities and are
frequently associated with low toxicity.29 Because of their
inherent affinity for several biological targets, coumarins can be
regarded as a privileged scaffold and represent an ideal
framework for the design of compounds able to interact with
different targets.30 Our strategy was to link the 2-hydroxy-4-
chlorophenyl of 1 (ring A) to selected coumarin backbones to
afford a new druglike scaffold, which has the potential to engage
in new interactions with the target and enable further exploration
of the Pf FabI active site. The design of the compounds was
carried out by taking advantage of the available crystal structures
of PfFabI in complex with 1 and derivatives thereof19 and was
supported by docking simulations. We designed two series of
molecules (Tables 1 and 2), hereafter referred to as series I (3−8)
and series II (9−15).
Docking of both series showed that we could replace the
lipophilic subunit of 1 with a chromenone function while keeping
relevant interactions within the PfFabI active site. Figure 2 shows
a schematic representation of docking of series I (Figure 2A) and
II (Figure 2B). In these models, ring A engages a stacking
interaction with the nicotinamide ring of NADH, with hydrogen
bonds to the 2′-hydroxyl group of the nicotinamide ribose and
with Tyr277. The 4-chloro atom of ring A was surrounded by
hydrophobic residues and made van der Waals contacts with the
side chains of Tyr267, Pro314, and Phe368. The coumarin
moiety occupied the same pocket of ring B as 1, lying on the
side chain of Met281 and suggesting a possible sulfur−arene
interaction.31
Figure 1. Pf FabI inhibitors 1 and 2.
is considered to be a prototypical lead compound that specifically
inhibits FabI in several species. In particular, triclosan has been
reported to act against a broad spectrum of bacteria,20 including
Escherichia coli,21 Mycobacterium tuberculosis,22 multidrug-
resistant Staphylococcus aureus,23 but also the unicellular parasite
P. falciparum.24 Triclosan inhibits P. falciparum with an IC50 of
around 1 μM 19,24,25 and exhibits a Ki of 50 nM against purified P.
falciparum FabI (Pf FabI).19 The structural basis of Pf FabI in
complex with 1 has been elucidated and allowed the identification
of structural features important for Pf FabI inhibition. In particular,
Docking solutions for compounds 3 and 6, in which the A ring
was inserted on positions 6 and 7 of the coumarin scaffold,
clustered in an almost identical fashion, maintaining the native
interactions of the A ring of 1 and suggesting a good shape-
complementarity between the coumarin portion and the binding
site. Furthermore, the coumarin-2-one function could form
H-bonds with both the backbone nitrogen of Ala219 and the side
chain of Asn218 (Figure 2A).
B
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Table 1. Inhibitory Activities of Series I (Compounds 3−8) and Reference Compounds: IC50 (μM) for Pf FAS-II Enzymes from
P. falciparum and in Vitro Antiplasmodial Activities against Drug Resistant P. falciparum K1 Strain and Activity on Mammalian L6
a
Cells (Cytotoxicity) (μM)
b
c
compd
R
PfFabI
PfFabG
PfFabZ
PfK1
L-6
SI
3
4
5
6
7
8
H
0.63
14.2
11.0
2.7
nd
nd
10.7
15.6
5.6
96.2
35.5
142.0
187.6
85.9
84.8
9.0
CH3
nd
nd
2.2
CH2CH3
H
nd
nd
25.6
20.1
9.0
nd
nd
9.3
CH3
0.25
0.27
0.05
>100
>100
>100
>100
9.5
CH2CH3
5.7
14.9
d
triclosan (1)
1.51
(−)-epigallocatechin gallate
chloroquine
0.32
0.03
0.23
podophyllotoxin
0.006
a
IC50 values represent the concentration of a compound that causes 50% growth inhibition and are the mean of three independent experiments for
the enzyme inhibition assay and are the average of at least two independent assays performed in duplicate for the cell assay. The errors for individual
b
c
d
measurements differed by less than 50%. Resistant to chloroquine and pyrimethamine. SI = IC50(L-6)/IC50(parasite). See ref 43.
Table 2. Inhibitory Activities of Series II (Compounds 9−15) and Reference Compounds: IC50 (μM) for Pf FAS-II Enzymes from
P. falciparum and in Vitro Antiplasmodial Activity against Drug Resistant P. falciparum K1 Strain and Activity on Mammalian L6
a
Cells (Cytotoxicity) (μM)
b
c
compd
R
R′
Pf FabI
Pf FabG
PfFabZ
PfK1
L-6
SI
9
H
H
5.5
>100
>100
99
>100
>100
16.3
90
15.0
108.4
134.2
143.9
127.9
51.53
96.3
7.2
10
11
12
13
14
15
H
6-OH
7-OH
5,7-di-OH
H
14.0
6.0
>15.0
>15.0
>15.0
>15.0
>15.0
8.0
>9.5
>9.6
>8.5
>3.4
>6.4
5.1
H
H
44
>100
>100
>100
>100
CH3
CH3
12.0
15.0
0.45
0.05
13
7-OH
28
10
41.0
d
triclosan (1)
1.51
(−)-epigallocatechin gallate
chloroquine
0.32
0.03
0.23
podophyllotoxin
0.006
a
IC50 values represent the concentration of a compound that causes 50% growth inhibition and are the mean of three independent experiments for
the enzyme inhibition assay and are the average of at least two independent assays performed in duplicate for the cell assay. The errors for individual
b
c
d
measurements differed by less than 50%. Resistant to chloroquine and pyrimethamine. SI = IC50(L-6)/IC50(parasite). See ref 43.
A relatively small and hydrophobic pocket formed by the side
chain of residues Val222, Tyr267, Met281, and Ile323 and
adjacent to position 4 of the coumarin moiety (Figure 2A)
suggested the possibility of introducing 4-methyl and 4-ethyl
substituents at this position (4, 5, 7, 8). Docking of 7 and 8
generated a single cluster of poses with the 4-substituents
inserted into this hydrophobic pocket, thus providing an
additional anchoring point within the binding site. Hydroxylated
derivatives were finally designed to explore the possibility of
establishing new H-bonds. The high score obtained with
7-monohydroxylated ligands (11, 12, 14) suggested a possible
network of H-bonds with the side chain nitrogen of Asn218 and
the main chain oxygen and nitrogen of Ala219.
Compounds 3−15 were then synthesized and biologically
tested to assess their antimalarial profile, namely, inhibitory activity
against multiple Pf FAS-II elongation enzymes (Pf FabI, Pf FabZ,
and Pf FabG) and in vitro growth inhibitory potential against
malaria parasites (drug resistant P. falciparum K1 strain).
Furthermore, their toxicity against mammalian cells was assessed
against a primary cell line derived from rat skeletal myoblasts (L6).
Chemistry. The Ullmann-type copper-catalyzed coupling
of brominated coumarin intermediates 16−21 and 34 with
C
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Figure 2. (A) Schematic representation of the backbone of compounds 3−8 (carbon atoms are white). The various substitutions, in which the A ring
was appended on the aryl ring of the coumarin scaffold, are labeled. (B) Predicted binding mode for 9−14 (carbon atoms in white). Several substituents
on the coumarin moiety were introduced and virtually tested for complementarity with the PfFabI binding site (carbon atoms in pink). A section of the
Connolly surface of the receptor is shown in blue.
a
Scheme 1. Synthesis of Series I Pf FabI Inhibitors (3−8)
a
Reagents and conditions: (i) 4-chloro-2-methoxyphenol, Cs2CO3, (CuOTf)·PhH, ethyl acetate, 1-naphthoic acid, activated 5 Å molecular sieves,
toluene, 110 °C, N2; (ii) BBr3, DCM, −70 °C, N2 atmosphere.
a
Scheme 2. Synthesis of Series II PfFabI Inhibitors (9−14)
a
Reagents and conditions: (i) triethylamine, Ac2O, 180 °C; (ii) BBr3, DCM, −70 °C, N2 atmosphere.
a
Scheme 3. Synthesis of Pf FabI Inhibitor 15
a
Reagents and conditions: (i) triethylamine, Ac2O, 180 °C; (ii) 4-chloro-2-methoxyphenol, Cs2CO3, (CuOTf)·PhH, ethyl acetate, 1-naphthoic acid,
activated 5 Å molecular sieves, toluene, 110 °C, N2 atmosphere; (iii) BBr3, DCM, −70 °C, N2.
4-chloro-2-methoxyphenol gave the desired diaryl ethers 22−27
and 35 (Schemes 1 and 3, respectively). Different synthetic
strategies were employed to obtain the suitable brominated
coumarin intermediates (16−21) (see the Experimental
Section). Compound 34 was obtained according to Scheme 3,
in which the 2-hydroxy-4-methoxybenzaldehyde was reacted
D
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Figure 3. View of the 2mFo − DFc electron density map contoured at 1.0σ for bound analogues 7 (A) and 11 (B) and cofactor NADH.
with sodium 2-(4-bromophenyl)acetate in the presence of
triethylamine. Similarly, 28−33 (Scheme 2) were prepared
by reaction of suitable methoxylated 2-hydroxybenzaldehyde and
2-hydroxyacetophenone with sodium 2-(4-chloro-2-methoxy-
phenoxy)acetate. The methoxy groups were removed by
treatment with BBr3 to afford the final compounds as phenol
derivatives (3−15).
Inhibition of Pf FabI. The ability of 3−15 to inhibit the
recombinant Pf FabI enzyme was investigated by using a
spectrophotometric assay (see the Experimental Section for
details). All data are shown in Tables 1 and 2. Compounds 3−8,
where ring A of 1 is attached to either the 6- or 7-position of the
coumarin scaffold, inhibited PfFabI with IC50 values in the low
micromolar to the submicromolar range. Among them, 6
exhibited moderate PfFabI inhibition (IC50 = 2.7 μM), while 7
and 8, obtained by appending the A ring of 1 at the 7-position of a
4-alkylcoumarin scaffold, showed the highest inhibitory activity
with IC50 values of 0.25 and 0.27 μM, respectively. A different
trend was observed for the 6-subtituted subset of compounds
(3−5). Derivative 3, bearing an unsubstituted coumarin subunit,
showed a strong inhibitory effect against PfFabI, with an IC50
value of 0.63 μM, while the inhibition was decreased in the
4-alkylcoumarin analogues 4 and 5 relative to 3.
With series II (9−15), we explored linking the A ring of 1 to
position 3 of several different coumarins. All compounds in this
subset displayed moderate activity toward Pf FabI (micromolar
to low-micromolar IC50 values), with the exception of 15
featuring a 3-arylcoumarin subunit, which turned out to be the
most active compound within series II (IC50 = 0.45 μM).
Derivative 9 and the 7-hydroxylated analogue 11 inhibited
PfFabI with IC50 values of 5.5 and 6.0 μM, respectively, and
potency was further reduced in the 6-hydroxy derivative (10, IC50 =
14.0 μM) and in the 5,7-dihydroxy disubstituted analogue 12
(IC50 = 44.0 μM). The introduction of a 4-methyl group in 9 and
11, affording compounds 13 and 14, respectively, caused a 2-fold
reduction in the activity compared to the parent compounds.
Conversely, a remarkable improvement in potency was obtained
by inserting a bridging phenyl group between position 3 of the
7-hydroxycoumarin scaffold and the A ring of 1 (compound 15),
lowering the IC50 value by 1 order of magnitude with respect to
the close analogue 11.
Pf FabG (IC50 > 100 μM), whereas 11−15 were able to
moderately inhibit Pf FabZ. The latter compounds are therefore
dual Pf FabI/PfFabZ inhibitors, targeting two enzymes in the
same pathway.
Antimalarial Activity against Multidrug Resistant
P. falciparum. The in vitro antiplasmodial activity of the designed
coumarin-based triclosan analogues (3−15) against multidrug
resistant P. falciparum K1 strain was determined by using the
3
established H-hypoxanthine method (see the Experimental
Section for details). As shown in Tables 1 and 2, the antimalarial
potential of compounds 3−8 and 15, endowed with the entire
diaryl ether function, proved to be substantially superior with
respect to compounds 9−14 of series II. Among series I,
derivatives 5 and 8 showed a good activity against the malaria
parasite (IC50 values of 5.6 and 5.7 μM, respectively). A common
chemical feature of these compounds is an ethyl substituent in
position 4 of the coumarin scaffold. The other compounds of the
series (i.e., 3, 6, 7, and 15) showed a moderate decrease in
potency with respect to the former ones. A further decrease in
antiplasmodial activity was observed for series II (compounds
9−14) whose IC50 values were superior to 15 μM.
The low activity of the compounds on BS parasites was not
fully unexpected. Indeed, FAS-II is only moderately active in BS
parasites, and therefore, the activities of 3−9 could also be due to
activity against other targets not belonging to the FAS-II
pathway. However, the aim of these experiments was to test the
ability of these molecules to act at an intracellular level, and
further experiments using the LS parasites will be conducted to
fully characterize the biological profile of this new series of
antimalarial compounds.
As a positive feature, all compounds showed low cytotoxicity
on mammalian cells (L6), indicating that the selected coumarin
scaffold, upon the designed functionalization, did not elicit toxic
effect. The compounds selectivity index (SI) was calculated as the
ratio of cell growth inhibition (IC50 values) for mammalian cells to
that for the selected parasite, according to the WHO/TDR
guidelines (see footnote c in the tables for detailed formula).
Crystal Structures of Pf FabI in Complex with 7 and 11.
To investigate the binding mode at an atomic level, we solved the
structures of PfFabI in complex with 7 (series I, PBD code 4IGE,
Figure 3A) and 11 (series II, PDB code 4IGF, Figure 3B), following
the protocol previously reported by Perozzo et al.19 The
corresponding crystallographic data and refinement statistics may
be found in the Supporting Information (Table S1).
Inhibition of PfFabG and PfFabZ. To analyze selectivity
with respect to other enzymes involved in parasite fatty acid
biosynthesis pathway, we tested a selection of the new molecules
(7−15) against Pf FabZ and Pf FabG, two enzymes also
belonging to the chain elongation cycle of pathway. Data are
reported in Tables 1 and 2. All compounds were inactive against
Comparison of the structures reported in this study with those
previously published19 revealed no major structural differences.
The overall protein and the active site residues were in the very
E
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Figure 4. Comparison between the predicted binding mode (carbon atoms in gray) and the experimentally determined binding mode (carbon atoms in
pink) for 7 (A) and 11 (B) in complex with Pf FabI and NADH. The RMSD calculated between the docking model and the crystal structure using non-
hydrogen atoms of 7 and 11 is 1.49 and 1.02 Å, respectively. A section of the Connolly surface of the receptor is shown in blue.
same position, highlighting a high degree of conservation
between the present and the previously reported crystallographic
structures (see Figures 1 and 2 in the Supporting Information).
In both complexes, the position of the cofactor NADH was
clearly defined by excellent electron density, showing NADH
binding in the typical extended conformation with both ribose
sugar rings in the C2′-endo conformation and the nicotinamide
group in the syn conformation.
The crystal structures of PfFabI in complex with 7 and 11
unambiguously revealed the binding mode of the inhibitors
showing strong and continuous difference density for 7 (Figure 3A)
in both monomers and for 11 in monomer B (Figure 3B).
Comparison of both structures with those of PfFabI in complex
with 1 and 219 revealed that the binding mode was very similar,
showing the same stacking interaction of ring A with the
nicotinamide ring of NADH and the same hydrogen bonding
pattern as observed in other structures, i.e., the phenolic hydroxyl
hydrogen bonded to the 2′-hydroxyl moiety of the nicotinamide
ribose and the oxygen atom of Tyr277 while van der Waals
interactions were formed via the side chains of Tyr267, Tyr277,
Pro314, Phe368, and Ile369. The ether oxygen atom of both
compounds interacted with the 2′-hydroxyl group of the
nicotinamide ribose at a distance of around 3.3 Å.
The coumarin ring systems of 7 and 11 were located in a
pocket formed by the pyrophosphate and the nicotinamide
group of NADH, the peptide backbone residues 217−231, and
the side chains of Asn218, Val222, Tyr277, Met281, Ala319, and
Ile323. Both coumarin moieties were oriented in a very similar
way as observed for the extended ring B of 2, tilted by about 10°
out of the plane found for ring B of 1. This allowed 7 to interact
with the backbone oxygen of Ala217 by H-bonding the ring
oxygen in the coumarin group. Interestingly, the methyl
substituent of the coumarin scaffold in 7 was predicted to fill
the small and hydrophobic pocket located next to position 4 of
the coumarin moiety, which is formed by the side chain of
residues of Val222, Tyr267, Met281, and Ile323, implying a small
rotation of the whole molecule toward this pocket. However, this
movement did not occur, as 7 maintained the conserved binding
mode with the methyl group only pointing to the pocket but not
filling it. This is probably due to the penalizing effect of losing the
H-bond formed between Ala217 and the coumarin group. As
observed for 2, compound 11 could also establish the same three
H-bonds through the hydroxyl group of the coumarin ring, the
side chain nitrogen of Asn218, and the main chain oxygen and
nitrogen of A219. Moreover, the ring oxygen in the coumarin
group of 11 could also interact with the backbone oxygen of
Ala217.
The docking binding mode and the pool of interactions
predicted for compounds 7 and 11 were confirmed and found to
be in good agreement with those detected in the crystal structure
of the ternary complex with Pf FabI and NADH (Figure 4A and
Figure 4B, respectively). In particular, after superimposition of
the protein backbone, we observed that 7 showed an RMSD of 1.49 Å
between the docking model and the crystal structure (Figure 4A),
while the RMSD was 1.02 Å for the docking model of 11 vs the
experimentally determined binding mode (Figure 4B).
CONCLUSIONS
■
In this study, the introduction of ring A of 1 to purposely selected
positions of a coumarin scaffold led to a new series of PfFabI
inhibitors that could be regarded as tools to explore the chemical
space of the target. Compounds of series I (3−8), together with
15, gave the best results in terms of both inhibition of PfFabI
enzyme and antiplasmodial actvity. In particular, derivatives 3, 7,
8, and 15, with submicromolar IC50 values, showed the highest
potency against Pf FabI by virtue of the well-established
capability of the diaryl ether function to interact with this
enzyme. These compounds also showed micromolar IC50 values
for inhibition of P. falciparum growth in the whole cell assays.
From the data obtained, some conclusions can be drawn: (i) the
presence of a 2-hydroxy-4-chlorophenyl-based diaryl ether
framework, as a capping fragment, plays a fundamental role in
the inhibition of both Pf FabI and parasite growth; (ii) the
coumarin scaffold is tolerated by PfFabI.
X-ray data suggested that potency toward Pf FabI resulted
from interactions between the enzyme active site and two
separate parts of the coumarin framework, such as the 2-one
function and the substituent at position 4 of the coumarin
scaffold. In particular, the coumarin-2-one function was
responsible for a penalizing orientation toward the pyrophos-
phate moiety of NADH (compounds 9−14) and was able to
interact favorably with nitrogen of Ala219 backbone and the side
chain of Asn218 (compounds 3−8). An improvement in potency
was achieved by introducing an alkyl substituent at position 4 of
the main backbone, thus providing an additional anchoring point
within the binding site.
F
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8.8 Hz, 1H), 7.75 (d, J = 2.2 Hz, 1H). MS (ESI+) m/z: 253 [M + H]+.
Exact mass: 251.9786.
These encouraging results allow us to consider this new class
of coumarin-based triclosan analogues as a promising starting
point for a drug discovery program aimed at exploring in more
depth the significance of the coumarin framework for interaction
with FabI from P. falciparum. For instance, compound 8 is a
7-Bromo-2H-chromen-2-one (19). To a mixture of 3-bromophe-
nol (1 g, 5.8 mmol) and malic acid (0.52 g, 3.9 mmol) at 0 °C was added
dropwise 98% H2SO4 (1.3 mL), and the resulting solution was heated at
120 °C for 6 h. After the mixture was cooled to room temperature,
crushed ice was added, and the precipitated solid was collected by
filtration. Purification by crystallization from ethanol gave the desired
compound 19 as a white solid (0.80 g, 80% yield), mp 121−123 °C. 1H
NMR (CDCl3, 200 MHz): δ 6.39 (d, J = 9.6 Hz, 1H), 7.11 (dd, J = 8.4 and
1.8 Hz, 1H), 7.15 (d, J = 1.8 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.55 (d, J =
9.6 Hz, 1H). MS (ESI+) m/z: 225 [M + H]+. Exact mass: 223.9473.
7-Bromo-4-methyl-2H-chromen-2-one (20). To a solution of
3-bromophenol (1 g, 5.81 mmol), ethyl 3-oxobutanoate (0.74 mL, 5.80
mmol), and ethyl alcohol (58 mL) at 0 °C was added dropwise a 70%
H2SO4 aqueous solution (0.62 mL, 11.6 mmol). The resulting solution
was stirred at room temperature overnight and then poured into crushed
ice. The solid formed was filtered and crystallized from toluene to afford
promising hit because of its Pf FabI inhibitory activity (IC50
=
0.27 μM), selectivity vs Pf FabG and PfFabZ, and antiplasmodial
activity (IC50 = 5.7 μM), which is only 4- to 5-fold less than that
of 1. Derivatives 7 and 15 also exhibited substantial antimalarial
activity. In particular, compound 15, because of its unique
structure, may represent a promising starting point for lead
optimization.
Finally, FabI enzymes are widely and intensively investigated
as potential antibacterial targets. Because of the high sequence
homology between Plasmodium and bacterial isoforms, the
inhibitors presented herein could also be relevant in the field of
antibacterial drug discovery.6
1
20 as a white solid (0.90 g, 90% yield), mp 118−120 °C. H NMR
(CDCl3, 200 MHz): δ 2.52 (s, 3H), 6.32 (s, 1H), 7.01−7.15 (m, 1H),
7.23 (s, 1H), 7.45 (dd, J = 2.1 and 8.4 Hz, 1H). MS (ESI+) m/z: 239
[M + H]+. Exact mass: 237.9629.
EXPERIMENTAL SECTION
■
Chemistry. General Procedures. Starting materials, unless
otherwise specified in the Experimental Section, were used as high-
grade commercial products. Solvents were of analytical grade. Melting
7-Bromo-4-ethyl-2H-chromen-2-one (21). Following the proce-
dure described for 18, starting from 1-(4-bromo-2-hydroxyphenyl)-
propan-1-one (2.5 g, 10.9 mmol) and methyl(triphenyl-
phosphoranylidene)acetate (5.69 g, 16.3 mmol) in benzene (15 mL),
compound 21 was obtained as colorless needles (1.5 g, 54% yield), mp
130−132 °C. 1H NMR (CDCl3, 200 MHz): δ 1.33 (t, J = 7.6 Hz, 3H),
2.80 (q, J = 7.8 Hz, 2H), 6.31 (s, 1H), 7.44−7.51 (m, 3H). MS (ESI+) m/
z: 253 [M + H]+. Exact mass: 251.9786.
3-(4-(4-Chloro-2-methoxyphenoxy)phenyl)-7-methoxy-2H-
chromen-2-one (34). Following the procedure described for 16,
starting from 1-(2-hydroxy-4-methoxyphenyl)benzaldehyde (0.4 g, 2.7
mmol) and sodium 2-(4-bromophenyl)acetate (0.68 g, 2.7 mmol),
compound 34 was obtained as a white solid (0.43 g, 48% yield), mp
184−187 °C. 1H NMR (CDCl3, 400 MHz): δ 3.90 (s, 3H), 6.86 (d, J =
2.0 Hz, 1H), 6.87 (dd, J = 2.0 and 8.4 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H),
7.50−7.59 (m, 4H), 7.77 (s, 1H). MS (ESI+) m/z: 331 [M + H]+. Exact
mass: 329.9892.
Sodium 2-(4-Chloro-2-methoxyphenoxy)acetate. A solution of
4-chloro-2-methoxyphenol (0.6 g, 3.85 mmol), 2-chloroacetic acid (0.40 g,
4.2 mmol), K2CO3 (1.16 g, 8.47 mmol), and KI (0.10 g) in acetone
(50 mL) was heated under reflux for 8 h. The solvent was removed
under reduced pressure, and the solid was suspended in CH2Cl2 and
washed with 2 N NaOH (2 × 20 mL) and then with brine (20 mL). The
organic layer was dried over anhydrous Na2SO4 and concentrated to
dryness to give the desired product (0.40 g, 90% yield), mp 126−132 °C.
The compound was then transformed into the sodium salt by adding a
10% NaOH in ethyl alcohol (40 mL) and stirring at room temperature
overnight. The solvent was removed under reduced pressure, and the
white solid was dried (0.45 g, 95% yield). 1H NMR (DMSO, 400 MHz):
δ 3.85 (s, 3H), 4.05 (s, 2H), 6.70 (d, J = 8.4 Hz, 1H), 6.80 (dd, J = 8.4 and
1.8 Hz, 1H), 6.85 (d, J = 1.8 Hz, 1H).
points were determined in open glass capillaries, using a Buchi
̈
1
apparatus, and are uncorrected. H NMR and 13C NMR spectra were
recorded on Varian Gemini spectrometers at 200, 300, or 400 MHz, and
chemical shifts are reported in parts per million (ppm δ value) relative to
the peak for tetramethylsilane (TMS) as internal standard. Standard
abbreviations indicating spin multiplicities are given as follows: s
(singlet), d (doublet), t (triplet), br (broad), q (quartet), or m
(multiplet). Mass spectra were recorded on a Waters ZQ 4000 apparatus
operating in electrospray mode (ES). Wherever analyses are only
indicated with element symbols, analytical results obtained for those
elements are within 0.4% of the theoretical values. Chromatographic
separations were performed on silica gel columns using the flash method
(Kieselgel 40, 0.040−0.063 mm, Merck). Reactions were followed by
thin layer chromatography (TLC) on precoated silica gel plates (Merck
silica gel 60 F254) and then visualized with a UV lamp. Satisfactory
elemental analyses were obtained for all new compounds, confirming
>95% purity. Compounds were named following IUPAC rules as
applied by Beilstein-Institute AutoNom (version 2.1), a PC-integrated
software package for systematic names in organic chemistry.
6-Bromo-2H-chromen-2-one (16). A mixture of 5-bromo-2-
hydroxybenzaldehyde (3.0 g, 14.9 mmol) and sodium acetate (1.22 g,
14.9 mmol) in acetic anhydride (2.82 mL, 29.8 mmol) was heated at
180 °C for 8 h. When the mixture was cooled, a 10% K2CO3 aqueous
solution was added, and the solid residue was filtered and crystallized
from ligroin to obtain compound 16 as yellowish needles (1.9 g, 56%
yield), mp 166−168 °C. 1H NMR (CDCl3, 200 MHz): δ 6.47 (d, J = 9.6
Hz, 1H), 7.22 (dd, J = 2.2 and 8.4 Hz, 1H), 7.24 (d, J = 2.2 Hz, 1H), 7.62
(d, J = 8.4 Hz, 1H), 7.66 (d, J = 9.6 Hz, 1H). MS (ESI+) m/z: 225
[M + H]+. Exact mass: 223.9473.
General Procedure for the Synthesis of 3-(4-Chloro-2-
methoxyphenoxy)-2H-chromen-2-one-Based Derivatives (28−33).
A mixture of sodium 2-(4-chloro-2-methoxyphenoxy)acetate (0.60 g,
2.36 mmol), triethylamine (0.69 mL, 4.95 mmol) in acetic anhydride
(0.60 g, 5.9 mmol), and the suitable 2-OH carbonyl compound
(1.18 mmol) was heated at 180 °C for 8 h. When the mixture was cooled,
a 10% K2CO3 aqueous solution was added, and the solution was
extracted with CH2Cl2 (3 × 20 mL). The combined organic layers were
dried over anhydrous Na2SO4 and concentrated to dryness to give a
crude product that was purified by flash chromatography on silica gel,
using a mixture of petroleum ether/ethyl acetate (80:10) as eluent.
3-(4-Chloro-2-methoxyphenoxy)-2H-chromen-2-one (28).
Starting from 2-hydroxybenzaldehyde (0.14 g, 1.18 mmol), the desired
6-Bromo-4-methyl-2H-chromen-2-one (17). Following the
procedure described for 16, starting from 1-(5-bromo-2-hydroxy-
phenyl)ethanone (4.10 g, 19.06 mmol), compound 17 was obtained as
1
colorless needles (2.20 g, 50% yield), mp 190−192 °C. H NMR
(CDCl3, 200 MHz): δ 2.43 (s, 3H), 6.33 (s, 1H), 7.20 (d, J = 8.8 Hz,
1H), 7.60 (dd, J = 2.2 and 8.8 Hz, 1H), 7.72 (d, J = 2.2 Hz, 1H). MS
(ESI+) m/z: 239 [M + H]+. Exact mass: 237.9629.
6-Bromo-4-ethyl-2H-chromen-2-one (18). A solution of 1-(5-
bromo-2-hydroxyphenyl)propan-1-one (1 g, 4.4 mmol) and methyl
(triphenylphosphoranylidene)acetate (2.3 g, 6.6 mmol), in benzene
(10 mL), was heated under reflux for 24 h. The solvent was removed
under reduced pressure and the crude product was purified by flash
column chromatography using a mixture of petroleum ether/ethyl
acetate (90:10) as eluent to afford 18 (2.1 g, 60% yield), mp 115−117 °C.
1H NMR (CDCl3, 200 MHz): δ 1.34 (t, J = 7.8 Hz, 3H), 2.79 (q, J =
7.8 Hz, 2H), 6.33 (s, 1H), 7.24 (d, J = 8.8 Hz, 1H), 7.60 (dd, J = 2.2 and
1
derivative 28 was obtained (0.26 g, 72% yield), mp 136−138 °C. H
NMR (CDCl3, 300 MHz): δ 3.80 (s, 3H), 6.40 (dd, J = 8.4 and 2.2 Hz,
1H), 6.80 (d, J = 2.2 Hz, 1H), 7.19 (d, J = 8.4 Hz, 1H), 7.20 (s, 1H),
G
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7.28−7.35 (m, 2H), 7.43−7.46 (m, 1H), 7.75 (d, J = 8.2 Hz, 1H). MS
(ESI+) m/z: 303/305 [M + H]+. Exact mass: 302.0346.
δ 1.30 (t, J = 7.8 Hz, 3H), 2.72 (q, J = 7.2 Hz, 2H), 3.83 (s, 3H), 6.31
(s, 1H), 6.92 (dd, J = 2.2 and 8.4 Hz, 1H), 6.98 (d, J = 2.2 Hz, 1H), 7.00
(d, J = 8.4 Hz, 1H), 7.09 (dd, J = 2.2 and 8.4 Hz, 1H), 7.18 (d, J = 2.2 Hz,
1H), 7.25 (d, J = 8.4 Hz, 1H). MS (ESI+) m/z: 331/333 [M + H]+. Exact
mass: 330.0557.
3-(4-Chloro-2-methoxyphenoxy)-6-methoxy-2H-chromen-2-
one (29). Starting from 2-hydroxy-5-methoxybenzaldehyde (0.36 g,
2.55 mmol), the desired derivative 29 was obtained (0.3 g, 36% yield),
1
mp 135−138 °C. H NMR (CDCl3, 300 MHz): δ 3.78 (s, 3H), 3.80
7-(4-Chloro-2-methoxyphenoxy)-2H-chromen-2-one (25).
Starting from 19 (0.6 g, 2.66 mmol), compound 25 was obtained (0.2
g, 36% yield), mp 130−132 °C. 1H NMR (CDCl3, 300 MHz): δ 3.72 (s,
3H), 7.25 (d, J = 9.6 Hz, 1H), 6.66 (d, J = 2.2 Hz, 1H), 6.79 (dd, J = 2.2
and 8.4 Hz, 1H), 6.82 (dd, J = 2.2 and 8.4 Hz, 1H), 6.95 (d, J = 2.2 Hz,
1H), 6.98 (d, J = 8.4, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.62 (d, J = 9.6 Hz,
1H). MS (ESI+) m/z: 303/305 [M + H]+. Exact mass: 302,0346.
7-(4-Chloro-2-methoxyphenoxy)-4-methyl-2H-chromen-2-
one (26). Starting from 20 (0.42 g, 1.7 mmol), compound 26 was
(s, 3H), 6.65 (d, J = 2.2 Hz, 1H), 6.80 (dd, J = 2.2 and 8.4 Hz, 1H), 7.05
(d, J = 2.2 Hz, 1H), 7.09 (dd, J = 2.2 and 8.4 Hz, 1H), 7.12 (d, J = 2.2 Hz,
1H), 7.18 (d, J = 8.4 Hz, 1H), 7.30 (s, 1H). MS (ESI+) m/z: 333/335
[M + H]+. Exact mass: 332.0452.
3-(4-Chloro-2-methoxyphenoxy)-7-methoxy-2H-chromen-2-
one (30). Starting from 2-hydroxy-4-methoxybenzaldehyde (0.58 g,
2.61 mmol), the desired derivative 30 was obtained (0.16 g, 74%, yield),
mp 125−127 °C. 1H NMR (CD3COCD3, 400 MHz): δ 3.83 (s, 3H),
3.93 (s, 3H), 6.93 (dd, J = 2.4 and 8.4 Hz, 1H), 7.08 (d, J = 2.4 Hz, 1H),
7.17 (s, 1H), 7.19 (d, J = 8.4 Hz, 1H), 7.36 (d, J = 1.8 Hz, 1H), 7.41
(dd, J = 1.8 and 8.2 Hz, 1H), 7.65 (d, J = 8.2 Hz, 1H). MS (ESI+) m/z:
333/335 [M + H]+. Exact mass: 332.0452.
3-(4-Chloro-2-methoxyphenoxy)-5,7-dimethoxy-2H-chro-
men-2-one (31). Starting from 2-hydroxy-4,6-dimethoxybenzaldehyde
(1.07 g, 5.9 mmol), the desired derivative 31 was obtained (0.32 g, 14%
yield), mp 129−130 °C. 1H NMR (CDCl3, 400 MHz): δ 3.81 (s, 3H),
3.85 (s, 3H), 3.88 (s, 3H), 6.41 (d, J = 2.2 Hz, 1H), 6.64 (d, J = 2.2 Hz,
1H), 6.90 (dd, J = 1.8 and 8.4 Hz, 1H), 7.0 (d, J = 8.4 Hz, 1H), 7.15 (d,
J = 2.2 Hz, 1H), 7.25 (s,, 1H). MS (ESI+) m/z: 363/365 [M + H]+. Exact
mass: 362.0557.
3-(4-Chloro-2-methoxyphenoxy)-4-methyl-2H-chromen-2-
one (32). Starting from 1-(2-hydroxyphenyl)ethanone (0.13 mL, 0.98
mmol), the desired derivative 32 was obtained (0.15 g, 30% yield), mp
128−130 °C. 1H NMR (CDCl3, 300 MHz): δ 2.18 (s, 3H), 3.93 (s, 3H),
6.64 (dd, J = 1.8 and 8.4 Hz, 1H), 6.68 (dd, J = 1.8 and 8.4 Hz, 1H), 6.97
(d, J = 1.8 Hz, 1H), 7.26−7.39 (m, 2H), 7.53−7.55 (m, 1H), 7.65 (d, J =
8.4 Hz, 1H). MS (ESI+) m/z: 317/319 [M + H]+. Exact mass: 316.0502.
3-(4-Chloro-2-methoxyphenoxy)-7-methoxy-4-methyl-2H-
chromen-2-one (33). Starting from 1-(2-hydroxy-4-methoxyphenyl)-
ethanone (0.14 g, 0.81 mmol), the desired derivative 33 was obtained
(0.07 g, 25% yield) mp 128−131 °C. 1H NMR (CDCl3, 300 MHz): δ
2.18 (s, 3H), 3.80 (s, 3H), 3.84 (s, 3H), 6.84 (d, J = 2.2 Hz, 1H), 6.88
(dd, J = 2.2 and 8.4 Hz, 1H), 7.06 (d, J = 2.2 Hz, 1H), 7.12 (dd, J = 2.2
and 8.4 Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H). MS
(ESI+) m/z: 347/349 [M + H]+. Exact mass: 346.0608.
General Procedure for the Ulmann-Type Synthesis of Diaryl
Ethers (22−27, 35).32 In an oven-dried three-neck flask, a mixture of
the bromocoumarin intermediate (2.5 mmol), 4-chloro-2-methoxyphe-
nol (5.0 mmol), Cs2CO3 (5.0 mmol), (CuOTf)·PhH (0.0625 mmol,
5.0 mol % Cu), ethyl acetate (0.0125 mmol, 5.0 mol %), 1-naphthoic
acid (5.0 mmol), and activated 5 Å molecular sieves (0.62 g) in toluene
(2 mL) was heated at 110 °C under N2 atmosphere for 24 h. When the
mixture was cooled, 2 N NaOH (50 mL) was added and the aqueous
layer was extracted with CH2Cl2 (3 × 50 mL). The combined organic
layers were washed with brine, dried over anhydrous Na2SO4, and
concentrated to dryness to give a crude product that was purified by flash
chromatography on silica gel using a mixture of petroleum ether/ethyl
acetate (90:10) as eluent.
1
obtained (0.3 g, 36% yield), mp 138−140 °C. H NMR (CDCl3, 300
MHz): δ 2.04 (s, 3H), 3.52 (s, 3H), 6.32 (s, 1H), 6.93 (dd, J = 2.2 and 8.4
Hz, 1H), 6.95 (d, J = 2.2 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 7.11 (dd, J =
2.2 and 8.4 Hz, 1H), 7.27 (d, J = 2.2 Hz, 1H), 7.29 (d, J = 8.4 Hz, 1H).
MS (ESI+) m/z: 317/319 [M + H]+. Exact mass: 316.0502.
7-(4-Chloro-2-methoxyphenoxy)-4-ethyl-2H-chromen-2-one
(27). Starting from 21 (0.5 g, 1.98 mmol), compound 27 was obtained
(0.35 g, 52% yield), mp 140−142 °C. 1H NMR (CDCl3, 300 MHz): δ
1.33 (t, J = 7.4 Hz, 3H), 2.80 (q, J = 7.4 Hz, 2H), 3.80 (s, 3H), 6.20 (s,
1H), 6.75 (d, J = 2.2 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.90 (dd, J = 2.2
and 8.4 Hz, 1H), 7.01 (dd, J = 2.2 and 8.4 Hz, 1H), 7.09 (d, J = 2.2 Hz,
1H), 7.15 (d, J = 8.4 Hz, 1H). MS (ESI+) m/z: 331/333 [M + H]+. Exact
mass: 330.0659.
3-(4-(4-Chloro-2-methoxyphenoxy)phenyl)-7-methoxy-2H-
chromen-2-one (35). Starting from 34 (0.33 g, 1 mmol), compound
1
35 was obtained (0.50 g, 60% yield), mp 176−179 °C. H NMR
(CDCl3, 300 MHz): δ 3.90 (s, 3H), 3.92 (s, 3H), 6.83 (d, J = 2.0 Hz,
1H), 6.92 (dd, J = 2.0 and 8.4 Hz, 1H), 7.04 (d, J = 2.2 Hz, 1H), 7.11 (dd,
J = 8.4 and 2.2 Hz, 1H), 7.19 (d, J = 8.4 Hz, 1H), 7.25 (d, J = 8.4 Hz, 2H),
7.36−7.44 (m, 3H), 7.77 (s, 1H). MS (ESI+) m/z: 409/411 [M + H]+.
Exact mass: 408.0765.
General Procedure for Methyl Ether Cleavage (3−15). A 1.0 M
solution of BBr3 in dichloromethane was slowly added to a solution
of the methoxylated derivative in dry dichloromethane maintained at
−70 °C under nitrogen atmosphere. The reaction mixture was stirred at
the same temperature for 1 h and then at room temperature for 4 h. The
mixture was cooled at 0 °C, and the reaction was quenched by adding
methanol. The solvent was removed under reduced pressure, and the
crude product was purified by flash column chromatography using a
mixture of petroleum ether/ethyl acetate (70:30) as eluent.
6-(4-Chloro-2-hydroxyphenoxy)-2H-chromen-2-one (3).
Starting from 22 (0.2 g, 0.66 mmol) and BBr3 (1.0 M, 0.86 mL),
1
compound 3 was obtained (0.18 g, 94% yield), mp 185−187 °C. H
NMR (CD3COCD3, 400 MHz): δ 6.42 (d, J = 9.6 Hz, 1H), 6.85 (dd, J =
1.8 and 8.4 Hz, 1H), 6.88 (d, J = 1.8 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H),
7.04 (d, J = 1.8 Hz, 1H), 7.20 (dd, J = 1.8 and 8.4 Hz, 1H), 7.26 (d, J = 8.4
Hz, 1H), 7.94 (d, J = 9.6 Hz, 1H). 13C NMR (CD3COCD3, 400 MHz):
δ 116.27, 118.95, 119.45, 119.53, 121.59, 122.96, 124.49, 131.67, 144.25,
145.27, 151.52, 152.64, 156.22, 161.78, 205.58. MS (ESI+) m/z:
289/291 [M + H]+. Exact mass: 288.0189.
6-(4-Chloro-2-methoxyphenoxy)-2H-chromen-2-one (22).
Starting from 16 (0.6 g, 2.66 mmol), compound 22 was obtained (0.2
g, 24% yield), mp 143−146 °C. 1H NMR (CDCl3, 400 MHz): δ 3.81 (s,
3H), 6.42 (d, J = 9.6 Hz, 1H), 6.93 (dd, J = 1.8 and 8.4 Hz, 1H), 6.99
(d, J = 1.8 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 7.22 (dd, J = 1.8 and 8.4 Hz,
1H), 7.43 (d, J = 1.8 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 9.6
Hz, 1H). MS (ESI+) m/z: 303/305 [M + H]+. Exact mass: 302.0346.
6-(4-Chloro-2-methoxyphenoxy)-4-methyl-2H-chromen-2-
one (23). Starting from 17 (0.58 g, 2.43 mmol), compound 23 was
6-(4-Chloro-2-hydroxyphenoxy)-4-methyl-2H-chromen-2-
one (4). Starting from 23 (0.3 g, 0.95 mmol) and BBr3 (1.0 M, 1.2 mL),
1
compound 4 was obtained (0.28 g, 96% yield), mp 242−244 °C. H
NMR (CD3COCD3, 400 MHz): δ 2.34 (s, 3H), 6.05 (s, 1H), 6.87 (dd,
J = 2.2 and 8.4 Hz, 1H), 7.02−7.05 (m, 2H), 7.32 (d, J = 2.2 Hz, 1H),
7.35 (dd, J = 2.2 and 8.4 Hz, 1H), 7.50 (d, J = 8.4 Hz, 1H), 8.90 (br, 1H).
13C NMR (CD3COCD3, 400 MHz): δ 24.6, 109.5, 115.4, 115.8, 116.9,
120.1, 121.0, 121.2, 127.5, 128.3, 141.4, 143.9, 147.5, 152.8, 155.0, 162.0.
MS (ESI+) m/z: 303/305 [M + H]+. Exact mass: 302.0346.
1
obtained (0.3 g, 39% yield), mp 120−122 °C. H NMR (CDCl3, 300
6-(4-Chloro-2-hydroxyphenoxy)-4-ethyl-2H-chromen-2-one
(5). Starting from 24 (0.15 g, 0.50 mmol) and BBr3 (1.0 M, 0.65 mL),
MHz): δ 2.37 (s, 3H), 3.78 (s, 3H), 6.12 (s, 1H), 6.93 (d, J = 2.2 Hz,
1H), 6.95 (dd, J = 2.2 and 8.4 Hz, 1H), 7.04 (d, J = 2.2 Hz, 1H), 7.11 (dd,
J = 2.2 and 8.4 Hz, 1H), 7.19 (d, J = 8.4 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H).
MS (ESI+) m/z: 317/319 [M + H]+. Exact mass: 316.0502.
1
compound 5 was obtained (0.14 g, 96% yield), mp 202−204 °C. H
NMR (CD3COCD3, 400 MHz): δ1.33 (t, J = 7.6 Hz, 3H), 2.90 (q, J =
7.6 Hz, 2H), 6.17 (s, 1H), 6.90 (dd, J = 2.0 and 8.4 Hz, 1H), 7.04 (d, J =
2.0 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H), 7.20 (dd, J = 2.0 and 8.4 Hz, 1H),
7.31 (d, J = 2.0 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 8.83 (br, 1H). 13C
6-(4-Chloro-2-methoxyphenoxy)-4-ethyl-2H-chromen-2-one
(24). Starting from 18 (0.3 g, 1.2 mmol), compound 24 was obtained
1
(0.16 g, 40% yield), mp 110−112 °C. H NMR (CDCl3, 300 MHz):
H
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NMR (CD3COCD3, 400 MHz): δ 11.1, 32.3, 109.7, 115.3, 115.9, 116.7,
120.3, 121.2, 121.5, 127.7, 128,5, 141.5, 144.0, 147.5, 152.7, 155.1, 162.5.
MS (ESI+) m/z: 317/319 [M + H]+. Exact mass: 316.0502.
125.68, 128.04, 128.98, 131.56, 140.25, 144.77, 149.56, 155.12, 160.21,
204.45. MS (ESI+) m/z: 321/323 [M + H]+. Exact mass: 320.0088.
3-(4-Chloro-2-hydroxyphenoxy)-4-methyl-2H-chromen-2-
one (13). Starting from 32 (0.15 g, 0.41 mmol) and BBr3 (1.0 M,
0.53 mL), compound 13 was obtained (0.11 g, 93% yield), mp 175−178 °C.
1H NMR (CD3COCD3, 400 MHz): δ 2.59 (s, 3H), 6.93 (dd, J = 2.2 and 8.4
7-(4-Chloro-2-hydroxyphenoxy)-2H-chromen-2-one (6).
Starting from 25 (0.20 g, 0.66 mmol) and BBr3 (1.0 M, 0.85 mL),
1
compound 6 was obtained (0.16 g, 84% yield), mp 207−209 °C. H
NMR (CD3COCD3, 400 MHz): δ 6.26 (d, J = 9.6 Hz, 1H), 6.77 (d, J =
2.2 Hz, 1H), 6.88 (dd, J = 2.2 and 8.4 Hz, 1H), 6.96 (dd, J = 2.2 and 8.4
Hz, 1H), 7.11 (d, J = 2.2 Hz, 1H), 7.15 (d, J = 8.4 Hz, 1H), 7.64 (d, J =
8.4 Hz, 1H), 7.94 (d, J = 9.6 Hz, 1H). 13C NMR (CD3COCD3, 400
MHz): δ 116.29, 119.65, 119.95, 120.33, 124.19, 124.96, 125.67, 133.67,
146.28, 147.28, 151.52, 152.64, 156.22, 161.77, 205.85. MS (ESI+) m/z:
289/291 [M + H]+. Exact mass: 288.0189.
7-(4-Chloro-2-hydroxyphenoxy)-4-methyl-2H-chromen-2-
one (7). Starting from 26 (0.30 g, 0. 95 mmol) and BBr3 (1.0 M, 1.2
mL), compound 7 was obtained (0.27 g, 95% yield), mp 205−208 °C.
1H NMR (CD3COCD3, 400 MHz): δ 2.40 (s, 3H), 6.20 (s, 1H), 6.79 (d,
J = 2.2 Hz, 1H), 6.93 (dd, J = 8.4 and 2.2 Hz, 1H), 6.97 (dd, J = 8.4 and
2.2 Hz, 1H), 7.12 (d, J = 2.2 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 7.73
(d, J = 8.4 Hz, 1H), 8.50 (br, 1H). 13C NMR (CD3COCD3, 400 MHz):
δ 18.50, 104.27, 113.20, 115.78, 118.40, 121.10, 124.32, 127.31, 131.42,
141.74, 151.11, 153.44, 155.90, 160.53, 161.84, 205.70. MS (ESI+) m/z:
303/305 [M + H]+. Exact mass: 302.0346.
Hz, 1H), 7.06 (d, J = 2.2 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.24−7.29 (m,
1H), 7.31 (dd, J = 8.4 and 0.8 Hz, 1H), 7.44−7.50 (m, 1H), 7.66 (dd, J = 8.0
and 1.8 Hz, 1H). 13C NMR (CD3COCD3, 400 MHz): δ 27.12, 111.45,
118.54, 122.68, 125.41, 125.18, 127.54, 128.29, 128.98, 131.52, 140.46,
146.05, 149.11, 154.85, 161.32, 205.32. MS (ESI+) m/z: 303/305 [M + H]+.
Exact mass: 302.0346.
3-(4-Chloro-2-hydroxyphenoxy)-7-hydroxy-4-methyl-2H-
chromen-2-one (14). Starting from 33 (0.06 g, 0.2 mmol) and BBr3
(1.0 M, 0.26 mL), compound 14 was obtained (0.04 g, 84% yield), mp
191−193 °C. 1H NMR (CD3COCD3, 400 MHz): δ 2.33 (s, 3H), 6.93
(dd, J = 2.2 and 8.4 Hz, 1H), 7.04 (d, J = 2.2 Hz, 1H), 7.24 (d, J = 8.4 Hz,
1H), 7.26−7.33 (m, 2H), 7.52 (d, J = 8.4 1H). 13C NMR (CD3COCD3,
400 MHz): δ 27.25, 110.12, 116.57, 124.71, 125.06, 126.47, 127.99,
129.09, 129.67, 130.12, 144.24, 146.05, 149.24, 153.45, 163.35, 205.32.
MS (ESI+) m/z: 319/321 [M + H]+. Exact mass: 318.0295.
3-(4-(4-Chloro-2-hydroxyphenoxy)phenyl)-7-hydroxy-2H-
chromen-2-one (15). Starting from 35 (0.50 g, 1.2 mmol) and BBr3
(1.0 M, 3.1 mL), compound 15 was obtained (0.4 g, 87% yield), mp
104−106 °C. 1H NMR (CD3COCD3, 400 MHz): δ 6.82 (d, J = 2.0 Hz,
1H), 6.88 (dd, J = 2.0 and 8.4 Hz, 1H), 6.94 (d, J = 2.2 Hz, 1H), 6.98 (dd,
J = 2.2 and 8.4 Hz, 1H), 7.12−7.17 (m,2H), 7.55 (d, J = 8.4 Hz, 2H),
7.75 (d, J = 8.4 Hz, 2H), 8.01 (s, 1H). 13C NMR (CD3COCD3, 400
MHz): δ 103.02, 113.04, 113.84, 118.25, 118.31, 122.95,123.01, 123.68,
124.68, 125.12, 126.55, 127.11, 128.98, 130.02, 137.55, 141.75, 147.52,
151.62, 156.11, 160.42, 204.02. MS (ESI+) m/z: 381/383 [M + H]+.
Exact mass: 380.0452.
Docking Models Building. The 3D coordinates of PfFabI were
retrieved from the Protein Data Bank (in complex with Triclosan; PDB
code 1nhg). The 3D structure of coumarin ligands was built, and the
geometry was optimized using the molecular modeling suite of
programs Sybyl 7.3 (Tripos Inc., St. Louis, MO). Molecular docking
was carried out using the default settings parameters of Gold, version
5.0.1.33 The binding site definition included the pyridine nitrogen in the
nicotinamide moiety of the NADH cofactor and every residue within
20 Å. Preliminary docking calculations on the triclosan/Pf FabI complex
were performed using both GoldScore and ChemScore fitness
functions. The ChemScore function allowed the crystallographic
triclosan/Pf FabI binding mode to be reproduced and was therefore
used to drive and rank the genetic algorithm search for the coumarin
analogues of triclosan. One-hundred poses were generated for each
ligand, and the docking outcomes were then submitted to cluster
analysis by means of the AClAP program.34,35
7-(4-Chloro-2-hydroxyphenoxy)-4-ethyl-2H-chromen-2-one
(8). Starting from 27 (0.30 g, 1.0 mmol) and BBr3 (1.0 M, 1.5 mL),
1
compound 8 was obtained (0.28 g, 95% yield), mp 195−198 °C. H
NMR (CD3COCD3, 400 MHz): δ 1.33 (t, J = 7.6 Hz, 3H), 2.87 (q, J =
7.6 Hz, 2H), 6.17 (s, 1H), 6.79 (d, J = 1.8 Hz, 1H), 6.94 (dd, J = 1.8 and
8.4 Hz, 1H), 6.98 (dd, J = 1.8 and 8.4 Hz, 1H), 7.09 (d, J = 1.8 Hz, 1H),
7.13 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 8.4 Hz, 1H), 9.32 (br, 1H). 13C
NMR (CD3COCD3, 400 MHz) δ: 10.85, 23.39, 102.61, 109.49, 111.74,
113.15, 116.63, 119.21, 122.61, 125.11, 129.65, 149.49, 154.29, 156.65,
159.11, 159.98, 202.56. MS (ESI+) m/z: 317/319 [M + H]+. Exact mass:
316.0502.
3-(4-Chloro-2-hydroxyphenoxy)-2H-chromen-2-one (9).
Starting from 28 (0.25 g, 0.82 mmol) and BBr3 (1.0 M, 1.1 mL),
1
compound 9 was obtained (0.22 g, 93% yield), mp 201−204 °C. H
NMR (CD3COCD3, 400 MHz): δ 6.93 (dd, J = 2.2 and 8.4 Hz, 1H),
7.08 (d, J = 2.2 Hz, 1H), 7.17 (s, 1H), 7.19 (d, J = 8.4 Hz, 1H), 7.29−7.31
(m, 1H), 7.34 (dd, J = 1.8 and 8.0 Hz, 1H), 7.48−7.52 (m, 1H), 7.59 (dd,
J = 1.2 and 8.0 Hz, 1H). 13C NMR (CD3COCD3, 400 MHz): δ 116.29,
119.65, 120.14, 122.48, 124.65, 124.68, 127.43, 129.39, 130.47, 142.62,
144.62, 150.64, 156.18, 161.77, 205.85. MS (ESI+) m/z: 289/291
[M + H]+. Exact mass: 288.0189.
3-(4-Chloro-2-hydroxyphenoxy)-6-hydroxy-2H-chromen-2-
one (10). Starting from 29 (0.35 g, 1.05 mmol) and BBr3 (1.0 M, 2.7
mL), compound 10 was obtained (0.30 g, 94% yield), mp 185−188 °C.
1H NMR (CD3COCD3, 400 MHz): δ 6.91 (dd, J = 2.2 and 8.4 Hz, 1H),
Protein Purification and Inhibition Assay. All enzymes used in
this study were expressed and purified exactly as outlined before,36 and
the corresponding inhibition assays were carried out without changes
according to published procedures.36−38
6.92 (d, J = 2.2 Hz, 1H), 6.96−6.99 (m, 2H), 7.03 (s, 1H), 7.05 (d, J = 2.4
1H), 7.15 (d, J = 8.4 Hz, 1H). 13C NMR (CD3COCD3, 400 MHz): δ
116.31, 118.05, 121.11, 122.68, 125.65, 125.68, 127.42, 128.53, 130.66,
140.08, 144.62, 149.92, 155.31, 161.88, 205.92. MS (ESI+) m/z: 305/
307 [M + H]+. Exact mass: 304.0139.
3-(4-Chloro-2-hydroxyphenoxy)-7-hydroxy-2H-chromen-2-
one (11). Starting from 30 (0.15 g, 0.45 mmol) and BBr3 (1.0 M, 1.2
mL), compound 11 was obtained (0.10 g, 73% yield), mp 195−196 °C.
1H NMR (CD3COCD3, 400 MHz): δ 6.69 (d, J = 1.8 Hz, 1H), 6.72 (s,
1H), 6.77 (dd, J = 1.8 and 8.4 Hz, 1H), 6.86−6.86 (m, 1H), 6.89 (dd, J =
1.8 and 8.4 Hz, 1H), 7.95 (dd, J = 1.8 and 8.4 Hz, 1H), 7.05 (d, J = 8.4
Hz, 1H). 13C NMR (CD3COCD3, 400 MHz): δ 116.32, 118.55, 121.42,
122.57, 125.42, 125.56, 127.12, 128.11, 131.05, 139.58, 144.52, 149.14,
155.57, 161.42, 205.54. MS (ESI+) m/z: 305/307 [M + H]+. Exact mass:
304.0139.
3-(4-Chloro-2-hydroxyphenoxy)-5,7-dihydroxy-2H-chro-
men-2-one (12). Starting from 31 (0.30 g, 0.82 mmol) and BBr3 (1.0
M, 3.2 mL), compound 12 was obtained (0.20 g, 76% yield), mp 185−
188 °C. 1H NMR (CD3COCD3, 400 MHz): δ 6.98 (dd, J = 2.2 and 8.4
Hz, 1H), 7.00 (d, J = 2.2 Hz, 1H), 7.15 (d, J = 8.4 Hz, 1H), 7.25 (s, 1H),
7.35 (d, J = 1.8 Hz, 1H), 7.59 (d, J = 1.8 Hz, 1H). 13C NMR
(CD3COCD3, 400 MHz): δ 114.22, 116.98, 120.48, 121.55, 124.98,
Crystallization, Data Collection, and Structure Solution.
Pf FabI suitable for crystallization was expressed and purified as
published elsewhere.19 Crystals of ternary complexes with enzyme,
NADH, and compounds 7 and 11 were prepared by soaking. To this
end, the inhibitors were dissolved in acetonitrile and the solution was
directly added to the drops containing crystals of binary complexes of
Pf FabI and NADH, and the resulting crystals were incubated for a week.
Crystals of PfFabI in complex with NADH were prepared using the
hanging drop vapor diffusion method as described recently.19
Data sets of two crystals of ternary Pf FabI complexes were collected
at 100 K as a consecutive series of 0.5° rotation images at the protein
beamline PXIII (X06DA) of the Swiss Light Source (SLS) in Villigen,
Switzerland, using a MAR225 CCD detector. The data sets were
processed with XDS,39 and the corresponding data set statistics are given
in Supporting Information Table S1.
Both crystals of PfFabI in complex with NADH and inhibitors 7 and
11, respectively, were isomorphous to each other and to the crystals of
PfFabI in complex with NAD and triclosan (PDB code 1NHG) or with
I
dx.doi.org/10.1021/jm400637m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
NADH and a triclosan analogue (PDB code 1NNU).19 For initial
phasing, it was therefore sufficient to properly place the protein chains A
and B from PDB entry 1NNU into the unit cells of the ternary PfFabI
complexes of this study by rigid-body refinement using the program
PHENIX.40 The crystal structure of PfFabI in complex with NADH and
compound 7 showed clear electron density for the cofactor and the
inhibitor at both active sites of chains A and B. Subsequent iterative
rounds of model building with COOT41 and restrained maximum-
likelihood refinement with BUSTER42 and PHENIX40 led to the final
model (R-factor of 0.16, free R-factor of 0.20) with very good
stereochemistry (Table S1). The crystal structure of PfFabI in complex
with NADH and 11 showed clear electron density maps for the bound
cofactor and inhibitor at the active site of chain B. In contrast, in chain A
only incomplete electron density of apparently partially bound cofactor
and inhibitor was visible. Thus, at active site A, both NADH and
compound 11 were tentatively modeled and refined with occupancies of
50%. The difference electron density map of the refined model still
shows some uninterpretable positive peaks in the vicinity of NADH and
11, presumably due to crystallization buffer components bound in the
absence of cofactor and inhibitor. Iterative rounds of model building
with COOT41 and restrained maximum-likelihood refinement with
BUSTER42 and PHENIX40 provided the final model (R-factor of 0.15,
free R-factor of 0.20) with very good stereochemistry (Table S1).
In Vitro Assay for P. falciparum K1. In vitro activity against
erythrocytic stages of P. falciparum was determined using the
[3H]hypoxanthine incorporation described recently.43 The compounds
and the standard drug chloroquine were tested against the chloroquine-
and pyrimethamine-resistant K1 strain. IC50 values were calculated from
graphically plotted dose−response curves.
ACP, acyl carrier protein; FabG, β-ketoacyl-ACP-reductase;
FabZ, β-hydroxacyl-ACP-dehydratase; FabI, enoyl-ACP-reductase
REFERENCES
■
(1) WHO. World Malaria Report 2010; World Health Organization:
Geneva, 2010; http://www.who.int/malaria/world_malaria_report_
2010/en/index.html.
(2) Noedl, H.; Se, Y.; Schaecher, K.; Smith, B. L.; Socheat, D.; Fukuda,
M. M. Evidence of artemisinin-resistant malaria in Western Cambodia.
N. Engl. J. Med. 2008, 359, 2619−2620.
(3) Ekland, E. H.; Fidock, D. A. In vitro evaluations of antimalarial
drugs and their relevance to clinical outcomes. Int. J. Parasitol. 2008, 38,
743−747.
(4) Greenwood, B. M.; Fidock, D. A.; Kyle, D. E.; Kappe, S. H.; Alonso,
P. L.; Collins, F. H.; Duffy, P. E. Malaria: progress, perils, and prospects
for eradication. J. Clin. Invest. 2008, 118, 1266−1276.
(5) Amino, R.; Giovannini, D.; Thiberge, S.; Gueirard, P.; Boisson, B.;
Dubremetz, J. F.; Prevost, M. C.; Ishino, T.; Yuda, M.; Menard, R. Host
cell traversal is important for progression of the malaria parasite through
the dermis to the liver. Cell Host Microbe 2008, 3, 88−96.
(6) Lu, H.; Tonge, P. J. Inhibitors of FabI, an enzyme drug target in the
bacterial fatty acid biosynthesis pathway. Acc. Chem. Res. 2008, 41, 11−
20.
(7) Hemmer, C. J.; Holst, F. G.; Kern, P.; Chiwakata, C. B.; Dietrich,
M.; Reisinger, E. C. Stronger host response per parasitized erythrocyte
in Plasmodium vivax or ovale than in Plasmodium falciparum malaria.
Trop. Med. Int. Health 2006, 11, 817−823.
(8) Baird, J. K.; Hoffman, S. L. Primaquine therapy for malaria. Clin.
Infect. Dis. 2004, 39, 1336−1345.
(9) Mazier, D.; Renia, L.; Snounou, G. A pre-emptive strike against
malaria’s stealthy hepatic forms. Nat. Rev. Drug Discovery 2009, 8, 854−
864.
(10) Tarun, A. S.; Vaughan, A. M.; Kappe, S. H. Redefining the role of
de novo fatty acid synthesis in Plasmodium parasites. Trends Parasitol.
2009, 25, 545−550.
(11) Vaughan, A. M.; O’Neill, M. T.; Tarun, A. S.; Camargo, N.;
Phuong, T. M.; Aly, A. S.; Cowman, A. F.; Kappe, S. H. Type II fatty acid
synthesis is essential only for malaria parasite late liver stage
development. Cell. Microbiol. 2009, 11, 506−520.
(12) Yu, M.; Kumar, T. R.; Nkrumah, L. J.; Coppi, A.; Retzlaff, S.; Li, C.
D.; Kelly, B. J.; Moura, P. A.; Lakshmanan, V.; Freundlich, J. S.;
Valderramos, J. C.; Vilcheze, C.; Siedner, M.; Tsai, J. H.; Falkard, B.;
Sidhu, A. B.; Purcell, L. A.; Gratraud, P.; Kremer, L.; Waters, A. P.;
Schiehser, G.; Jacobus, D. P.; Janse, C. J.; Ager, A.; Jacobs, W. R., Jr.;
Sacchettini, J. C.; Heussler, V.; Sinnis, P.; Fidock, D. A. The fatty acid
biosynthesis enzyme FabI plays a key role in the development of liver-
stage malarial parasites. Cell Host Microbe 2008, 4, 567−578.
(13) Ralph, S. A.; van Dooren, G. G.; Waller, R. F.; Crawford, M. J.;
Fraunholz, M. J.; Foth, B. J.; Tonkin, C. J.; Roos, D. S.; McFadden, G. I.
Tropical infectious diseases: metabolic maps and functions of the
Plasmodium falciparum apicoplast. Nat. Rev. Microbiol. 2004, 2, 203−
216.
ASSOCIATED CONTENT
* Supporting Information
■
S
Crystallographic data, elemental analysis results, crystal
structures of PfFabI in complex with compounds 7 and 11.
This material is available free of charge via the Internet at http://
pubs.acs.org.
Accession Codes
The coordinates for PfFabI in complex with 7 and those for
PfFabI in complex with 11, along with the observed structure
factors, have been deposited with the Protein Data Bank with
entry codes 4IGE and 4IGF, respectively.
AUTHOR INFORMATION
Corresponding Authors
*F.B: phone, +39-051-2099732; fax, +39-051-2099734; e-mail,
federica.belluti@unibo.it.
■
*R.P.: phone, +41-22-3793371; fax, +41-22-3793360; e-mail,
remo.perozzo@unige.ch.
Author Contributions
∞F.B. and R.P. contributed equally.
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of the
manuscript.
(14) Fichera, M. E.; Roos, D. S. A plastid organelle as a drug target in
apicomplexan parasites. Nature 1997, 390, 407−409.
(15) Colizzi, F.; Recanatini, M.; Cavalli, A. Mechanical features of
Plasmodium falciparum acyl carrier protein in the delivery of substrates.
J.Chem. Inf. Model. 2008, 48, 2289−2293.
Notes
The authors declare no competing financial interest.
(16) Zhang, Y. M.; Lu, Y. J.; Rock, C. O. The reductase steps of the type
II fatty acid synthase as antimicrobial targets. Lipids 2004, 39, 1055−
1060.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the excellent support of the beamline
scientists of the X06DA and X06SA beamlines at the SLS,
Villigen, Switzerland. The University of Bologna, Italy, is
gratefully acknowledged for financial support.
(17) Surolia, A.; Ramya, T. N.; Ramya, V.; Surolia, N. “FAS”t inhibition
of malaria. Biochem. J. 2004, 383, 401−412.
(18) Waller, R. F.; Ralph, S. A.; Reed, M. B.; Su, V.; Douglas, J. D.;
Minnikin, D. E.; Cowman, A. F.; Besra, G. S.; McFadden, G. I. A type II
pathway for fatty acid biosynthesis presents drug targets in Plasmodium
falciparum. Antimicrob. Agents Chemother. 2003, 47, 297−301.
(19) Perozzo, R.; Kuo, M.; Sidhu, A. S.; Valiyaveettil, J. T.; Bittman, R.;
Jacobs, W. R., Jr.; Fidock, D. A.; Sacchettini, J. C. Structural elucidation
ABBREVIATIONS USED
LS, liver stage; BS, blood stage; FAS-II, type II fatty acid synthase;
PfFAS-II, Plasmodium falciparum type II fatty acid synthase;
■
J
dx.doi.org/10.1021/jm400637m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
of the specificity of the antibacterial agent triclosan for malarial enoyl
acyl carrier protein reductase. J. Biol. Chem. 2002, 277, 13106−13114.
(20) Bhargava, H. N.; Leonard, P. A. Triclosan: applications and safety.
Am. J. Infect. Control 1996, 24, 209−218.
(21) Heath, R. J. Bacterial fatty-acid biosynthesis: an antibacterial drug
target waiting to be exploited. Drug Discovery Today 2001, 6, 715.
(22) McMurry, L. M.; McDermott, P. F.; Levy, S. B. Genetic evidence
that InhA of Mycobacterium smegmatis is a target for triclosan.
Antimicrob. Agents Chemother. 1999, 43, 711−713.
(23) Heath, R. J.; Li, J.; Roland, G. E.; Rock, C. O. Inhibition of the
Staphylococcus aureus NADPH-dependent enoyl-acyl carrier protein
reductase by triclosan and hexachlorophene. J. Biol. Chem. 2000, 275,
4654−4659.
(24) Surolia, N.; Surolia, A. Triclosan offers protection against blood
stages of malaria by inhibiting enoyl-ACP reductase of Plasmodium
falciparum. Nat. Med. 2001, 7, 167−173.
(25) McLeod, R.; Muench, S. P.; Rafferty, J. B.; Kyle, D. E.; Mui, E. J.;
Kirisits, M. J.; Mack, D. G.; Roberts, C. W.; Samuel, B. U.; Lyons, R. E.;
Dorris, M.; Milhous, W. K.; Rice, D. W. Triclosan inhibits the growth of
Plasmodium falciparum and Toxoplasma gondii by inhibition of
apicomplexan Fab I. Int. J. Parasitol. 2001, 31, 109−113.
(26) Chhibber, M.; Kumar, G.; Parasuraman, P.; Ramya, T. N.; Surolia,
N.; Surolia, A. Novel diphenyl ethers: design, docking studies, synthesis
and inhibition of enoyl ACP reductase of Plasmodium falciparum and
Escherichia coli. Bioorg. Med. Chem. 2006, 14, 8086−8098.
(27) Freundlich, J. S.; Anderson, J. W.; Sarantakis, D.; Shieh, H. M.; Yu,
M.; Valderramos, J. C.; Lucumi, E.; Kuo, M.; Jacobs, W. R., Jr.; Fidock,
D. A.; Schiehser, G. A.; Jacobus, D. P.; Sacchettini, J. C. Synthesis,
biological activity, and X-ray crystal structural analysis of diaryl ether
inhibitors of malarial enoyl acyl carrier protein reductase. Part 1: 4′-
Substituted triclosan derivatives. Bioorg. Med. Chem. Lett. 2005, 15,
5247−5252.
(38) Tasdemir, D.; Brun, R.; Perozzo, R.; Donmez, A. A. Evaluation of
̈
antiprotozoal and plasmodial enoyl-ACP reductase inhibition potential
of turkish medicinal plants. Phytother. Res. 2005, 19, 162−166.
(39) Kabsch, W. Automatic processing of rotation diffraction data from
crystals of initially unknown symmetry and cell constants. J. Appl.
Crystallogr. 1993, 26, 795−800.
(40) Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis, I.
W.; Echols, N.; Headd, J. J.; Hung, L. W.; Kapral, G. J.; Grosse-
Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R. J.;
Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H.
PHENIX: a comprehensive Python-based system for macromolecular
structure solution. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66,
213−221.
(41) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and
development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010,
66, 486−501.
(42) Bricogne, G.; Blanc, E.; Brandl, M.; Flensburg, C.; Keller, P.;
Paciorek, W.; Roversi, P.; Sharff, A.; Smart, O. S.; Vonrhein, C.;
Womack, T. O. BUSTER, version 2.11.2; Global Phasing Ltd.:
Cambridge, U.K., 2011.
(43) Tasdemir, D.; Sanabria, D.; Lauinger, I. L.; Tarun, A.; Herman, R.;
Perozzo, R.; Zloh, M.; Kappe, S. H.; Brun, R.; Carballeira, N. M. 2-
Hexadecynoic acid inhibits plasmodial FAS-II enzymes and arrests
erythrocytic and liver stage Plasmodium infections. Bioorg. Med. Chem.
2010, 18, 7475−7485.
(28) Freundlich, J. S.; Yu, M.; Lucumi, E.; Kuo, M.; Tsai, H. C.;
Valderramos, J. C.; Karagyozov, L.; Jacobs, W. R., Jr.; Schiehser, G. A.;
Fidock, D. A.; Jacobus, D. P.; Sacchettini, J. C. Synthesis and biological
activity of diaryl ether inhibitors of malarial enoyl acyl carrier protein
reductase. Part 2: 2′-Substituted triclosan derivatives. Bioorg. Med. Chem.
Lett. 2006, 16, 2163−2169.
(29) Borges, F.; Roleira, F.; Milhazes, N.; Santana, L.; Uriarte, E.
Simple coumarins and analogues in medicinal chemistry: occurrence,
synthesis and biological activity. Curr. Med. Chem. 2005, 12, 887−916.
(30) DeSimone, R. W.; Currie, K. S.; Mitchell, S. A.; Darrow, J. W.;
Pippin, D. A. Privileged structures: applications in drug discovery. Comb.
Chem. High Throughput Screening 2004, 7, 473−494.
(31) Meyer, T. E.; Cusanovich, M. A. Discovery and characterization of
electron transfer proteins in the photosynthetic bacteria. Photosynth. Res.
2003, 76, 111−126.
(32) Cristau, H.-J.; Cellier, P. P.; Hamada, S.; Spindler, J.-F.; Taillefer,
M. A general and mild Ullmann-type synthesis of diaryl ethers. Org. Lett.
2004, 6, 913−916.
(33) Jones, G.; Willett, P.; Glen, R. C. Molecular recognition of
receptor sites using a genetic algorithm with a description of desolvation.
J. Mol. Biol. 1995, 245, 43−53.
(34) Bottegoni, G.; Cavalli, A.; Recanatini, M. A comparative study on
the application of hierarchical-agglomerative clustering approaches to
organize outputs of reiterated docking runs. J. Chem. Inf. Model. 2006,
46, 852−862.
(35) Bottegoni, G.; Rocchia, W.; Recanatini, M.; Cavalli, A. AClAP,
autonomous hierarchical agglomerative cluster analysis based protocol
to partition conformational datasets. Bioinformatics 2006, 22, e58−e65.
(36) Tasdemir, D.; Lack, G.; Brun, R.; Ruedi, P.; Scapozza, L.; Perozzo,
R. Inhibition of Plasmodium falciparum fatty acid biosynthesis:
evaluation of FabG, FabZ, and FabI as drug targets for flavonoids. J.
Med. Chem. 2006, 49, 3345−3353.
(37) Kirmizibekmez, H.; Calis, I.; Perozzo, R.; Brun, R.; Donmez, A. A.;
Linden, A.; Ruedi, P.; Tasdemir, D. Inhibiting activities of the secondary
metabolites of Phlomis brunneogaleata against parasitic protozoa and
plasmodial enoyl-ACP reductase, a crucial enzyme in fatty acid
biosynthesis. Planta Med. 2004, 70, 711−717.
K
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