Design and synthesis of chalcone derivatives as inhibitors of the ferredoxin - Ferredoxin-NADP+ reductase interaction of Plasmodium falciparum: Pursuing new antimalarial agents

Article abstract of DOI:10.3390/molecules191221473

Some chalcones have been designed and synthesized using Claisen-Schmidt reactions as inhibitors of the ferredoxin and ferredoxin-NADP⁺ reductase interaction to pursue a new selective antimalaria agent. The synthesized compounds exhibited inhibition interactions between PfFd-PfFNR in the range of 10.94%-50%. The three strongest inhibition activities were shown by (E)-1-(4-aminophenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (50%), (E)-1-(4-aminophenyl)-3-(2,4-dimethoxyphenyl)prop-2-en-1-one (38.16%), and (E)-1-(4-aminophenyl)-3-(2,3-dimethoxyphenyl)prop-2-en-1-one (31.58%). From the docking experiments we established that the amino group of the methoxyamino chlacone derivatives plays an important role in the inhibition activity by electrostatic interaction through salt bridges and that it forms more stable and better affinity complexes with FNR than with Fd.

Full text of DOI:10.3390/molecules191221473

Molecules 2014, 19, 21473-21488; doi:10.3390/molecules191221473
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Design and Synthesis of Chalcone Derivatives as Inhibitors of
+
the Ferredoxin — Ferredoxin-NADP Reductase Interaction of
Plasmodium falciparum: Pursuing New Antimalarial Agents
Hery Suwito 1,2,*, Jumina , Mustofa , Pratiwi Pudjiastuti , Much Zaenal Fanani ,
1
3
2
4
5
5
5
5
Yoko Kimata-Ariga , Ritsuko Katahira , Toru Kawakami , Toshimichi Fujiwara ,
5
6
2,4
Toshiharu Hase , Hasnah Mohd Sirat and Ni Nyoman Tri Puspaningsih
1
Department of Chemistry, Faculty of Science and Mathematics, University of Gajah Mada,
Jogjakarta 55281, Indonesia
2
3
Department of Chemistry, Faculty of Science and Technology, Airlangga University,
Surabaya 60115, Indonesia
Department of Pharmacology and Therapy, Faculty of Medicine, University of Gajah Mada,
Jogjakarta 55281, Indonesia
4
5
6
Institute of Tropical Disease, Airlangga University, Surabaya 60115, Indonesia
Institute of Protein Research, Osaka University, 3-2 Yamadoaka, Suita-Shi, Osaka 656-0871 Japan
Department of Chemistry, Faculty of Science, University Technology Malaya,
Johor Bahru 81310, Malaysia
*
Author to whom correspondence should be addressed; E-Mail: herys08032002@yahoo.com;
Tel./Fax: +62-31-592-2427.
External Editor: Derek J. McPhee
Received: 10 September 2014; in revised form: 13 November 2014 / Accepted: 11 December 2014 /
Published: 19 December 2014
Abstract: Some chalcones have been designed and synthesized using Claisen-Schmidt
+
reactions as inhibitors of the ferredoxin and ferredoxin-NADP reductase interaction to
pursue a new selective antimalaria agent. The synthesized compounds exhibited inhibition
interactions between PfFd-PfFNR in the range of 10.94%–50%. The three strongest inhibition
activities were shown by (E)-1-(4-aminophenyl)-3-(4-methoxyphenyl)prop-2-en-1-one
(
(
50%), (E)-1-(4-aminophenyl)-3-(2,4-dimethoxyphenyl)prop-2-en-1-one (38.16%), and
E)-1-(4-aminophenyl)-3-(2,3-dimethoxyphenyl)prop-2-en-1-one (31.58%). From the
docking experiments we established that the amino group of the methoxyamino chlacone
derivatives plays an important role in the inhibition activity by electrostatic interaction

Molecules 2014, 19
21474
through salt bridges and that it forms more stable and better affinity complexes with FNR
than with Fd.
Keywords: methoxyamino chalcones; antimalarial; PfFd-PfFNR inhibitor
1
. Introduction
Malaria is considered a global health problem. In year 2012, there were an estimated 207 million
malaria cases and the disease was responsible for 627,000 deaths [1]. Although numerous antimalarial
drugs have been used, their applications are limited due to toxicity and resistance [2]. High rates of
resistance of Plasmodium falciparum to sulfadoxine-pyrimethamin (SP) and chloroquine have been
identified in several endemic areas in Indonesia [3]. Mutations in the pfdhfr and pfdhps genes of
Plasmodium falciparum isolated from Banjar district, South Kalimantan Province, Indonesia associated
with the SP resistance has recently been reported [4]. Resistance to Artemisinin-based Combination
Therapies (ACTs) has also already been reported in four countries of the greater Mekong subregion:
Cambodia, Myanmar, Thailand, and Vietnam [1]. Based on the mentioned situation, the need for new
antimalarial agents has motivated the research to find synthetic molecules which are able to answer
the problem.
Plasmodium falciparum possesses a non-photosynthetic plastid organelle called apicoplast which is
very important for the parasite’s survival. This respiration organelle is Plasmodium falciparum-specific
and it is not present in humans [5]. During the respiration process, electron transfer to ferredoxin (PfFd)
+
catalyzed by ferredoxin NADP reductase (PfFNR) occurs in the apicoplast [6]. The interaction of
PfFNR-PfFd proceeds through an intermolecular electrostatic interaction between acidic amino acid
residues of PfFd with basic amino acid residues of PfFNR. There are two acidic regions of PfFd that
dominantly contribute to the electrostatic interaction with PfFNR, those are Asp26, Glu29, Glu34, and
the others include Asp65 and Glu66 [7]. Therefore targeting inhibition of the interaction of these proteins
could open new possibilities to find new and selective antimalarial compounds.
Chalcones are natural products that can also be obtained synthetically using a relatively simple
synthesis procedure. The general method applied to synthesize chalcones is the Claisen-Schmidt
reaction, while a modern alternative to synthesize chalcones uses the palladium-catalyst cross coupling
reactions of styryltrifluoroborates with benzoyl chlorides [8]. Chalcone derivatives are well known for
their broad spectrum of pharmacological activities, inclusing radical scavenger [9], antihepatotoxic [10],
anticancer [11], and antimalarial properties [12]. Alkoxylated chalcone derivatives exhibited higher
antimalarial activity compared to hydroxylated chalcones [13].
The synthesis and bioactivity of the prepared compounds were already reported. The interaction of
compounds 1‒3 with bovine serum albumin (BSA)—a protein mainly responsible for the transportation
of a number compounds in a living system—has been studied, and it was found that compound 1 was
the most reactive [14]. Furthermore, compound 3 was used as intermediate in the synthesis of phthalimide
derivatives as analgesic and anti-inflammatory agents [15]. Continuous-flow hydration-condensation
reactions between phenylacetylene and benzaldehyde derivatives using Amberlyst 15 as heterogeneous
catalyst was applied for the synthesis of compounds 8, 10, and 12 in excellent yield [16]. Compound 10

Molecules 2014, 19
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exhibited good anticancer activity toward the A549, PC3, MCF-77, HT-29, and WRL68 cancer cell
lines [17], while compound 12 showed potential anticancer, anti-inflammatory, and antioxidant
activity [18]. Based on the background, herein we report the synthesis of methoxychalcone derivatives
designed to inhibit the PfFd-PfFNR interaction and analyze the interaction through docking experiments.
2
. Results and Discussion
2
.1. Design and Synthesis of Chalcone Derivatives
According to the electrostatic interaction between the acidic amino acid residues of PfFd with the
basic amino acid residues of PfFNR [7] and the important role of the methoxy group for antimalarial
activity [13], we designed and then synthesized methoxyamino chalcone derivatives. The amino group
is assumed to form an electrostatic interaction with the acidic amino acid residues of PfFd to hinder the
PfFd-PfFNR electrostatic interaction. To prove this assumption, we then synthesized methoxybromo
chalcone derivatives, as bromo substituted on an aromatic group is known as an electron withdrawing
group, while the amino group is known as an electron donating group. The role of the methoxy group
was studied by synthesizing chalcone 17. The synthesis of chalcone derivatives was accomplished
using the Claisen-Schmidt reaction. This reaction can take place under various reaction
conditions [12,13,19–21]. In our research, the chalcones were prepared by the reaction of equimolar
amounts of acetophenone and benzaldehyde derivatives in ethanol using a 40% NaOH solution as
catalyst. By applying these reaction conditions, good to excellent yields were obtained (Table 1).
Table 1. Structure of the prepared chalcones.
Compd
R
NH
NH
NH
NH
NH
NH
NH
H
H
H
H
H
R
OCH
H
1
R
2
R
H
H
OCH
H
OCH
H
H
H
H
OCH
H
OCH
H
OCH
OCH
H
3
R
H
H
H
H
H
OCH
H
H
H
H
H
H
OCH
H
H
OCH
H
4
R
5
1
2
3
4
5
6
7
8
9
2
2
2
2
2
2
2
3
H
OCH
H
OCH
H
H
H
H
OCH
H
OCH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3
H
3
OCH
OCH
OCH
H
OCH
H
3
3
3
3
3
3
3
3
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
H
3
OCH
OCH
OCH
H
OCH
OCH
H
3
3
3
3
3
H
3
Br
Br
Br
H
3
3
3
3
H
H
3
H

Molecules 2014, 19
21476
The molecular structures of the synthesized compounds were confirmed by FT-IR, HRESI-MS or
1
13
1
ESI-MS, and H- and C-NMR spectral data. The H-NMR coupling constant analyses indicated that
all hydrogen atoms of the olefinic carbon–carbon bond were in a trans conformation. The existence of
a carbonyl group conjugated with the olefinic carbon–carbon bond was evident from the infra-red spectra
as the carbonyl peak was observed at a lower wavenumber than a normal carbonyl peak (around
−1
13
1
1
650–1660 cm ) and from the C-NMR spectra. The existence of a bromine atom in compounds 14,
5 and 16 was detected by the appearance in their mass spectra of two molecular peaks in the [M+H]+
at m/z 317.3 and 319.2 (compound 14), 347.3 and 349.3 (compound 15), and at m/z 347.1 and 349.2
(
compound 16) with an intensity of 1:1. The existence of methoxy groups was evidenced by the peaks
1
at chemical shifts between 3.6 until 3.9 ppm in the H-NMR spectra [22].
2.2. Production, Characterization and Kinetic Analysis of PfFd and PfFNR
The protein PfFNR was isolated from the clone E coli TG1 pTrc99A, whereas protein PfFd was
produced from the clone E coli JM105 pTrc99A (both from the collection of the Laboratory of
Regulation and Biological Reaction IPR Osaka University-Japan). Confirmation of protein purity was
determined from their UV-Vis spectrum profiles at λ between 240–650 nm (Figure 1).
Figure 1. UV-Vis spectrum profile of the proteins PfFd and PfFNR.
The red colored PfFd showed peaks at 423, 334, and 276 nm, whereas the yellow colored PfFNR
showed peaks at 455, 389, and 276 nm, which indicated the specific peaks for the family protein
containing 2Fe-2S and flavoprotein, respectively [6,7]. The absorbance ratios Abs276/423 and Abs276/455
were then used to determine the purity of each protein, which resulted in 1.95 for PfFd and 6.66 for
PfFNR. These values were close to the results from a previous report [7], 1.75 and 7.0.
The kinetic parameter determined in this study was the Michaelis constant (K ). The basis for the
M
kinetic parameter determination procedure was in accordance with Scheme 1. The number of reduced
Cyt-c is equivalent to the number of electrons transferred by PfFNR from NADPH to PfFd, which was
observed spectrometrically through the absorbance exchange by adding oxidized-Cyt-c as indicator at

Molecules 2014, 19
21477
5
50 nm. Glucose-6-phosphate dehydrogenase (G6PDH) and glucose-6-phosphate (G6P) were added as
−1
a NADPH regeneration system. The K value obtained from this research was 0.80 µM , which was
M
the same value as the one reported before [6,23].
Scheme 1. Chain reaction cycle applied in the inhibition assay.
2
.3. Inhibition of PfFd-PfFNR Interaction by Synthesized Compounds
The inhibition assay was performed under the same conditions used in the K
M
determination
employing the following conditions: the concentration of PfFd was 1 µM, the concentration of PfFNR
was 20 nM, and the concentration of tested compounds was 100 µM. A 5 mM solution of DMSO was
used as control solution. The % inhibition was calculated according to the following equation, and the
results are listed in Table 2:
ꢈꢉ
ꢈꢅ
ꢈꢉ
ꢊ ꢋꢆꢂꢅꢌꢆꢍ − ꢇ ꢊ ꢎꢏꢐꢑꢍꢒ
ꢈꢅ
ꢇ
%
ꢀꢁꢂℎꢃꢄꢃꢅꢃꢆꢂ =ꢀ
× 100%
(1)
ꢈꢉ
ꢇ
ꢊ ꢋꢆꢂꢅꢌꢆꢍ
ꢈꢅ
Table 2. Inhibition (%) of electron transfer of the tested chalcones (100 µM) to PfFd.
Tested Compound dA/dt % Inhibition
Control-1
0.076
0.064
0.059
0.038
0.052
0.047
0.064
0.073
0.057
0.061
0.060
0.068
0.075
0.068
0.072
0.064
0.063
0.050
0.060
0.053
-
1
15.79
22.37
50.00
31.58
38.16
-
2
3
4
5
Control-2
6
7
−14.06
10.94
4.69
1
1
0
2
6.25
Control-3
-
8
9
−10.29
0.00
1
1
1
1
1
1
1
3
4
5
6
7
−5.88
5.88
7.35
26.47
11.76
22.06

Molecules 2014, 19
21478
The results showed that some of the tested compounds were able to inhibit the electron transfer by
between 4.68%–50% (compounds 1‒5, 7, 10, 12‒17), while compound 9 showed no activity.
Compounds 6, 8, and 11 exhibited a negative inhibition, which means that these compounds accelerated
the electron transfer from PfFNR to PfFd. In general, these results gave us the following information:
(
1) methoxyamino chalcone derivatives exhibited better inhibition of electron transfer than
methoxybromo chalcones and methoxy chalcones with no amino group; (2) the amino group played an
important role in the inhibition activity, whereas the existence of a methoxy group gave only minor
contributions to the inhibition activity. The effect of ortho- or meta-substituted amino groups on ring A
will be investigated in the future.
2
.4. In Silico Analysis of the Protein—Synthesized Compounds Interaction
The molecular interaction between both proteins and synthesized compounds was studied by docking
experiments. Because the molecular structure of PfFd-PfFNR complex is not available in the Protein
Data Bank (PDB), we took the 3D-structure of PfFd from PDB with access code 1IUE to study the
interaction of the tested compounds with Plasmodium falciparum ferredoxin, whereas the interaction of
PfFNR with the synthesized compounds was studied by taking the 3D-structure of maize-FNR obtained
from the structure of maize Fd-FNR complex (PDB access code 1GAQ) [24]. The 3D-structure of maize
FNR was used for the docking experiment because of its high homology to the PfFNR (82%) and it was
obtained after the maize-Fd structure was deleted from the complex structure.
The downloaded structure of PfFd (1IUE) contained no ligand, so that it was impossible to define the
binding position and run a docking validation, which was to be used subsequently in the docking
experiment. Therefore, the grid box used for docking experiment was made as follow. The position of
the grid box was derived from the position of the amino acid residues of PfFd involved dominantly in
the interaction with PfFNR; Asp26, Glu29, Glu34, Asp65, and Glu66 [7], while the dimension of grid
box was made, so that it could cover all these amino acid residues.
Figure 2. Docking result of compound 3 generated by AutoDock4 and viewed by PyMol.
(
A) Docking pose of compound 3 into PfFd. The magenta color is the surface of amino acid
residues of PfFd involved in the interaction with PfFNR, (B) Grid box used in docking
experiment into maize leaf-FNR. Dark blue color is the surface of active amino acid residues
in the interaction with maize leaf-Fd, (C) docking pose of compound 3 on maize-FNR and
attached by maize-Fd. Yellow: maize leaf-FNR, red: maize leaf-Fd, and blue: compound 3.
Grid size: X: 26; Y: 42; Z: 34. Spacing 1 Å,
Grid center: X: 27.45; Y: 13.558; Z: 47.392.
(
A)
(B)
(C)

Molecules 2014, 19
21479
The same preparation was conducted for docking experiments to maize leaf-FNR. The position of the
grid box was derived from the position of amino acid residues of maize leaf-FNR involved in the
interaction with maize leaf-Fd; Lys88, Lys91, Lys33, Glu154, and Lys304 [24]. To verify the docking
condition, a docking experiment of compound 3 to maize leaf-FNR was performed, and then the maize
leaf-Fd were put to the docking result. The docking results are shown in Figure 2. The docking results
of the synthesized compounds into PfFd and maize-FNR are tabulated in Table 3. The docking of
compounds 14‒16 into both proteins was not performed.
Table 3. The docking results of synthesized compounds into PfFd and maize-FNR.
Protein PfFd
Amino Acid
Protein Maize Leaf-FNR
Amino Acid Residues
Amino Acid
Residues Form
H-Bonding
Asn30
Amino Acid Residues
Comp ∆G Energy
∆G Energy
Residues Form
H-Bonding
Tyr37
Form Electrostatic
Form Electrostatic
Interaction
Tyr32
(
Kcal/mol)
(Kcal/mol)
Interaction
---
1
2
3
4
5
−2.45
−2.96
−2.71
−2.45
−2.43
−3.68
−3.78
−4.00
−3.50
−4.09
Arg30, Asn32
Arg30, Tyr37
Arg30,
---
Asn30, Leu94
Lys304
Tyr32
Glu34
Glu34
---
Lys304, Asp307
Lys304, Asp307
---
Lys304, Arg305
Lys304, Arg305
Asn30, leu94,
Thr148
Asn32
6
−2.49
Arg30, Tyr37
---
−3.55
---
7
8
9
−2.79
−2.47
−2.90
−2.75
−2.32
−2.48
−2.57
-
Tyr37
Glu34
---
−4.10
−3.99
−4.33
−4.09
−3.84
−3.76
−4.09
-
Lys304(2x)
Lys301, Lys304
Val311
Asp307
---
Asn32, Tyr37
---
---
---
---
---
---
---
---
---
---
---
1
0
1
2
3
4
5
6
7
---
Lys304, Arg305
Lys304
---
1
1
1
1
1
1
1
---
---
---
Lys304
---
---
Lys304
---
---
Not observed
Not observed
Not observed
Lys304
---
-
---
-
---
-
---
-
---
−2.65
---
−4.03
---
In general, the docking results provide us with the information that the affinity of synthesized
compounds toward maize leaf-FNR was bigger than toward PfFd as shown by the lower maize
leaf-FNR–synthesized compounds binding energy. Beside affinity, the inhibitory activity of the
synthesized compounds can also be reflected from the interaction of the protein with the tested
compounds (H-bonding, electrostatic interactions through salt bridges).
2
.5. Docking into PfFd
Visualization of docking results employing PyMol program exhibited that all methoxyamino chalcone
derivatives (compounds 1–7) formed hydrogen bonds with amino acid residues of PfFd, while the others
did not show hydrogen bond interactions. In this article we report only the visualization of the
interactions of compounds 3‒5 that showed high inhibitory activity with PfFd and maize leaf-FNR,
displayed in Figure 3. From the docking results the following observations were noted: (1) the acidic

Molecules 2014, 19
21480
amino acid residue of PfFd involved in electrostatic acid-base interactions with the amino groups of the
synthesized chalcones was Glu34. This result was in accordance with the results of a previous study
which showed that the acidic region of PfFd conferred a larger contribution for electrostatic
interactions [7]. With PfFNR it was Asp26/Glu29/Glu34 [7]; (2) due to its dominant involvement of
Tyr37 of PfFd in the formation of hydrogen bonds with the tested compounds, we assumed that this
amino acid residue functions as modulator in the electron transfer process; (3) the amino groups of the
tested compounds played an active role in the inhibition of electron transfer to PfFd. The amino group
in the tested compounds was able to build electrostatic interactions with the acidic residue Glu34 of PfFd
and consequently, the PfFd-PfFNR interaction was inhibited; (4) the existence of a methoxy group
played an insignificant role in the electron transfer process to PfFd (comparing the inhibition activity of
compounds 1–7 to 8–13 and 17); (5) the presence of a bromine atom as an electron withdrawing group
played a minor role in the transfer electron process; (6) however, for compounds 6, 8, and 11 which
showed acceleration of transfer electron property, this still remains to be explored.
Figure 3. Docking pose and 2D presentation of the molecular interaction of the synthesized
chalcones on PfFd and maize leaf-FNR. (a) PfFd-compound 3, (b) PfFd-compound 4, and
(
(
c) PfFd -compound 5, (d) maize leaf-FNR-compound 3, (e) maize leaf-FNR-compound 4,
f) maize leaf-FNR-compound 5.
(
a)
(
b)

Molecules 2014, 19
21481
Figure 3. Cont.
(
c)
(
d)
(
e)
H
N
H2N
HN
Arg305
Lys304
C
H2C
H2C
H2C
H2C
CH
H
N
CH2
CH2
CH2 NH2
H2N
H
O
OCH3
C
OH
O
CH
CH3
O
C
H
HO
O
N
H
OH
C
O
O
C
H2
C
CH
OH
Asp307
H2N
(
f)

Molecules 2014, 19
.6. Docking into Maize Leaf-FNR
The docking results showed that the tested compounds formed more hydrogen bonding and
21482
2
electrostatic interactions with maize leaf-FNR than with PfFd. Particularly interesting is the electrostatic
interaction between the amino groups of the tested compound with the carboxyl group of non-acidic
amino acid residues of FNR which was observed in methoxyamino chalcone derivatives 1–7. This can
explain the inhibition mechanism of the tested compounds through inhibition of electron flow from FNR
to Fd, which is in accordance with the electron flow during respiration process in the apicoplasts of
P falciparum. Based on the docking results analysis, we assume that the basic amino groups of the
synthesized compounds interact better with the active amino acid residues of FNR than with the active
amino acid residues of Fd. This interaction leads to a good three dimensional complementarity between
the FNR structure and the synthesized compounds.
3. Experimental Section
3.1. General Information
The chemicals used in the research were pro analysis grade. Melting points were measured with a
Fisher John melting point apparatus and are uncorrected. The purity of the synthesized compounds were
checked by thin layer chromatography on silica gel GF254 plates; the eluent was a mixture of n-hexane/ethyl
acetate in 3:2 ratio and the spots were identified by UV (254 nm). The mass spectra were recorded by
HRESI-MS (Waters LCT Premier XE, Waters Corp., Milford, MA, USA) or on ESI-MS (LCQ-DECA
XP Plus, Thermo-Finnigan, San Diego, CA, USA) spectrometers; NMR spectra were recorded in CDCl
3
1
13
on a NMR Bruker 400 MHz instrument (400.13 MHz for H and 100.61 MHz for C, Bruker, Billerica,
MA, USA). The IR spectra were recorded on a Spectrum One FT-IR spectrophotometer (Perkin Elmer,
Waltham, MA, USA). The UV-Vis spectra were measured on a UV-2500 PC UV-Vis spectrophotometer
(
(
Shimadzu, Kyoto, Japan). The inhibition rates and enzyme activity were recorded on a microplate reader
SH-1000 Lab Microplate Reader, Corona Electric Co., Ltd., Ibaraki, Japan).
3.2. General Procedure for Chalcone Synthesis
A mixture of acetophenone derivative (6 mmol) and benzaldehyde derivative (6 mmol) was dissolved
in ethanol (30 mL), then NaOH 40% solution (6 mL) was added dropwise, while the temperature was
kept under 10 °C, the reaction mixture was stirred under this condition for 1 hour, then the stirring was
continued at room temperature for 4 hours. Thereafter the reaction mixture was poured into ice-water;
the precipitated solid was filtered off, and recrystallized from aqueous ethanol. For compound 17, the
synthesis was conducted according to procedure described by Durst and Gokel [19].
(
E)-1-(4-Aminophenyl)-3-(2-methoxyphenyl)prop-2-en-1-one (1): yellow crystals (1.14 g; 75%), m.p
+
1
04–106 °C, Rf = 0.42 (n-hexane/ethyl acetate: 3/2); HRESI-MS [M+H] calculated for C16
H
16NO
2
−1
2
54.1181, found 254.1180; IR (KBr, cm ): 3445 and 3344 (-NH
2
); 1637 (C=O), 1596 (C=Cchain), 1173
1
(
C-O-Caryl alkyl ether); H-NMR (CDCl
3
) δ 8.11 (d, 1H, J = 15.6 Hz); 7.67 (d, 1H, J = 15.6 Hz); 7.01 (t,
1
H); 7.38 (s, 1H); 6.97 (s, 1H,); 6.95 (s, 1H); 7.96 (d, 1H, J = 8.4 Hz); 6.72 (d, 1H, J = 8.8 Hz); 3.93 (s,

Molecules 2014, 19
21483
1
3
3
1
H); 4.19 (s, br, 2H); C-NMR (CDCl
3
) δ 141.0; 121.3; 125.7; 159.2; 114.2; 128.9; 120.9; 135.2; 127.9;
32.0; 114.7; 154.2; 114.7; 132.0; 56.2; 189.70.
(
E)-1-(4-Aminophenyl)-3-(3-methoxyphenyl)prop-2-en-1-one (2): yellow crystals (1.31 g; 86%), m.p
+
1
18–120 °C, Rf = 0.47 (n-hexane/ethyl acetate: 3/2); HRESI-MS [M+H] calculated for C16
H
16NO
2
−1
2
54.1181, found 254.1181; IR (KBr, cm ): 3439 and 3338 (-NH
2
), 1645 (C=O), 1610 (C=Cchain), 1173
1
(
C-O-Caryl alkyl ether); H-NMR (CDCl
3
) δ 7.77 (d, 1H, J = 15.6 Hz); 7.54 (d, 1H, J = 15.6 Hz); 7.17 (t, 1H,
J = 2 Hz); 7.25 (d, 1H, J = 7.6 Hz); 7.35 (t, 1H, J = 8 Hz); 7.95 (d, 1 H, J = 8.8 Hz); 6.72 (d, 1H,
1
3
J = 8.8 Hz); 7.95 (d, 1 H, J = 8.8 Hz); 3.88 (s, 3H), 4.21 (s, br, 2 H). C-NMR (CDCl
3
) δ 143.04; 122.44;
1
36.76; 115.83; 159.94; 113.36; 129.85; 120.91; 128.56; 131.10; 119.95; 151.14; 119.95; 55.35; 188.12.
(
E)-1-(4-Aminophenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (3): yellow crystals (1.138 g, 75%); m.p
+
1
2
40–142 °C; Rf = 0.42 (n-hexane/ethyl acetate: 3/2); HRESI-MS [M+H] calculated for C16
H
16NO
2
−1
54.1181, found 254.1183; IR (KBr, cm ): 3467 and 3328 (-NH
2
), 1631 (C=O), 1598 (C=Cchain), 1162
1
(
C-O-Caryl alkyl ether); H-NMR (CDCl
3
) δ 7.78 (d, 1H, J = 15.6 Hz); 7.46 (d, 1H, J = 15.6 Hz); 7.61 (d,
1
H, J = 8.4 Hz); 6.72 (d, 1H, J = 8.4 Hz); 7.61 (d, 1H, J = 8.4 Hz); 7.95 (d, 1H, J = 8.8 Hz); 6.95 (d, 1H,
1
3
J = 8.8 Hz); 7.95 (d, 1H, J = 8.8 Hz); 3.87 (s, 3H); 4.21 (s, br, 2H). C-NMR (106.61 MHz, CDCl
3
) δ
1
42.97; 119.81; 128.83; 129.96; 114.36; 161.33; 128.10; 130.96; 113.94; 150.99; 130.96; 55.59; 188.23.
(
E)-1-(4-Aminophenyl)-3-(2,3-dimethoxyphenyl)prop-2-en-1-one (4): yellow crystals (1.358 g, 80%);
+
m.p = 140–142 °C; Rf = 0.42 (n-hexane/ethyl acetate: 3/2); HRESI-MS [M+H] calculated for
−
1
C
17
H
17NO
3
283.1287, found 283.1286; IR (KBr, cm ): 3466 dan 3427 (-NH
2
), 1631 (C=O), 1602
1
(
C=Cchain), 1169 (C-O-Caryl alkyl ether); H-NMR (CDCl
3
) δ 8.08 (d, 1H, J = 15.6 Hz); 7.63 (d, 1H,
= 8.0 Hz); 7.11 (t, 1H, J = 8 Hz); 6.60 (d, 1H, J = 1.2 Hz;
= 8.0 Hz); 7.96 (d, 1H, J = 8.8 Hz); 6.72 (d, 1H, J = 8.8 Hz); 7.96 (d, 1H, J = 8.8 Hz); 3.91 (s, 3H);
J = 15.6 Hz); 6.97 (dd, J
1
= 1.2 Hz; J
2
1
J
3
1
2
1
3
.89 (s, 3H); 4.16 (s, br, 2H). C-NMR (CDCl
3
) δ 131.6; 124.1; 137.9; 152.5; 153.2; 113.9; 123.8;
19.7; 129.6; 131.1; 113.9; 151.1; 61.3; 55.9; 188.5.
(
E)-1-(4-Aminophenyl)-3-(2,4-dimethoxyphenyl)prop-2-en-1-one (5): yellow crystals (1.66 g, 98%); m.p
+
1
48–150 °C; Rf = 0.49 (CHCl
3
/ethyl acetate: 3/2); HRESI-MS [M+H] calculated for C17
H
17NO
3
−1
2
83.1287, found 283.1288; IR (KBr, cm ): 3425 and 3349 (-NH
2
), 1634 (C=O), 1597 (C=Cchain), 1171
1
(
C-O-Caryl alkyl ether); H-NMR (CDCl
3
) δ 8.05 (d, 1H, J = 15.6 Hz); 6.51 (d, 1H, J = 15.6 Hz); 7.58 (d,
= 1.2 Hz, J = 8.8 Hz); 7.56 (d, 1H, J = 8.8 Hz); 7.94 (d, 1H, J = 8 Hz);
.71 (d, 1H, J = 8 Hz); 3.91 (s, 3H); 3.87 (s, 3H); 4.16 (s, br, 2H). C-NMR (CDCl
17.58; 162.64; 138.81; 160.19; 113.75; 130.61; 120.47; 130.93; 113.91; 150.74; 55.55; 55.47; 189.70.
1
6
1
H, J = 1.2 Hz); 6.55 (dd, 1H, J
1
2
1
3
3
) δ 105.32; 98.48;
(
E)-1-(4-Aminophenyl)-3-(2,5-dimethoxyphenyl)prop-2-en-1-one (6): orange crystals (1.58 g, 90%);
+
m.p = 130–132 °C; Rf = 0.39 (n-hexane/ethyl acetate: 3/2); HRESI-MS [M+H] calculated for
−1
C
17
H17NO
3
283.1287, found 283.1284; IR (KBr, cm ): 3445 and 3348 (-NH
2
), 1664 (C=O), 1607
1
(
C=Cchain), 1175 (C-O-Caryl alkyl ether); H-NMR (CDCl
3
) δ 8.05 (d, 1H, J = 15.6 Hz); 7.59 (d, 1H,
J = 15.6 Hz); 7.19 (d, 1H, J = 8 Hz); 6.93 (dd, 1H, J = 8.8 Hz); 6.90 (s, 1H); 7.94 (d, 1H, J = 8.8 Hz);
1
3
6
.71 (d, 1H, J = 8.8 Hz); 7,94 (d, 1H, J = 8.8 Hz); 3.89 (s, 3H); 3,84 (s, 3H); 4.19 (s, br, 2H). C-NMR

Molecules 2014, 19
21484
(CDCl
3
) δ 138.45; 125.09; 123.22; 150.97; 116.65; 112.55; 153.23; 113.77; 131.09; 131.54; 113.93;
1
53.56; 56.19; 55.87; 188.70.
(
E)-1-(4-Aminophenyl)-3-phenylprop-2-en-1-one (7): yellow crystals (0.59 g, 42%); m.p = 90–92 °C;
+
Rf = 0.50 (n-hexane/ethyl acetate: 3/2); HRESI-MS [M+H] calculated for C15
H
13NO 224.1075, found
−1
1
2
7
24.1073; IR (KBr, cm ): 3339 and 3213 (-NH
2
), 1628 (C=O), 1603 (C=Cchain); H-NMR (CDCl
3
) δ
.81 (d, 1H, J = 15.6 Hz); 7.51 (d, 1H, J = 15.6 Hz); 7.65 (m, 1H); 7.43 (m, 1H); 7.96 (d, 1H,
1
3
J = 8.8 Hz); 6.72 (d, 1H, J = 8.8 Hz); 4.21 (s, br, 2H). C-NMR (CDCl
28.9; 130.1; 128.3; 131.1; 113.9; 151.1; 188.16.
3
) δ 143.1; 122.1; 135.4; 128.5;
1
(
(
E)-3-(2-Methoxyphenyl)-1-phenylprop-2-en-1-one (8): yellow oily liquid (0.52 g, 27%); Rf = 0.78
+
−1
1
CHCl
3
); ESI-MS [M+H] 239.2; IR (KBr, cm ) 1661 (C=O), 1601 (C=Cchain); H-NMR (CDCl
3
) δ
8
1
1
.21 (d, 1H, J = 17.2 Hz); 7.65 (d, 1H, J = 17.2 Hz); 8.05 (s, 2H); 7.46 (d, 2H); 7.62 (s, 1H); 7.53 (d,
13
H); 7.33 (t, 1H); 6.96 (t, 1H); 6.88 (d, 1H); 3.78 (s, 3H); C-NMR (CDCl
3
) δ 128.38; 128.45; 128.98;
38.31; 190.71; 122.42; 140.13; 126.61; 158.62; 111.10; 131.76; 120.60; 132.49; 55.32.
(
(
E)-3-(3-Methoxyphenyl)-1-phenylprop-2-en-1-one (9): yellow oily liquid (1.31 g, 86%); Rf = 0.47
+
−1
n-hexane/ethyl acetate = 3/2); ESI-MS [M+H] 293.3; IR (KBr, cm ) 1663 (C=O), 1605 (C=Cchain);
) δ 7.54 (d, 1H, J = 15.7 Hz); 7.88 (d, 1H, J = 15.7 Hz); 8.02 (d, 2H); 7.46 (d, 2H); 7.55
1H-NMR (CDCl
3
1
3
(
s, 1H); 7.13 (s, 1H); 6.92 (d, 1H); 7.28 (t, 1H); 7.18 (d, 1H); 3.71 (s, 3H); C-NMR (CDCl ) δ 137.9;
3
1
28.4; 128.5; 132.7; 190.1; 122.0; 144.5; 136.0; 113.4; 159.8; 116.1; 129.8; 120.9; 55.1.
(
E)-3-(4-Methoxyphenyl)-1-phenylprop-2-en-1-one (10): pale yellow crystals (1.09 g, 57%);
+
−1
m.p = 118–120 °C; Rf = 0.62 (CHCl
3
); ESI-MS [M+H] 239.3; IR (KBr, cm ) 1657 (C=O), 1599
1
(
(
(
C=Cchain); H-NMR (CDCl
3
) δ 7.77 (d, 1H, J = 13.45 Hz); 7.42 (d, 1H, J = 13.45Hz); 7.92 (d, 2H); 7.47
1
3
d, 2H); 7.54 (s, 1H); 7.56 (d, 2H, J = 6.72 Hz); 6.92 (d, 2H, J = 6.72 Hz); 3.83 (s, 3H); C-NMR
CDCl ) δ 138.6; 128.7; 130.4; 132.7; 190.7; 119.9; 144.9; 127.7; 128.6; 114.6; 161.8; 55.6.
3
(
[
E)-3-(2,3-Dimethoxyphenyl)-1-phenylprop-2-en-1-one (11): yellow oily liquid (0.89 g, 41%); ESI-MS
+
−1
1
M+H] 269.2; IR (KBr, cm ) 1663 (C=O), 1603 (C=Cchain); H-NMR (CDCl ) δ 8.11 (d, 1H,
3
J = 15 Hz); 7.57 (d, 1H, J = 15 Hz); 8.06 (d, 2H); 7.43 (t, 2H); 7.53 (d, 1H); 6.89 (d, 1H); 7.03 (t, 1H);
1
3
7
.23 (d, 1H); 3.77 (s, 3H); 3.81 (s, 3H); C-NMR (CDCl
3
) δ 138.1; 128.5; 128.4; 132.7; 190.5; 124.2;
1
39.4; 128.9; 148.8; 153.1; 114.14; 123.2; 119.4; 55.7; 61.2.
(
E)-3-(2,4-Dimethoxyphenyl)-1-phenylprop-2-en-1-one (12): yellow crystals (1.10 g, 51%);
+
−1
m.p = 172–174 °C; Rf = 0.52 (n-hexane/CHCl
3
= 3/7); ESI-MS [M+H] 269.3; IR (KBr, cm ) 1651
1
(
(
C=O), 1581 (C=Cchain); H-NMR (CDCl
3
) δ 8.05 (d, 1H, J = 15.55 Hz); 5.22 (d, 1H, J = 15.55 Hz); 8.02
d, 2H); 7.50 (d, 2H); 7.56 (d, 1H); 6.46 (d, 1H); 6.51 (d, 1H); 6.53 (d, 1H); 3.84 (s, 3H); 3.88 (s, 3H);
) δ 139.06; 128.68; 128.66; 132.51; 191.46; 120.63; 140.78; 117.33; 131.20; 105.58;
63.23; 98.65; 160.62; 55.72; 55.77.
13C-NMR (CDCl
3
1
(
E)-3-(2,5-Dimethoxyphenyl)-1-phenylprop-2-en-1-one (13): yellow oily liquid (1.55 g, 72%);
+
−1
Rf = 0.39 (n-hexane/ethyl acetate = 3/2); ESI-MS [M+H] 269.2; IR (KBr, cm ) 1660 (C=O), 1601
1
(
C=Cchain); H-NMR (CDCl
3
) δ 8.08 (d, 1H, J = 15.68 Hz); 7.53 (d, 1H, J = 15.68 Hz); 7.95 (d, 2H);

Molecules 2014, 19
21485
7
3
1
.42 (d, 2H); 7.12 (d, 1H); 7.48 (d, 1H, J = 7.84 Hz); 6.75 (d, 1H, J = 7.84 Hz); 6.85 (d, 1H); 3.70 (s,
1
3
H); 3.74 (s, 3H); C-NMR (CDCl
3
) δ 138.63; 128.74; 128.41; 132.54; 191.30; 122.66; 140.38; 124.65;
17.13; 153.67; 113.54; 112.29; 153.53; 56.30; 56.03.
(
E)-1-(4-Bromophenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (14): pale yellow crystals (0.64 g, 68%);
+
m.p = 170–172 °C; Rf = 0.62 (n-hexane/ethyl acetate = 8/2); ESI-MS [M+H] 317.3 and 319.2; IR (KBr,
−1
1
cm ) 1655 (C=O), 1593 (C=Cchain); H-NMR (CDCl
3
) δ 7.77 (d, 1H, J = 18 Hz); 7.46 (d, 1H, J = 18 Hz);
7
.85 (d, 2H, J = 9 Hz); 7.63 (d, 2H, J = 9 Hz); 7.61 (d, 2H, J = 9 Hz); 6.94 (d, 2H, J = 9 Hz); 3.84
1
3
(
s, 3H); C-NMR (CDCl
62.06; 55.7.
3
) δ 127.8; 132.07; 130.60; 137.4; 189.6; 119.3; 145.5; 127.6; 130.2; 114.68;
1
(
E)-1-(4-Bromophenyl)-3-(2,4-dimethoxyphenyl)prop-2-en-1-one (15): yellow crystals (0.91 g, 87%);
+
m.p = 133–135; Rf = 0.50 (n-hexane/ethyl acetate = 9/1); ESI-MS [M+H] 347.3 and 349.3; IR (KBr,
−
1
1
cm ) 1651 (C=O), 1587 (C=Cchain); H-NMR (CDCl
3
) δ 8.02 (d, 1H, J = 15.8 Hz); 7.46 (d, 1H,
= 8.6 and
) δ 137.8; 130.2;
J = 15.8 Hz); 7.60 (d, 2H, J = 8.5 Hz); 7.84 (d, 2H, J = 8.5 Hz); 6.45 (s, 1H); 6.51 (dd, 1H, J
1
1
3
J
2
= 2.2 Hz); 7.54 (d, 1H, J = 8.6 Hz); 3.83 (s, 3H); 3.88 (s, 3H); C-NMR (CDCl
3
1
31.9; 127.5; 190.3; 120.0; 141.4; 117.1; 131.4; 105.7; 163.8; 98.6; 160.7; 55.7; 55.8.
(
E)-1-(4-Bromophenyl)-3-(2,5-dimethoxyphenyl)prop-2-en-1-one (16): pale yellow crystals (1.02 g,
+
9
8%); m.p = 112–114 °C, Rf = 0.68 (CHCl
3
/ethyl acetate = 6/4); ESI-MS [M+H] 347.1 and 349.2; IR
−1
1
(
KBr, cm ) 1666 (C=O), 1599 (C=Cchain); H-NMR (CDCl
3
) δ 8.05 (d, 1H, J = 15.9 Hz); 7.56 (d, 1H,
= 9.09 and
) δ 137.37; 135.68;
32.06; 130.31; 190.26; 122.78; 141.02; 127.85; 153.60; 117.68; 114.06; 153.69; 112.65; 56.07; 56.32.
J = 15.9 Hz); 7.86 (d, 2H, J = 9.09 Hz); 6.49 (d, 1H, J = 9.09 Hz); 6.95 (dd, 1H, J
1
1
3
J
2
= 3 Hz); 7.18 (d, 1H, J = 3 Hz); 3.61 (s, 3H); 3.66 (s, 3H); C-NMR (CDCl
3
1
(
[
(
E)-1,3-diphenyl-2-propen-1-one (17): pale yellow crystals (1.18 g, 90%); m.p = 57–59 °C; ESI-MS
+
−1
1
M+H] 209.3; IR (KBr, cm ) 1664 (C=O), 1607 and 1574 (C=Cchain); H-NMR (CDCl
3
) δ 7.99–8.04
m, 2H); 7.81 (d, 1H, J = 18.18 Hz); 7.55 (d, 1H, J = 18.18 Hz); 7,61–7.66 (m, 3H); 7.48–7.52 (m, 2H);
7
1
.38–7.42 (m, 3H); 13C-NMR (CDCl
) δ 191.0; 145.08; 133.38; 135.6; 133.1; 129.1; 128.8; 128.7;
3
28.6; 122.2.
3
.3. Preparation of of PfFNR and PfFd in E. coli
Production and purification procedure of PfFd and PfFNR from recombinant E. coli was carried out
according to the protocol reported by Kimata-Ariga et al. [7]. Enzymatic activity analysis of
NADPH-dependent ferredoxin reduction by FNR was performed as reported by Onda et al. [25] with
little modification. Shortly, enzymatic analysis was performed under the following reaction conditions:
solution A (200 µM Cyt-c, 3 mM G6P, 3 U/mL G6PDH, 100 mM NaCl, 50 mM tris-HCl pH 7.5,
2
5 nM FNR), PfFd in concentration 0–5 µM PfFd, and 200 mM NADPH.
3.4. Inhibitory Activity Assay
Inhibition activity of the electron transfer from PfFNR to PfFd was determined by using a chain
reaction with addition of Cyt-c as an electron acceptor (Equation (1)). The inhibition test was performed

Molecules 2014, 19
21486
as reported by Onda et al. [25]. The solution of prepared compound (4 µL, 100 µM), NADPH (4 µL,
2
2
00 µM), and PfFd (12 µL, 1 mM) were mixed in 96-well plate, then solution A (180 µL, composed of
00 µM Cyt-c, 3 mM G6P, 3 U/mL of G6PDH, 100 mM NaCl, 50 mM Tris-HCl pH 7.5, and 25 nM
PfFNR) was added to the reaction mixture and mixed well. The rate of absorbance change was then
observed for 5 min in 10 second intervals at 550 nm using a microplate reader.
3
.5. Ligand Docking Analysis
The ligand docking pose was analyzed through molecular docking experiments. The 3D structure of
PfFd used as docking target was downloaded from the Protein Data Bank (access code 1IUE) and the
structure of maize Fd-FNR was downloaded from Protein Data Bank (access code 1GAQ). Preparation
of ligand and macromolecule was performed using AutoDock Tools 1.5.6 [26], whereas the docking
experiment was carried out using AutoDock4 [27]. The dimension of the gridbox was made so that it
covered the binding site of PfFNR involving to amino acid residues involved in the PfFNR-PfFd
interaction as reported by Kimata-Ariga et al. [7]. Docking results were visualized by the PyMol program.
4
. Conclusions
In conclusion, we have designed and synthesized in good to excellent yield chalcone derivatives using
Claisen-Schmidt reactions. The methoxyamino chalcone derivatives displayed promising antimalarial
activity. Based on the docking results, the synthesized chalcones preferred to interact with FNR by
electrostatic interaction through salt bridges between the amino groups of the synthesized chalcones and
the carboxyl groups of active amino acid residues of FNR rather than with Fd.
Acknowledgments
The authors acknowledge to the Ministry of Research and Technology of Republic Indonesia for the
research supporting fund through the Riset Insentif Nasional (INSINAS) scheme.
Author Contributions
The contributions of the listed authors to this work are described in the following. Hery Suwito carried
out the synthetic work and prepared the manuscript. Pratiwi Pudjiastuti and Yoko Kimata-Ariga
performed the inhibition assay. Much Zaenal Fanani conducted the protein isolation and in silico work.
Ritsuko Katahira, Toshimichi Fujiwara, and Toru Kawakami recorded the NMR spectra. Hasnah Mohd Sirat
contributed her ideas through valuable discussions and spectra of the substances. Ni Nyoman Tri Puspaningsih
contributed her ideas through valuable discussion and scientific input. Toshiharu Hase took part in
valuable discussion, funding for spectra of the substances and bioactivity assay. Jumina contributed
development of the study concept, valuable discussions, and proofreading. Mustofa contributed
scientific input and proofreading.
Conflicts of Interest
The authors declare that there is no conflict of interest.

Molecules 2014, 19
21487
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Sample Availability: Samples of the compounds 1–17 are not available from the authors.
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