Synthesis, biological evaluation and molecular docking of novel chalcone-coumarin hybrids as anticancer and antimalarial agents

Article abstract of DOI:10.1016/j.ejmech.2014.07.087

A new series of chalcone-coumarin derivatives (9-19) linked by the 1,2,3-triazole ring were synthesized through the azide/alkyne dipolar cycloaddition. Hybrid molecules were evaluated for their cytotoxic activity against four cancer cell lines (e.g., HuCC

Full text of DOI:10.1016/j.ejmech.2014.07.087

European Journal of Medicinal Chemistry 85 (2014) 65e76
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, biological evaluation and molecular docking of novel
chalconeecoumarin hybrids as anticancer and antimalarial agents
,
,
, c
Ratchanok Pingaew a *, Amporn Saekee a, Prasit Mandi b c, Chanin Nantasenamat b
,
Supaluk Prachayasittikul b, Somsak Ruchirawat d f, Virapong Prachayasittikul c
, e,
a Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok 10110, Thailand
b Center of Data Mining and Biomedical Informatics, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand
c Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand
d Chulabhorn Research Institute, Bangkok 10210, Thailand
e Program in Chemical Biology, Chulabhorn Graduate Institute, Bangkok 10210, Thailand
f Center of Excellence on Environmental Health and Toxicology (EHT), CHE, Ministry of Education, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of chalconeecoumarin derivatives (9e19) linked by the 1,2,3-triazole ring were synthesized
through the azide/alkyne dipolar cycloaddition. Hybrid molecules were evaluated for their cytotoxic
activity against four cancer cell lines (e.g., HuCCA-1, HepG2, A549 and MOLT-3) and antimalarial activity
toward Plasmodium falciparum. Most of the synthesized hybrids, except for analogs 10 and 16, displayed
cytotoxicity against MOLT-3 cell line without affecting normal cells. Analogs (10, 11, 16 and 18) exhibited
higher inhibitory efficacy than the control drug, etoposide, in HepG2 cells. Significantly, the high cyto-
toxic potency of the hybrid 11 was shown to be non-toxic to normal cells. Interestingly, the chalcone
Received 13 March 2014
Received in revised form
19 July 2014
Accepted 24 July 2014
Available online 24 July 2014
Keywords:
Chalcone
Coumarin
Triazole
Molecular docking
Cytotoxicity
Antimalarial activity
ecoumarin 18 was the most potent antimalarial compound affording IC50 value of 1.60
docking suggested that the cytotoxicity of reported hybrids could be possibly due to their dual inhibition
of - and -tubulins at GTP and colchicine binding sites, respectively. Furthermore, falcipain-2 was
identified to be a plausible target site of the hybrids given their antimalarial potency.
© 2014 Elsevier Masson SAS. All rights reserved.
mM. Molecular
a
b
1. Introduction
erythrocyte membranes have been disclosed [14]. Structur-
eeactivity relationship (SAR) elucidation of antimalarial chalcones
Chalcones (1,3-diaryl-2-propen-1-ones) belonging to the flavo-
noid class of natural products have drawn considerable interest
owing to their pharmacological properties [1e6]. Naturally-
occurring chalcones and their synthetic analogs displayed signifi-
cant cytotoxic activity against various cancer cells. One of the most
widely proposed anticancer mechanisms of chalcones is its pre-
vention of tubulin polymerization by binding to the colchicine-
binding site [5e7]. The natural chalcone, licochalcone A (Fig. 1),
isolated from Chinese licorice roots was revealed to exhibit po-
tential antimalarial activity [8]. Numerous chalcones have been
extensively studied for their antimalarial activity, and the results
showed that the activity was exerted by plasmodial aspartate
protease [9], cysteine protease [10e12] and cyclin-dependent
protein kinase (CDK), Pfmrk [13] inhibitors. In addition, inhibition
of new permeation pathways induced by malarial parasite in the
demonstrated that the presence of the a,b-unsaturated ketone
linker [11] and E configuration [15] are critical for their activities in
which the alkoxylated chalcones displayed superior antimalarial
activity than those of the hydroxylated analogs [14,16].
Pharmacological applications of synthetic and natural couma-
rins (Fig.1) have previously been reviewed [17e20]. For example, 7-
hydroxycoumarin was shown to inhibit the release of cyclin D1,
which is overexpressed in many types of cancers [19]. Daphnetin,
the Chinese herbal product, exhibited potency against malarial
parasite both in vitro and in vivo [21].
The design and development of new bioactive agents based on
the molecular hybridization strategy, involving the integration of
two or more pharmacophoric units having different mechanisms of
action in the same molecule, is a rationally attractive approach
[22,23]. These combined pharmacophores probably offer some
advantages such as in overcoming drug resistance [24] as well as
improving their biological potency [25]. Such hybrid approaches
had previously reported the coupling of chalcones with various
bioactive compounds including nucleosides [26], quinolines
* Corresponding author.
E-mail address: ratchanok@swu.ac.th (R. Pingaew).
http://dx.doi.org/10.1016/j.ejmech.2014.07.087
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

66
R. Pingaew et al. / European Journal of Medicinal Chemistry 85 (2014) 65e76
Fig. 1. Bioactive chalcone and coumarins.
[26e28], pyrrolobenzodiazepines [29], ciprofloxacin [30],
b
-lactam
tubulin and falcipain-2, have been carried out to gain insight into
the biological results.
[31], nitric oxide donors [32,33], thiolactone [12] and isatin [12]. In
addition, a number of new chalconeecoumarin hybrids affording
anticancer, antimalarial, vasorelaxant, anti-inflammatory, antioxi-
dant and trypanocidal activities have been documented
[10,34e39]. For instance, hybrid (1) (Fig. 2) has been found to exert
significant cytotoxic activity against the paclitaxel-resistant cancer
cells [37]. Derivative (2) has shown cytotoxic activity against cer-
2. Results and discussion
2.1. Chemistry
The novel target chalconeecoumarin hybrids (9e19) were
synthesized in three steps (Scheme 1). Initially, chalcones 6 were
synthesized using the base-catalyzed ClaiseneSchmidt condensa-
vical carcinoma cells (IC50 ¼ 3.59
mM) without affecting normal
fibroblast NIH3T3 cells [38]. Additionally, conjugate (3) exhibited
antimalarial activity against both chloroquine sensitive and
chloroquine-resistant strains with IC50 values of 3.1 and 4 mg/mL,
respectively [39]. Such chalcone analogs (1e3) constitute coumaryl
group, but the different substituents are pyridyl and phenyl groups
on the 2-propen-1-one core structure.
Triazole moiety is commonly found in diverse bioactive com-
pounds. Its distinct property is its metabolically stable heterocyclic
ring that is capable of forming hydrogen bonds. Furthermore, it can
be used as linkers of various molecules as to give rise to new
compounds and which might increase their bioavailability [40].
The reported cytotoxic and antimalarial activities of chalcone
analogs led to our rational design and synthesis of new molecular
hybrids of chalcone and coumarin tethered by the triazole ring. In
this article, the synthetic approaches have been designed as follows
(Fig. 3):
tion of various aromatic aldehydes
4 with two amino-
acetophenones 5 (3- or 4-NH2). Subsequently, azotization reaction
of the aminochalcones 6 using sodium nitrite and sodium azide in a
mixture of glacial acetic acid and concentrated hydrochloric acid
afforded the corresponding azidochalcones 7. The azide/alkyne
dipolar cycloaddition, represented as the Click-type reaction, is the
key route to the synthesis of 1,2,3-triazole linkage. Finally, the
azides 7 readily underwent cycloaddition with alkynes (8) to afford
the novel desired hybrid molecules (9e19) in moderate to good
yields (50e88%). The starting alkynes (8) were derived from the
alkylation of either 4-hydroxycoumarin or 7-hydroxycoumarin
with propargyl bromide.
Structures of the hybrids (9e19) were characterized by 1H NMR
spectra, which showed the existence of the singlet of methine
proton at
formed. In addition, triazole methylene ether linker displayed the
singlet of methylene proton at 5e6 ppm. The chalcone part was
d 8e10 ppm indicating that the triazole linkage was
(i) use 1,3-diphenylpropenone (rings A and B) as the core
structure
(ii) vary methoxy groups substituted on ring B (2,3-dimethoxy-,
3,4-dimethoxy-, 2,3,4-trimethoxy- or 3,4,5-trimethoxy
substituents)
(iii) vary the position of 1,2,3-triazole ring on ring A (3- or 4-
position)
(iv) vary coumaryl ring substituted on the triazole moiety (4-
chromenone or 7-chromenone).
d
shown to be in the trans-configuration of double bond as repre-
sented by the coupling constant (J) values of 15e16 Hz. Structures of
all obtained compounds were further supported by 13C NMR, IR and
HRMS. Such spectral data using the chalconeecoumarin hybrid 9 as
an example are provided below.
The hybrid 9 showed molecular ion [MþNa]þ peak at 532.1464
corresponding to the molecular formula of C29H23N3NaO6. The IR
spectra of the compound 9 exhibited absorption bands of C]O
groups of coumarin and chalcone moieties at 1723 and 1660 cmꢀ1
,
respectively. In the 1H NMR spectra, two singlets at
d 3.92 and
Herein, a variety of novel target chalconeecoumarin molecules
containing triazole linker (9e19) were synthesized and evaluated
for their cytotoxic and antimalarial activities. In addition, molecular
docking studies of these compounds toward their target proteins,
3.94 ppm were assigned to two methoxy protons on ring B of the
chalcone part. The methylene protons connected to the triazole
moiety appeared as a singlet at
d 5.35 ppm. Two protons of the
Fig. 2. Chalcone containing coumarin derivatives.

R. Pingaew et al. / European Journal of Medicinal Chemistry 85 (2014) 65e76
67
2.2. Biological activities
2.2.1. Cytotoxic activity
Cytotoxicity of the synthesized chalconeecoumarin hybrids
(9e19) were assayed against human cancer cell lines; HuCCA-1
(cholangiocarcinoma), HepG2 (hepatocellular carcinoma), A549
(lung carcinoma) and MOLT-3 (lymphoblastic leukemia), as sum-
marized in Table 1. These compounds were also evaluated for non-
cancerous cell line (Vero) derived from African green monkey
kidney.
Results showed that most of the tested hybrid molecules exerted
significant cytotoxicity, especially against MOLT-3 cancer cell line,
except for the hybrid 11. Among these, triazole hybrid 16 was
shown to be the most potent cytotoxic compound against MOLT-
Fig. 3. Newly designed chalconeecoumarin hybrids linked by triazole.
propenone moiety were observed as two doublets at
d
7.38 and
3 cells with an IC50 of 0.53
mM. Aside from MOLT-3 cells, com-
7.82 ppm with J value of 15.6 Hz. Two doublets at
d 6.25 and
pounds 9e11 and 15e17 also showed inhibitory effect against
HuCCA-1 cells. However, tri-substituted methoxy hybrids 12, 13, 14
and 19 selectively inhibited MOLT-3 cells with IC50 in the range of
7.62 ppm with J value of 9.5 Hz were attributed to two protons of
coumarin ring at positions 3 and 4. The rest of aromatic protons of
this ring at C-6 and C-8 appeared as multiplet at
and a proton at position 5 was found to be displayed as a doublet at
7.39 ppm with J value of 8.9 Hz. The aromatic ring B protons of
chalcone at position 500, 200 and 600 were observed at
6.89 (d,
d 6.92e6.98 ppm,
3.89e79.49
noted for 2,3-dimethoxy analogs 10, 11 and 16, in which 11 was the
most potent compound (IC50 ¼ 7.95 M). The investigated chalco-
mM. Significant cytotoxic activity toward A549 cells was
d
m
d
neecoumarin hybrids bear a variety of substituents on ring B (2,3-,
3,4-, 2,3,4- and 3,4,5-methoxys) and ring A (3- and 4-substituted
triazoles) of the chalcone moiety while the coumarins linked to
triazoles as a series of 7- (9e14) and 4- (15e19) substituted cou-
marins. SAR investigation of the compounds revealed that the most
potent and promising cytotoxicity depended on the substitution
pattern of substituents on rings A and B as well as coumaryl moi-
eties. Apparently, compounds with 2,3-dimethoxy groups on ring B
and 3-substituted ring A of 4-oxycoumaryl series exhibited higher
inhibitory potency (HuCCA-1 cells) than the corresponding 3,4-
J ¼ 8.3 Hz), 7.16 (s) and 7.25 (d, J ¼ 8.3 Hz) ppm, respectively. The
aromatic protons at position 50 and 20 of chalcone (ring A) were
visible as a triplet at
singlet at
8.32 ppm, respectively, whereas position 40 and 60
appeared as two doublets at
8.01 (J ¼ 8.0 Hz) and 8.07 (J ¼ 7.7 Hz)
ppm. A singlet of a methine proton of the triazole ring appeared at
8.20 ppm. In the 13C NMR spectra, two methoxy carbons (at C-300
and C-400) of chalcone part (ring B) were observed at
56.1 ppm
d 7.68 ppm with J value of 7.9 Hz and as a
d
d
d
d
whereas a methylene carbon adjacent to triazole ring was noted at
62.3 ppm. Two carbonyl groups of ketone and lactone were seen at
188.9 and 161.0 ppm, respectively. Nine quaternary aromatic car-
bons (ArC) were observed at chemical shift 113.2, 127.5, 137.3, 140.2,
144.1, 149.4, 151.9, 155.8 and 161.2 ppm. Eleven tertiary aromatic
carbons (ArCH) and four olefinic carbons appeared at chemical shift
102.2, 110.3, 111.2, 112.7, 113.6, 119.0, 120.1, 121.3, 123.6, 124.3, 128.7,
129.0, 130.2, 143.2 and 146.5 ppm.
dimethoxy (ring B) as observed for 16 (IC50 ¼ 2.36
(IC50 ¼ 38.57 M). Similar cytotoxic effect against HuCCA-1 was
noted for 7-oxycoumaryl analogs 10 (IC50 ¼ 11.13 M) and 9
M). Obviously, 2,3-dimethoxy (ring B), 3-substituent
mM) and 15
m
m
(IC50 ¼ 33.37
m
(ring A) of 4-coumaryl analog 16 exerted higher cytotoxicity to-
wards all of the tested cancer cells as compared to its 7-coumaryl
analog (10). In addition, other 4-coumaryl derivatives exerted
Scheme 1. Synthesis of chalconeecoumarin hybrids (9e19) through the Click reaction.

68
R. Pingaew et al. / European Journal of Medicinal Chemistry 85 (2014) 65e76
M) of hybrid compounds (9e19).
Table 1
Cytotoxic and antimalarial activities (IC50
,
m
Compound
A-ring
B-ring
Cancer cell linesa
HuCCA-1
Antimalarialb
Vero cell line
HepG2
A549
MOLT-3
9
10
11
12
13
14
15
16
3-Triazole
3-Triazole
4-Triazole
3-Triazole
4-Triazole
4-Triazole
3-Triazole
3-Triazole
3-Triazole
4-Triazole
4-Triazole
3,4-DiOMe
2,3-DiOMe
2,3-DiOMe
2,3,4-TriOMe
2,3,4-TriOMe
3,4,5-TriOMe
3,4-DiOMe
33.37 0.84
11.13 0.40
4.81 0.92
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
38.57 1.48
2.36 0.14
6.13 0.06
Non-cytotoxic
Non-cytotoxic
ND
Non-cytotoxic
15.70 2.00
8.18 0.76
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
4.26 0.29
Non-cytotoxic
22.85 0.51
Non-cytotoxic
30.16 0.50
0.79 0.08
ND
Non-cytotoxic
31.40 1.00
7.95 3.04
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
18.06 1.07
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
ND
3.12 0.11
5.16 0.69
Non-cytotoxic
3.89 0.28
22.50 3.63
79.49 1.44
3.91 0.34
0.53 0.08
1.13 0.18
12.33 1.78
6.58 1.78
0.051 0.002
ND
Inactive
6.63
Non-cytotoxic
35.03
Inactive
13.03
Inactive
Inactive
Inactive
4.73
7.47
1.60
Inactive
ND
ND
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
3.91
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
ND
2,3-DiOMe
17
18
19
2,3,4-TriOMe
2,3,4-TriOMe
3,4,5-TriOMe
Etoposidec
Doxorubicinc
Dihydroartemisininec
Ellipticinec
0.83 0.07
ND
ND
0.44 0.01
ND
ND
ND
ND
1.94
ND
ND
0.0011
ND
ND
Non-cytotoxic ¼ IC50 > 50
m
g/mL.
Inactive ¼ IC50 > 10
mg/mL.
Vero cell line ¼ African green monkey kidney cell line.
ND, not determined.
a
Cancer cell lines comprise the following: HuCCA-1 human cholangiocarcinoma cell line, HepG2 human hepatocellular carcinoma cell line, A549 human lung carcinoma cell
line, MOLT-3 human lymphoblastic leukemia cell line.
b
Antimalarial against Plasmodium falciparum.
Etoposide, doxorubicin, ellipticine and dihydroartemisinine were used as reference drugs.
c
better cytotoxic activity than 7-coumaryls were shown to be in the
following order 17 > 12, 18 > 13 and 19 > 14. Interestingly, HepG2
cells inhibitory action was observed for hybrids containing 2,3-
dimethoxy (10, 11 and 16) and 2,3,4-trimethoxy (18) on ring B.
Such compounds (10, 11, 16 and 18) were shown to be more potent
anticancer agents as compared to etoposide, the reference drug.
covalent bonds between E64 and the falcipain-2 active site were
disrupted and re-docked via non-covalent docking. Colchicine and
E64 could be re-docked to their respective target proteins with a
root mean squared deviation (RMSD) of 0.697 Å (Fig. 4A) and
1.157 Å (Fig. 4B), respectively.
Particularly, the most potent hybrid 16 (IC50 of 4.26
m
M) displayed
2.3.1. Molecular docking of chalconeetriazoleecoumarin
7-fold stronger activity than the etoposide (IC50 ¼ 30.16
mM). So far,
derivatives to
As previously mentioned, the cytotoxic activity of chalcone is
often associated with its ability to inhibit -tubulin formation via
a- and b-tubulin complexes
the 2,3-dimethoxy derivatives 10 and 16 containing triazole ring at
position 3 showed cytotoxicity against the Vero cell with IC50 of
35.03 and 3.91 mM, respectively. It should be noted that the hybrid
11 showed cytotoxicity in a broad spectrum of cancer cells (i.e.,
HuCCA-1, HepG2 and A549 cells) with IC50 values in the range of
4.81e8.18 mM without affecting normal cells. Molecular docking of
the anticancer hybrids to their target proteins, tubulins have been
described herein.
b
binding of compounds to the colchicine binding site [5e7]. Mo-
lecular docking was performed to investigate the binding modal-
ities of chalconeetriazoleecoumarin derivatives against tubulin.
Results suggested that all chalconeetriazoleecoumarin analogs
could snugly occupy the colchicine binding site of
b-tubulin as
shown in Fig. 4C. Binding energies for these compounds are in the
range of ꢀ9.6 to ꢀ10.8 kcal/mol (Table 2). It was observed that all
chalconeetriazoleecoumarin derivatives afforded stronger binding
energies than that of the colchicine found in the co-crystallized
structure thereby suggesting promising inhibitory effect for this
set of compounds. The observed tubulin binding energies for these
compounds are in accordance with their cytotoxic activities, in
which the more potent compounds as seen for the dimethoxy an-
alogs (9e11, 15 and 16) displayed lower binding energies (ꢀ10.5
to ꢀ10.8 kcal/mol) than the trimethoxy analogs (12e14 and 17e19)
with the binding energies in the range of ꢀ9.6 to ꢀ10.3 kcal/mol.
Particularly, the lowest cytotoxic activity of compound 14 showed
the highest tubulin binding energy of ꢀ9.6 kcal/mol. Interestingly,
docking poses of these compounds revealed that they could also
2.2.2. Antimalarial activity
Antimalarial activity of the hybrids (9e19) was examined
against Plasmodium falciparum (K1, multidrug resistant strain) as
shown in Table 1. The synthesized hybrids of 2,3-dimethoxy (10 and
16) and 2,3,4-trimethoxy (12, 17 and 18) showed significant anti-
malarial activity with IC50 values in the range of 1.60e13.03 mM. At
this point, it seemed reasonable that the presence of methoxy
groups at ortho- and para-positions of chalcones (ring B) played
crucial role for antimalarial activity. In addition, a series of 4-
coumarinoxymethyl derivatives showed superior potency than
the corresponding 7-coumarinoxymethyl analogs as seen for
16 > 10, 17 > 12 and 18 > 13. Notably, the 2,3,4-trimethoxy analog
18 was shown to be the most potent compound (IC50 ¼ 1.60
mM)
occupy the GTP binding site of
colchicine binding site of -tubulin. These findings suggest that
chalconeetriazoleecoumarin derivatives could plausibly act as
dual site inhibitors of - and -tubulin complexes. Molecular
modeling analysis of the crystal structure revealed that the co-
crystallized colchicine interacts with -tubulin via the formation
a-tubulin that is adjacent to the
and was non-toxic to normal cells. Results from the molecular
docking of antimalarial analogs are discussed hereafter.
b
a
b
2.3. Validation of docking protocol
b
Co-crystallized ligands, colchicine and trans-epoxysuccinyl-L-
leucylamido-(4-guanidino)butane (E64), were re-docked to their
target proteins, tubulin and falcipain-2, as to validate the docking
protocol. Owing to limitations of the available docking software,
of hydrogen bonds involving a methoxy group to Cys241 and
engaging in hydrophobic interactions with Leu255 and Ala316
(Fig. 4A). The GTP binds
a-tubulin via the formation of hydrogen
bonding network between the phosphate moiety to Ala12, Asp69,

R. Pingaew et al. / European Journal of Medicinal Chemistry 85 (2014) 65e76
69
Table 2
b
-tubulin, which are located inside the colchicine binding site. In
addition, ring B of chalcone moiety of the compound could form
hydrophobic interaction with Ala12 and Tyr224 together with
interaction with Tyr224 of -tubulin, which constitutes the GTP
Binding energy of chalconeetriazoleecoumarin derivatives (9e19).
Binding energy (kcal/mol)a
Tubulin
pep
Compounds
a
Falcipain-2
binding pocket. The less potency of trimethoxy analogs (13, 14, 18
and 19) compared to the 2,3-dimethoxy analog (11) may be due to
the steric reason in this hydrophobic area. In addition, such inter-
action was strengthened by hydrogen bonding as afforded by the N
9
10
11
12
13
14
15
16
ꢀ10.8
ꢀ10.6
ꢀ10.5
ꢀ10.2
ꢀ9.8
ND
ꢀ8.6
ND
ꢀ8.9
ND
ND
atom at position 2 of triazole ring to Asn101 of
a-tubulin and
ꢀ9.6
pecation interaction of chalcone ring A to the positively-charged
ꢀ10.6
ꢀ10.7
ꢀ10.3
ꢀ10.1
ꢀ10.0
ꢀ9.4
ND
side chain of Lys254 from b-tubulin. These finding suggested that
ꢀ8.3
ꢀ8.1
ꢀ8.4
ND
ND
ꢀ7.3
the cytotoxicity of chalconeetriazoleecoumarin derivatives could
be possibly due to their dual inhibition of - and -tubulins.
17
18
19
a
b
Colchicine
E64b
ND
2.3.2. Molecular docking of chalconeecoumarin derivatives to
falcipain-2
a
ND, not determined.
b
E64 was covalently bound to falcipain-2. Binding energy was obtained from
non-covalent docking.
Many chalcone derivatives have been revealed to inhibit ma-
larial cysteine protease [10e12], the enzyme plays an essential role
in the degradation of host hemoglobin into small peptides as nu-
trients occurring within the acidic food vacuole of the parasite.
Accordingly, molecular docking was performed against the cysteine
protease-like falcipain-2 using selected compounds (i.e., 10, 12, 16,
17 and 18) that have been shown to exert antimalarial activity. The
crystal structure of falcipain-2 was co-crystallized with inhibitor
E64, which was covalently-bonded to the enzyme as to block
substrates from reaching the catalytic triad as defined by Gln36,
Cys42 and His174 residues [41]. Docking results suggested that all
selected compounds could bind the active site of falcipain-2
(Fig. 4D) with binding energies in the range of ꢀ8.1 to ꢀ8.9 kcal/
mol (Table 2). Unfortunately, due to the limitation of available
docking software in performing covalent docking, therefore the
Ser140, Thr145, Ser178 and Glu183 while the guanine ring forms
hydrogen bonds with Asn206 and Asn288 as well as
with Tyr224. Moreover, hydrophobic interaction between Ala12
and Tyr224 of -tubulin with GTP were also observed. These
aforementioned residues could be the essential site for ligand
binding as to inhibit microtubule formation.
Compound 11, which provided cytotoxicity against a broad
spectrum of cancer cells (i.e., HuCCA-1, HepG2 and A549 cells)
without affecting normal cells, could interact with both colchicine
pep stacking
a
and GTP binding sites of b- and a-tubulins, respectively (Fig. 5A).
The coumarin moiety of the compound could occupy the hydro-
phobic pocket as defined by Leu248, Ala316 and Ala354 residues of
Fig. 4. Molecular docking of chalconeetriazoleecoumarin ligands to tubulin. Re-docking poses of colchicine to tubulin (A) and E64 to falcipain-2 (B) are shown with original ligand
structures colored in green while re-docking poses colored in red. Docking poses of chalconeetriazoleecoumarin ligands to tubulin (C) are shown with colchicine colored in green,
GTP colored in yellow, ligands colored in orange and blue. Docking poses of chalconeetriazoleecoumarin ligands to falcipain-2 (D) are shown with E64 colored in green and ligands
colored in orange. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

70
R. Pingaew et al. / European Journal of Medicinal Chemistry 85 (2014) 65e76
Fig. 5. Two-dimensional schematics of the protein-ligand interaction of compound 11 to tubulin (A) and compound 18 to falcipain-2 (B). The amino acid residues such as Ala12A, A
is denoted as -tubulin; and Ala316B, B is denoted as -tubulin.
a
b
binding energy of E64 was obtained by disrupting the covalent
bond and re-docked non-covalently. Owing to these inherent dif-
ferences in the binding mechanism, thus it cannot be assumed that
the chalconeetriazoleecoumarins could possess higher potency
than E64 by judging from their binding energies. Compound 18,
which had the highest antimalarial activity, could bind the active
site of falcipain-2 via the interaction scheme shown in Fig. 5B. As
such, the coumarin moiety of the compound could occupy the
binding site of falcipain-2 via hydrophobic interaction with Cys42
falcipain-2 active site. The docking results suggested that the
antimalarial activity of the chalconeetriazoleecoumarin de-
rivatives might be due to their inhibitions of falcipain-2.
Furthermore, metabolic pathways in intracellular parasites (i.e.,
P. falciparum) and cancer cells are more sensitive to oxidative stress
than normal cells [42]. It could be presumably explained that
dimethoxychalcone moieties might be metabolized to catechols,
then to phenoxy radicals and ultimately to superoxide anions,
which are toxic to the cells [5].
and Trp206 residues together with the formation of
pep stacking
with Trp206. Such interaction afforded strong stability because ring
A and ring B of chalcone moiety formed hydrophobic interaction
with Gly40 and Leu84, respectively. Moreover, 3,4-dimethoxy
substituents of chalcone ring B provided hydrogen bonding with
Ser149 thereby resulting in tight binding of compound 18 to the
3. Conclusions
Eleven conjugated molecules (9e19) derived from chalcones
and coumarins linked by 1,2,3-triazole ring have been successfully
synthesized via the Click reaction. Cytotoxic activity testing

R. Pingaew et al. / European Journal of Medicinal Chemistry 85 (2014) 65e76
71
revealed that most of the hybrids displayed cytotoxicity against
1H, CH]CHCO). 13C NMR (75 MHz, CDCl3)
d
55.9, 61.3, 114.1, 114.6,
MOLT-3 cell line. In particular, the chalconeecoumarin 16 was the
119.0, 119.4, 119.6, 123.9, 124.2, 129.2, 129.4, 139.3, 139.4, 146.7,
149.0, 153.2, 191.0. HRMS-TOF: m/z HRMS-TOF: m/z [MþNa]þ
306.1113 (Calcd for C17H17NNaO3: 306.1101).
most potent cytotoxic agent (IC50 ¼ 0.53
mM). Compounds 10, 11, 16
and 18 displayed higher cytotoxic potency against HepG2 cells than
the control drug, etoposide. The hybrid 16 was shown to be the
most potent one (IC50 ¼ 4.26
m
M), but unfortunately it was toxic
4.2.3. (E)-1-(4-aminophenyl)-3-(2,3-dimethoxyphenyl)prop-2-en-
1-one (6c)
toward non-cancerous (Vero) cells. Significantly, the analog 11
displayed the second most potent activity (HepG2) with IC50 value
Yellow solid. 65%. mp 133e134 ꢁC (135.6e136.8 ꢁC [43]). IR
of 8.18
activity results concluded that the conjugate 18 exhibited the most
M) without affecting non-cancerous
mM and non-toxic to Vero cells. Importantly, antimalarial
(UATR) cmꢀ1: 3453, 3351, 3228, 1623, 1602, 1579, 1478. 1H NMR
(300 MHz, CDCl3)
d
3.90, 3.91 (2s, 6H, 2 ꢂ OCH3), 4.17 (br s, 2H,
potent activity (IC50 ¼ 1.60
m
NH2), 6.72 (d, J ¼ 8.7 Hz, 2H, C3-ArH, C5-ArH), 6.97 (d, J ¼ 8.0 Hz,1H,
C-40-ArH), 7.10 (t, J ¼ 8.0 Hz, 1H, C-50-ArH), 7.29 (d, J ¼ 8.0 Hz, 1H, C-
60-ArH), 7.62 (d, J ¼ 15.8 Hz, 1H, CH]CHCO), 7.95 (d, J ¼ 8.6 Hz, 2H,
C2-ArH, C6-ArH), 8.07 (d, J ¼ 15.8 Hz, 1H, CH]CHCO). 13C NMR
cells. Docking results suggested that the cytotoxic and antima-
larial activities of the hybrids might be due to their inhibition of
tubulin and falcipain-2, respectively. This study provides novel
chalconeecoumarin hybrids as potential lead molecules for further
structural optimization as anticancer and antimalarial agents.
(75 MHz, CDCl3)
d 55.9, 61.3, 113.8, 113.9, 119.6, 123.8, 124.1, 128.7,
129.6, 131.1, 138.0, 148.8, 151.0, 153.2, 188.5. HRMS-TOF: m/z
[MþH]þ 284.1285 (Calcd for C17H18NO3: 284.1281).
4. Experimental
4.2.4. (E)-1-(3-aminophenyl)-3-(2,3,4-trimethoxyphenyl)prop-2-
en-1-one (6d)
4.1. Chemistry
Yellow solid. 62%. mp 94e95 ꢁC. IR (UATR) cmꢀ1: 3458, 3364,
Column chromatography was carried out using silica gel 60
(70e230 mesh ASTM). Analytical thin-layer chromatography (TLC)
was performed on silica gel 60 F254 aluminum sheets. 1H and 13C
NMR spectra were recorded on a Bruker AVANCE 300 NMR spec-
trometer (operating at 300 MHz for 1H and 75 MHz for 13C). FTIR
spectra were obtained using a universal attenuated total reflectance
attached on a PerkineElmer Spectrum One spectrometer. High
resolution mass spectra (HRMS) were recorded on a Bruker Dal-
tonics (microTOF). Melting points were determined using a Griffin
melting point apparatus and were uncorrected.
3233, 1653, 1627, 1577, 1494. 1H NMR (300 MHz, CDCl3)
d 3.85 (br s,
2H, NH2), 3.91, 3.93, 3.96 (3s, 9H, 3 ꢂ OCH3), 6.74 (d, J ¼ 8.8 Hz, 1H,
C50-ArH), 6.90 (dd, J ¼ 8.0, 1.9 Hz, 1H, C4-ArH), 7.29 (t, J ¼ 7.8 Hz, 1H,
C5-ArH), 7.34 (t, J ¼ 1.9 Hz, 1H, C2-ArH), 7.40 (d, J ¼ 8.7 Hz, 2H, C6-
ArH, C60-ArH), 7.53 (d, J ¼ 15.8 Hz, 1H, CH]CHCO), 7.99 (d,
J ¼ 15.8 Hz, 1H, CH]CHCO). 13C NMR (75 MHz, CDCl3)
d 56.1, 60.9,
61.4, 107.6, 114.5, 118.8, 119.2, 121.7, 122.1, 123.8, 129.4, 139.7, 139.8,
142.5, 146.8, 153.8, 155.7, 191.1. HRMS-TOF: m/z [MþH]þ 314.1390
(Calcd for C18H20NO4: 314.1387).
4.2.5. (E)-1-(4-aminophenyl)-3-(2,3,4-trimethoxyphenyl)prop-2-
en-1-one (6e)
4.2. General procedure for the synthesis of aminochalcones (6)
Yellow solid. 80%. mp 125e126 ꢁC. IR (UATR) cmꢀ1: 3458, 3343,
To a stirred solution of benzaldehyde 4 (5 mmol) and amino-
acetophenone 5 (5 mmol) in ethanol (15 mL) at 5 ꢁC, then 40% KOH
(10 mL) was added dropwise, and stirred at room temperature
overnight. The reaction mixture was neutralized with 2 M HCl, and
then extracted with ethyl acetate (3 ꢂ 30 mL). The combined
organic phases were washed with water (25 mL), dried over
anhydrous sodium sulfate and evaporated to dryness. The crude
product was purified using silica gel column chromatography and
eluted with 12% acetoneehexane.
3234, 1633, 1595, 1580, 1494. 1H NMR (300 MHz, CDCl3)
d 3.87, 3.88,
3.91 (3s, 9H, 3 ꢂ OCH3), 4.15 (br s, 2H, NH2), 6.67 (d, J ¼ 8.7 Hz, 2H,
C3-ArH, C5-ArH), 6.69 (d, J ¼ 8.8 Hz,1H, C50-ArH), 7.35 (d, J ¼ 8.8 Hz,
1H, C60-ArH), 7.55 (d, J ¼ 15.7 Hz,1H, CH]CHCO), 7.90 (d, J ¼ 8.7 Hz,
2H, C2-ArH, C6-ArH), 7.93 (d, J ¼ 15.7 Hz, 1H, CH]CHCO). 13C NMR
(75 MHz, CDCl3)
d 56.1, 60.9, 61.4, 107.6, 113.9, 121.5, 122.5, 123.7,
128.9, 131.0, 138.4, 142.5, 150.9, 153.6, 155.4, 188.6. HRMS-TOF: m/z
[MþH]þ 314.1395 (Calcd for C18H20NO4: 314.1387).
4.2.6. (E)-1-(4-aminophenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-
4.2.1. (E)-1-(3-aminophenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-
1-one (6a)
en-1-one (6f)
Yellow solid. 72%. mp 156e157 ꢁC (160 ꢁC [44]). IR (UATR) cmꢀ1
:
Yellow solid. 57%. mp 68e69 ꢁC. IR (UATR) cmꢀ1: 3448, 3362,
3457, 3361, 3234, 1628, 1604, 1584, 1504. 1H NMR (300 MHz, CDCl3)
3225, 1652, 1621, 1574, 1508. 1H NMR (300 MHz, CDCl3)
d
2.80 (br s,
d
3.91, 3.94 (2s, 9H, 3 ꢂ OCH3), 4.20 (br s, 2H, NH2), 6.72 (d,
2H, NH2), 3.95, 3.96 (2s, 6H, 2 ꢂ OCH3), 6.91 (d, J ¼ 8.3 Hz, 1H, C50-
ArH), 6.95 (d, J ¼ 7.7 Hz,1H, C4-ArH), 7.16 (d, J ¼ 1.4 Hz,1H, C20-ArH),
7.23 (d, J ¼ 8.3 Hz, 1H, C60-ArH), 7.30 (t, J ¼ 7.2 Hz, 1H, C5-ArH), 7.35
(d, J ¼ 15.4 Hz, 1H, CH]CHCO), 7.38 (s, 1H, C2-ArH), 7.42 (d,
J ¼ 7.5 Hz,1H, C6-ArH), 7.75 (d, J ¼ 15.6 Hz,1H, CH]CHCO). 13C NMR
J ¼ 8.5 Hz, 2H, C3-ArH, C5-ArH), 6.87 (s, 2H, C20-ArH, C60-ArH), 7.43
(d, J ¼ 15.5 Hz, 1H, CH]CHCO), 7.72 (d, J ¼ 15.5 Hz, 1H, CH]
CHCO),7.95 (d, J ¼ 8.5 Hz, 2H, C2-ArH, C6-ArH). 13C NMR (75 MHz,
CDCl3) d 56.2, 61.0, 105.5, 113.9, 121.4, 128.6, 130.9, 131.1, 140.1, 143.3,
151.1, 153.5, 188.0. HRMS-TOF: m/z [MþH]þ 314.1390 (Calcd for
(75 MHz, CDCl3)
d
56.0, 110.1, 111.1, 114.8, 119.2, 119.6, 120.3, 123.1,
C18H20NO4: 314.1387).
127.9, 129.4, 139.6, 144.8, 146.0, 149.2, 151.4, 190.8. HRMS-TOF: m/z
[MþNa]þ 306.1102 (Calcd for C17H17NNaO3: 306.1101).
4.3. General procedure for the synthesis of azidochalcones (7)
To cold solution of aminochalcone (3 mmol) in
4.2.2. (E)-1-(3-aminophenyl)-3-(2,3-dimethoxyphenyl)prop-2-en-
1-one (6b)
a
6
HCl:CH3COOH (3:3 mL) at 0 ꢁC, a solution of sodium nitrite
(9 mmol) in water (5 mL) was added. The stirred reaction mixture
was maintained for 15 min and then added dropwise a solution of
sodium azide (9 mmol) in water (5 mL). The reaction mixture was
allowed to stir at room temperature for 0.5 h, then the precipitate
was filtered, washed with cold water and recrystallized from
methanol.
Yellow oil. 41%. IR (UATR) cmꢀ1: 3462, 3367, 3229, 1656, 1623,
1575, 1477. 1H NMR (300 MHz, CDCl3)
d 2.90 (br s, 2H, NH2), 3.86,
3.87 (2s, 6H, 2 ꢂ OCH3), 6.87 (d, J ¼ 7.9 Hz, 1H, C4-ArH), 6.94 (d,
J ¼ 8.1 Hz, 1H, C40-ArH), 7.07 (t, J ¼ 8.0 Hz, 1H, C50-ArH), 7.21e7.28
(m, 2H, C5-ArH, C60-ArH), 7.31 (s, 1H, C2-ArH), 7.37 (d, J ¼ 7.6 Hz,1H,
C6-ArH), 7.53 (d, J ¼ 15.9 Hz, 1H, CH]CHCO), 8.05 (d, J ¼ 15.9 Hz,

72
R. Pingaew et al. / European Journal of Medicinal Chemistry 85 (2014) 65e76
4.3.1. (E)-1-(3-azidophenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-
one (7a)
4.3.6. (E)-1-(4-azidophenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-
en-1-one (7f)
Yellow solid. 77%. mp 75e76 ꢁC. IR (UATR) cmꢀ1: 2105, 1660,
Yellow solid. 90%. mp 70e71 ꢁC. IR (UATR) cmꢀ1: 2122, 1658,
1576, 1511. 1H NMR (300 MHz, CDCl3)
d
3.92, 3.94 (2s, 6H, 2 ꢂ OCH3),
1599, 1579, 1502. 1H NMR (300 MHz, CDCl3)
d 3.92, 3.94 (2s, 6H,
6.89 (d, J ¼ 8.3 Hz, 1H, C50-ArH), 7.13 (d, J ¼ 1.9 Hz, 1H, C20-ArH),
7.19e7.24 (m, 2H, C4-ArH, C60-ArH), 7.31 (d, J ¼ 15.6 Hz, 1H, CH]
CHCO), 7.47 (t, J ¼ 7.9 Hz, 1H, C5-ArH), 7.64 (d, J ¼ 1.9 Hz, 1H, C2-
ArH), 7.74 (d, J ¼ 7.9 Hz, 1H, C6-ArH), 7.75 (d, J ¼ 15.6 Hz, 1H,
3 ꢂ OCH3), 6.88 (s, 2H, C20-ArH, C60-ArH), 7.15 (d, J ¼ 8.1 Hz, 2H, C3-
ArH, C5-ArH), 7.39 (d, J ¼ 15.5 Hz, 1H, CH]CHCO), 7.75 (d,
J ¼ 15.5 Hz,1H, CH]CHCO), 8.05 (d, J ¼ 8.1 Hz, 2H, C2-ArH, C6-ArH).
13C NMR (75 MHz, CDCl3)
d 56.3, 61.0, 105.8, 119.1, 120.9, 130.3,
CH]CHCO). 13C NMR (75 MHz, CDCl3)
d
56.0, 110.1, 111.1, 118.8,
130.5, 134.9, 140.6, 144.7, 145.1, 153.5, 188.7. HRMS-TOF: m/z HRMS-
119.6, 122.9, 123.4, 124.8, 127.6, 130.0, 140.2, 140.9, 145.8, 149.3,
TOF: m/z [MþH]þ 340.1288 (Calcd for C18H18N3O4: 340.1292).
151.7, 189.6. HRMS-TOF: m/z [MþH]þ 310.1200 (Calcd for
C
17H16N3O3: 310.1186).
4.4. General procedure for the synthesis of propynyloxy derivatives
(8aeb)
4.3.2. (E)-1-(3-azidophenyl)-3-(2,3-dimethoxyphenyl)prop-2-en-
1-one (7b)
A propargyl bromide (2.4 mmol) was added to a suspension of
an appropriate coumarin (2 mmol) and potassium carbonate
(4 mmol) in acetone (15 mL). The suspension was heated under
reflux for 2 h. The reaction was allowed to cool and then concen-
trated under reduced pressure. Water (30 mL) was added and
extracted with EtOAc (3 ꢂ 30 mL). The organic extracts were
combined and washed with water (20 mL) and brine (20 mL). The
organic layer was dried over anhydrous sodium sulfate, filtered and
concentrated in vacuo. The crude product was purified by column
chromatography.
Yellow solid. 78%. mp 73e74 ꢁC. IR (UATR) cmꢀ1: 2109, 1663,
1577, 1479. 1H NMR (300 MHz, CDCl3)
d 3.87, 3.88 (2s, 6H,
2 ꢂ OCH3), 6.97 (dd, J ¼ 8.1, 1.3 Hz, 1H, C40-ArH), 7.09 (t, J ¼ 7.9 Hz,
1H, C50-ArH), 7.18e7.28 (m, 2H, C4-ArH, C60-ArH), 7.45 (t, J ¼ 8.0 Hz,
1H, C5-ArH), 7.48 (d, J ¼ 15.8 Hz, 1H, CH]CHCO), 7.65 (t, J ¼ 1.8 Hz,
1H, C2-ArH), 7.75 (d, J ¼ 7.7 Hz, 1H, C6-ArH), 8.09 (d, J ¼ 15.8 Hz, 1H,
CH]CHCO). 13C NMR (75 MHz, CDCl3)
d 55.9, 61.3, 114.5, 118.9,
119.7, 123.1, 123.2, 124.2, 125.0, 128.9, 130.0, 140.0, 140.5, 140.9,
153.3, 189.9. HRMS-TOF: m/z [MþH]þ 310.1191 (Calcd for
1H NMR spectra of 7-(propynyloxy)-2H-chromen-2-one (8a)
[45] and 4-(propynyloxy)-2H-chromen-2-one (8b) [46] were
consistent with those reported in the literatures.
C
17H16N3O3: 310.1186).
4.3.3. (E)-1-(4-azidophenyl)-3-(2,3-dimethoxyphenyl)prop-2-en-
1-one (7c)
4.5. General procedure for the synthesis of chalconeecoumarin
hybrids (9e19)
Orange-yellow solid. 69%. mp 82e83 ꢁC. IR (UATR) cmꢀ1: 2122,
1660, 1601, 1577, 1479. 1H NMR (300 MHz, CDCl3)
d 3.91 (s, 6H,
To a stirred solution of azidochalcone 7 (0.2 mmol) and alkyne 8
(0.22 mmol) in t-BuOH:H2O (3:3 mL), CuSO4$5H2O (0.22 mmol)
and sodium ascorbate (0.5 mmol) was added. The reaction mixture
was stirred at room temperature for 2 h and then concentrated
under reduced pressure. The residue was added water (10 mL) and
extracted with dichloromethane (3 ꢂ 20 mL). The combined
organic phases were washed with water (20 mL), dried over
anhydrous sodium sulfate and evaporated to dryness. The crude
product was purified using silica gel column chromatography and
eluted with methanol:dichloromethane (1:50).
2 ꢂ OCH3), 7.00 (dd, J ¼ 8.0, 1.3 Hz, 1H, C40-ArH), 7.12 (t, J ¼ 8.0 Hz,
1H, C50-ArH), 7.15 (d, J ¼ 8.7 Hz, 2H, C3-ArH, C5-ArH), 7.29 (d,
J ¼ 8.0 Hz,1H, C60-ArH), 7.59 (d, J ¼ 15.9 Hz, 1H, CH]CHCO), 8.07 (d,
J ¼ 8.7 Hz, 2H, C2-ArH, C6-ArH), 8.12 (d, J ¼ 15.9 Hz,1H, CH]CHCO).
13C NMR (75 MHz, CDCl3)
d 55.9, 61.3, 114.3, 119.0, 119.7, 123.2,
124.2, 129.1, 130.5, 135.0, 139.8, 144.6, 149.0, 153.3, 189.1. HRMS-
TOF: m/z [MþH]þ 310.1195 (Calcd for C17H16N3O3: 310.1186).
4.3.4. (E)-1-(3-azidophenyl)-3-(2,3,4-trimethoxyphenyl)prop-2-
en-1-one (7d)
Yellow solid. 93%. mp 65e66 ꢁC. IR (UATR) cmꢀ1: 2107, 1660,
4.5.1. (E)-7-((1-(3-(3-(3,4-dimethoxyphenyl)acryloyl)phenyl)-1H-
1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (9)
1576, 1495. 1H NMR (300 MHz, CDCl3)
d 3.91, 3.94, 3.98 (3s, 9H,
3 ꢂ OCH3), 6.75 (d, J ¼ 8.8 Hz, 1H, C50-ArH), 7.23 (dd, J ¼ 7.8, 1.8 Hz,
1H, C4-ArH), 7.41 (d, J ¼ 8.8 Hz, 1H, C60-ArH), 7.50 (t, J ¼ 7.8 Hz, 1H,
C5-ArH), 7.52 (d, J ¼ 15.8 Hz, 1H, CH]CHCO), 7.68 (t, J ¼ 1.8 Hz, 1H,
C2-ArH), 7.78 (d, J ¼ 7.8 Hz, 1H, C6-ArH), 8.03 (d, J ¼ 15.8 Hz, 1H,
Starting from azidochalcone 7a and alkyne 8a. Yellow solid. 65%.
mp 132e133 ꢁC. IR (UATR) cmꢀ1: 1723, 1660, 1611, 1576, 1509, 1464.
1H NMR (300 MHz, CDCl3)
d
3.92, 3.94 (2s, 6H, 2 ꢂ OCH3), 5.35 (s,
2H, CH2O), 6.25 (d, J ¼ 9.5 Hz, 1H, C3), 6.89 (d, J ¼ 8.3 Hz, 1H, C500-
ArH), 6.92e6.98 (m, 2H, C6 and C8), 7.16 (s, 1H, C200-ArH), 7.25 (d,
J ¼ 8.3 Hz,1H, C600-ArH), 7.38 (d, J ¼ 15.6 Hz,1H, CH]CHCO), 7.39 (d,
J ¼ 8.9 Hz, 2H, C5), 7.62 (d, J ¼ 9.5 Hz, 1H, C4), 7.68 (t, J ¼ 7.9 Hz, 1H,
C50-ArH), 7.82 (d, J ¼ 15.6 Hz,1H, CH]CHCO), 8.01 (d, J ¼ 8.0 Hz, 1H,
C40-ArH), 8.07 (d, J ¼ 7.7 Hz, 1H, C60-ArH), 8.20 (s, 1H, CHN), 8.32 (s,
CH]CHCO). 13C NMR (75 MHz, CDCl3)
d 56.1, 60.9, 61.4, 107.6, 118.8,
120.9, 121.8, 122.9, 124.1, 124.9, 129.9, 140.3, 140.8, 141.0, 142.5,
154.0, 156.0, 189.9. HRMS-TOF: m/z [MþH]þ 340.1299 (Calcd for
C
18H18N3O4: 340.1292).
1H, C20-ArH). 13C NMR (75 MHz, CDCl3)
d 56.1, 62.3, 102.2, 110.3,
4.3.5. (E)-1-(4-azidophenyl)-3-(2,3,4-trimethoxyphenyl)prop-2-
en-1-one (7e)
111.2, 112.7, 113.2, 113.6, 119.0, 120.1, 121.3, 123.6, 124.3, 127.5, 128.7,
129.0, 130.2, 137.3, 140.2, 143.2, 144.1, 146.5, 149.4, 151.9, 155.8,
161.0, 161.2, 188.9. HRMS-TOF: m/z [MþNa]þ 532.1464 (Calcd for
Yellow solid. 93%. mp 115e116 ꢁC. IR (UATR) cmꢀ1: 2126, 1654,
1598, 1584, 1494. 1H NMR (300 MHz, CDCl3)
d
3.92, 3.94, 3.97 (3s,
C29H23N3NaO6: 532.1479).
6H, 3 ꢂ OCH3), 6.75 (d, J ¼ 8.8 Hz, 1H, C50-ArH), 7.15 (d, J ¼ 8.5 Hz,
2H, C3-ArH, C5-ArH), 7.41 (d, J ¼ 8.8 Hz, 1H, C60-ArH), 7.56 (d,
J ¼ 15.7 Hz, 1H, CH]CHCO), 8.02 (d, J ¼ 15.7 Hz, 1H, CH]CHCO),
8.06 (d, J ¼ 8.5 Hz, 2H, C2-ArH, C6-ArH). 13C NMR (75 MHz, CDCl3)
4.5.2. (E)-7-((1-(3-(3-(2,3-dimethoxyphenyl)acryloyl)phenyl)-1H-
1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (10)
Starting from azidochalcone 7b and alkyne 8a. Pale yellow solid.
d
56.1, 60.9, 61.4, 107.6, 119.0, 120.9, 122.0, 124.0, 130.4, 135.3, 140.3,
61%. mp 131e132 ꢁC. IR (UATR) cmꢀ1: 1726, 1663, 1610, 1587, 1576,
142.6, 144.4, 153.9, 155.9, 189.1. HRMS-TOF: m/z HRMS-TOF: m/z
1506, 1478. 1H NMR (300 MHz, CDCl3)
(s, 2H, CH2O), 6.26 (d, J ¼ 9.5 Hz, 1H, C3), 6.93e7.00 (m, 2H, C6, C8
d
3.88 (s, 6H, 2 ꢂ OCH3), 5.35
[MþH]þ 340.1283 (Calcd for C18H18N3O4: 340.1292).

R. Pingaew et al. / European Journal of Medicinal Chemistry 85 (2014) 65e76
73
and C400-ArH), 7.10 (t, J ¼ 7.9 Hz, 1H, C500-ArH), 7.28 (dd, J ¼ 7.9,
1.2 Hz, 1H, C600-ArH), 7.39 (d, J ¼ 9.3 Hz, 1H, C5), 7.55e7.71 (m, 3H,
C4, C50-ArH and CH]CHCO), 8.05e8.14 (m, 2H, C40-ArH and C60-
ArH), 8.18 (d, J ¼ 15.9 Hz, 1H, CH]CHCO), 8.25 (s, 1H, CHN), 8.36 (t,
J ¼ 15.5 Hz, 1H, CH]CHCO), 7.99 (d, J ¼ 9.6 Hz, 1H, C4), 8.14 (d,
J ¼ 8.4 Hz, 2H, C30-ArH and C50-ArH), 8.39 (d, J ¼ 8.4 Hz, 2H, C20-ArH
and C60-ArH), 9.16 (s, 1H, CHN). 13C NMR (75 MHz, DMSO-d6)
d 56.7,
60.6, 62.0, 102.1, 107.2, 113.2, 113.3, 113.4, 120.5, 121.5, 123.8, 126.9,
130.1, 130.6, 130.9, 137.9, 139.9, 140.4, 144.0, 144.7, 145.6, 153.6,
155.8, 160.7, 161.5, 188.5. HRMS-TOF: m/z [MþNa]þ 562.1569 (Calcd
for C30H25N3NaO7: 562.1585).
J ¼ 1.8 Hz, 1H, C20-ArH). 13C NMR (75 MHz, CDCl3)
d 56.0, 61.4, 62.3,
102.2, 112.7, 113.2, 113.6, 114.7, 119.7, 120.2, 121.2, 122.8, 124.3, 124.4,
128.7, 128.8, 129.0, 130.3, 137.3, 139.9, 141.2, 143.2, 144.1, 149.2,
153.3, 155.8, 161.0, 161.2, 189.3. HRMS-TOF: m/z [MþNa]þ 532.1488
(Calcd for C29H23N3NaO6: 532.1479).
4.5.7. (E)-4-((1-(3-(3-(3,4-dimethoxyphenyl)acryloyl)phenyl)-1H-
1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (15)
4.5.3. (E)-7-((1-(4-(3-(2,3-dimethoxyphenyl)acryloyl)phenyl)-1H-
1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (11)
Starting from azidochalcone 7a and alkyne 8b. Yellow solid. 62%.
mp 201e202 ꢁC. IR (UATR) cmꢀ1: 1713, 1660, 1622, 1609, 1568, 1510,
Starting from azidochalcone 7c and alkyne 8a. White solid. 80%.
1454. 1H NMR (300 MHz, CDCl3)
d
3.96, 3.98 (2s, 6H, 2 ꢂ OCH3), 5.47
mp 228e229 ꢁC. IR (UATR) cmꢀ1: 1741, 1659, 1621, 1608, 1577, 1479.
(s, 2H, CH2O), 5.94 (s, 1H, C3), 6.93 (d, J ¼ 8.3 Hz, 1H, C500-ArH), 7.20
(s, 1H, C200-ArH), 7.24e7.37 (m, 3H, C6, C8 and C600-ArH), 7.44 (d,
J ¼ 15.6 Hz, 1H, CH]CHCO), 7.57 (t, J ¼ 8.4 Hz, 1H, C7), 7.73 (t,
J ¼ 7.9 Hz, 1H, C50-ArH), 7.87 (d, J ¼ 7.9 Hz, 1H, C5), 7.87 (d,
J ¼ 15.8 Hz,1H, CH]CHCO), 8.03 (d, J ¼ 8.0 Hz, 1H, C40-ArH), 8.13 (d,
J ¼ 7.7 Hz, 1H, C60-ArH), 8.33 (s, 1H, CHN), 8.39 (s, 1H, C20-ArH). 13C
1H NMR (300 MHz, DMSO-d6)
d
3.82, 3.85 (2s, 6H, 2 ꢂ OCH3), 5.40
(s, 2H, CH2O), 6.32 (d, J ¼ 9.5 Hz, 1H, C3), 7.06 (dd, J ¼ 8.6, 2.4 Hz,1H,
C6), 7.15e7.24 (m, 3H, C8 and C400-ArH, C500-ArH), 7.67 (d, J ¼ 8.8 Hz,
2H, C5 and C600-ArH), 7.93e8.08 (m, 3H, C4 and CH]CHCO), 8.15 (d,
J ¼ 8.7 Hz, 2H, C30-ArH and C50-ArH), 8.37 (d, J ¼ 8.7 Hz, 2H, C20-ArH
and C60-ArH), 9.17 (s, 1H, CHN). 13C NMR (75 MHz, DMSO-d6)
d
56.3,
NMR (75 MHz, CDCl3) d 56.1, 62.6, 91.3, 110.2, 111.2, 115.4, 116.8,
61.5, 62.1, 102.1, 113.2, 113.3, 113.4, 115.8, 119.7, 120.5, 123.1, 123.8,
124.8, 128.6, 130.1, 130.9, 137.8, 139.0, 139.9, 144.0, 144.7, 148.9,
153.3, 155.8, 160.7, 161.5, 188.6. HRMS-TOF: m/z [MþNa]þ 532.1480
(Calcd for C29H23N3NaO6: 532.1479).
118.9,120.1, 121.8, 123.1,123.7,124.0,124.4,127.4,128.8,130.3,132.6,
137.2, 140.2, 142.6, 146.6, 149.4, 151.9, 153.4, 162.6, 164.9, 188.8.
HRMS-TOF: m/z [MþNa]þ 532.1458 (Calcd for C29H23N3NaO6:
532.1479).
4.5.4. (E)-7-((1-(3-(3-(2,3,4-trimethoxyphenyl)acryloyl)phenyl)-
1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (12)
4.5.8. (E)-4-((1-(3-(3-(2,3-dimethoxyphenyl)acryloyl)phenyl)-1H-
1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (16)
Starting from azidochalcone 7d and alkyne 8a. Yellow solid. 74%.
Starting from azidochalcone 7b and alkyne 8b. White solid. 88%.
mp 120e121 ꢁC. IR (UATR) cmꢀ1: 1725, 1657, 1611, 1578, 1494. 1H
mp 137e138 ꢁC. IR (UATR) cmꢀ1: 1713, 1663, 1622, 1608, 1587, 1575,
NMR (300 MHz, CDCl3)
d
3.92, 3.95, 3.99 (3s, 9H, 3 ꢂ OCH3), 5.40 (s,
1478. 1H NMR (300 MHz, CDCl3)
d
3.92 (s, 6H, 2 ꢂ OCH3), 5.47 (s, 2H,
2H, CH2O), 6.30 (d, J ¼ 9.5 Hz, 1H, C3), 6.76 (d, J ¼ 8.8 Hz, 1H, C500-
ArH), 6.97e7.03 (m, 2H, C6 and C8), 7.41e7.47 (m, 2H, C5 and C600-
ArH), 7.58 (d, J ¼ 15.8 Hz,1H, CH]CHCO), 7.67 (d, J ¼ 9.3 Hz, 2H, C4),
7.71 (t, J ¼ 7.9 Hz, 1H, C50-ArH), 8.02e8.10 (m, 2H, C40-ArH and C60-
ArH), 8.09 (d, J ¼ 15.8 Hz, 1H, CH]CHCO), 8.23 (s, 1H, CHN), 8.36 (t,
CH2O), 5.94 (s, 1H, C3), 7.02 (dd, J ¼ 8.1, 1.2 Hz, 1H, C400-ArH), 7.14 (t,
J ¼ 8.0 Hz, 1H, C500-ArH), 7.25e7.37 (m, 3H, C6, C8 and C600-ArH),
7.57 (dt, J ¼ 7.9, 1.5 Hz, 1H, C7), 7.63 (d, J ¼ 15.6 Hz, 1H, CH]CHCO),
7.74 (t, J ¼ 7.9 Hz, 1H, C50-ArH), 7.85 (dd, J ¼ 7.9, 1.4 Hz, 1H, C5),
8.08e8.17 (m, 2H, C40-ArH and C60-ArH), 8.19 (d, J ¼ 15.9 Hz, 1H,
CH]CHCO), 8.32 (t, J ¼ 1.7 Hz, 1H, CHN), 8.38 (s, 1H, C20-ArH). 13C
J ¼ 1.7 Hz, 1H, C20-ArH). 13C NMR (75 MHz, CDCl3)
d 56.1, 61.0, 61.5,
62.3, 102.2, 107.7, 112.7, 113.2, 113.6, 120.1, 120.4, 121.2, 121.6, 124.2,
128.7, 129.0, 130.2, 137.2, 140.3, 141.6, 142.5, 143.3, 144.1, 154.0,
155.8, 156.3, 161.0, 161.2, 189.3. HRMS-TOF: m/z [MþNa]þ 562.1585
(Calcd for C30H25N3NaO7: 562.1585).
NMR (75 MHz, CDCl3) d 55.9, 61.4, 62.6, 91.3, 114.7, 115.4, 116.8,
119.7, 120.2, 121.7, 122.7, 123.2, 124.0, 124.3, 124.5, 128.7, 128.9,
130.3, 132.6, 137.2, 140.0, 141.2, 149.2, 153.3, 153.4, 162.6, 164.9,
189.3. HRMS-TOF: m/z [MþNa]þ 532.1476 (Calcd for C29H23N3NaO6:
532.1479).
4.5.5. (E)-7-((1-(4-(3-(2,3,4-trimethoxyphenyl)acryloyl)phenyl)-
1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (13)
4.5.9. (E)-4-((1-(3-(3-(2,3,4-trimethoxyphenyl)acryloyl)phenyl)-
1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (17)
Starting from azidochalcone 7e and alkyne 8a. Yellow solid. 50%.
mp 221e222 ꢁC. IR (UATR) cmꢀ1: 1737, 1658, 1623, 1605, 1499, 1464.
Starting from azidochalcone 7d and alkyne 8b. Yellow solid. 79%.
1H NMR (300 MHz, CDCl3)
d
3.92, 3.94, 3.98 (3s, 9H, 3 ꢂ OCH3), 5.39
mp 198e199 ꢁC. IR (UATR) cmꢀ1: 1713, 1660, 1622, 1609, 1579, 1565,
(s, 2H, CH2O), 6.30 (d, J ¼ 9.6 Hz, 1H, C3), 6.76 (d, J ¼ 8.8 Hz, 1H, C500-
ArH), 6.96e7.02 (m, 2H, C6 and C8), 7.40e7.47 (m, 2H, C5 and C600-
ArH), 7.58 (d, J ¼ 15.8 Hz,1H, CH]CHCO), 7.67 (d, J ¼ 9.5 Hz,1H, C4),
7.93 (d, J ¼ 8.8 Hz, 2H, C30-ArH and C50-ArH), 8.06 (d, J ¼ 15.8 Hz,1H,
CH]CHCO), 8.20 (d, J ¼ 8.8 Hz, 2H, C20-ArH and C60-ArH), 8.22 (s,
1494, 1464. 1H NMR (300 MHz, CDCl3)
d 3.93, 3.95, 3.97 (3s, 9H,
3 ꢂ OCH3), 5.47 (s, 2H, CH2O), 5.94 (s, 1H, C3), 6.76 (d, J ¼ 8.8 Hz, 1H,
C500-ArH), 7.28 (dt, J ¼ 7.6, 1.0 Hz, 1H, C6), 7.35 (d, J ¼ 8.4 Hz, 1H, C8),
7.44 (d, J ¼ 8.8 Hz, 1H, C600-ArH), 7.58 (dt, J ¼ 7.8, 1.6 Hz, 1H, C7), 7.59
(d, J ¼ 15.8 Hz, 1H, CH]CHCO), 7.73 (t, J ¼ 7.9 Hz, 1H, C50-ArH), 7.85
(dd, J ¼ 7.9, 1.5 Hz, 1H, C5), 8.09 (d, J ¼ 15.8 Hz, 1H, CH]CHCO),
8.08e8.16 (m, 2H, C40-ArH and C60-ArH), 8.32 (s, 1H, CHN), 8.38 (t,
1H, CHN). 13C NMR (75 MHz, CDCl3)
d 56.1, 60.9, 61.4, 62.2, 102.2,
107.7, 112.7, 113.2, 113.7, 120.2, 120.6, 121.0, 121.7, 124.2, 129.0, 130.2,
130.6, 138.8, 139.4, 141.3, 142.5, 143.2, 144.2, 154.0, 155.8, 156.2,
161.0, 161.2, 189.3. HRMS-TOF: m/z [MþH]þ 540.1767 (Calcd for
J ¼ 1.7 Hz, 1H, C20-ArH). 13C NMR (75 MHz, CDCl3)
d 56.1, 61.0, 61.5,
62.6, 91.3, 107.7, 115.4, 116.8, 120.2, 120.3, 121.6, 121.7, 123.2, 124.0,
124.2, 124.3, 128.9, 130.3, 132.6, 137.1, 140.3, 141.7, 142.5, 153.4,
154.0, 156.3, 162.6, 165.0, 189.3. HRMS-TOF: m/z [MþNa]þ 562.1591
(Calcd for C30H25N3NaO7: 562.1585).
C
30H26N3O7: 540.1765).
4.5.6. (E)-7-((1-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)-
1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (14)
Starting from azidochalcone 7f and alkyne 8a. Yellow solid. 87%.
4.5.10. (E)-4-((1-(4-(3-(2,3,4-trimethoxyphenyl)acryloyl)phenyl)-
1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (18)
mp 223e224 ꢁC. IR (UATR) cmꢀ1: 1705, 1662, 1606, 1580, 1502. 1H
NMR (300 MHz, DMSO-d6)
d
3.72, 3.87 (2s, 9H, 3 ꢂ OCH3), 5.40 (s,
Starting from azidochalcone 7e and alkyne 8b. Yellow solid. 70%.
2H, CH2O), 6.32 (d, J ¼ 9.5 Hz, 1H, C3), 7.07 (d, J ¼ 8.6 Hz, 1H, C6),
7.22 (s, 1H, C8), 7.26 (s, 2H, C200-ArH and C600-ArH), 7.67 (d,
J ¼ 8.6 Hz, 1H, C5), 7.75 (d, J ¼ 15.5 Hz, 1H, CH]CHCO), 7.95 (d,
mp 151e152 ꢁC. IR (UATR) cmꢀ1: 1717, 1660, 1621, 1606, 1563, 1494,
1464. 1H NMR (300 MHz, CDCl3)
d
3.88, 3.91, 3.95 (3s, 9H, 3 ꢂ OCH3),
5.43 (s, 2H, CH2O), 5.90 (s, 1H, C3), 6.73 (d, J ¼ 8.8 Hz, 1H, C500-ArH),

74
R. Pingaew et al. / European Journal of Medicinal Chemistry 85 (2014) 65e76
7.25 (t, J ¼ 7.2 Hz, 1H, C6), 7.31 (d, J ¼ 8.3 Hz, 2H, C8), 7.40 (d,
J ¼ 8.8 Hz, 1H, C600-ArH), 7.54 (t, J ¼ 7.3 Hz, 1H, C7), 7.55 (d,
J ¼ 15.8 Hz, 1H, CH]CHCO), 7.80 (d, J ¼ 7.8 Hz, 1H, C5), 7.92 (d,
J ¼ 8.6 Hz, 2H, C30-ArH and C50-ArH), 8.03 (d, J ¼ 15.8 Hz, 1H, CH]
CHCO), 8.18 (d, J ¼ 8.6 Hz, 2H, C20-ArH and C60-ArH), 8.29 (s, 1H,
and untreated conditions (CPMU) as shown by the following
equation;
% parasite growth ¼ CPMT=CPMU ꢂ 100
CHN). 13C NMR (75 MHz, CDCl3)
d 56.1, 61.0, 61.5, 62.5, 91.4, 107.7,
4.8. Cytotoxicity assay: primate cell line (Vero)
115.4,116.8,120.3,120.6,121.7,123.1,124.0,124.2,130.3,132.7,139.0,
139.3, 141.4, 153.4, 154.0, 156.2, 162.5, 164.9, 189.3. HRMS-TOF: m/z
[MþH]þ 540.1776 (Calcd for C30H26N3O7: 540.1765).
The cytotoxicity assay was performed using the Green Fluo-
rescent Protein (GFP) detection method [52]. The GFP-expressing
Vero cell line was generated in-house by stably transfecting the
African green monkey kidney cell line (Vero, ATCC CCL-81), with
pEGFP-N1 plasmid (Clontech). The cell line was maintained in a
minimal essential medium supplemented with 10% heat-
4.5.11. (E)-4-((1-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)-
1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (19)
Starting from azidochalcone 7f and alkyne 8b. Yellow solid. 86%.
mp 229e230 ꢁC. IR (UATR) cmꢀ1: 1712,1687,1659,1623,1606, 1583,
inactivated fetal bovine serum, 2 mM L-glutamine, 1 mM so-
1505, 1455. 1H NMR (300 MHz, DMSO-d6)
d 3.72, 3.88 (2s, 9H,
dium pyruvate, 1.5 g/L sodium bicarbonate and 0.8 mg/mL
geneticin, at 37 ꢁC in a humidified incubator with 5% CO2. The
3 ꢂ OCH3), 5.56 (s, 2H, CH2O), 6.22 (s, 1H, C3), 7.27 (s, 2H, C200-ArH
and C600-ArH), 7.36 (t, J ¼ 7.7 Hz, 1H, C6), 7.42 (d, J ¼ 8.3 Hz, 1H, C8),
7.67 (dt, J ¼ 8.3, 1.4 Hz, 1H, C7), 7.76 (d, J ¼ 15.5 Hz, 1H, CH]CHCO),
7.85 (dd, J ¼ 7.8, 1.2 Hz, 1H, C5), 7.96 (d, J ¼ 15.6 Hz, 1H, CH]CHCO),
8.18 (d, J ¼ 8.6 Hz, 2H, C30-ArH and C50-ArH), 8.40 (d, J ¼ 8.7 Hz, 2H,
C20-ArH and C60-ArH), 9.25 (s, 1H, CHN). 13C NMR (75 MHz, DMSO-
assay was carried out by adding 45
mL of cell suspension at
3.3 ꢂ 104 cells/mL to each well of 384-well plates containing 5
mL
of test compounds previously diluted in 0.5% DMSO, and then
incubating for 4 days at 37 ꢁC incubator with 5% CO2. Fluores-
cence signals were measured by using SpectraMax M5 micro-
plate reader (Molecular Devices, USA) in the bottom reading
mode with excitation and emission wavelengths of 485 and
535 nm, respectively. Fluorescence signal at day 4 was subtracted
with background fluorescence at day 0. IC50 values were derived
from doseeresponse curves, using 6 concentrations of 3-fold
serially diluted samples, by the SOFTMax Pro software (Molecu-
lar device). Ellipticine and 0.5% DMSO were used as a positive
and a negative control, respectively.
d6)
d 56.7, 60.6, 63.2, 92.0, 107.2, 115.5, 116.9, 120.6, 121.5, 123.5,
124.1, 124.7, 130.6, 130.9, 133.4, 138.0, 139.9, 143.1, 145.6, 153.3,
153.6, 162.0, 164.8, 188.5. HRMS-TOF: m/z [MþNa]þ 562.1581 (Calcd
for C30H25N3NaO7: 562.1585).
4.6. Cytotoxic assay: cancer cell lines
The cells suspended in the corresponding culture medium were
inoculated in 96-well microtiter plates (Corning Inc., NY, USA) at a
density of 10,000e20,000 cells per well, and incubated for 24 h at
37 ꢁC in a humidified atmosphere with 95% air and 5% CO2. An equal
volume of additional medium containing either the serial dilutions
of the test compounds, positive control (etoposide and/or doxoru-
bicin), or negative control (DMSO) was added to the desired final
concentrations, and the microtiter plates were further incubated
for an additional 48 h. The number of surviving cells in each well
was determined using MTT assay [47,48] (for adherent cells:
HuCCA-1, HepG2, and A549 cells) and XTT assay [49] (for sus-
pended cells: MOLT-3 cells). The IC50 value is defined as the drug (or
compound) concentration that inhibits cell growth by 50% (relative
to negative control).
4.9. Molecular docking
Molecular docking was performed to investigate the binding
modalities of ligands toward possible targets comprising of
falcipain-2 (PDB id 3BPF), a cysteine protease from P. falciparum, as
well as a- and b-tubulin complexes (PDB id 1SA0) from Bos taurus.
Protein structures were prepared for docking by adding essential
hydrogen atoms and modeling missing side chains using the WHAT
IF web server version 10.1a [53]. Furthermore, non-polar hydrogen
atoms were merged, Gasteiger atomic charges were assigned, and
atom type of receptors were specified using AutoDock Tools version
1.5.6. [54,55]. Chalconeetriazoleecoumarin ligands were con-
structed using Marvin Sketch version 6.1.4 [56] and geometrically
optimized with Gaussian 09 [57] at the B3LYP/6-31G(d) level of
theory. Ligand structures were prepared for docking by merging
non-polar hydrogen atoms and defining rotatable bonds using
AutoDock Tools version 1.5.6. Partial atomic charges calculated by
Gaussian 09 were assigned to ligands for further use in the docking
process. A grid box size of 25.0, 25.0, 25.0 Å was generated and
allocated at the center of the receptor binding site using x, y and z
coordinates of ꢀ91.5906, 5.0925 and ꢀ21.4080 for falcipain-2 and
4.7. Antimalarial assay: radioisotope techniques
P. falciparum (K1, multidrug resistant strain) was cultivated
in vitro conditions, according to Trager & Jensen (1976) [50], in
RPMI 1640 medium containing 20 mM HEPES (N-2-
hydroxyethylpiperazine-N0-2-ethanesulfonic
acid),
32
mM
NaHCO3 and 10% heat activated human serum with 3% erythro-
cytes, in humidified 37 ꢁC incubator with 3% CO2. The culture was
passaged with fresh mixture of erythrocytes and medium for every
day to maintain cell growth. Quantitative assessment of antima-
larial activity in vitro was determined by microculture radioisotope
techniques based upon the methods described by Desjardins et al.
118.4155, 89.6890 and 6.2141 for
a- and b-tubulin complexes.
Molecular docking simulations were performed using AutoDock
Vina as part of the PyRx 0.8 software [58]. Co-crystallized ligands
were re-docked as validation of the docking protocol. Docked
structures were visualized using PyMOL [59]. Two-dimensional
schematic representation of protein-ligand interaction was gener-
ated using PoseViewWeb version 1.97.0 [60].
(1979) [51]. Briefly, a mixture of 200
parasitemia at the early ring stage was pre-exposed to 25
medium containing a test sample dissolved in 1% DMSO (0.1% final
mL of 1.5% erythrocytes with 1%
mL of the
concentration) for 24 h. Subsequently, 25
mL of [3H] hypoxanthine
(Amersham, USA) in culture medium (0.5
mCi) was added to each
Acknowledgments
well and the plates were incubated for an additional 24 h. Levels of
incorporated radioactive labeled hypoxanthine, indicating parasite
growth, were determined using the Top Count microplate scintil-
lation counter (Packard, USA). The percentage of parasite growth
was calculated using the signal count per minute of treated (CPMT)
This project is supported by the research grants from Srina-
kharinwirot University under the Government Budget (B.E. 2559).
We would like to thank the Thailand Research Fund, the office of
the Higher Education Commission, and Srinakharinwirot University

R. Pingaew et al. / European Journal of Medicinal Chemistry 85 (2014) 65e76
75
[25] R. Pingaew, S. Prachayasittikul, S. Ruchirawat, V. Prachayasittikul, Synthesis
and cytotoxicity of novel 4-(4-(substituted)-1H-1,2,3-triazol-1-yl)-N-phene-
thylbenzenesulfonamides, Med. Chem. Res. 23 (2014) 1768e1780.
[26] E.M. Guantai, K. Ncokazi, T.J. Egan, J. Gut, P.J. Rosenthal, P.J. Smith, K. Chibale,
Design, synthesis and in vitro antimalarial evaluation of triazole-linked chal-
cone and dienone hybrid compounds, Bioorg. Med. Chem. 18 (2010)
8243e8256.
(Grant No. MRG5680001) for financial support. Great supports
from the office of the Higher Education Commission and Mahidol
University under the National Research Universities Initiative are
appreciated. We are also indebted to Chulabhorn Research Institute
for recording mass spectra and bioactivity testing.
[27] K.V. Sashidhara, S.R. Avula, G.R. Palnati, S.V. Singh, K. Srivastava, S.K. Puri,
J.K. Saxena, Synthesis and in vitro evaluation of new chloroquine-chalcone
hybrids against chloroquine-resistant strain of Plasmodium falciparum, Bio-
org. Med. Chem. Lett. 22 (2012) 5455e5459.
Appendix A. Supplementary data
[28] K.V. Sashidhara, M. Kumar, R.K. Modukuri, R.K. Srivastava, A. Soni,
K. Srivastava, S.V. Singh, J.K. Saxena, H.M. Gauniyal, S.K. Puri, Antiplasmodial
activity of novel keto-enamine chalcone-chloroquine based hybrid pharma-
cophores, Bioorg. Med. Chem. 20 (2012) 2971e2981.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.07.087.
[29] A. Kamal, S. Prabhakar, M.J. Ramaiah, P.V. Reddy, Ch.R. Reddy, A. Mallareddy,
N. Shankaraiah, T.L.N. Reddy, S.N.C.V.L. Pushpavalli, M. Pal-Bhadra, Synthesis
and anticancer activity of chalcone-pyrrolobenzodiazepine conjugates linked
via 1,2,3-triazole ring side-armed with alkane spacers, Eur. J. Med. Chem. 46
(2011) 3820e3831.
[30] M. Abdel-Aziz, S.-E. Park, G.E.-D.A.A. Abuo-Rahma, M.A. Sayed, Y. Kwon, Novel
N-4-piperazinyl-ciprofloxacin-chalcone hybrids: synthesis, physicochemical
properties, anticancer and topoisomerase I and II inhibitory activity, Eur. J.
Med. Chem. 69 (2013) 427e438.
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