Skeletal hybridization and PfRIO-2 kinase modeling for synthesis of α-pyrone analogs as anti-malarial agent

Article abstract of DOI:10.1016/j.ejmech.2013.10.028

The pharmacophoric hybridization and computational design approach were applied to generate a novel series of α-pyrone analogs as plausible anti-malarial lead candidate. A putative active site in flexible loop close to wing-helix domain of PfRIO2 kinase was explored computationally to understand the molecular basis of ligand binding. All the synthesized molecules (3a-g) exhibited in vitro antimalarial activity. Oxidative stress induced by 3a-d were calculated and found to be significantly higher in case of 3b. Therefore, 3b, which shown most significant result was identified as promising lead for further SAR study to develop potent anti-malarials.

Full text of DOI:10.1016/j.ejmech.2013.10.028

European Journal of Medicinal Chemistry 70 (2013) 607e612
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Skeletal hybridization and PfRIO-2 kinase modeling for synthesis of
-pyrone analogs as anti-malarial agent
a
Afsana Parveen a, Arnish Chakraborty b, Ananda Kumar Konreddy a,
**
,
a,
*
Harapriya Chakravartya, Ashoke Sharon a, Vishal Trivedi b , Chandralata Bal
a Department of Applied Chemistry, Birla Institute of Technology, Mesra, Ranchi 835215, India
b Malaria Research Group, Department of Biotechnology, Indian Institute of Technology, Guwahati 781039, Assam, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The pharmacophoric hybridization and computational design approach were applied to generate a novel
series of -pyrone analogs as plausible anti-malarial lead candidate. A putative active site in flexible loop
Received 13 July 2013
Received in revised form
19 September 2013
Accepted 11 October 2013
Available online 23 October 2013
a
close to wing-helix domain of PfRIO2 kinase was explored computationally to understand the molecular
basis of ligand binding. All the synthesized molecules (3aeg) exhibited in vitro antimalarial activity.
Oxidative stress induced by 3aed were calculated and found to be significantly higher in case of 3b.
Therefore, 3b, which shown most significant result was identified as promising lead for further SAR study
to develop potent anti-malarials.
Keywords:
Anti-malarial
Ó 2013 Elsevier Masson SAS. All rights reserved.
Pyrone analogs
Plasmodium falciparum
RIO2 kinase
Molecular modeling
Induced fit docking
1. Introduction
defined ATP binding pocket and is significantly different than the
human RIO-2 kinase [4]. Further, the absence of activation loop and
Malaria is a protozoal parasitic disease that causes millions of
death worldwide every year. In addition, the treatment is becoming
more and more difficult because of emergence of drug-resistant
malaria. Artemisinin combination therapy (ACT) has been useful
over past decades [1], but the recent emergence of ACT resistance
[2] provides strong motivation to discover new potential antima-
larial drugs. Parasite growth and ability to maintains stress is linked
to the down-stream signaling and ribosome biogenesis [3]. RIO like
kinase, RIO-1 (PFL1490w) and RIO-2 kinase (PFD0975w) are pre-
sent in the malaria kinome with uncharacterized function (www.
plasmodb.org). Structural characterization of PFD0975w, the RIO-
2 kinase of Plasmodium Falciparum (PfRIO-2 kinase) indicates N-
terminal DNA binding winged helix domain (1e84), a linker region
(85e147) and C-terminal kinase domain (148e275). It has a well
distinguished structural features of PfRIO-2 kinase provides an
opportunity to explore it as a new drug target [4]. Recently, natu-
rally occurring bioactive molecules and selected heterocycles were
screened against PfRIO-2 kinase, which allow us to delineate
pharmacological points required for inhibitor development against
PfRIO-2 kinase [5,6].
Febrifugine (I, Fig. 1), a natural product with high anti-malarial
activity [7]. was isolated in 1947 from the roots of a Chinese
shrub, Chang Shan. The 4-quinazolinone moiety, 10-amino and C-20
as well as C-300O-functionalities are crucial for the anti-malarial
activity of febrifugine [8]. Argentilactone (II, Fig. 1), isolated from
the roots and the leaves of Raimondia cf. monoica was found to have
ED50 of 0.1
mg/mL against Plasmodium falciparum (P. falciparum) [9].
It was the first report of
a
-pyrone derivative having anti-malarial
activity. However, due to toxicity [9,10] both these molecules
couldn’t develop as a clinical drug. Recently, 4(1H)-quinolone ester
derivatives (III, Fig. 1) with two close keto groups have been
considered as promising anti-malarials needing further SAR and
optimization [11]. In our ongoing anti-malarial lead identification
project, we intended to apply hybridization of the identified
Abbreviations: RIO2 kinase, right open reading frame 2 kinase; PfRIO2 kinase,
Plasmodium falciparum RIO2 kinase; ATP, adenosine triphosphate; DNA, deoxy-
ribonucleic acid; w-HTH, wing helix domain.
* Corresponding author. Tel.: þ91 651 2276531; fax: þ91 651 2275401.
** Corresponding author. Tel.: þ91 361 2582217; fax: þ91 361 258 2249.
pharmacophores (Fig. 1) and generated a novel
a-pyrone skeleton
E-mail
addresses:
vtrivedi@iitg.ernet.in,
Vishalash_1999@yahoo.com
(IV, Fig. 1). In the current work, we carried out detailed in-silico
(V. Trivedi), chandralata.bal@gmail.com, cbal@bitmesra.ac.in (C. Bal).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.10.028

608
A. Parveen et al. / European Journal of Medicinal Chemistry 70 (2013) 607e612
Fig. 1. Structures of reported potent antimalarials (IeIII), identified pharmacophores (red) for hybridization and newly generated hybridized skeleton (blue). (For interpretation of
the references to color in this figure legend, the reader is referred to the web version of this article.)
studies (modeling, docking and binding mode analysis) of our
newly generated -pyrone derivatives (IV, Fig. 1) to analyze
whether they could be potential antimalarials exploiting PfRIO2
kinase. The synthesized molecules were evaluated for their anti-
malarial potential. One lead molecule of this novel class was
identified for further optimization and mode of action study.
S1, S2, S3 and S4 (Fig. 2a). The S1 is found within the flexible loop
region and close to DNA binding region (wing helix Domain). The
S1 site comprised of E104, S105, D106, I107, K121, L125, R127, L163,
P174, S187, I189, G191, P193, N227, N230, K232, I242, D243, P245.
The S2 subsite is the major groove and known for ATP binding and
contains major residues K2, D4, I5, F8, D79, F80, L83, I95, Y108,
L120, I122, and M183. The subsite S3 found near to C-terminal was
surrounded by S129, F130, R131, I133, N135, Y139, G141, and K142.
The amino acid residues L250, R251, H252, V253, A255, K256, P282
formed the S4 subsite. The S3 and S4 are very small and close to
surface, which may not be suitable for inhibitor binding. Since the
active site is not yet identified, we studied the ligand interactions to
S1 and S2 sites of PfRIO2. The proteineligand interaction study was
carried out with two complex models of the protein. The 1st
complex comprised both ATP and DNA, while in the 2nd complex
only DNA was present. Therefore, in the 2nd complex, ligand can
a
2. Results and discussion
2.1. Molecular modeling
Recently, we developed and explored the 3D structural aspect of
PfRIO2 kinase [4,12] as plausible novel anti-malarial drug target to
support our anti-malarial drug discovery program. In the present
study, the binding sites were identified using SiteMap module of
Schrödinger Suite. Four binding sites were identified and named as
Fig. 2. a) The SiteMap analysis of PfRIO2 kinase showing the four locations as plausible ligand binding site. b) The surface diagram showing the binding of 3b into S1 site. c) The
docked pose of 3b in the S1 site. d) Ligandereceptor interaction diagram showing the major hot spot residues of flexible loop interacting with 3b.

A. Parveen et al. / European Journal of Medicinal Chemistry 70 (2013) 607e612
609
utilize the full site including the space of ATP. The ligand structures
were built using Maestro interface of and their conformations were
optimized by LigPrep. The optimized ligands were docked by
default protocols to S1 and S2 sites of the above mentioned two
types of PfRIO2 complex using Glide [13]. However the standard
docking results didn’t show significant interactions and binding
energy score. The poor results obtained may be due to the
requirement of additional enzyme flexibility within the site. Thus
the induced fit docking (IFD) protocols [14] were implemented to
conduct the docking experiment followed by side chain refinement
using Prime. The prime refinement ensures protein side chain
flexibility according to ligand fitting into the active site. This was
necessary because the actual conformation of the active site in
PfRIO2 is not yet known. When the receptor flexibility was
considered, the ligandeprotein interactions were found significant
in S1 site. It is interesting to note that the molecular docking and
interaction profile were found better in the 1st complex model.
Therefore, all the docking experiments were conducted on this
complex using S1 active site located in the flexible loop region.
Fig. 2b is a surface diagram showing binding of 3b (the most active
compound) in the S1 site. Docked pose of 3b (Fig. 2c) was created to
have a more closer look into its binding mode into the flexible loop
region near to the DNA binding region. Ligandereceptor in-
teractions were studied in detail and shown in Fig. 2d. This analysis
highlighted the major hot spot residues in the flexible loop (hy-
drophobic: Tyr139, Leu151, Trp148, Tyr31, Ile133, Phe130; acidic:
Asp 138; polar/hydrophilic: Ser129, Thr132, Asn136, Asn135; basic:
Arg137, Arg131, Lys142, His29) interacting with 3b. Among them
interestingly the same were found significant in biological
screening. In absence of any ligand or inhibitor reported for PfRIO2
kinase, chloroquine, a potent antimalarial was selected for possible
comparison. The poor binding score (Table 1) of chloroquine with
PfRIO2 limits the possibility of chloroquineePfRIO2 binding. The
significant binding score of some of the synthesized compounds
suggest the plausible anti-malarial activity through PfRIO2 kinase
inhibition. However more specific biological screening is necessary
in future to confirm the mode of action of these class of compounds
(Scheme 1).
2.2. Chemistry
The required a-pyrone C-3 carboxylic acids (1aeb) were syn-
thesized in two steps from appropriate acetophenones following
synthetic methodology reported by our group [15]. Coupling of
respective amines with the carboxyl group of 1aeb was carried out
using HATU as a coupling reagent [16]. The coupled products (2ae
e) were obtained in more than 70% yield. The methylthio group in
2aed were replaced with appropriate secondary amines [17] by
heating in dioxane to yield 3aed. Compounds 3eeg were prepared
by heating appropriate intermediates (2a, b, e) with H2O2 and
catalytic amount of acetic acid in dioxane. All the synthesized
compounds were characterized by spectral analysis.
2.3. Anti-malarial activity
Antimalarial potentials of 3a-g were evaluated in an in-vitro
schizonticidal inhibition assay. All the compounds were found to
block life-cycle stage transitions (Table 2) of Plasmodium falciparum.
The compound 3d was parasitostatic while others were para-
sitocidal. The minimum killing concentration (MKC) was the con-
centration of compounds required to kill parasite present in the
one of the hydrophobic residues Phe130 also forms a p/p stacking
with bromophenyl ring of 3b. Dg4 (black block in Fig. 2d) indicates
the presence of DNA in proximity. The IFD dockings were further
scored by extra precision scoring function within the S1 active site
to evaluate the energetic preference of compounds and the binding
profile results are summarized in Table 1. The negative eMBrAcE
(Multi-ligand Bimolecular Association with Energetic) score sug-
gests binding of the ligand with PfRIO2 kinase to lower the energy
of PfRIO2eligand complex. This result also supports the suitability
of S1 to be considered as active site. The energetic scores are based
on semi-empirical calculation and may not correlate exactly with
biological activity, they reveal the possibly of the ligand binding in
this novel target enzyme. Ligand binding affinity was also esti-
mated by Glide Emodel score (Table 1). Emodel score is the com-
bination of GlideScore (GScore) and nonbonded interaction energy.
The four molecules 3ae3d shown favorable energy score and
culture. Glutathione (GSH),
a non-proteinous thiol plays an
important role as a cellular redox buffer [18]. The cell health de-
pends on balance between generation and elimination of reactive
oxygen/nitrogen species (ROS/RNS), which maintains the redox
balance of the cell and proper function of redox-sensitive signaling
proteins. Moderate levels of ROS/RNS may function as signals to
promote cell proliferation and survival, whereas severe increase of
ROS/RNS can induce cell death [19]. GSH is known to directly bind
with some ROS species or assist ROS as a source of reductive power
for certain antioxidant systems [20]. Therefore, depletion in GSH
will imbalance the level of ROS inside cell leading to oxidative
stress. Lipids are most susceptible target of oxidative modification
through peroxidation that generates lipid radicals, which can
further attack the subsequent lipid molecules and propagate as a
chain reaction [21]. The lipid peroxides can form adduct with
oxidized proteins which in turn can inactivate 26S and 20S pro-
teasomes leading to accumulation of damaged protein and cell
death [22]. The lipid peroxidation related cell death is also associ-
ated with formation of malondialdehyde (MDA) and 4-hydroxy-2-
nonenal (HNE) [23]. In addition, severe damage to the membrane
structure might induce membrane perturbation and compromised
cellular integrity. As most of our compounds were parasitocidal, we
were curious whether they are able to affect the intracellular ROS
level. Therefore, to explore such a possibility, we choose 3aed for
study. Compounds 3aec are parasitocidal with very good MKC and
Schinzoticidal Activity while 3d was parasitostatic (Table 2). The
parasite cultures were treated with the sub-lethal concentration of
3aed individually and change in intracellular ROS, lipid peroxida-
tion (LP) were measured (Table 3). Untreated parasite culture was
used to calculate change in oxidative stress indices (ROS and LP)
and expressed as fold ꢀ SD. It was found that cells treated with 3ae
d had higher ROS level and consequently higher lipid peroxidation
Table 1
In-silico binding analysis by Glide and eMBrAcE score.
S. No
Compound
Dockinga
eMBrAcE scoreb
(energy difference)
Glide Emodel
score
VdW
Electrostatic
Total (DE)
1
2
3
4
5
6
7
8
3a
3b
3c
3d
3e
3f
3g
ꢁ72.8
ꢁ78.9
ꢁ74.8
ꢁ76.4
ꢁ62.4
ꢁ69.5
ꢁ67.6
ꢁ42.4
ꢁ181.8
ꢁ178.3
ꢁ190.2
ꢁ218.2
ꢁ160.4
ꢁ167.8
ꢁ187.7
ꢁ157.5
ꢁ139.0
ꢁ185.3
ꢁ142.9
ꢁ172.3
ꢁ140.1
ꢁ233.4
ꢁ90.4
ꢁ140.7
ꢁ319.3
ꢁ222.9
ꢁ172.0
ꢁ107.9
ꢁ282.9
ꢁ207.9
ꢁ78.7
Chloroquine
ꢁ81.6
a
Emodel score: the minimized poses are rescored using Schrödinger’s pro-
prietary GlideScore scoring function and the binding affinity can be estimated by
Glide Emodel score, which is the combination of GlideScore (GScore) and non-
bonded interaction energy.
b
eMBrAcE (Multi-Ligand Bimolecular Association with Energetics): (
D
ETotal
¼
Ecomplex ꢁ Eligand ꢁ Eprotein); VdW: van der Waals interaction.
D
E ¼ energy difference
(kcal/mol).

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A. Parveen et al. / European Journal of Medicinal Chemistry 70 (2013) 607e612
Scheme 1. Reagents and conditions: i) R2NH2, HATU, DMF, rt, 6 h; ii) R3H, Dioxane, 70 ꢂC, 4 h; iii) H2O2, CH3COOH, dioxane, 85 ꢂC, 4 h.
compared to untreated cells. The results are indicative of several
damages to the cellular structure through peroxidation of mem-
brane lipids and may affect proliferation machinery inside the cell.
4. Experimental section/materials and methods
4.1. Computational studies
It explains parasiticidal action of most of the a-pyrone derivatives
tested in the current study. However, a further detailed study is
required to understand the molecular mechanism as well as the
cellular machinery involved in the anti-malarial action of these
new class of compounds. Also, a PfRIO2 kinase specific assay is
needed to understand whether the activity is governed by PfRIO2
kinase.
In absence of PfRIO2 co-crystal structure, our previously
described model of PfRIO2 kinase complex with short chain of DNA
including ATP was used for current modeling studies [12]. The
modeling was carried out using the latest modeling suite from
Schrödinger [24]. The Protein Preparation Wizard (PPW) was uti-
lized iteratively to check for any errors related to bond-order, metal
charges (þ2 for Mn ion), proton assignment, or H-bonding to
ensure the chemical correctness of model. The SiteMap program
[25,26] can successfully suggest possible binding sites [27].
Therefore, this method was used and “four site points” were dis-
closed as possible ligand binding site in PfRIO2 kinase (Fig. 2a). The
S1 active site was found optimum for ligand binding and used for
docking studies. The 3D structure of compounds is treated in Lig-
Prep module of Schrödinger using OPLS2005 force field followed by
conformational search analysis through MacroModel [28] using
MMFFs force field to obtain the low energy conformer. SiteMap
analysis followed by molecular docking [14] of conformationally
optimized molecules in S1 active site was used to identify the
binding mode of compounds (Fig. 2d). Several docked poses were
generated and the best docked poses with lowest Emodel Score
were selected for comparative analysis (Table 1). The Emodel en-
ergy score has been further verified by the eMBrAcE module of
Schrödinger [28]. This module has been used to determine the
binding energy differences through minimization using OPLS2005
3. Summary and conclusion
The site identification followed by modeling studies revealed a
novel active site within the flexible loop region of PfRIO2 kinase,
which can interact with appropriate ligand. The binding mode of
designed compounds with target protein was analyzed by IFD
molecular docking. Overall, the pharmacophoric hybridization and
modeling approach were explored to generate a novel series of a-
pyrone molecules for synthesis. All the synthesized molecules
showed in vitro antimalarial activity. One of the synthesized com-
pounds, 3b, with most significant result was identified as promising
lead for further SAR study. The detailed molecular modeling and
chemical modifications are in process to convert this preliminary
lead into antimalarial drug candidate.
Table 2
Anti-malarial activities of different
a
-pyrone analogs (3aeg).
Comp Code
Schinzoticidal Activity Nature of inhibition MKC (
m
M ꢀ SE)
Table 3
IC50 M ꢀ SE)
(m
Effect of 3aed on antioxidant system of malaria parasite.
3a
3b
3c
3d
3e
3f
0.859 ꢀ 0.59
0.337 ꢀ 0.19
0.765 ꢀ 0.28
1.568 ꢀ 0.48
1.42 ꢀ 0.37
0.75 ꢀ 0.33
0.90 ꢀ 0.23
Parasitocidal
Parasitocidal
Parasitocidal
Parasitostatic
Parasitocidal
Parasitocidal
Parasitocidal
ND
1.00 ꢀ 0.002
0.21 ꢀ 0.026
1.99 ꢀ 0.30
NA
38.9 ꢀ 3.82
3.90 ꢀ 0.59
2.39 ꢀ 0.33
ND
Compounds
Change in ROS
Change in lipid
peroxidation (fold ꢀ SD)
level (fold ꢀ SDa)
Untreated
1
1
3a
3b
3c
3d
1.66 ꢀ 0.14
1.87 ꢀ 0.21
1.20 ꢀ 0.18
1.99 ꢀ 0.20
11.03 ꢀ 0.45
4.49 ꢀ 0.26
9.03 ꢀ 0.37
4.88 ꢀ 0.66
3g
Chloroquine 0.078 ꢀ 0.012
a
ND ¼ Not determined.
Standard deviation.

A. Parveen et al. / European Journal of Medicinal Chemistry 70 (2013) 607e612
611
force field. The calculations were performed with GB/SA continuum
water solvation model. The eMBrAcE minimization was performed
for 5000 steps or until the energy difference between subsequent
conformations was 0.05 kJ molꢁ1. The energetic calculations pro-
vided energy difference in terms of VdW, electrostatic and total
7.94 (d, J ¼ 8 Hz, 1H, ArH), 8.30e8.32 (d, J ¼ 8 Hz, 1H, ArH), 8.50e8.54
(m, 2H, ArH), 9.88 (s, 1H, NH), 11.22 (s, 1H, ArOH).
4.2.3. General procedure for the synthesis of 3eeg
To a solution 2H-pyran-4-methylthio-3-carboxamide (2aeb, e,
3.3 mmol) in dioxane, 30% (w/w in H2O) Hydrogen Peroxide solu-
tion (6.6 mmol), acetic acid 2e3 drops was added at room tem-
perature. The resulting reaction mixture was stirred at 85 ꢂC for 4 h.
The reaction was monitored by TLC for completion. The reaction
mixture was diluted with cold water (25 ml) and extracted with
DCM, concentrated under reduced pressure. The crude was column
purified to get 3eeg.
(
D
E ¼ Ecomplex ꢁ Eligand ꢁ Eprotein). The implicit-water model was
used during minimization, conformational search, molecular
docking, SiteMap analysis, and energetic calculation.
4.2. Chemistry
4.2.1. General
All reactions were carried out in oven-dried glassware under
nitrogen atmosphere. The chemicals and solvents were purchased
from Spectrochem, Across, Rankem or SigmaeAldrich. Melting
points were recorded on Veego melting point apparatus. Analytical
thin layer chromatography (TLC) was performed on precoated
plates (silica gel 60 F-254) purchased from Merck Inc. Purification
by gravity column chromatography was carried out on silica gel
(100e200 mesh). Electro UV/Vis spectrophotometer was used for
recording the UV spectra. 1H/13C NMR were obtained from a Varian
(400 MHz) spectrometer or Bruker spectrometer using CDCl3 or
DMSO-d6, as solvents. Peaks are recorded with the following ab-
breviations: s, singlet; bs, broad singlet; d, doublet; t, triplet; q,
quartet; m, multiplet; J, coupling constant (hertz).
4.2.3.1. 4-Hydroxy-2-oxo-6-phenyl-N-(p-tolyl)-2H-pyran-3-þcarbo-
xamide (3e). Yield: 35%; mp: 198e199 ꢂC; MS-ESI (m/z): [M þ 1]
322.1; UV (MeOH) lmax 247 nm, 340 nm; IR: (KBr) cmꢁ1 1545, 1699,
2906, 3041, 3234; 1H NMR (400 MHz, CDCl3):
d 2.35 (s, 3H, CH3), 6.65
(s, 1H, CH), 7.17e7.19 (d, J ¼ 8.0 Hz, 2H, ArH), 7.50e7.55 (m, 5H, ArH),
7.87e7.89 (d, J ¼ 6.8 Hz, 2H, ArH), 10.91 (s, 1H, NH).
4.2.3.2. N-(4-Bromophenyl)-4-hydroxy-2-oxo-6-phenyl-2H-pyran-
3-carboxamide (3f). Yield: 40%; mp: 220e222 ꢂC; MS-ESI (m/z):
[Mþ] 386.1, [Mþ þ 2]: 388.2; UV (MeOH) lmax 253 nm, 342 nm; IR:
(KBr) cmꢁ1 1597, 1628, 1697, 3101, 3238; 1H NMR (400 MHz, CDCl3):
d
6.67 (s, 1H, CH), 7.48e7.57 (m, 7H, ArH), 7.88e7.90 (d, J ¼ 6.8 Hz,
2H, ArH), 11.02 (s, 1H, NH).
4.2.2. General procedure for the synthesis of 3aed
To a solution of an appropriate 2H-pyran-4-methylthio-3-
carboxamide (2aed, 3.3 mmol) in dioxane, appropriate secondary
amine (6.7 mmol) was added and stirred for 4 h at 70 ꢂC. The re-
action was monitored by TLC for completion. The solvent was
removed under reduced pressure. The ice cold water was added
and stirred for 2 h at 0e5 ꢂC. The resulting precipitate was filtered
and purified by column chromatography to obtain pure compound.
4.2.3.3. N-(4-Fluorophenyl)-4-hydroxy-2-oxo-6-phenyl-2H-pyran-3-
carboxamide (3g). Yield: 30%; mp > 260 ꢂC; MS-ESI (m/z): [Mþ ꢁ 1]
323.9; UV (MeOH) lmax 236 nm, 340 nm; IR: (KBr) cmꢁ1 1547, 1637,
1697, 3066, 3236; 1H NMR : (400 MHz, DMSO-d6):
d 7.02 (s, 1H, CH),
7.14e7.19 (m, 2H, ArH), 7.50e7.55 (m, 3H, ArH), 7.61e7.64 (m, 2H,
ArH), 7.86e7.88 (d, J ¼ 6.8 Hz, 2H, ArH), 10.40 (s, 1H, NH).
4.3. Anti-malarial activity
4.2.2.1. 2-Oxo-6-phenyl-4-(piperidin-1-yl)-N-(p-tolyl)-2H-pyran-3-
carboxamide (3a). Yield: 55%; mp: 133e135 ꢂC; MS-ESI (m/z): [Mþ]
388.6; UV (MeOH) lmax 256.8 nm, 316 nm; IR: (KBr) cmꢁ1 1635,
4.3.1. In-vitro schizonticidal inhibition assay
The in vitro antimalarial assay was carried out in 96-well
microtitre plates as described previously [29,30]. In brief, test
compound was incubated with ring stage synchronized
P. falciparum (3D7) parasitised cell. After 42 h incubation, the blood
smears from each well was prepared to record maturation of ring
stage parasites into trophozoites and schizonts. MS-Excel Sheet
based HN-NonLin program (www.malaria.farch.net) was used to
calculate IC50 of 3aeg compounds based on their schizont inhibi-
tion profile using regression analysis. To determine the nature of
parasite growth inhibition (parasitostatic/parasitocidal), the com-
pounds were removed and the cultures were washed 3 times with
albumax II free RPMI-1640 and incubated in complete media with
fresh hematocrit for another 72 h. A thin smear was prepared and
number of RBCs containing viable parasite was counted. The min-
imum concentration of compounds giving no viable parasite was
used to calculate minimum killing concentration (MKC) of com-
pounds with parasitocidal activity.
1674, 2856, 2937, 3275; 1H NMR (400 MHz, DMSO-d6):
d 1.70 (bs,
6H, 3CH2) 2.42 (s, 3H, ArCH3), 3.75 (bs, 4H, 2NCH2), 7.50 (s, 1H, CH),
7.55e7.59 (m, 2H, ArH), 8.0e8.05 (m, 5H, ArH), 8.45e8.52 (m, 2H,
ArH), 10.82 (s, 1H, NH).
4.2.2.2. N-(4-Bromophenyl)-2-oxo-6-phenyl-4-(piperidin-1-yl)-2H-
pyran-3-carboxamide (3b). Yield: 78%; mp: 210e211 ꢂC; MS-ESI (m/
z): [Mþ þ 2] 455.0, [Mþ] 453.3; UV (MeOH) lmax 259 nm, 324 nm; IR:
(KBr) cmꢁ1 1639, 1692, 2843, 2939, 3105, 3325; 1H NMR (400 MHz,
CDCl3): d 1.75 (bs, 6H), 3.56 (bs, 4H), 6.67 (s, 1H), 7.41e7.43 (m, 2H),
7.47e7.50 (m, 3H), 7.57e7.59 (m, 2H), 7.83e7.85 (m, 2H),10.68 (s,1H).
4.2.2.3. N-(4-Hydroxyphenyl)-2-oxo-6-phenyl-4-(piperidin-1-yl)-
2H-pyran-þ3-carboxamide (3c). Yield: 67%; mp: 134e135 ꢂC; MS-ESI
(m/z): [M þ 1] 391.20; UV (MeOH) lmax 254 nm, 307 nm; IR:
(KBr) cmꢁ1 1633, 1666, 2895, 2937, 3184 (broad); 1H NMR
(400 MHz, DMSO-d6):
d 1.75 (bs, 6H, 3CH2), 3.55 (bs, 4H, 2NCH2),
5.40 (s, 1H, ArOH), 6.65 (s, 1H, CH), 6.79e6.81 (m, 2H, ArH), 7.48e
7.50 (m, 5H, ArH), 7.82e7.84 (m, 2H, ArH), 10.36 (s, 1H, NH).
4.3.2. Measurements of oxidative stress indices
Parasite cultures were treated with 3a-d separately. The intra-
cellular ROS was measured by a fluorescent probe (20,70-dichloro-
fluorescein diacetate) and lipid peroxidation was measured as
described previously [29,31].
4.2.2.4. N-(4-Hydroxyphenyl)-6-(4-methoxyphenyl)-4-(4-methyl-
piperazin-1-yl)-2-oxo-2H-pyþran-3-carboxamide (3d). Yield: 31%; mp:
>245 ꢂC; MS-ESI (m/z): [M þ 1] 436.36; UV (MeOH) lmax 239 nm,
313 nm; IR: (KBr) cmꢁ11560, 1612, 1726, 2949, 3066, 3294 (broad); 1H
Acknowledgments
NMR (400 MHz, DMSO-d6):
d 2.82 (bs, 3H, NCH3), 3.35e3.42 (m, 8H,
4CH2), 4.12 (s, 3H, OCH3), 7.17e7.19 (d, J ¼ 8 Hz, 1H, ArH), 7.55 (s, 1H,
This research received funding from Department of Biotech-
nology (DBT), New Delhi, India through grant no. BT/PR13436/
CH), 7.58e7.62 (m, 2H, ArH), 7.76e7.78 (d, J ¼ 8 Hz, 1H, ArH), 7.92e

612
A. Parveen et al. / European Journal of Medicinal Chemistry 70 (2013) 607e612
J. Mirsalis, R.K. Guy, Lead optimization of 3-carboxyl-4(1H)-quinolones to
deliver orally bioavailable antimalarials, J. Med. Chem. 55 (2012) 4205e4219.
[12] D. Chouhan, A. Sharon, C. Bal, Molecular and structural insight into Plasmo-
dium falciparum RIO2 kinase, J. Mol. Model. 19 (2013) 485e496.
[13] R.A. Friesner, R.B. Murphy, M.P. Repasky, L.L. Frye, J.R. Greenwood,
T.A. Halgren, P.C. Sanschagrin, D.T. Mainz, Extra precision glide: docking and
scoring incorporating a model of hydrophobic enclosure for proteinꢁligand
complexes, J. Med. Chem. 49 (2006) 6177e6196.
MED/12/450/2009 & BT/41/NE/TBP/2010. AP is thankful to UGC,
India for MANF Junior Research Fellowship. AK and HC acknowl-
edges BIT Mesra for Institute Research Fellowship. AC acknowl-
edges the financial support in the form of a fellowship from IIT-
Guwahati. Authors acknowledge Dr. Reddy’s Institute of Life Sci-
ences, Hyderabad for NMR-Mass Facility and Central Instrument
Facility, BIT Mesra for analytical support.
[14] W. Sherman, T. Day, M.P. Jacobson, R.A. Friesner, R. Farid, Novel procedure for
modeling ligand/receptor induced fit effects, J. Med. Chem. 49 (2006) 534e
553.
[15] S. Karampuri, P. Bag, S. Yasmin, D.K. Chouhan, C. Bal, D. Mitra,
D. Chattopadhyay, A. Sharon, Structure based molecular design, synthesis and
biological evaluation of alpha-pyrone analogs as anti-HSV agent, Bioorg. Med.
Chem. Lett. 22 (2012) 6261e6266.
[16] L.A. Carpino, 1-Hydroxy-7-azabenzotriazole. An efficient peptide coupling
additive, J. Am. Chem. Soc. 115 (1993) 4397e4398.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.10.028.
[17] A. Sharon, P.R. Maulik, C. Vithana, Y. Ohashi, V.J. Ram, Synthesis of biphe-
nanthrenyls and role of CeH/X noncovalent interactions in conformational
control, Eur. J. Org. Chem. 2004 (2004) 886e893.
References
[18] M. Marí, A. Morales, A. Colell, C. García-Ruiz, J.C. Fernández-Checa, Mito-
chondrial glutathione, a key survival antioxidant, Antioxid. Redox Signal. 11
(2009) 2685e2700.
[19] D. Trachootham, W. Lu, M.A. Ogasawara, N.R.-D. Valle, P. Huang, Redox
regulation of cell survival, Antioxid. Redox Signal. 10 (2008) 1343e1374.
[20] V.I. Lushchak, Glutathione homeostasis and functions: potential targets for
medical interventions, J. Amino Acids 2012 (2012) 736837.
[21] A.W. Girotti, Lipid hydroperoxide generation, turnover, and effector action in
biological systems, J. Lipid Res. 39 (1998) 1529e1542.
[22] D. Poppek, T. Grune, Proteasomal defense of oxidative protein modifications,
Antioxid. Redox Signal. 8 (2006) 173e184.
[23] I. Pinchuk, E. Schnitzer, D. Lichtenberg, Kinetic analysis of copper-induced
peroxidation of LDL, Biochim. Biophys. Acta 1389 (1998) 155e172.
[24] Schrödinger Suite, LLC, New York, NY, 2012.
[25] SiteMap-V-2.5, V 2.5, Schrödinger, LLC, New York, NY, 2011.
[26] T.A. Halgren, Identifying and characterizing binding sites and assessing
druggability, J. Chem. Inf. Model. 49 (2009) 377e389.
[27] M. Nayal, B. Honig, On the nature of cavities on protein surfaces: application
to the identification of drug-binding sites, Proteins 63 (2006) 892e906.
[28] MacroModel-V-9.9, Schrödinger, LLC, New York, NY, 2011.
[29] V. Trivedi, P. Chand, K. Srivastava, S.K. Puri, P.R. Maulik, U. Bandyopadhyay,
Clotrimazole inhibits hemoperoxidase of Plasmodium falciparum and induces
oxidative stress. Proposed antimalarial mechanism of clotrimazole, J. Biol.
Chem. 280 (2005) 41129e41136.
[30] 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 chloroquineechalcone
hybrids against chloroquine-resistant strain of Plasmodium falciparum, Bioorg.
Med. Chem. Lett. 22 (2012) 5455e5459.
[1] I. Hastings, How artemisinin-containing combination therapies slow the
spread of antimalarial drug resistance, Trends Parasitol. 27 (2011) 67e72.
[2] A.P. Phyo, S. Nkhoma, K. Stepniewska, E.A. Ashley, S. Nair, R. McGready, S. Al-
Saai, A.M. Dondorp, K.M. Lwin, P. Singhasivanon, Emergence of artemisinin-
resistant malaria on the western border of Thailand: a longitudinal study,
The Lancet. 379 (2012) 1960e1966.
[3] S. Kumar, U. Bandyopadhyay, Free heme toxicity and its detoxification sys-
tems in human, Toxicol. Lett. 157 (2005) 175e188.
[4] V. Trivedi, S. Nag, In silico characterization of atypical kinase PFD0975w from
plasmodium kinome: a suitable target for drug discovery, Chem. Biol. Drug
Des. 79 (2012) 600e609.
[5] S. Nag, D. Chouhan, S.N. Balaji, A. Chakraborty, K. Lhouvum, C. Bal, A. Sharon,
V. Trivedi, Comprehensive screening of heterocyclic compound libraries to
identify novel inhibitors for PfRIO-2 kinase through docking and substrate
competition studies, Med. Chem. Res. (2013) 1e8.
[6] S. Nag, K. Prasad, V. Trivedi, Identification and screening of antimalarial
phytochemical reservoir from northeastern Indian plants to develop PfRIO-2
kinase inhibitor, Eur. Food Res. Technol. 234 (2012) 905e911.
[7] J.B. Koepfli, J.F. Mead, J.A. Brockman, An alkaloid with high antimalarial ac-
tivity from Dichroa Febrifuga1, J. Am. Chem. Soc. 69 (1947), 1837e1837.
[8] S. Jiang, Q. Zeng, M. Gettayacamin, A. Tungtaeng, S. Wannaying, A. Lim,
P. Hansukjariya, C.O. Okunji, S. Zhu, D. Fang, Antimalarial activities and
therapeutic properties of febrifugine analogs, Antimicrob. Agents Chemother.
49 (2005) 1169e1176.
[9] D. Carmona, J. Saez, H. Granados, E. Perez, S. Blair, A. Angulo, B. Figadere,
Antiprotozoal 6-substituted-5,6-dihydro-alpha-pyrones from Raimondia cf.
monoica, Nat. Prod. Res. 17 (2003) 275e280.
[10] P.L. Chien, C.C. Cheng, Structural modification of febrifugine. Some methyl-
enedioxy analogs, J. Med. Chem. 13 (1970) 867e870.
[11] Y. Zhang, J.A. Clark, M.C. Connelly, F. Zhu, J. Min, W.A. Guiguemde, A. Pradhan,
L. Iyer, A. Furimsky, J. Gow, T. Parman, F. El Mazouni, M.A. Phillips, D.E. Kyle,
[31] S.N. Balaji, V. Trivedi, Methemoglobin incites primaquine toxicity through
single-electron oxidation and modification, J. Basic Clin. Physiol. Pharmacol.
24 (2013) 105e114.