Synthesis of novel α-pyranochalcones and pyrazoline derivatives as Plasmodium falciparum growth inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.05.069

Both the lack of a credible malaria vaccine and the emergence and spread of parasites resistant to most of the clinically used antimalarial drugs and drug combination have aroused an imperative need to develop new drugs against malaria. In present work, α

Full text of DOI:10.1016/j.bmcl.2010.05.069

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4675–4678
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of novel a-pyranochalcones and pyrazoline derivatives as Plasmodium
falciparum growth inhibitors
a
a
b
b
b
Gajanan Wanare a, , Rahul Aher , Neha Kawathekar , Ravi Ranjan , Naveen Kumar Kaushik , Dinkar Sahal
*
a Department of Pharmacy, Shri G.S. Institute of Technology and Science, 23—Park Road, Indore, MP 452003, India
b Malaria Research Laboratory, International Center for Genetic Engineering & Biotechnology, New Delhi 110067, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 December 2009
Revised 4 May 2010
Accepted 18 May 2010
Available online 26 May 2010
Both the lack of a credible malaria vaccine and the emergence and spread of parasites resistant to most of
the clinically used antimalarial drugs and drug combination have aroused an imperative need to develop
new drugs against malaria. In present work, a-pyranochalcones and pyrazoline analogs were synthesized
to discover chemically diverse antimalarial leads. Compounds were tested for antimalarial activity by
evaluation of the growth of malaria parasite in culture using the microtiter plate based SYBR-Green-I
assay. The (E)-3-(3-(2,3,4-trimethoxyphenyl)-acryloyl)-2H-chromen-2-one (Ga6) turned out to be the
Keywords:
Antimalarial activity
Coumarine
most potent analog of the series, showing IC50 of 3.1
lg/ml against chloroquine-sensitive (3D7) strain
and IC50 of 1.1 g/ml against chloroquine-resistant field isolate (RKL9) of Plasmodium falciparum. Cyto-
l
toxicity study of the most potent compounds was also performed against HeLa cell line using the MTT
assay. All the tested compounds showed high therapeutic indices suggesting that they were selective
in their action against the malaria parasite. Furthermore, docking of Ga6 into active site of falcipain
enzyme revealed its predicted interactions with active site residues. This is the first instance wherein
chromeno-pyrazolines have been found to be active antimalarial agents. Further exploration and optimi-
zation of this new lead could provide novel, antimalarial molecules which can ward off issues of cross-
resistance to drugs like chloroquine.
Chalcone
a-Pyranochalcones
Chromeno-pyrazolines
Plasmodium falciparum
Falcipain
SYBR-Green-I assay
Ó 2010 Elsevier Ltd. All rights reserved.
Malaria, a major cause of morbidity and mortality affecting
third-world countries,1 is one of the foremost health and develop-
mental challenges facing mankind. Malaria is responsible for up to
500 million clinical cases and 3 million deaths annually.2 The WHO
estimates that malaria kills an African child every 30 s.3 In the ab-
sence of an effective malaria vaccine, prevention and treatment re-
lies mainly on drugs. Even as a number of drugs and drug
combinations are in clinical use for malaria, many of these are
becoming less effective due to widespread parasite resistance (like
recent cases of falciparum malaria resistance to artesunate–meflo-
quine combination).4 This means that there is an imperative need
to develop new drugs with different mechanisms of action to help
preclude issues of cross-resistance.
Chalcone is an exceptional chemical template having multifari-
ous biological activities. Antimalarial property of some chalcone
derivatives is derived from their ability to inhibit the parasitic cys-
teine proteases like the falcipain.5 These proteases catabolize glo-
bin into small peptides within the acidic food vacuole of the
intra-erythrocytic malaria parasite. Without cysteine protease ac-
tion osmotic swelling occurs, food vacuolar functions are impaired,
and the parasite is starved to death.
The literature on chalcones (Fig. 1) as antimalarials was ana-
lyzed to provide a meaningful overview of the structural require-
ments for chalcones as antimalarials. Some of the important
points to be considered are5,6 (i) the influence of B ring on the
activity is related to size considerations and bulky substitutions
are favorable, while the A ring may be more important in influenc-
ing hydrophobicity; (ii) presence of chloro and fluoro substitutions
on ring A does not necessarily increase the activity and the influ-
ence of these halogen atoms on ring A seems to depend on the kind
of substitution on ring B; (iii) Steric and hydrophobic factors, par-
ticularly substituent on ring A must be width–limited so that the
molecular width along X-axis of chalcone derivatives is small;
(iv) high electron density at the C3, C4 of ring B might play a favor-
able role in activity; (v) requirement of hydrogen bond acceptor at
C4 of ring B in a particular orientation to provide stronger and
effective hydrogen bonding.
* Corresponding author. Tel.: +91 9098616130.
E-mail addresses: gajananwanare@gmail.com, gajananwanare@yahoo.co.in
(G. Wanare).
Figure 1. General structure of chalcone.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.069

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G. Wanare et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4675–4678
As a result, it was decided to undertake the synthesis and anti-
malarial evaluation of -pyranochalcones. The acetophenone part
in chalcone was replaced with the acetyl coumarines in -pyran-
ochalcones. This was done in order to satisfy the structural require-
ments of limited bulk and availability of hydrogen bond acceptor at
C4 of ring B.
The antimalarial activity of all of the compounds was deter-
a
mined by SYBR-Green-I in vitro assay.11,12 The results are shown
in Table 2 indicate that eight of the 13 compounds evaluated,
showed significant antimalarial activity against the chloroquine-
sensitive (3D7) strain. Interestingly, six of these eight compounds
a
showed good promise (IC50 < 15 lg/ml) also against RKL9, the
The
acetyl-coumarin 3 (Scheme 1). The latter was prepared from
salicylaldehyde using
reported method,7 with necessary
modification to improve yield and appearance. This was fol-
lowed by condensation of 3-acetyl-coumarin 3 with substituted
benzaldehydes by using novel solvent-free method involving a
heterogeneous catalyst, silica sulfuric acid (synthetic details
a
-pyranochalcones Ga1–Ga8 were synthesized from 3-
chloroquine-resistant field isolate of P. falciparum. It is noteworthy
that the resistance index of all the tested compounds is higher than
1 (for Ga6, its 2.8). This is due to their surprisingly higher potencies
against chloroquine-resistant strain than that against chloroquine-
sensitive one (Table 2). The same is not true for most of the previ-
ously reported chalcones and even for the artemisinin derivatives.
1
a
The compound Ga1 (IC503D7 = 3.1
Ga6 (IC503D7 = 3.1 g/ml; IC50RKL9 = 1.1
tent analogs of the series. The most active compounds (IC50
l
g/ml; IC50RKL9 = 1.9
lg/ml) and
are provided in Supplementary material). The
a
-pyranochal-
l
l
g/ml) were the most po-
RKL9
cones Gb1–Gb2 were synthesized from 6-acetyl-5-hydroxy-4-
methyl-2H-chromen-2-one 13. Compound 13 was synthesized
using Pechmann condensation as reported by Sethna et al.8
<
10 lg/ml, i.e., Ga1, Ga2, Ga6, Gc, and Gd) were further evaluated
for cytotoxicity by MTT assay method13 against HeLa cell line. All
tested compounds were found to be devoid of cytotoxicity at inhib-
itory concentrations. In general, their cytotoxicity appeared to be
(Scheme 2). Treatment of
a-pyranochalcone Ga3 with hydrazine
hydrate and phenyl hydrazine in presence of acetic acid9 affor-
ded pyrazolines, Gc and Gd (Scheme 3). Similarly, pyrazoline Ge
was synthesized from diphenylchalcone 20. The p-hydroxyace-
tophenone in base catalyzed Claisen–Schmidt condensation with
p-flourobenzaldehyde afforded the diphenylchalcone 20
(Scheme 4).10 The synthesized compounds are summarized in
Table 1.
at much higher concentrations (TC50HeLa P 100
lg/ml) than the
concentrations responsible for their antimalarial activity. This indi-
cates their safety in the mammalian system. Furthermore, these
compounds were found to possess good therapeutic indices
(TC50HeLa/IC50RKL9 = 10–90.9) showing their selectivity towards
malaria parasite.
Scheme 1. Synthesis of 3-acetyl coumarine (3) and its chalcones (Ga1–Ga8). Reagents and condition: (i) piperidine, ethanol, rt, stirring; (ii) silica sulfuric acid, solvent-free.
Scheme 2. Synthesis of 6-acetyl-5-hydroxy-4-methyl-coumarin (13) and its chalcones (Gb1–Gb2). Reagents and conditions: (iii) AlCl3/dry PhNO2, 125–130 °C; (iv) silica
sulfuric acid, solvent-free, 80 °C.
Scheme 3. Synthesis of a-pyranopyrazoline (Gc–Gd). Reagents and conditions: (v) hydrazine hydrate, glacial acetic acid, reflux; (vi) methanol, phenyl hydrazine, few drops of
acetic acid, reflux.
Scheme 4. Synthesis of diphenyl chalcone (20) and its pyrazoline (Ge). Reagents and conditions: (vii) NaOH/dry methanol, rt, stirring; (viii) hydrazine hydrate, glacial acetic
acid, reflux.

G. Wanare et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4675–4678
4677
Table 1
Molecular formula, molecular weight, melting point and% yields of synthesized compounds
Compound code
R
Molecular formula
C21H18O6
Molecular weight
Melting point (°C)
Yield (%)
Ga1
Ga2
Ga3
Ga4
Ga5
Ga6
Ga7
Ga8
Gb1
Gb2
Gc
3,4,5-TriCH3O–
3-MeO-4-H–
3,4-DiMeO–
4-MeO–
2,5-DiMeO–
2,3,4-TriMeO–
2,4,5-TriMeO–
2-MeO–
366.36
322.31
336.34
306.31
336.34
336.34
366.36
306.31
340.76
366.36
392.4
155–156
171–172
151–152
153–154
114–115
131–132
155–156
143–144
195–198
160–162
210–212
148–150
228–231
75.88
79.12
74.4
79.98
72.0
74.64
67.69
68.55
61.05
69.6
C
C
C
19H14O5
20H16O5
19H14O4
C20H16O5
C
C
C
C
C
21H18O6
21H18O6
19H14O4
19H13ClO4
21H18O6
2-Cl
2,5-DiMeo
a
C22H20N2O5
C26H22N2O4
C17H13FN2O2
81.8
77.42
71.88
a
a
Gd
Ge
426.46
296.3
a
Structures are provided in Schemes 3 and 4.
Table 2
The in vitro antimalarial activity against chloroquine-sensitive (3D7) strain and chloroquine-resistant field isolate (RKL9) of P. falciparum; and cytotoxicity against HeLa cell line
Compound
Antimalarial activity against P. falciparum (
l
g/ml)
Cytotoxic activity against HeLa cell line (lg/ml)
3D7
3D7
RKL9
RKL9
HeLa
RKL9
IC50
IC80
IC50
Resistance index (IC503D7/IC50
)
TC50
TC50HeLa/IC50
Ga1
Ga2
Ga3
Ga4
Ga5
Ga6
Ga7
Ga8
Gb1
Gb2
Gc
3.1
11
26.7
>100
2.7
6
1.9
4.9
a
a
a
1.1
a
a
13.5
a
1.6
2.2
—
—
—
2.8
—
—
1.5
—
1.1
1.3
—
>100
>100
a
a
a
100
a
a
>50
>20
—
—
—
90.9
—
—
—
—
>10
>13
—
—
—
24
100
a
11.3
6.2
a
a
25
a
14
3.1
>100
>100
20
>100
10
a
a
9
7.6
a
>100
>100
a
a
a
Gd
10
44
a
Ge
>100
20.63c
4.51c
CQb
36.11c
8.46c
206.37c
4.51c
0.1
1
Artemisinin
a
b
c
Not done.
Chloroquine diphosphate.
ng/ml.
The antimalarial activity of chalcones with different substitu-
tion patterns has been previously reported.14–18 Albeit, they were
found to act through a variety of different mechanisms,14–18 the
inhibition of falcipain by newly designed chromenochalcones
was apparent from the docking study of most active compound
Ga6 in the active site of falcipain enzyme. The docking of most ac-
tive compound, Ga6 was performed by GLIDE19 module of Schrö-
dinger Suite 2009 with protein, falcipain enzyme (PDB ID: 3BPF).
The default settings with standard precision mode in the GLIDE
module were used for docking. It can be inferred from the docking
study that the compound Ga6 seems to possess good affinity for ac-
tive site residues of 3BPF. The Fig. 2 depicts the type of predicted
interaction and fit of the molecule with the surface cleft of falcipain
enzyme. The hydrogen bonding interaction with Cys 42 as pre-
dicted by the docked pose of Ga6 is in agreement with the previ-
ously discussed requirement of hydrogen bonding acceptor in
ring B.
Figure 2. The proposed interactions of Ga6 with active site residue Cys 42 of
falcipain enzyme revealed from docking study.
The high activity of trimethoxy-derivatives (Ga1 and Ga6) is
also in accord with discussed influence of hydrophobicity and ste-
ric bulk on the ring A. Among the disubstituted compounds, the
monohydroxylated compound Ga2 is more active than the dime-
thoxy-derivatives. Between the 6-acetyl-coumarin derivatives,
ochalcones derivatives. Although this initial study involved only
a limited number of compounds, it has provided a platform that
is well worth studying for further detailed SAR of pyranochalcones.
This is the first report wherein chromeno-pyrazolines were
found to be potent antimalarials. Among the pyrazolines, diphenyl
pyrazoline Ge possessed insignificant antiplasmodial potential,
the halogenated derivatives (Gb1, IC50RKL9 = 13.5
l
g/ml) exhibited
greater activity than the methoxylated derivative (Gb2,
IC503D7 > 100
g/ml). These results offer new possibilities for fur-
ther improvements in the antimalarial performance of -pyran-
l
whereas
a-pyranopyrazolines, Gc and Gd were found to be signif-
a
icantly active against both strains of the malarial parasite. This

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G. Wanare et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4675–4678
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These results revealed that the compounds synthesized by us
exhibited promising antimalarial activity against both chloro-
quine-sensitive strain (3D7) and chloroquine-resistant field isolate
(RKL9) of P. falciparum with meaningful SAR. A comparatively
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a-pyranopyrazolines in the present study suggests that
active -pyranopyrazoline leads can be further optimized for
a
building potent and chemically diversified antimalarial drugs with
less chances of developing cross-resistance.
Acknowledgments
The authors (G.W. and R. A.) acknowledge the financial support
from Ministry of HRD, Govt. of India in the form of fellowships. The
authors are thankful to Dr. V. S. Chauhan, Director, ICGEB, New Del-
hi for providing state-of-the-art laboratory facility for biological
evaluation. The authors are grateful to Dr. R. C. Pillai, NIMR, New
Delhi for providing chloroquine-resistant field isolate (RKL9) of P.
falciparum for screening. The authors also thank Dr. R. A. Joshi
and Dr. R. R. Joshi, National Chemical Laboratory (NCL), Pune for
their technical assistance in spectral characterization and analysis.
The authors also appreciate the provision of evaluation version of
Schrödinger Suite 2009 from Schrödinger Inc., USA.
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Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.05.069.
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