Synthesis and in vitro evaluation of new chloroquine-chalcone hybrids against chloroquine-resistant strain of Plasmodium falciparum

Article abstract of DOI:10.1016/j.bmcl.2012.07.028

The control of malaria has been complicated with increasing resistance of malarial parasite against existing antimalarials. Herein, we report the synthesis of a new series of chloroquine-chalcone based hybrids (8-22) and their antimalarial efficacy agains

Full text of DOI:10.1016/j.bmcl.2012.07.028

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5455–5459
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro evaluation of new chloroquine-chalcone hybrids
against chloroquine-resistant strain of Plasmodium falciparum q
Koneni V. Sashidhara a, , Srinivasa Rao Avula a, Gopala Reddy Palnati a, Shiv Vardan Singh b,
⇑
c
b
Kumkum Srivastava c, , Sunil K. Puri , J. K. Saxena
⇑
a Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow 226 001, India
b Biochemistry Division, CSIR-Central Drug Research Institute, Lucknow 226 001, India
c Parasitology Division, CSIR-Central Drug Research Institute, Lucknow 226 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The control of malaria has been complicated with increasing resistance of malarial parasite against exist-
ing antimalarials. Herein, we report the synthesis of a new series of chloroquine-chalcone based hybrids
(8–22) and their antimalarial efficacy against both chloroquine-susceptible (3D7) and chloroquine-resis-
tant (K1) strains of Plasmodium falciparum. Most of the compounds showed enhanced antimalarial activ-
ity as compared to chloroquine in chloroquine-resistant (K1) strain of Plasmodium falciparum.
Furthermore, to unfold the mechanism of action of these synthesized hybrid molecules, we carried out
hemin dependent studies, in which three compounds were found to be active.
Received 14 May 2012
Revised 27 June 2012
Accepted 9 July 2012
Available online 14 July 2012
Keywords:
Synthesis
Quinoline
Ó 2012 Elsevier Ltd. All rights reserved.
Chalcones
Antimalarial agents
The magnitude of human suffering and fatalities associated
with malaria are well known.1 The causal agent of the most lethal
form of malaria, Plasmodium falciparum, has developed resistance
to a multitude of drugs including the efficacious, and cheap drug
Chloroquine (CQ) (1, Fig. 1), that was once used as a first line of de-
fence against infection in most endemic regions.2 This develop-
ment of resistance has in part been responsible for the global rise
of malaria.3 Chloroquine induces heme accumulation in cytosol
and causes membrane degradation of the parasite which finally
contributes to cell death.4 It is known that the resistance develops
as a result of decreased accumulation of the drug in the parasite,
due to enhanced efflux, reduced uptake and/or unavailability of
drug in parasite food vacuole. Artemisinin and its semi-synthetic
derivatives are renowned for their potent antimalarial activity
and are only choice to treat resistant malaria parasites.5 However,
recently, first cases of clinical resistance were reported for the arte-
misinin class of antimalarials from the Thai–Cambodian border.6
To overcome the challenges of multi-drug resistance in
P. falciparum, many approaches currently being adopted contain
optimisation of treatment with available drugs including combina-
tion therapy, developing analogues of the existing drugs and
evaluation of drug resistance reversers (chemo sensitizers). Among
these approaches, the medicinal chemistry hybridization, which
involves the rational design of new chemical entities by the fusion
of two drugs, both active compounds and/or pharmacophoric units
with interesting and complimentary activities is an attractive strat-
egy. Additionally, different and/or dual mode of action may reduce
undesired side effects.7 The 7-chloroquinoline moiety present in
several established antimalarials such as chloroquine and amodia-
quine (1 & 2, Fig. 1), is thought to confer antimalarial potency to
these compounds by facilitating binding to heme and consequently
inhibiting hemozoin formation.8 A number of chloroquine analogs
and derivatives maintain significant activity against chloroquine
resistant strains of P. falciparum,9 indicating that the resistance
mechanism is compound specific and not associated to modifica-
tion in the structure of the drug target. On the other hand, chal-
cones (1,3-diaryl-2-propen-1-ones), which are the bio precursors
of flavonoids, have been a prosperous source of inspiration for
medicinal chemists for many years and their analogs display a
broad range of biological activities including antimalarial activ-
ity.10 They have shown antimalarial activity by inhibiting either
plasmodial aspartate proteases or cysteine proteases,11 in addition
they were also shown to inhibit the parasite-induced channels12
and cause pronounced membrane perturbations of the erythro-
cytes.13 Plasmodial aspartate proteases and cysteine proteases
are the attractive targets in malarial chemotherapy to overcome
the drug resistance.14 Cysteine protease falcipains are essential
for degradation of hemoglobin during erythrocytic parasite
q
Part XXI in the series, ‘Advances in drug design and discovery’. CSIR-CDRI
number 8281.
⇑
Corresponding authors. Tel.: +91 9919317940; fax: +91 5222623405.
E-mail
addresses:
kv_sashidhara@cdri.res.in,
sashidhar123@gmail.com
(K.V. Sashidhara), kumkum_srivastava@cdri.res.in (K. Srivastava).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.028

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K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5455–5459
OH
N
N
HN
HN
Cl
N
N
Cl
Chloroquine (1)
Amodiaquine (2)
O
Br
HO
OH
O
HN
NH
N
O
OCH3
O
Cl
O
Licochalcone-A (3)
Our previous potent keto-enamine
O
O
chalcone-chloroquine based hybrid (4)
O
N
Cl
HN
N
Cl
Most active chloroquine-chalcone hybrid
in this study (13)
Figure 1. Chemical structures of some potent antimalarials and our synthesized potent hybrids.
OH
NH2
Cl
N
HN
N
N
HN
ii
i
Cl
Cl
Cl
N
5
6
iii
R
O
O
O
O
N
N
HN
N
Cl
HN
Cl
iv
Cl
Cl
N
7
8-22
Scheme 1. Synthesis of chloroquine-chalcone hybrids. Reagents and conditions: (i) Hydrazine hydrate, ethanol, reflux, 8 h; (ii) 4-hydroxybenzaldehyde, ethanol, reflux,
2–3 h; (iii) 4-chlorophenacyl bromide, acetonitrile, K2CO3, rt, 2 h; (iv) Different substituted aldehydes, 10% methanolic KOH, rt.
development. Licochalcone-A (3, Fig. 1) was the first chalcone re-
ported with potent antimalarial activity against chloroquine resis-
tant P. falciparum.15 Subsequently, several synthetic chalcones
were synthesized and reported as significant antimalarial agents.16
As part of our ongoing drug discovery program in developing
potential antimalarial agents,17 we recently designed and synthe-
sized a series of potent novel keto-enamine chalcone-chloroquine
based hybrids.17a Inspired by these encouraging results, herein,
we report, design and synthesis of CQ-chalcone based hybrid
molecules and their antimalarial evaluation against resistant strain
of P. falciparum. The synthesis of target (8–22) and intermediate
(5–7) compounds was performed as outlined in the Scheme 1.
Synthesis of the compound 5 was achieved by the nucleophilic
substitution of the 4-Chloro of 4,7-dichloroquinoline with
hydrazine hydrate. The resulting compound 5 was easily con-
densed with 4-hydroxy benzaldehyde affording the compound 6.

K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5455–5459
5457
Table 1
In vitro antimalarial activity against chloroquine sensitive strain 3D7, chloroquine resistant strain K1 of P. falciparum, in vitro cytotoxicity of compounds using VERO cell line and
LogP values
Compd
R
Molecular weight
In vitro antimalarial activity IC50 (nM)
Selectivity index (SI)
LogP
3D7
K1
7
8
449
537
36.70
78.77
125.87
800.36
5.41
7.19
110.20
97.87
96.91
2364.07
9
551
567
111.68
48.09
1624.96
2050.61
7.67
7.06
CH
3
OCH
10
3
OCH
3
11
597
250.41
125.19
668.89
6.93
H CO
3
OCH
3
12
13
597
627
84.03
97.77
82.93
1442.69
837.293
6.93
6.81
OCH
3
OCH
3
123.50
OCH
H CO
3
3
OCH
3
OCH
14
15
627
627
90.49
95.31
89.50
727.88
970.21
6.81
6.81
3
H CO
3
OCH
OCH
3
OCH
3
122.61
3
16
17
18
527
555
571
57.30
29.06
144.69
114.70
102.07
199.49
962.25
5.75
7.34
7.74
O
6199.63
1210.36
F
Cl
F
19
20
21
573
605
138.81
295.42
128.12
170.22
NA
1040.86
559.50
7.50
8.30
8.05
F
Cl
Cl
2
NO
582
582
314.86
1341.02
22
NO
>500
7.68
NA
NA
8.05
3.73
2
CQ
463+
30612
IC50: Concentration corresponding to 50% growth inhibition of the parasite.
SI: IC50 values of cytotoxic activity/IC50 values of antimalarial activity.
LogP: Calculated by Chem Draw Ultra software.
NA: Not active.
Furthermore, the nucleophilic substitution on compound 6 by
using 4-chlorophenacyl bromide in the presence of potassium
carbonate, furnished compound 7 in quantitative yields. Finally,
chloroquine-chalcone hybrid compounds (8–22).18 The detailed
reaction conditions are illustrated in Scheme 1. The structures of
all the new synthesized compounds were confirmed by 1H NMR,
13C NMR, IR spectroscopy and mass spectrometry. The purity of
these compounds was ascertained by TLC analysis. (See Supple-
mentary data).
the target compounds were synthesized by
a base (KOH)
catalyzed Claisen-Schmidt condensation of compound 7 with
the appropriate aldehydes at room temperature to furnish

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K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5455–5459
Table 2
Inhibition of in vitro b-hematin formation
Table 3
Inhibition of hydrogen peroxide mediated hemin
decomposition
Compd
Inhibition of b-hematin
formation IC50
(lg/mL)
Compd
Percentage of
remaining hemin
7
8
9
16.4
5.66
5.58
17.6
25.0
3.46
3.52
10.6
10.8
3.74
15.6
8.38
ND
Control
Chloroquine
12
13
16
43
65
59
63
53
10
11
12
13
14
15
16
17
18
19
20
21
22
CQ
Results were expressed as the percentage of remained
hemin in the reaction mixture. Data are the mean of
three different experiments in triplicate.
further tested for their cytotoxicity against VERO cells using MTT
assay.20 Among all the synthesized hybrid derivatives, compound
17, which was more potent against CQ-sensitive 3D7 strain
showed good selectivity index of 6199.63 in comparison with other
derivatives. While the most potent compound 13 having IC50
82.93 nM against CQ-resistant strain showed selectivity index of
837.293. Rest of compounds showed SI values ranging between
387 and 2364. Compounds with selectivity index (SI) value greater
than 50 are generally considered safe. Although these hybrid com-
pounds have exhibited higher IC50 values against 3D7 strain but
unlike CQ they have exhibited lower IC50 values against K1 strain,
these findings make these hybrid compounds worth pursuing as
promising leads.
5.91
5.72
12.8
3.75
Data are the mean of three different experiments in
triplicate.
The IC50 represents the concentration of compound
that inhibit b-hematin formation by 50%.
ND: Not Done.
All the synthesized chloroquine-chalcone based hybrids (8–22)
and intermediate compound 7 were evaluated for their in vitro
antimalarial efficacy against 3D7 (CQ sensitive) and K1 (CQ resis-
tant) strains of P. falciparum.19 Most of the compounds showed po-
tent antimalarial activity as compared to CQ against K1 strain, on
the other hand some of the compounds showed comparable activ-
ity to CQ against 3D7 strain. The results have been summarized in
Table 1. Among 16 tested compounds, 13 was found to be the most
potent compound of the series with IC50 value of 82.93 nM against
chloroquine resistant K1 strain. It was almost sixfold more potent
than chloroquine. Five compounds of the series (9, 10, and 12–14)
showed IC50 in the range of 80–100 nM and six compounds (7, 8,
11, and 15–17) showed IC50 ranging between 100–130 nM. The
structure-activity relationship studies for these compounds sug-
gest that the presence of either methyl or methoxy functionalities
on phenyl ring improves their activity. The compounds 9 and 10
consisting methyl and methoxy groups on phenyl ring showed
IC50 97.87 and 96.91 nM, respectively. The replacement of electron
releasing groups with those of electron withdrawing groups like Cl
and NO2 on phenyl ring resulted in reduced antimalarial activity as
shown by compounds 18, 20, 21, 22 (Table 1), however, in the case
of compound 17 which consists of fluorine on phenyl ring the
activity was found to be comparable to compounds with electron
releasing groups. The substitution pattern as well as number of
substituent’s on the phenyl ring also altered the activity profile,
wherein compound 13 consisting three methoxy groups on phenyl
ring at 2nd, 3rd, and 4th positions showed IC50 value of 82.93 nM,
whereas the compound 15 consisting three methoxy groups at 3rd,
4th, and 5th positions showed IC50 value 122.61 nM, suggesting
that steric factor also played a crucial role in the antimalarial activ-
ity of these hybrids against CQ-resistant K1 strain. In the case of
chloroquine sensitive 3D7 strain, compound 17 containing fluorine
group on phenyl ring showed maximum antimalarial activity with
IC50 value of 29.06 nM, while, the incorporation of an extra fluorine
group on phenyl ring (compound 19), resulted in the reduction of
antimalarial activity (IC50 = 138.81 nM). In case of compound 21
with a nitro group at meta position of phenyl ring showed an
IC50 value of 128.12 nM. While changing the position of nitro group
from meta to para (compound 22), we observed a sharp decrease in
antimalarial activity (IC50 >500 nM). These compounds were
Furthermore, to unfold the mechanism of action of these syn-
thesized hybrid molecules, the b-hematin inhibitory activity of
all the molecules were carried out.21 The results have been sum-
marized in Table 2. The compounds 12, 13 consisting dimethoxy
and trimethoxy groups on phenyl ring and compound 16 consist-
ing furan ring showed stronger b-hematin inhibitory activities
(3.46, 3.52 and 3.75
lg/mL, respectively) as compared to chloro-
quine (3.75 g/mL). These three compounds were further analyzed
l
for hydrogen peroxide mediated hemin degradation.22 The results
have been summarized in Table 3. The compounds 12, 13 and 16
were found to be significantly protective against hemin peroxida-
tion in comparison to control groups. We observed the positive
correlation between these two mechanisms that is the inhibition
of b-hematin formation in the digestive vacuole of parasite leads
to release of excessive free hemin and further its accumulation in
cytoplasm,23 where H2O2 mediated secondary depredation of
hemin would take place. These chloroquine-chalcone hybrids were
analyzed with the assumption that their mode of action matches
that of chloroquine to prevent heme polymerization in the food
vacuole of the parasite. As most of these compounds showed sig-
nificant activities against CQ resistant strain (K1) of malarial para-
site but only few of them acted through inhibition of heme
polymerization in the parasite, it might be possible that these hy-
brids may also affect other resistance reversal mechanism.
In summary, a novel series of chloroquine-chalcones based hy-
brid antimalarials were designed and synthesized. The synthesized
molecules showed significant in vitro antimalarial activity espe-
cially against CQ resistant strain (K1). Three molecules were found
to be most efficacious to inhibit in vitro b-hematin formation.
Activity results indicated that compounds 12, 13 and 16 could be
utilized as lead molecules for further chemical modifications to en-
hance their therapeutic potential as antimalarial agents in the
emergence of rapid resistance against existing antimalarials. Fur-
ther pharmacological and pharmacokinetic development of these
hybrid molecules is currently in progress.
Acknowledgments
The authors are grateful to the Director, CDRI, Lucknow, India
for constant encouragement in drug development program, S.P.

K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5455–5459
5459
the reaction mixture was evaporated to dryness, the solid obtained was
neutralised with dil HCl and extracted with ethyl acetate. The combined
organic extracts were dried (Na2SO4), and concentrated in vacuo, and the
residue was further purified by column chromatography (SiO2, 1% to 2% CH3OH
in CH2Cl2 as eluant) afforded compound (13) as Yellow solid; yield: 76%; mp
218–220 °C; IR (KBr, cm1): 3455, 3018, 2906, 1582, 1360, 1216, 766; 1H NMR
(CDCl3 + CD3OD, 300 MHz) d: 8.44 (s, 1H), 8.10–8.05 (m, 2H), 7.85–7.83 (m,
3H), 7.68–7.64 (m, 4H), 7.45 (d, J = 8.4 Hz, 2H), 7.40–7.37 (m, 2H), 7.03 (d,
J = 8.5 Hz, 2H), 6.53 (s, 1H), 3.92 (s, 3H), 3.87 (s, 3H), 3.65 (s, 3H); 13C NMR
(CDCl3, 75 MHz) d: 191.1, 157.5, 156.0, 153.5, 150.3, 148.1, 146.3, 143.6, 142.2,
139.4, 135.9, 131.0, 129.5, 129.0, 128.9, 127.2, 126.1, 125.8, 125.0, 122.4, 119.3,
116.2, 115.8, 108.0, 102.0, 62.0, 62.1, 56.3; ESI-MS (m/z): 628 (M+H)+;
HRMS(m/z): calcd for C34H27Cl2N3O5 (M+H) +: 628.1406, Found: 628.1396.
(b) Ducki, S.; Rennison, D.; Woo, M.; Kendall, A.; Chabert, J. F. D.; McGown, A.
T.; Lawrence, N. J. Bioorg. Med. Chem. 2009, 17, 7698.
Singh for technical support, SAIF for NMR, IR, and Mass spectral
data. S.R.A and G.R.P. are thankful to CSIR, New Delhi, India for
financial support. This work was supported by EMPOWER grant
(OLP0007) of CSIR-CDRI to KVS. This is CSIR-CDRI communication
number 8281.
Supplementary data
Supplementary data (spectral data of all the compounds) asso-
ciated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2012.07.028.
19. In vitro antimalarial assay: The compounds were dissolved in DMSO at 5 mg/
mL. Twofold serial dilutions of test samples were made in 96 well plates and
incubated with 1.0% parasitized cell suspension containing 0.8% parasitaemia
(Asynchronous culture with more than 80% ring stages). The plates were
incubated at 37 °C in CO2 incubator in an atmosphere of 5% CO2 and air
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buffer pH (5.1), 50
compound/drug in
l
a
L plasma, 100
total reaction volume of 1.0 mL. The control tube
lM hemin as the substrate and 1–20 lg
contained all reagents except compound. The reaction mixture in triplicate
was incubated at 37 °C for 16 h in a rotary shaker. The reaction was stopped by
centrifugation at 10,000 rpm for 10 min at 30 °C. The pellet was suspended in
100 mM Tris–HCl buffer pH (7.4) containing 2.5% SDS. The pellet obtained after
centrifugation was washed thrice with distilled water (TDW) to remove free
hemin attached to b-hematin. The pellet was solubilized in 50 lL of 2 N NaOH
and volume was made up to 1.0 mL with TDW. Absorbance was measured at
400 nm. The 50% inhibitory concentration (IC50) was determined using non-
linear regression analysis dose response curves (Pandey, A.V.; Singh, N.;
Tekwani, B.L.; Puri, S.K.; Chauhan, V.S. Assay of beta-hematin formation by
malaria parasite. J. Pharm. Biomed. Anal. 1999, 20, 203).
22. Measurement of hydrogen peroxide-mediated hemin degradation: Hydrogen
peroxide-mediated hemin degradation was also evaluated in the presence/
absence of compounds by using the method of Loria et al. (1999) with some
modifications. The inhibition was monitored in 96 well ELISA plate with a total
16. (a) Mishra, N.; Arora, P.; Kumar, B.; Mishra, L. C.; Bhattacharya, A.; Awasthi, S.
K.; Bhasin, V. K. Eur. J. Med. Chem. 2008, 43, 1530; (b) Liu, M.; Wilairat, P.; Go,
M. L. J. Med. Chem. 2001, 44, 4443.
17. (a) Sashidhara, K. V.; Kumar, M.; Modukuri, R. K.; Srivastava, R. K.; Soni, A.;
Srivastava, K.; Singh, S. V.; Saxena, J. K.; Gauniyal, H. M.; Puri, S. K. Bioorg. Med.
Chem. 2012, 20, 2971; (b) Sashidhara, K. V.; Kumar, A.; Dodda, R. P.; Krishna, N.
N.; Agarwal, P.; Srivastava, K.; Puri, S. K. Bioorg. Med. Chem. Lett. 2012, 22, 3926.
18. (a) Representative procedure for synthesis of (Z)-1-(4-chlorophenyl)-2-(4-((E)-(2-
(7-chloroquinolin-4-yl)hydrazono)methyl)phenoxy)-3-(2,3,4-trimethoxyphenyl)-
prop-2-en-1-one (13).
reaction volume of 200
NaOH), 180 g Bovine serum albumin (in 0.2 M Sodium acetate buffer pH 5.1),
20 mM of H2O2 and 20 of Chloroquine/Compounds. The plates were
lL for each well consisting 25 lM Hemin (in 0.1 N
l
l
M
incubated to equilibrate for 10 min. at room temperature after addition of
hemin and bovine serum albumin. The peroxidative reaction was initiated by
the addition of H2O2 and followed by measuring the decrease in absorption at
the Soret band (405 nm) after 30 min. incubation at room temperature. Control
groups (TDW instead of H2O2) were also included in each experiment. Results
were expressed as the percentage of remained hemin in the reaction mixture.
Data are the mean of three different experiments in triplicate (Loria. P.; Miller,
S.; Foley, M.; Tilley, L. Biochem. J. 1999, 339, 363).
To
a solution of 7 (449 mg, 0.001 mol) in 10% methanolic KOH, 2,3,4-
trimethoxy benzaldehyde (196 mg, 0.001 mol) was added and stirred at
room temperature for 3 h. After completion of reaction (monitored by TLC),
23. Ginsburg, H.; Ward, S. A.; Bray, P. G. Parasitol. Today 1999, 15, 357.