Synthesis and exploration of novel curcumin analogues as anti-malarial agents

Article abstract of DOI:10.1016/j.bmc.2007.12.054

Curcumin, a major yellow pigment and active component of turmeric, has been shown to possess anti-inflammatory and anti-cancer activities. Recent studies have indicated that curcumin inhibits chloroquine-sensitive (CQ-S) and chloroquine-resistant (CQ-R) Plasmodium falciparum growth in culture with an IC₅₀ of ～3.25 μM (MIC = 13.2 μM) and IC₅₀ 4.21 μM (MIC = 14.4 μM), respectively. In order to expand their potential as anti-malarials a series of novel curcumin derivatives were synthesized and evaluated for their ability to inhibit P. falciparum growth in culture. Several curcumin analogues examined show more effective inhibition of P. falciparumgrowth than curcumin. The most potent curcumin compounds 3, 6, and 11 were inhibitory for CQ-S P. falciparum at IC₅₀ of 0.48, 0.87, 0.92 μM and CQ-R P. falciparum at IC₅₀ of 0.45 μM, 0.89, 0.75 μM, respectively. Pyrazole analogue of curcumin (3) exhibited sevenfold higher anti-malarial potency against CQ-S and ninefold higher anti-malarial potency against CQ-R. Curcumin analogues described here represent a novel class of highly selective P. falciparum inhibitors and promising candidates for the design of novel anti-malarial agents.

Full text of DOI:10.1016/j.bmc.2007.12.054

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2894–2902
Synthesis and exploration of novel curcumin analogues
as anti-malarial agents
Satyendra Mishra,a Krishanpal Karmodiya,b Namita Suroliab and Avadhesha Suroliaa,c,
*
aMolecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India
bMolecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560 064, India
cNational Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India
Received 15 November 2007; revised 27 December 2007; accepted 28 December 2007
Available online 1 January 2008
Abstract—Curcumin, a major yellow pigment and active component of turmeric, has been shown to possess anti-inflammatory and
anti-cancer activities. Recent studies have indicated that curcumin inhibits chloroquine-sensitive (CQ-S) and chloroquine-resistant
(CQ-R) Plasmodium falciparum growth in culture with an IC50 of ꢀ3.25 lM (MIC = 13.2 lM) and IC50 4.21 lM (MIC = 14.4 lM),
respectively. In order to expand their potential as anti-malarials a series of novel curcumin derivatives were synthesized and
evaluated for their ability to inhibit P. falciparum growth in culture. Several curcumin analogues examined show more effective
inhibition of P. falciparumgrowth than curcumin. The most potent curcumin compounds 3, 6, and 11 were inhibitory for CQ-S
P. falciparum at IC50 of 0.48, 0.87, 0.92 lM and CQ-R P. falciparum at IC50 of 0.45 lM, 0.89, 0.75 lM, respectively. Pyrazole ana-
logue of curcumin (3) exhibited sevenfold higher anti-malarial potency against CQ-S and ninefold higher anti-malarial potency
against CQ-R. Curcumin analogues described here represent a novel class of highly selective P. falciparum inhibitors and promising
candidates for the design of novel anti-malarial agents.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
is inexpensive, and demonstrates little toxicity across a
diverse population represents the ideal choice for treat-
ing malaria.
Malaria is caused by the protozoa of the genus Plasmo-
dium. Because of its prevalence, virulence, and drug
resistance, it is the most serious and widespread para-
sitic disease. It has an overwhelming impact on public
health in developing regions of the world.1–3 Attempts
to control mosquito vector and the use of anti-malarial
drugs notwithstanding, there are yet some 2.5–3 million
fatalities, mostly children and women annually from
malaria.4 Not clearly evident in the mortality statistics
are the vast range of problems associated with the mil-
lions of cases suffering from morbidity derived from
malaria infection. In part, the extent of the economic
and public health problems relevant to malaria pertains
to difficulties in the chemotherapy of this protozoan
disease.5 As a result, there is an urgent need for new
efficient anti-malarial agents.6,7 Thus, identification of
an anti-malarial agent that is easy to synthesize/isolate,
Curcumin is a b-diketone constituent of the turmeric
that is obtained from the powdered root of Curcuma
longa Linn. It is used as a spice to give a specific flavor
and yellow color to curry, which is consumed in trace
quantities daily by nearly a billion people. Curcumin
has been used by traditional medicine for liver disease
(jaundice), indigestion, urinary tract diseases, rheuma-
toid arthritis, and insect bites. This phytochemical
has also been demonstrated to possess both anti-cancer
and anti-angiogenic properties. Its anti-tumor proper-
ties include growth inhibition and apoptosis induction
in a variety of cancer cell lines in vitro, as well as the
ability to inhibit tumorigenesis in vivo.8–14 Moreover,
pyrazole analogues of curcumin were synthesized and
investigated for lipoxygenase inhibitory activity,15
cytotoxic activity16,17, and anti-oxidant activity.18 Re-
cently, curcumin has been shown to have synergistic ef-
fects with artemisinin against Plasmodium berghei
in vivo.19,20 Curcumin is also potent against both chlo-
roquine (CQ)-susceptible (CQ-S) and -resistant (CQ-R)
Keywords: Plasmodium falciparum; Curcumin; Antimalarial; Pyrazole.
*
Corresponding author. Tel.: +91 11 26717102; fax: +91 11
26717104; e-mail: surolia@nii.res.in
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.12.054

S. Mishra et al. / Bioorg. Med. Chem. 16 (2008) 2894–2902
2895
Plasmodium falciparum strains, and the parasiticidal ef-
3.1. Anti-malarial activity and structure–activity
relationship of curcumin and its analogues against
chloroquine sensitive (CQ-S) and chloroquine resistant
fect is at least partially due to the generation of reac-
tive oxygen species (ROS), and down-regulation of
the Pf GCN5 HAT activity.21 The P. falciparum gen-
eral control non-derepressed 5 (PfGCN5) is a HAT
that preferentially acetylates K9 and K14 of histone
H3. Knoevenagel condensates of curcumin analogues
were found to be capable of inhibiting NF-jb activa-
tion.22 In addition, there are no reports on develop-
ment of resistance to curcumin against anti-cancer
activity.23 In view of the above, we have synthesized
curcumin analogues to improve its potency as an
anti-malarial agent. Isoxazole derivative of curcumin
(2), pyrazole derivatives of curcumin (3–8), Knoevena-
gel condensate derivatives of curcumin (9–11), di-O-
acetyl curcumin (12), and di-O-methylcurcumin (13),
thus synthesized exhibit anti-malarial activity against
both CQ-R and CQ-S P. falciparum.
(CQ-R) P. falciparum
Based on the above-mentioned curcumin properties, we
used its scaffold to develop novel anti-malarial agents.
All the synthesized curcumin analogues were checked
on P. falciparum cultures to determine the IC50 values
for inhibitors. Each test was performed in triplicate
and the IC50 reported represent the result of at least
two repetitions. Curcumin inhibits CQ-S P. falciparum
at IC50 of 3.25 0.6 lM and CQ-R P. falciparum at
IC50 of 4.21 0.8, in a similar range as reported earlier
for chloroquine-resistant P. falciparum cultures in a dose
dependent manner with an IC50 of ꢀ5 lM;19 while its
minimum inhibitory concentration (MIC) against CQ-
S
and CQ-R was found to be 13.2 1.3 and
14.4 1.6 lM. MIC was read as the lowest concentra-
tion that resulted in no malarial parasite growth. The
slight difference in IC50 between our results and others
could be due to variations in culture conditions, genetic
background of the parasite, developmental stages, drug
purity, solvents for dissolving the drug, etc.
2. Chemistry
The isoxazole derivative of curcumin was prepared
as depicted in Scheme 1. Isoxazole derivative (2) of
curcumin was synthesized by treatment of curcumin
with hydroxylamine hydrochloride in acetic acid at
85 ꢁC for 6 h.18 Pyrazole derivatives of curcumin were
synthesized as reported previously (Scheme 1).15 Pyra-
zole derivatives (3–8) were prepared by heating
curcumin (1) for 8 h with hydrazine hydrate, phen-
ylhydrazine, 2-fluorophenylhydrazine hydrochloride,
3-nitrophenylhydrazine hydrochloride, 2, 4-dichloro-
phenylhydrazine hydrochloride, 4-methoxyphenylhy-
drazine hydrochloride in acetic acid in good yield.
Compounds 9–11 reported in Table 1 were synthesized
according to Scheme 2. Knoevenagel condensates of
curcumin (9–11) were prepared by treatment of curcu-
min with aromatic aldehyde (benzaldehyde, 4-hydroxy-
benzaldehyde, 4-hydroxy-3-methoxybenzaldehyde) in
presence of triphenylphosphane (as catalyst) to yield
respective compounds. Di-O-acetyl and di-O-methyl
curcumin were synthesized as previously reported.16,24
Acetylation of curcumin (1) with acetic anhydride in
presence of pyridine gave di-O-acetyl derivative (12).
Di-O-methyl curcumin derivative 13 was synthesized
by methylation of curcumin (1) with dimethyl sulfate
(Scheme 3). All the synthesized derivatives were charac-
terized by 1H NMR, 13C NMR, and Electrospray–
mass spectrometry [ESI–MS].
All the compounds mentioned in the manuscript except-
ing curcumin used here as the reference compound have
been reported for the first time for their anti-malarial
activity. While, compounds (4–11) are novel and have
been synthesized and characterized by us for the first
time, compounds (2, 3, 12, and 13)15–18 have been syn-
thesized previously, but have been checked for their
anti-malarial activity for the first time in this study.
Starting from curcumin structure, we have investigated
the effect of different substituents on N-phenylpyrazole
and Knoevenagel condensates of curcumin.
3.2. Pyrazole versus isoxazole
When isoxazole group in isoxazole derivative of curcu-
min (2) was replaced by a pyrazole group, it led to a
moderate increase of anti-malarial potency against
CQ-S and CQ-R. Thus converting the keto-enol moiety
(1) to the corresponding pyrazole (3) led to increased
anti-malarial potency against P. falciparum in culture
(Table 1) and replacing –NH group of pyrazole deriva-
tive of curcumin (3) by –O isoxazole derivative (2) re-
sults in decrease of potency. It therefore implies that
the presence of –NH group in pyrazole derivative (3)
is necessary for its potency. Pyrazole derivative of curcu-
min (3) inhibited the CQ-S and CQ-R P. falciparum cul-
tures at nanomolar concentrations with IC50 of 0.48 and
0.45 lM, respectively.
3. Results and discussion
While curcumin itself has recently been shown to exhi-
bit anti-malarial activity, prior to these studies com-
pounds designed around the scaffold of curcumin
have not been explored systematically for their anti-
malarial activity against both CQ-S and CQ-R
P. falciparum. A correlation between their structure
and anti-malarial activity has not been attempted so
far. Moreover, this study attempts for the first time a
structure–activity relation for the anti-malarial activity
of curcumin scaffold.
3.3. Effect of electron withdrawing group on N-phenyl
curcumin pyrazole
Electron withdrawing substituents at N-phenyl curcu-
min pyrazole system affect the anti-malarial potency of
corresponding compounds. Previous reports show that
electron withdrawing groups enhance the biological
activity25,26 while electron donating groups show
marked decrease in activity. Similarly, in our case elec-

2896
S. Mishra et al. / Bioorg. Med. Chem. 16 (2008) 2894–2902
NH
N
ii
H3CO
OCH3
OH
3
HO
O
O
N
O
H3CO
HO
OCH3
OH
H3CO
HO
OCH3
OH
i
2
1
R3
R2
R1
N
N
H3CO
OCH3
OH
iii
4-8
HO
Compound
R
1
R
2
R
3
4
5
6
7
8
H
H
H
Cl
H
H
H
NO2
H
H
F
H
Cl
H
OCH3
Scheme 1. Synthesis of pyrazole, isoxazole, and N-(substituted) phenylpyrazole analogues of curcumin. Reagents and conditions: (i) NH2-OHÆHCl,
CH3COOH/85 ꢁC 6 h; (ii and iii) hydrazinehydrate, phenylhydrazine, 2-fluorophenylhydrazine hydrochloride, 3-nitrophenylhydrazine hydrochlo-
ride, 2, 4-dichlorophenylhydrazine hydrochloride, 4-methoxyphenylhydrazine hydrochloride, CH3COOH/8 h, reflux.
tron withdrawing substituted compounds (5–7) have
shown improved activity while compounds with electron
donating group substitutions show drastic reduction in
anti-malarial activity as is evident from the activities
of unsubstituted phenylpyrazole curcumin (4) and
substituted phenylpyrazole (5–8).
We investigated the role played by various electron
withdrawing and electron donating groups on phenyl-
pyrazole curcumin analogues. No significant correla-
tion between Hammett r and the IC50 values was
found for the substituted phenylpyrazole compounds.
From the above outcome it is concluded that nature
of substitutions on N-phenylpyrazole derivative affects
the potency of compounds. 3-Nitro phenylpyrazole
curcumins (6) showed better inhibition as compared
to curcumin and other N-phenylpyrazole curcumin
against both CQS and CQR (Table 1). Thus, the ring
substituents affect the potency of the phenylpyrazole
derivatives.
While a dramatic decrease in potency was observed when
the unsubstituted phenylpyrazole derivative was replaced
by electron donating 4-methoxyphenyl group (4 vs 8),
activity was enhanced in both CQ-S and CQ-R strains
when 3-nitrophenyl substitution was performed (4 vs
6), The most active 3-nitrophenylpyrazole derivative of
curcumin (Table 1 and Fig. 1) exhibits tenfold more po-
tency as compared with N-phenylpyrazole curcumin
and four to fivefold more potency as compared to curcu-
min against CQ-S and CQ-R strains, respectively. We
also explored the effect of the introduction of other elec-
tron withdrawing groups viz. 4-fluoro and 2, 4-dichloro
at N-phenylpyrazole system, in order to increase the
anti-malarial potency of N-phenylpyrazole. When 4-flu-
oro and 2, 4-dichloro substituents were introduced, the
resulting compounds 5 and 7 display two to fourfold
more potencies, respectively, relative to the parent N-
phenyl curcumin pyrazole against CQ-S and CQ-R.
Although 4-fluoro (5) and 2, 4-di-chloro (7) substituted
N-phenyl pyrazole derivative have also electron with-
drawing properties, they are weaker than the nitro substi-
tuted derivative. Consequently compounds 5 and 7 are
less potent than nitro substituted N-phenylpyrazole cur-
cumin (6). Also compound 5 has identical potency to cur-
cumin and 7 has lower potency compared to that of
curcumin (Table 1). Lower inhibition by compound (7)
was perhaps due to steric hindrance caused by the pres-
ence of two chlorine atoms. Comparisons between 5
and 8 suggest the effect of electron withdrawing and elec-
tron donating groups on unsubstituted phenylpyrazole
curcumin (4).
3.4. Substitution on Knoevenagel condensates of curcumin
Prior to this study some Knoevenagel condensates of
curcumin were synthesized and found to be capable of
inhibiting NF-jb activation.22 In the present communi-
cation we have synthesized a number of novel Knoeve-
nagel condensates of curcumin and evaluated their
anti-malarial activity against CQ-S and CQ-R. Com-
pounds 9, 10, and 11 are Knoevenagel condensate deriv-
atives of curcumin and inhibit CQ-S cultures at
IC50 = 3.89 lM (MIC 16.5 lM), IC50 = 5.85 lM
(MIC = 23.7), and IC50 = 0.920 lM (MIC 5.2 lM),
respectively. Their inhibitory potencies for CQ-R cul-
tures also lie in a similar range (Table 1). The most ac-
tive compound amongst Knoevenagel condensates of
curcumin was found to be 11 (4-hydroxy-3-methoxy-
benzylidene derivative of curcumin) suggesting an aryl
hydroxy pharmacophore at the methylene center (C4)
containing methoxy group at meta position is contribut-
ing significantly to the enhanced anti-malarial potency
(Table 1). Compounds 9 (benzylidene derivative of cur-
cumin) and 10 (4-hydroxy-benzylidene derivative of cur-
cumin) were as effective as curcumin. Therefore, the
presence of methoxy group (electron donor group) at

S. Mishra et al. / Bioorg. Med. Chem. 16 (2008) 2894–2902
2897
Table 1. IC50 and minimum inhibitory concentration (MIC) values of curcumin and its derivatives on cultures of Plasmodium falciparum
Compound ID
Structure
P. falciparuma,b IC50 (lM)
P. falciparuma,b MIC (lM)
O
O
H CO
OCH
OH
3
3
1
3.25 0.6
(4.21 0.8)
13.2 1.3
(14.4 1.6)
HO
N
N
O
H CO
OCH
OH
3
3
2
3
8.44 2.1
(7.92 2.4)
27.3 6.7
(25.3 4.5)
HO
NH
H CO
OCH
OH
3
3
0.48 0.04
(0.45 0.07)
3.9 0.8
(3.6 0.63)
HO
N
N
4
5
8.48 0.8
(9.10 0.9)
22.4 6.1
(25.0 6.6)
H CO
OCH
3
3
HO
OH
F
2.42 0.4
(2.10 0.36 )
14.3 1.4
(12.9 1.6)
N
N
H CO
OCH
OH
3
3
HO
NO
2
6
7
0.87 0.07
(0.89 0.10)
4.9 0.7
(5.1 0.81)
N
N
N
H CO
OCH
OH
3
3
HO
Cl
N
Cl
4.65 0.7
(4.80 0.9)
21.81 5.7
(21.4 5.4)
H CO
3
OCH
OH
3
HO
OCH
N
3
8
9
22.60 2.7
(24.56 3.8)
35.80 3.2
(41.20 4.6)
N
H CO
OCH
OH
3
3
HO
O
O
H CO
OCH
OH
3
3
3.89 0.7
(4.12 0.6)
16.5 1.7
(17.20 1.9)
CH
HO
O
O
O
H CO
OCH
OH
3
3
10
11
5.85 0.9
(5.36 1.0)
23.7 7.3
(23.10 6.1)
CH
HO
HO
HO
O
H CO
3
OCH
OH
3
CH
OCH
0.92 0.09
(0.75 0.06)
5.2 0.9
(4.80 0.63)
(continued on next page)
HO
3

2898
S. Mishra et al. / Bioorg. Med. Chem. 16 (2008) 2894–2902
Table 1 (continued)
Compound ID
Structure
P. falciparuma,b IC50 (lM)
P. falciparuma,b MIC (lM)
O
O
O
O
H CO
OCH
OAc
3
3
12
13
2.34 0.4
(2.51 0.5)
14.9 1.1
(15.30 1.0)
AcO
H CO
OCH
OCH
3
3
3
7.86 1.1
(8.4 1.2)
29.6 8.7
29.1 8.1
H CO
3
a Data are expressed as means SD from at least three different experiments in duplicate.
b Values in parantheses indicate the activities of these derivatives against a CQ resistant strain MP-14 of P. falciparum.
O
O
O
O
H CO
3
OCH
OH
H CO
3
OCH
OH
3
3
a
CH
HO
HO
R
3
R
1
1
R
R3
H
OH
OH
9-11
2
Compound
R1
R2
9
10
11
H
H
H
H
H
OCH3
Scheme 2. Synthesis of curcumin Knoevenagel condensates (9–11). Reagents and conditions: (a) benzaldehyde, 4-hydroxybenzaldehyde, 4-hydroxy-
3-methoxy-benzaldehyde (vanillin), triphenylphosphine (catalytic amount), 75–80 ꢁC/ 3.5 h.
O
O
O
O
OCH
OR
H CO
3
OCH
OH
H CO
3
3
3
a or b
HO
RO
R = -Ac = 12
R = -CH = 13
1
3
Scheme 3. Synthesis of di-acetyl and di-methyl curcumin. Reagents and conditions: (a) acetyl chloride/pyridine, rt, overnight; (b) di-methyl sulfate,
K2CO3in benzene, reflux.
the meta position of 4-hydroxy-3-methoxy-benzylidene
derivative of curcumin appears to play an important
role for the potency of this class of compounds.
100
80
60
40
20
0
To understand the significance of phenolic OH group of
curcumin in its anti-malarial activity, di-O-acetyl (12)
and di-O-methyl (13) derivatives of curcumin have also
been tested for their anti-malarial activity. The di-O-
acetyl curcumin shows almost same potency as curcu-
min against P. falciparum culture. Reason may be that
acetyl groups are attached to curcumin by ester bonds,
which are biodegradable, and are apparently easily
cleaved by esterases in the parasite to release the parent
molecule curcumin. Hence the potency of compound 12
was equal to that of the parent molecule. Whereas meth-
ylation of the –OH resulted in the loss of potency be-
cause alkyl groups are not cleavable. From the above
results (Table 1) it is apparent that two unsubstituted
phenolic groups are necessary for curcumin’s anti-
malarial activity.
1
2
3
4
log (nM οf Compound 6)
Figure 1. Inhibition of the growth of the parasite in red blood cell
cultures. 3-Nitro-phenylpyrazole curcumin (Compound 6) was assayed
for parasite culture growth inhibition of P. falciparum by incubating
the cultures with different concentrations of the inhibitors in DMSO
(final concentration, 0.05%). The cultures were checked for growth
inhibition by microscopic examination by assessing the parasitemia.
The average of three data sets has been plotted, and the error bars are
shown.
Among all the derivatives tested, pyrazole derivative of
curcumin (3) showed the highest potency against both
CQ-S and CQ-R P. falciparum with an IC50 of 0.48

S. Mishra et al. / Bioorg. Med. Chem. 16 (2008) 2894–2902
2899
and 0.45 lM, respectively (Table 1). The inhibitory po-
tency of compound 3 was six to sevenfold higher against
CQ-S and ninefold higher against CQ-R than that ob-
served for curcumin. 3-nitrophenylpyrazole curcumin
(6) and 4-(4-hydroxy-3-methoxy-benzylidene) derivative
(11) of curcumin also showed an enhanced (three to five-
folds) anti-malarial potency in P. falciparum cultures. It
was found that curcumin analogues inhibited both CQ-
R and CQ-S P. falciparum cultures in a similar fashion,
indicating that curcumin and its analogues are able to
bypass the mechanisms that the parasite has evolved to-
ward CQ resistance. Given the structural differences be-
tween chloroquine and curcumin derivatives, these
findings can therefore easily be rationalized.
400 MHz Bruker NMR spectrometers using tetrameth-
ylsilane as internal standard and the chemical shifts
are reported in (d) units. Multiplicities are reported as
follows: s (singlet), d (doublet), t (triplet), q (quartet),
m (multiplet), and br (broadened). Coupling constants
are reported as a J value in Hertz (Hz).The sample con-
centration in each case was approximately 10 mg in
chloroform-d/methanol-d (0.6 mL). Mass spectra were
recorded on an Electrospray–MS (Bruker Daltonis, es-
quire 3000plus) instrument. All solvents used were of
spectral grade or distilled prior to use.
5.1. Isoxazole analogue of curcumin (2)
The isoxazole analogue was prepared by treating curcu-
min 0.736 g (2 mmol) with hydroxylamine hydrochlo-
ride, 0.139 g (2.2 mmol), in acetic acid at 85 ꢁC for 6 h
(Scheme 1). The reaction mixture was evaporated to
dryness; residue was dissolved in dichloromethane and
washed with water. The crude product was chromato-
graphed on a silica gel column, eluted with hexane/ethyl
acetate, 40:60. Rf = 0.75 (DCM/MeOH 9:1), 0.65
(DCM/MeOH 9:1) mp 162 ꢁC (lit. 162–163 ꢁC). Yield:
Although the specific mechanism by which curcumin
and its derivatives enact their activity against the human
malaria parasite is currently under study,15–17 their effi-
cacy in doing so, coupled with their relatively straight-
forward chemical structure and amenability to
chemical substitutions, make them attractive lead com-
pounds for structure modification and drug develop-
mental studies.
1
0.556 g (76%), H NMR (300 MHz, CDCl3): d 3.90 (s,
6H, OCH3), 6.68 (s, 1H, C4-H), 6.86–6.92 (m, 4H),
6.99–7.11 (m, 4H, Ar-H), 7.14 (d, 1H, J = 12.2 Hz),
7.2 (d. 2H, J = 12.2 Hz). 13C NMR (300 MHz, CDCl3):
169.6, 163.2, 151.5, 151.1, 149.3, 149.2, 138.2, 135.6,
130.1, 129.8, 122.8, 121.6, 120.5, 120.1, 117.4, 116.2,
115.9, 115.3, 98.7, 56.7. ESI–MS m/z [M+H]+366.1.
4. Conclusion
This work attempts to rationalize the structure–activity
correlation for curcumin scaffold as an anti-malarial
agent. The design strategy was focused on the develop-
ment of curcumin analogue with high anti-malarial
activity especially against CQ-R strains. Prior to these
studies compounds designed around the scaffold of cur-
cumin, excepting the usage of curcumin by itself, have
not been evaluated for their anti-malarial activity
against P. falciparum culture. We have now evaluated
the anti anti-malarial activity of curcumin and its deriv-
atives against this parasite.
5.2. General procedure for the preparation of N-(substi-
tuted)phenylcurcumin pyrazole compounds (3–8)
Curcumin (1.2 mmol) was dissolved in glacial acetic
acid (5 mL), and different hydrazine derivatives (1.5 mmol)
(hydrazinehydrate, phenylhydrazine, 4-fluorophenylhy-
drazine hydrochloride, 3-nitrophenylhydrazine hydro-
chloride, 2, 4-dichlorophenylhydrazine hydrochloride,
4-methoxyphenylhydrazine hydrochloride) were added
to the solution. The solution was refluxed for 8 h, and
then the solvent was removed in vacuo. Residue was dis-
solved in ethyl acetate and washed with water. Organic
portion was collected, dried over sodium sulfate, and con-
centrated in vacuo. Crude product was further purified by
column chromatography. All the products (3–8) were pre-
pared by using the same procedure.
From the results shown in Table 1 one can say that pyr-
azole curcumin (3), 3-nitrophenylpyrazole curcumin (6),
and 4-hydroxy-3-methoxy-benzylidene derivative (11) of
curcumin were more potent than curcumin (1) itself.
These compounds (3, 6 and 11) inhibited at nanomolar
concentration. Their enhanced potencies are probably
due to their increased cellular uptake and/or retention
by the parasite in addition to their greater efficacies of
inhibiting the target itself. The high potencies of some
of the curcumin derivatives reported here together with
the possibility of synthesizing a number of derivatives
around ‘curcumin’ scaffold open up new opportunities
for anti-malarial therapy.
5.2.1. Curcumin pyrazole (3). The silica gel eluent was
hexane/ethyl acetate, 60:40. Rf = 0.50 (DCM/MeOH
9:1), yield: 71%, mp 214 ꢁC (lit. 211–214). 1H NMR
(400 MHz, CD3OD): d 3.85 (s, 6H, 2 · OCH3), 6.65 (s,
1H, C4-H), 6.91 (d, 2H, J = 15.8 Hz, C2-H and, C6-H),
7.04 (d, 2H, J = 15.8 Hz, C1-H & C7-H), 7.16–7.21 (m,
6H, Ar-H). 13C NMR (400 MHz, CD3OD): 151.8,
151.4, 148.8, 148.2, 136.9, 136.4, 131.0, 129.8, 124.6,
124.2, 122.7, 122.6, 117.1, 116.7, 113.8, 110.9, 99.2,
56.5. ESI–MS m/z [M+H]+365.20.
5. Experimental
Reagents were purchased from Aldrich and were used as
received. Reaction progress was monitored by TLC
using Merck silica gel 60 F254 with detection by UV.
Column chromatography was performed using Merck
silica gel 100–200 mesh. Melting points were determined
5.2.2. N-Phenylpyrazole curcumin (4). The silica gel elu-
ent was hexane/ethyl acetate, 70:30. Rf = 0.72 (DCM/
MeOH 9:1), yield: 65%, mp 89 ꢁC. 1H NMR
(300 MHz, CDCl3): d 3.89 (s, 6H, 2 · OCH3), 6.79 (s,
in Pyrex capillary tube using Buchi Melting Point B-540
¨
apparatus. H NMR spectra was recorded on 300 and
1

2900
S. Mishra et al. / Bioorg. Med. Chem. 16 (2008) 2894–2902
1H, C4-H), 6.91 (d, 2H, J = 15.8 Hz, C2-H and, C6-H),
7.04 (d, 2H, J = 15.8 Hz, C1-H and C7-H), 7.15–25 (m,
hyde)
(5 mmol),
curcumin
(6.5 mmol),
and
triphenylphosphine (20 mol%]) was stirred at 75–80 ꢁC
for 3.5 h. The progress of the reaction was monitored
by TLC. After complete conversion, as indicated by
TLC, the reaction mixture was diluted with water and
extracted with ethyl acetate (3 · 10 mL). The combined
organic extracts were concentrated in vacuo and the
resulting product was directly charged onto a small silica
gel column and eluted with a mixture of ethyl acetate/
hexane to afford pure product. All the products (9–11)
were prepared using the same procedure.
6H,
Ar-H),
7.48–7.54(m,
5H,
Ar-H).
13C
NMR(300 MHz, CDCl3): 155.6, 151.9, 151.4, 147.5,
146.5, 142.1, 138.4, 133.4, 131.5, 129.6, 129.1, 127.6,
125.4, 125.1, 121.0, 120.6, 120.1, 114.5, 113.7, 108.2,
107.1, 100.1, 55.6, 55.4. ESI–MS m/z [M+H]+441.2.
5.2.3. N-(4-Fluorophenylpyrazole) curcumin (5). The silica gel
eluent was hexane/ethyl acetate, 70:30. Rf = 0.75 (DCM/
1
MeOH 9:1), yield: 62%. mp 65 ꢁC. H NMR (300 MHz,
CDCl3): d 3.89 (s, 6H, 2 · OCH3), 6.79 (s, 1H, C4-H),
6.98 (d, 2H, J = 15.6 Hz, C2-H and, C6-H), 7.06 (d, 2H,
J = 15.6 Hz, C1-H and C7-H), 7.10–7.22 (m, 6H, Ar-H),
7.45–7.69 (m, 4H, Ar-H). 13C NMR (300 MHz, CDCl3):
157.8, 154.1, 148.7, 148.2, 145.8, 145.2, 139.5, 135.8, 132.3,
129.8, 129.2, 124.7, 122.1, 117.9, 117.7, 113.5, 110.2, 110.6,
107.6, 56.8. ESI–MS m/z [M+H]+459.10.
5.3.1. 4-Benzylidene curcumin (9). The silica gel eluent
was ethyl acetate/ hexane, 15:85. Rf = 0.62 (DCM/
MeOH 9:1), yield: 58%, mp 120 ꢁC. 1H NMR
(300 MHz, CDCl3): d 3.89 (s, 6H, 2 · OCH3), 6.68–
7.15 (m, 6H, Ar-H), 7.18 (d, 2H, J = 15.0 Hz, C2-H
and, C6-H), 7.85(d, 2H, J = 15.0 Hz, C1-H and C7-H),
7.35–7.48 (m, 5H, Ar-H), 8.16 (s, 1H, =CH–Ar). 13C
NMR(300 MHz, CDCl3): 184.7, 165.9, 149.8, 147.5,
145.8, 142.1, 131.2, 129.0, 128.4, 128.1, 127.2, 124.4
123.1, 117.2, 111.9, 56.5. ESI–MS: m/z [M+H]+457.23.
5.2.4. N-(3-Nitrophenylpyrazole) curcumin (6). The silica
gel eluent was hexane/ethyl acetate, 70:30. Rf = 0.65
(DCM/MeOH 9:1), yield: 46%, mp 93 ꢁC. 1H NMR
(300 MHz, CDCl3): d 3.90 (s, 6H, OCH3), 6.80 (s, 1H,
C4-H), 7.02 (d, 2H, J = 14.8 Hz, C2-H and, C6-H),
7.10 (d, 2H, J = 14.8 Hz, C1-H and C7-H), 7.15–7.32
(m, 6H, Ar-H), 7.65–7.71 (m, 1H, Ar-H), 7.98–8.12 (m,
1H, Ar-H), 8.22–8.26 (m, 1 H, Ar-H), 8.43 (d, 1H,
J = 8.2 Hz, Ar-H). 13C NMR(300 MHz, CDCl3): 157.3,
153.2, 152.1, 150.8, 147.4, 146.7, 142.5, 139.0, 134.2,
132.8, 130.4, 130.1, 126.1, 124.8, 124.5, 122.0, 121.3,
120.7, 116.9, 116.5, 115.4, 113.1, 112.7, 105.2, 56.8,
56.6. ESI–MS m/z [MÀH]+(484.1), [M+H]+486.1.
5.3.2. 4-(4-Hydroxybenzylidene) curcumin (10). The silica
gel eluent was ethyl acetate/hexane, 20:80. Rf = 0.55
(DCM/MeOH 9:1), yield: 60%, mp 93 ꢁC. 1H NMR
(300 MHz, CDCl3): d 3.89 (s, 6H, 2 · OCH3), 6.77–
7.15 (m, 8H, Ar-H), 7.22 (d, 2H, J = 15.2 Hz, C2-H
and, C6-H), 7.68 (d, 2H, J = 8.6 Hz), 7.84(d, 2H,
J = 15.2 Hz, C1-H and C7-H), 8.06 (s, 1H, =CH-Ar).
13C NMR (300 MHz, CDCl3): 184.3, 165.7, 156.8,
149.4, 1467.9, 146.8, 142.1, 130.5, 127.9, 126.5, 125.6,
123.1, 117.2, 115.9, 111.9, 56.1. ESI–MS m/z
[MÀH]+471.2.
5.2.5. N-(2,4-Dichlorophenylpyrazole) curcumin (7). The
silica gel eluent was hexane/ethyl acetate, 70:30.
Rf = 0.70 (DCM/MeOH 9:1), yield: 54%, mp 142 ꢁC.
5.3.3. 4-(4-Hydroxy-3-methoxybenzylidene) curcumin
(11). The silica gel eluent was ethyl acetate/hexane,
20:80. Rf = 0.60 (DCM/MeOH 9:1), yield: 65%, mp
96 ꢁC. 1H NMR (300 MHz, CDCl3): d 3.87 (s, 3H,
OCH3), 3.89 (s, 6H, 2 · OCH3), 6.77–7.13 (m, 9H, Ar-
H), 7.21 (d, 2H, J = 15.0 Hz, C2-H and, C6-H), 7.83(d,
2H, J = 15.0 Hz, C1-H and C7-H), 8.01 (s, 1H, =CH–
Ar). 13C NMR (300 MHz, CDCl3): 184.3, 165.7, 149.8,
149.3, 1467.9, 147.6, 146.8, 146.5, 142.1, 130.5, 128.6,
128.1, 126.4, 125.6, 123.1, 117.1, 116.1, 111.6, 56.1,
55.9. ESI–MS m/z [M+H]+503.2.
1H NMR (300 MHz, CDCl3):
d
3.89 (s, 6H,
2 · OCH3), 6.81 (s, 1H, C4-H), 7.01 (d, 2HJ = 15.6 Hz,
C2-H and, C6-H), 7.08 (d, 2H, J = 15.6 Hz, C1-H and
C7-H), 7.18–7.31 ( m, 6H, Ar-H), 7.45 (m, 1H, Ar-H),
7.58 (m, 1H, Ar-H), 7.78 (m, 1H, Ar-H). 13C NMR
(300 MHz, CDCl3): 156.6, 154.1, 147.7, 147.0, 144.2,
142.8, 135.8, 132.3, 129.8, 129.2, 124.7, 122.1, 117.9,
117.7, 113.5, 110.2, 110.6, 107.6, 56.8, 56.4. ESI–MS
m/z [M]+509.38.
5.2.6. N-(4-Methoxyphenylpyrazole) curcumin (8). The sil-
ica gel eluent was hexane/ethyl acetate, 80:20. Rf = 0.65
(DCM/MeOH 9:1), yield: 35%, mp 106 ꢁC. 1H NMR
(400 MHz, CDCl3): d 3.89 (s, 6H, 2 · OCH3), 3.90(s,
3H, OCH3), 6.79 (s, 1H, C4-H), 6.98 (d, 2H,
J = 16.2 Hz, C2-H and, C6-H), 7.04 (d, 2H, J = 16.2 Hz,
C1-H and C7-H), 7.09–7.20(m, 6H, Ar-H), 7.28–7.48 (m,
4H, Ar-H). 13C NMR(400 MHz, CDCl3): 158.6, 154.3,
148.5, 148.0, 146.2, 146.4, 137.8, 132.3, 130.5, 129.8,
129.2, 124.7, 122.1, 117.9, 117.7, 113.5, 110.2, 110.6,
107.6, 56.8, 55.4. ESI–MS m/z [M+H]+471.10.
5.4. Di-O-acetylcurcumin (12)
A solution of curcumin 0.736 g (2 mmol) in anhydrous
pyridine was treated with anhydrous Ac2O (0.28 mL;
3 mmol) and stirred overnight. It was then poured in
crushed ice, extracted with carbon tetrachloride, and
the organic layer was concentrated in vacuo. Organic
layer was washed with 1% NaHCO3until effervescence
ceased. The organic layer was dried over Na2SO4 and
crystallized from absolute ethanol. Compound 12 was
1
identified on the basis of H NMR data comparison
5.3. General procedure for preparation of various curcu-
min Knoevenagel condensates (9–11)
with literature values, yield 85% (0.768 g), Rf = 0.90
(DCM/MeOH 9:1), mp 170 ꢁC (lit. 170–172 ꢁC). 1H
NMR (400 MHz, CDCl3): d 2.3 (s, 6H, acetyl), 3.89 (s,
6H, 2 · OCH3), 4.71 (s, 2H, C4-H), 6.9 (d, 2H,
J = 6.2 Hz, C2-H and C6-H), 7.15–7.34 (m, 6H, Ar-H),
A mixture of aromatic aldehyde (benzaldehyde, 4-hy-
droxy-benzaldehyde, 4-hydroxy-3-methoxy-benzalde-

S. Mishra et al. / Bioorg. Med. Chem. 16 (2008) 2894–2902
2901
7. 58 (d, 2H, J = 6.0 Hz, C1-H and C7-H). 13C NMR
(300 MHz, CDCl3): d 184.6, 183.4, 169.4, 150.4, 150.0,
149.8, 141.3, 140.3, 129.8, 128.7, 123.5, 122.6, 121.8,
111.6, 111.2, 101.4, 65.9, 55.9, 55.8. ESI–MS m/z
[M+H]+453.1, [M+Na]+ 475.0.
techniques as described above for the microscopic exam-
ination. The semi-automated microdilution technique of
Desjardins et al., which is based on [3H]-hypoxanthine
uptake by parasites, was used to assess the sensitivity
of the parasites to various inhibitors.30 Briefly, synchro-
nized parasites were cultured in 96-well plates (Nunc,
Copenhagen, Denmark) at 2–3% hematocrit and at an
initial parasitemia of 1–2%, with varying concentrations
of the inhibitors, and addition of the inhibitor in fresh
medium every 24 h for 48 h. All additions were done in
duplicate. Inhibitor stocks were made in sterile DMSO
and dilutions made such that the final concentration of
DMSO in the parasite culture did not exceed 0.05%. Par-
asites synchronized at the ring stage were cultured in the
presence of varying concentrations of the inhibitors for
the first 48 h and then incubated with [3H]-hypoxanthine
(1 lCi/well) for the next 36 h and harvested. They were
then harvested using a Nunc cell harvester onto glass fi-
ber filters, washed, and subjected to liquid scintillation
counting (Hewlett-Packard). IC50s were calculated from
plots of relative percent parasitemia versus log concen-
tration of the respective inhibitors, fitted to non-linear
regression analysis using Sigma Plot 2000 software.
The IC50 values using this method were similar to those
obtained using microscopic examination.
5.5. Di-O-methylcurcumin (13)
Curcumin 0.736 g (2 mmol) in dry benzene (100 mL)
was refluxed with di-methyl sulfate (10 mL) over anhy-
drous potassium carbonate (1 g) for 24 h, with stirring.
After evaporation of benzene, the residue was stirred
with warm aqueous sodium hydrogen carbonate. The
mixture was extracted with chloroform, and the extracts
were washed with water, dried, and evaporated. The res-
idue was purified by column chromatography (ethyl ace-
tate/hexane, 80:20). Rf = 0.85 (DCM/MeOH 9:1), yield
72% (0.570 g), mp 129–130 ꢁC (lit. 128–130 ꢁC). 1H
NMR (400 MHz, CDCl3): d 3.90 (s, 6H, 2 · OCH3),
3.92 (s, 6H, 2 · OCH3), 4.65 (s, 2H, C4-H), 6.98 (d,
2H, J = 12.4 Hz, C2-H and C6-H), 7.05–7.32 (m, 6H,
Ar-H), 7.61 (d, 2H, J = 12.4 Hz, C1-H and C7-H). 13C
NMR (300 MHz, CDCl3): d 183.2, 151.0, 149.2, 140.4,
128.1, 122.6, 122.0, 111.1, 109.8, 101.3, 56.0. ESI–MS
m/z [M+H]+ 397.2.
5.6. Assessment of inhibition of growth of P. falciparum
and determination of IC50 of the compounds
Acknowledgments
The experiments were performed using two strains of
P. falciparum, FCK2 strain (chloroquine-sensitive,
IC50, 18 nM), and MP-14 (chloroquine-resistant, IC50,
300 nM for chloroquine), isolates from Karnataka and
Maharashtra states of India. P. falciparum was cultured
using standard techniques27,28 and synchronized using
5% sorbitol29 at 4% hematocrit in RPMI 1640 medium
(Invitrogen, CA, USA) supplemented with 10% human
serum, 0.225% sodium bicarbonate, and 0.01 mg/mL
gentamicin. Growth inhibition was monitored using
microscopic examination of the parasites by standard
Giemsa staining. Typically uninfected or infected (1–
2% parasitemia, ring stage) red blood cells (2% hemato-
crit) were added to the culture medium in the wells of a
96-well plate (Nunc, Roskilde, Denmark), and different
concentrations of inhibitor in DMSO did not exceed
0.05%. The experiment started with the synchronized
parasite culture in the early trophozoite stage and inhib-
itors were added up to fourth day. DMSO (0.05%) was
used as the solvent control. The IC50 was calculated
from a plot of relative percent parasitemia versus log
concentration of the inhibitor by fitting it to non-linear
regression analysis using Sigma Plot 2000 software (Sy-
stat Software Inc., CA, USA). The MIC of the curcumin
derivatives is defined as the minimum concentration at
which more than 99% of the parasites, relative to the
control, were inhibited from developing to schizonts.
This work was supported by a grant from the Depart-
ment of Science and Technology (DST, Government
of India) under PRDSF to N.S. and also by a grant
from the Centre of Excellence, DBT, to A.S. A.S. is
J.C. Bose fellow of the Department of Science and Tech-
nology. S.M. acknowledges DBT, Government of India,
for postdoctoral fellowship. K.K. acknowledges the
CSIR, Government of India, for a senior research fel-
lowship. We do not have any competing financial inter-
ests with regard to this work.
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