Stilbene-chalcone hybrids: Design, synthesis, and evaluation as a new class of antimalarial scaffolds that trigger cell death through stage specific apoptosis

Article abstract of DOI:10.1021/jm201216y

Novel stilbene-chalcone (S-C) hybrids were synthesized via a sequential Claisen-Schmidt-Knoevenagel-Heck approach and evaluated for antiplasmodial activity in in vitro red cell culture using SYBR Green I assay. The most potent hybrid (11) showed IC₅₀

Full text of DOI:10.1021/jm201216y

Article
pubs.acs.org/jmc
Stilbene−Chalcone Hybrids: Design, Synthesis, and Evaluation as a
New Class of Antimalarial Scaffolds That Trigger Cell Death through
Stage Specific Apoptosis
Naina Sharma,†,∥ Dinesh Mohanakrishnan,‡,∥ Amit Shard,† Abhishek Sharma,†,§ Saima,† Arun K. Sinha,*,†
and Dinkar Sahal*,‡
†Natural Plant Products Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur (H.P.) 176061, India
‡Malaria Research Laboratory, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi
110067, India
S
* Supporting Information
ABSTRACT: Novel stilbene−chalcone (S-C) hybrids were
synthesized via a sequential Claisen−Schmidt−Knoevenagel−
Heck approach and evaluated for antiplasmodial activity in in
vitro red cell culture using SYBR Green I assay. The most
potent hybrid (11) showed IC50 of 2.2, 1.4, and 6.4 μM against
3D7 (chloroquine sensitive), Indo, and Dd2 (chloroquine
resistant) strains of Plasmodium falciparum, respectively.
Interestingly, the respective individual stilbene (IC50 > 100
μM), chalcone (IC50 = 11.5 μM), or an equimolar mixture of
stilbene and chalcone (IC50 = 32.5 μM) were less potent than
11. Studies done using specific stage enriched cultures and
parasite in continuous culture indicate that 11 and 18 spare the schizont but block the progression of the parasite life cycle at the
ring or the trophozoite stages. Further, 11 and 18 caused chromatin condensation, DNA fragmentation, and loss of
mitochondrial membrane potential in Plasmodium falciparum, thereby suggesting their ability to cause apoptosis in malaria
parasite.
e.g., trioxaquine7a and ferroquine7b comprising 1,2,4-trioxane-
INTRODUCTION
■
(4-aminoquinoline) and ferrocene−chloroquine moieties,
respectively, are under clinical trials as hybrid antimalarial
agents. In order to further reduce the probability of cross-
resistance,8 there is also an urgent need to explore conceptually
newer hybrid scaffolds, as the various prevalent hybrid
Malaria, one of the most devastating infectious diseases, affects
almost half of the global population and poses a major
socioeconomic hazard to humanity at large.1 The above grim
scenario is further aggravated by the persistent tendency of
Plasmodium falciparum, the most common malarial parasite, to
rapidly develop resistance against any newly introduced drug.2
In fact some recent reports have already indicated tolerance of
this parasite toward artemisinin and its derivatives,3 the current
World Health Organization prescribed gold standards against
chloroquine (CQ) resistant malaria.4
antimalarials are structurally analogous to existing drugs.2b
In this context, hydroxy substituted stilbenes and chalcones,
two important classes of plant secondary metabolites, are
promising candidates, as these individually possess multifarious
pharmacological profiles9 including antimalarial activities.10
Even as stilbenes and chalcones share overlapping biosynthetic
pathways, it seems that nature never attempted to form a
hybrid of the two. To the best of our knowledge, the
hybridization of these two pharmacophores into novel scaffolds
and evaluation of their biological activities have not yet been
reported. Herein, we describe a one-pot approach for the
synthesis of such hydroxystilbene−chalcone hybrids and
evaluation of their antimalarial activity against both CQ
sensitive and resistant strains of Plasmodium falciparum.
Recently the concept of hybrid antimalarials5 has attracted
much attention for tackling the alarming problem of drug
resistance, as these molecules often act on multiple therapeutic
targets because of the presence of two different, covalently
fused pharmacophores.6 Some hybrid molecules (Figure 1),
Received: September 13, 2011
Published: November 18, 2011
Figure 1. Some representative hybrid antimalarial drugs.
© 2011 American Chemical Society
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Figure 2. Retrosynthetic approaches for stilbene−chalcone hybrids.
a
Scheme 1
a
Reagents and conditions: (i) NaOH, MeOH at room temp, 8 h; (ii) Pd(PPh3)4, DMF, piperidine, LiCl, reflux, 14 h.
benzaldehyde in the reaction mixture (Figure 2) could lead
to the formation of the respective hydroxychalcone as side
product, besides the formation of the expected acetylstilbene.
Moreover, the subsequent step, i.e., Claisen−Schmidt con-
densation, could also lead to lower reaction performance due to
the presence of hydroxy substitution on acetylstilbene (Figure
2). On the other hand, the above concerns were clearly
circumvented in the second approach (route II, Figure 2), and
thus, it was adopted for the synthesis of various stilbene−
chalcone hybrids.
Initially, the nonhydroxylated S-C hybrids (1−7, Scheme 1)
were synthesized via a sequential Claisen−Schmidt condensa-
tion between 4-iodo-/bromoacetophenone and various aro-
matic/heteroaromatic benzaldehydes followed by Heck cou-
pling of the resulting iodo-/bromochalcones with styrene or p-
chlorostyrene. In order to further expand the scope of the
above hybrid molecules (1−7), the introduction of hydroxy
RESULTS AND DISCUSSION
■
Chemistry. In order to realize a facile access toward
stilbene−chalcone hybrids from readily available substrates, two
contrasting retrosynthetic approaches were visualized (Figure
2). Route I involved a Knoevenagel condensation−decarbox-
ylation sequence11 to afford hydroxylated styrene which can be
further coupled with bromocetophenone/iodoacetophenone
via Heck coupling, leading to the formation of 4-acetylstilbene.
Subsequently, the above 4-acetylstilbene can undergo Claisen−
Schmidt condensation with substituted benzaldehydes to give
the respective S-C hybrid. In the second approach, a Claisen−
Schmidt condensation reaction provides bromo/iodo substi-
tuted chalcone which is subjected to Heck coupling.
After a comparative evaluation of the above two strategies,
route I involving an initial Knoevenagel−Doebner condensa-
tion step was not deemed to be favorable, as the presence of
both p-iodoacetophenone and excess hydroxy substituted
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a
Scheme 2
a
Reagents and conditions: (i) NaOH, MeOH at room temp, 8 h; (ii) Pd(PPh3)4, DMF, piperidine, LiCl, reflux, 14 h.
a
Scheme 3
a
Reagents and conditions: (i) dimethyl sulfate, K2CO3, acetone, reflux, 24 h; (ii) acetic anhydride, sodium acetate, DMAP, DMF at room temp, 3 h;
(iii) p-toluenesulfonyl chloride, pyridine, cat. DMAP, DMF at room temp, 2 h; (iv) 3,5-dichlorobenzenesulfonyl chloride, pyridine, cat. DMAP,
DMF at room temp, 2 h.
a
Scheme 4
a
Reagents and conditions: (i) Pd(PPh3)4, DMF, piperidine, LiCl, reflux, 14 h.
a
Scheme 5
a
Reagents and conditions: (i) NaOH, MeOH at room temp, 8 h.
substitution at the stilbene ring was envisaged, as such
functional groups are known to impart enhanced biological
activities.9 In this context, the Heck coupling of iodochalcone
with hydroxystyrene seemed an attractive synthetic route.
However, the hydroxystyrenes are known to be prone to
polymerization and many of them, e.g., canolol (4-hydroxy-3,5-
dimethoxystyrene), are also not commercially available. There-
fore, it was decided to undertake in situ synthesis of hydroxy
substituted styrenes using our11 modified Knoevenagel
decarboxylation approach followed by their Heck coupling
with iodochalcones in the same pot. As illustrated in Scheme 1,
the above methodology was found to be quite useful for the
synthesis of diverse hydroxy substituted stilbene−chalcone
hybrids (8−13) from readily available benzaldehydes. The
above success encouraged us to explore a one-pot Claisen−
Schmidt/Knoevenagel−decarboxylation−Heck coupling se-
quence; however, the desired product (11) was obtained in
comparatively lower yield (10%) compared to 33% (in the case
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Figure 3. Antimalarial profile of nonhydroxylated stilbene−chalcone hybrids.
Figure 4. In vitro antimalarial potency of S-C hybrid derivatives against P. falciparum 3D7.
of two-step synthesis of 11) (see Supporting Information for
details).
All the synthesized compounds were fully characterized by
1H and 13C NMR and further confirmed through HRMS
analysis.
Interestingly, this approach developed for hybrid molecules
(8−13, A−HCCH−CO−B) was also successfully applied for
the synthesis of stilbene−chalcone hybrids possessing reverse
arrangement of the α,β-unsaturated carbonyl group (A−CO−
HCCH−B) across the rings A and B via condensation
between 4-iodobenzaldehyde and substituted acetophenones
followed by Heck coupling (14−16, Scheme 2).
In order to introduce further diversity in the S-C hybrids
synthesized as above, the hydroxy substitution on compound
11 was modified into methylated and acetylated12a derivatives
(17 and 18, Scheme 3). Similarly, sulfonyl derivatives12b,c of 11
were prepared using p-toluenesulfonyl chloride and 3,5-
dichlorobenzenesulfonyl chloride (19 and 20, Scheme 3) in
DMF at room temperature.
On the other hand, the hydroxystilbene 21 and distyr-
ylbenzene 22 (Scheme 4) were synthesized via our earlier
reported palladium catalyzed domino Knoevenagel−Heck
reaction11c between the appropriate hydroxybenzaldehyde,
malonic acid, and haloarenes.
Similarly 23 (chalcone) and 24, a bis-chalcone (Scheme 5),
were synthesized via condensation between 2,4,5-trimethox-
ybenzaldehyde and p-methoxyacetophenone or 1,3-diacetyl-
benzene, respectively.
Antiplasmodial Activity and Structure−Activity Rela-
tionships. The stilbene−chalcone hybrid compounds synthe-
sized as above were screened for in vitro antimalarial potency
against Pf 3D7 (CQ sensitive Plasmodium falciparum strain)
using microtiter based SYBR Green I fluorescence assay.13
Initially, the nonhydroxylated S-C hybrids (Figure 3, 1−7) were
screened against Pf 3D7. The rings B and C were kept constant,
while the substitution on ring A was varied.
It is evident from Figure 3 that a progressive increase in
electron density on ring A resulted in progressive enhancement
in antiplasmodial potency, with the 2,4,5-trimethoxy substituted
S-C hybrid (3) displaying the most promising activity (IC50
=
18 μM) followed by dimethoxy (2, IC50 = 29.5 μM) and
monomethoxy (1, IC50 > 100 μM). On the other hand, the
replacement of ring A with heteroaromatic moieties like
thiophene, furan, and indole (Figure 3, 4−6) led to only
moderate activities (IC50 of 36.3, >100, and 25.6 μM,
respectively). Having realized the importance of electron
releasing groups on ring A, we retained the 2,4,5-trimethoxy
substitution pattern while probing further structure−activity
relationship (SAR) studies. To begin with, we decided to
evaluate the antiplasmodial effect of structural variations on
ring C of the S-C hybrids. The presence of an electron
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withdrawing (p-Cl) group on ring C resulted in loss of activity
(Figure 3, 7, IC50 > 100 μM). In view of the immense
pharmacological importance of hydroxy substituted stilbenes,14
we were inspired to evaluate the antimalarial profiles of various
S-C hybrids possessing a hydroxylated C ring. Gratifyingly, our
above premise was confirmed, as the hydroxy S-C hybrids
(Figure 4) were in general found to display comparatively
enhanced potency with antimalarial activity, progressively
increasing upon an increase in electron density at ring C, (8,
9, 10, and 11 with IC50 of 10, 5.3, 4.2, and 2.2 μM,
respectively). In particular, 11, possessing high electron density
in both rings A and C, showed the best antimalarial activity
(IC50 = 2.2 μM) against the Pf 3D7 strain. Further the declining
potency in trimethoxy positional isomers 12 (IC50 = 4.9 μM)
and 13 (IC50 = 16 μM) indicated the importance of 2,4,5-
trimethoxy substitution on ring A (11) for the most potent
antimalarial effect.
In order to discern the role of relative positions of rings A
and B in the above scaffolds (A−CCH−CO−B), the S-C
hybrids possessing chalcone moiety with reversal in rings A and
B (A−CO−HCC−B) were also evaluated for antiplasmodial
activity (Figure 4, 14−16). Interestingly, such a study revealed
the beneficial role of the presence of a strong electron
withdrawing (carbonyl) group adjacent to the stilbene moiety,
as the S-C hybrids possessing an exocyclic olefinic bond
adjacent to the stilbene moiety generally displayed lower
activity, with 14 possessing an IC50 of 4.3 μM compared to 11
(IC50 = 2.2 μM). The above observation was further reinforced
when 15′ and 16′ (isomers of 15 and 16, respectively,
synthesized by a similar procedure as given in Scheme 1),
possessing carbonyl group adjacent to the B ring, displayed
higher potency (IC50 of 15 and 34 μM, respectively; Figure 5)
in comparison to 15 and 16 (IC50 pf 50 and 66 μM,
respectively; Figure 4).
Figure 6. Antimalarial activity of various derivatives of the most potent
S-C hybrid 11.
have observed that several S-C hybrids exhibited stronger
antimalarial activity against CQ resistant than CQ sensitive
strain of Plasmodium falciparum, giving resistance indices less
than 1 (Table 1). Significantly, the most active S-C hybrid (11)
that displayed IC50 = 2.2 μM against Pf 3D7 displayed better
potency against the Plasmodium falciparum CQ resistant strain
(INDO, IC50 = 1.4 μM) with a resistance index of 0.64 (Table
1). Similarly, 11 was also found to be active against another Pf
CQ resistant strain (Dd2, IC50 = 6.4 μM, resistance index of
2.9).
In addition, the active compounds were analyzed for their
cytotoxic behavior against two mammalian cell lines, viz., HeLa
and fibroblast L929. Therapeutic indices {TI = [IC50(HeLa) or
IC50(L929)]/IC50(Pf 3D7)} with values from 4.7 to >46.5
indicated that the most active compounds 11 and 18 (TI > 42
for HeLa and L929) were also relatively nontoxic.
In order to ascertain the specific benefit of merged stilbene−
chalcone hybrid, the respective individual stilbene (4-hydroxy-
3,4′,5-trimethoxystilbene, 21), chalcone (1-(4-methoxyphenyl)-
3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one, 23), and their
equimolar mixture were screened for antimalarial activity
(Figure 7). In addition, some related scaffolds like dimeric
stilbene (22) and dimeric chalcone (24) were also tested to
evaluate the effect of extended conjugation. Significantly, both
the individual stilbene/chalcone 21/23 (IC50 >100 μM/11.5
μM) and the equimolar mixture of chalcone and stilbene (IC50
= 32.5 μM) displayed significantly decreased potency against
Plasmodium falciparum, thereby demonstrating the signifi-
cance15 of hybridized S-C scaffold (11) for antimalarial activity.
Moreover, the 4-hydroxy analogue of chalcone 23, i.e., 1-(4-
hydroxyphenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one
(25; see Supporting Information), was found to be less potent
(IC50 = 61 μM) than both 11 and 23, thereby suggesting that a
4-OH substitution has a potentiating effect on S-C hybrid but
an opposite effect on chalcone. Interestingly, the distyrylben-
zene 22 (IC50 = 11.0 μM) also turned out to be inactive, while
IC50 = 2.8 μM for bischalcone (24) suggests it to be nearly
equipotent to the S-C hybrid (11). It is pertinent to mention
that the S-C hybrid (11) represents a more promising scaffold
over bis-chalcone (24), since the probability of resistance
against homodimeric pharmacophores is likely to be higher
than is the case with hybrid pharmacophores.
Figure 5. In vitro antimalarial potency of positional isomers of 15 and
16.
Having identified the core framework of an S-C hybrid (11)
responsible for potent antiplasmodial activity, we were
encouraged to evaluate the effect of further diversification on
the above motif by installing various functional groups on the
hydroxyl group of 11 (Figure 6, 17−20). However, methylation
of hydroxy group in 11 led to decreased activity (17, IC50 = 5.2
μM), while acetylation (18, IC50 = 2.2 μM) caused no change
in potency (Figure 6). On the other hand, the derivatization of
11 into p-tolylsulfonyl analogues resulted in drastically reduced
antimalarial activity (19, 20; IC50 > 100 μM). Since progressive
increments in the number of electron donating methoxy groups
on rings A and C led to molecules with progressively increasing
antimalarial potency, it is apparent that electron density in rings
A and C is a determinant of antimalarial potency of S-C
hybrids.
Mechanistic Studies. In spite of some suggestions that
chalcones may target plasmodial proteases16,17 or cyclins,18 our
knowledge of the antimalarial targets of chalcones is
incomplete. The knowledge about mechanistic details of their
antimalarial action is also minimal. Here we have attempted to
Finally, some of the potent hybrid compounds from the
above series (with IC50 ranging from 2.2 to 5.2 μM against CQ
sensitive Pf 3D7 strain) were screened for their antimalarial
potency against two CQ resistant strains (Pf Dd2 and Pf
INDO) {Table 1, Supporting Information (Figure 1)}. We
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Table 1. Antimalarial and Antifalcipain Potency, Resistance, and Therapeutic Indices of Potent Stilbene−Chalcone Hybrids
Figure 7. Antimalarial potency of S-C hybrid (11) compared to potencies of their individual components (21 and 23), homodimers (22 and 24),
and equimolar mixture of stilbene and chalcone.
study the mechanism of antimalarial action of two S-C hybrids
(11 and 18), found to be the most potent in the series of
compounds studied by us. Microscopy based stage specificity
experiments, where each of the stages of the parasite life cycle
was treated with drugs, have indicated that 11 and 18 were
specific for ring and trophozoite stage parasites and did not
affect the schizont stage, egress of merozoites, or the process of
merozoite invasion (Figure 8A). These microscopy based
results, which found confirmation also from the SYBR Green
fluorescence assay (Figure 8B), suggest that some molecular
targets common to ring and trophozoite stages may be involved
in the antimalarial properties of S-C hybrids.
For instance, falcipain-2, a cysteine protease involved in
hemoglobin degradation pathway of P. falciparum, is found in
both rings and trophozoites19 and some early studies have
reported that chalcones and their derivatives target the
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Figure 8. Specificity of 11 and 18 for rings and trophozoites. Highly
synchronized stage enriched Pf 3D7 cultures were treated with IC100
values of each compound (11, 6 μM; 18, 10 μM) for 48 h (rings), 24 h
(trophozoites), and 8 h (schizonts). The parasites were washed with
RPMI to remove the drug pressure and incubated for 48 h with drug
free media. The percentages of inhibition of growth were determined
by Giemsa staining (A) and fluorescence based SYBR Green I assay
(B). Blue and red bars indicate effects of 11 and 18, respectively. The
inability of 11 and 18 to inhibit schizont stage progression indicates
that they do not affect either egress of merozoites or their ability to
invade healthy red cells.
Figure 9. Schizonts treated with 11/18 are trapped at ring stage. (A)
Highly synchronized schizont stage Pf 3D7 cultures were grown
without (control) or with test compounds at their respective IC100 (11,
6 μM, middle panel; 18, 10 μM, bottom panel). All cultures were
washed free of drug using cRPMI after 8 h. The drug treated cultures
were further incubated with drug free (d−) or drug supplemented (d+)
complete RPMI for an additional 48 h. Cultures were Giemsa stained
and microscopically examined for parasitemia and stage of growth.
Note that 8 h of drug exposure causes no effect on the transition of
schizont to ring stage. However, reincubation with drug causes arrest
at ring stage. (B) Quantitative microscopic data obtained using auto
count have been plotted to show that schizont progression to rings is
unaffected by the presence of drug for 8 h. Continuous drug exposure
(solid lines) versus 8 h of drug exposure (first time window) followed
by drug withdrawal and growth for 48 h (second time window) in drug
free RPMI (dotted lines) indicates that in the presence of drug the
schizont successfully transits to ring stage and parasitemia increases
steadily (in the absence of drug beyond 8 h) but rings are killed when
the drug is present in the second time window.
hemoglobin degradation pathway by inhibiting falcipain-2.16 In
order to find if falcipain-2 may be a target for the antimalarial
action of S-C hybrids, we examined anti-falcipain-2 potency of
all those hybrids whose antimalarial IC50 was ≤10 μM. The
enzyme inhibitory data of the potent compounds against
falcipain-2 activity {Table 1, Supporting Information (Figure
2)} indicate that compounds 11 and 18 with antimalarial IC50
of 2.2 μM exhibit anti-falcipain-2 IC50 values of 37.5 and 25
μM, respectively. The generally high IC50 values (19−100 μM)
of S-C hybrids against falcipain-2 and lack of a direct relation
between anti-falcipain-2 potency and antimalarial potency
suggest that the true targets of S-C hybrids may be different
from falcipain 2. Similarly Dominguez et al.20 also reported that
the hybrid quinolinylchalcones failed to inhibit the falcipain-2
activity (IC50 > 100 μM) even as they inhibited the growth of
the malaria parasite with an IC50 ∼ 1 μM.
To find the activity of compounds against egress and
invasion of P. falciparum at blood stage, 11 (6 μM) and 18 (10
μM) were incubated for 8 h with schizont stage parasites. As
shown in Figure 9, 11 and 18 failed to inhibit schizont growth,
merozite egress, or RBC invasion. Hence, we predicted that in a
continuous culture starting with schizonts, continuous drug
pressure for an additional 48 h should stall growth of parasite at
ring stage while the culture subjected to drug pressure for only
8 h followed by drug withdrawal during the 48 h time window
must grow to the same extent as the untreated control culture.
This was indeed found to be the case as shown by results in
Figure 9 suggesting that schizonts, egress of merozoites, and
their ability to invade fresh red blood cells are unaffected while
rings are very sensitive to the action of 11 and 18.
(Figure 10B), 11, which caused complete inhibition of growth
at 18 h, was found to be faster acting than 18, which caused
similar growth inhibition only at 24 h.
Apoptosis, a well-known programmed cell death (PCD)
mechanism in multicellular organisms, is not well studied in
unicellular organisms.21,22 But recent studies have revealed that
unicellular organisms like leishmania, yeast, bacteria, blastocys-
tis, trypanosomes, and trichomonas also undergo PCD and
show apoptotic features resembling multicellular organisms.23,24
Thus, Picot et al.25 reported chloroquine induced oligonucleo-
somal DNA fragmentation suggesting PCD in nonsynchronized
erythrocytic stages of CQ sensitive P. falciparum strain. Recent
studies have reported that CQ, etoposide (topoisomerase II
inhibitor), and staurosporine (protein kinase C inhibitor)
induced PCD in CQ sensitive and resistant strains of P.
falciparum with hallmark features of apoptosis including nuclear
chromatin condensation, DNA fragmentation, loss of mito-
chondrial membrane potential, and changes in plasma
membrane permeability.26−28 Lopez et al.29 described that the
trophozoite and schizont stage-specific antimalarial Solanum
Studies on kinetics of action of 11 and 18 on ring stage
parasites suggest that 11, which caused complete inhibition of
growth at 48 h, was slower in action than 18, which caused
similar inhibition of growth at 24 h (Figure 10A). However, in
an analogous experiment with trophozoite stage parasite culture
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Figure 10. Microscopic examination of the effect of exposure of 11 and 18 (each at its IC100) for variable time durations to ring and trophozoite
stages. Synchronized cultures of P. falciparum 3D7 were incubated with IC100 of 11 (6 μM) and 18 (10 μM) for different time periods (d+, solid red
boundaries) followed by transition to drug free medium (d−). Total incubations (with and without drug pressure) for ring and trophozoite stages
were 96 and 72 h, respectively. In circles on the left margin, colored and colorless sections represent periods of drug and drug free exposures,
respectively.
nudum steroids induced PCD in P. falciparum with features of
apoptosis. The finding of a putative P. falciparum metacaspase
(Pf MCA-1)28 has provided further support for the occurrence
of PCD in P. falciparum. However, Nyakeriga et al.30 have
reported that chloroquine, atovaquone, and etoposide treat-
ments caused nonapoptotic cell death because these agents
failed to show any features of apoptosis. Likewise, another
report suggested that CQ and S-nitroso-N-acetylpenicillamine
(SNAP) treated blood stage P. falciparum died by autophagic-
like cell death without display of any apoptotic features.31
Metazoan apoptotic features including chromatin condensation,
nuclear DNA fragmentation, movement of phosphatidylserine
from the inner to the outer lamellae of the cell membrane, and
caspase-like protein activity have been observed in mosquito
midgut stage parasite of P. berghei (ookinete) both in vivo and
in vitro.32,33 Against this background, in the present study, we
have found that compounds 11 and 18 at their respective IC100
caused apoptotic cell death in P. falciparum 3D7 strain. To the
best of our knowledge, this is the first report of apoptotic cell
death caused by stilbene−chalcone hybrids in P. falciparum.
Treated parasites showed features of apoptotic cell death
including cell shrinkage, chromatin condensation, DNA
fragmentation, and loss of mitochondrial membrane potential
(Figure 11). Ring and trophozoite stage parasites treated with
11/18 showed crisis forms with condensed nuclei at 24 and 18
h, respectively, and the resulting reduction in parasitemia at 48
h (Figure 11). DNA fragmentation or degradation is one of the
major features of apoptosis.34 Hoescht 33342 staining29 further
confirmed the nuclear morphological changes including
chromatin condensation and fragmentation in P. falciparum.
Indeed the results of Hoescht 33342 staining also showed
distinct nuclear condensation in both ring and trophozoite
stage parasites at 18 h. Similar observations have earlier been
made by Lopez et al. and Meslin et al.28,29 At a later time point
(24 h), we observed clear internucleosomal fragmentation in
trophozoite stage parasites but not in ring stage parasites. This
may be due to the small size of nucleus in rings vs a significantly
larger nucleus in trophozoites. It is not clear why
internucleosomal fragmentation so clearly visible in Hoescht
33342 staining was not apparent in Giemsa staining (Figure
11). The loss of mitochondrial membrane potential is a major
characteristic of apoptosis. Our results have shown that the
compounds 11 and 18 disrupted mitochondrial membrane
potential (ΔΨm) of ring and trophozoite stage P. falciparum
similar to CQ, atovaquone, etoposide,28 and Solanum nudum
steroids.29 The results of JC-1 staining indicative of loss of ΔΨm
displayed strong evidence of apoptosis in P. falciparum by S-C
hybrids. Thus, our data have shown high percentage of
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Figure 11. Induction of apoptosis in P. falciparum by S-C hybrids. S-C-hybrid-treated erythrocyte stage parasites showed features of apoptosis
including chromatin condensation/DNA fragmentation and loss of mitochondrial membrane potential (ΔΨm). Panels A and B show the effects of
11 (6 μM) and 18 (10 μM) at different time points on the ring and trophozoite stage parasites, respectively. DNA degradation was observed in both
11and 18 treated ring and trophozoite parasites by Giemsa staining. The arrow marks show the DNA fragmentation in trophozoite stage parasites
treated with 11 and 18 by Hoechst 33342 staining. The loss of mitochondrial membrane potential was monitored by JC-1 stain. High ΔΨm in
control mitochondria causes association with the dye in an aggregated state leading to red-orange fluorescence emission. 11 and 18 treated parasites
lead to dissipation of ΔΨm, and the dye is now partitioned into cytosol, resulting in green fluorescence.
chromatin condensation, nucleus fragmentation, and loss of
ΔΨm in S-C hybrids treated parasites. The high therapeutic
indices (>45) of 11 and 18 suggest that these compounds may
gain specificity by choosing to specifically target the apoptotic
machinery of the malaria parasite. The above results assume
significance, as apoptotic plasmodium cell death has been
recognized to be a novel and promising target for developing
newer antimalarials.26,35
modifying the lead candidates into clinically useful antimalarial
compounds.
EXPERIMENTAL SECTION
■
Materials and Methods. All the starting materials were reagent
grade. The palladium catalysts were purchased from Acros, Aldrich,
and Merck and used as such. The substituted benzaldehydes,
acetophenones, and all other reagents were obtained from commercial
sources (Merck, Lancaster). The solvents used for isolation/
purification of compounds were obtained from commercial sources
(Merck) and used without further purification. Column chromatog-
raphy was performed using silica gel (Merck, 60−120 mesh size). The
chromatographic solvents are mentioned as v/v ratios. All the
synthesized compounds were fully characterized by 1H and 13C
NMR and further confirmed through HRMS analysis. 1H (300 MHz),
13C (75.4 MHz), and two-dimensional NMR spectra were recorded on
CONCLUSION
■
We have for the first time hybridized the hydroxystilbene and
chalcone pharmacophores through a facile Claisen−Schmidt/
Knoevenagel−decarboxylation/Heck approach. Significantly,
the obtained hybrid compounds were found to provide a new
and promising class of antimalarial agents against CQ resistant
strains of P. falciparum. The SAR analysis revealed that a 2,4,5-
trimethoxy substitution on ring A and 4-hydroxy-3,5-dimethoxy
substitution on ring C were critical for the stilbene−chalcone
hybrids to display potent antiplasmodial activity. We have
found these hybrids to have promising resistance and
therapeutic indices. Further, our data suggest that (a) the S-C
hybrids are far more potent than the physical mixture of the
constituent pharmacophores, (b) the antimalarial activity of S-
C hybrids may not be due to inhibition of falcipain-2, and (c) S-
C hybrids cause stage-specific death resembling apoptosis in
rings and trophozoites without perturbing the progression of
schizonts into rings. The observation that S-C hybrids caused
signs of programmed cell death in Plasmodium is significant,
since it suggests Plasmodium-specific new drug targets for this
class of molecules. Moreover, the above S-C hybrids also
displayed low cytotoxicities, thereby opening the possibility of
a Bruker Avance-300 spectrometer. The following abbreviations were
used to designate chemical shift multiplicities: s = singlet, d = doublet,
t = triplet, m = multiplet, q = quartet. The 13C NMR spectra are
proton decoupled. The melting points were determined on a digital
Barnsted Electrothermal 9100 apparatus and are uncorrected. HRMS-
ESI spectra were determined using micromass Q-TOF Ultima
spectrometer and reported as m/z (relative intensity). The purity of
the test compounds was determined by HPLC analysis using a
Shimadzu LC-20 instrument equipped with a photodiode array
detector (CBM 20A) and a Purospher-Star RP-18e column (4.6 mm ×
150 mm, 5 μm, Merck). Methanol−acetonitrile (70:30, v/v) (solvent
A) and TFA (0.05%) (solvent B) were used as mobile phases. The
above analysis indicated purity of ≥95%.
Representative Procedure for the Synthesis of Stilbene−
Chalcone Hybrid 1 via Claisen−Schmidt Condensation−Heck
Coupling Reaction (Scheme 1 and Figure 3). To a solution of 4-
iodoacetophenone (3.2 mmol) in methanol (15 mL) were added 10%
aqueous NaOH (6 mmol) and p-methoxybenzaldehyde (0.4 g, 3
mmol). The reaction mixture was stirred until complete consumption
305
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Journal of Medicinal Chemistry
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1
of starting material. The methanol was evaporated from the above
mixture, and the obtained precipitates were washed with dilute HCl,
excess water, dried in air, and finally recrystallized with methanol to
obtain pure 4-iodo-4′-methoxychalcone (0.85 g, yield 80%).
°C; H NMR (CD3COCD3, 300 MHz), δ (ppm) 11.45 (1H, s), 8.07
(3H, d, J = 8.2 Hz), 7.92 (1H, s), 7.71 (3H, t, J = 7.2 Hz), 7.58−7.52
(3H, m), 7.47 (1H, d, J = 7.8 Hz), 7.41 (2H, t, J = 7.3 Hz), 7.32 (1H,
d, J = 6.7 Hz), 7.24−7.15 (3H, m); 13C NMR (75.4 MHz, CDCl3/
DMSO, 9:1), δ (ppm) 188.7, 140.3, 137.7, 136.9, 135.9, 135.4, 131.6,
130.1, 127.9, 127.3, 126.8, 125.9, 125.7, 122.2, 118.9, 116.0, 112.8, and
112.2. HRMS-ESI: m/z [M + H]+ for C25H18ONCl, calculated
384.1149; observed 384.1129.
(2E)-1-{4-[(E)-2-(4-Chlorophenyl)ethenyl]phenyl}-3-(2,4,5-
trimethoxyphenyl)prop-2-en-1-one (7). Yellow solid (35%
yield); mp 118−120 °C; 1H NMR (CDCl3, 300 MHz), δ (ppm)
8.14 (1H, d, J = 15.7 Hz), 8.03 (4H, d, J = 7.9 Hz), 7.62 (1H, d, J = 7.9
Hz), 7.51−7.42 (3H, m), 7.35 (2H, d, J = 8.4 Hz), 7.12 (2H, d, J = 6.0
Hz), 6.53 (1H, s), 3.95 (3H, s), 3.91 (6H, s) ; 13C NMR (75.4 MHz,
CDCl3), δ (ppm) 180.0, 155.1, 153.0, 143.7, 141.3, 140.5, 135.7, 134.2,
130.6, 130.1, 129.5, 129.4, 129.0, 128.7, 128.3, 126.9, 120.6, 116.0,
111.9, 97.3, 57.0, 56.8, and 56.5. HRMS-ESI: m/z [M + H]+ for
C26H23O4Cl, calculated 435.1357; observed 435.1372.
Representative Procedure for the Synthesis of Hydroxyl
Stilbene−Chalcone Hybrid 8 via Knoevenagel Condensation−
Decarboxylation−Heck Sequence (Scheme 1 and Figure 4).
To a solution of 4-iodoacetophenone (3.2 mmol) in methanol (15
mL) were added 10% aqueous NaOH (6 mmol) and 2,4,5-trimethoxy
benzaldehyde (3 mmol). The reaction mixture was stirred until
complete consumption of starting material. The methanol was vacuum
evaporated from above mixture, and the obtained precipitates were
washed with dilute HCl, excess water, dried in air, and finally
recrystallized with methanol to obtain pure substituted 4-iodochal-
cone. In the second step, to the stirred mixture of malonic acid (8.4
mmol) and piperidine (10.60 mmol) in DMF (20 mL) were added 4-
hydroxybenzaldehyde (2.12 mmol), substituted 4-iodochalcone (0.5 g,
1.18 mmol), Pd(PPh3)4 (0.035 mmol), and LiCl11c (0.09 mmol), and
the mixture was refluxed for 14 h. The above mixture was cooled to
room temperature and filtered through Celite. The filtrate was poured
into water (100 mL, acidified with dilute HCl, pH 5−6) and extracted
with ethyl acetate (2 × 40 mL). The combined organic layer was
washed with water (2 × 15 mL), brine (1 × 10 mL), dried over
Na2SO4, and vacuum evaporated. The residue was subsequently
purified by column chromatography on silica gel (60−120 mesh size)
using hexane/ethyl acetate (9.3:0.7) to give product 8.
In the second step, to the solution of 4-iodo-4′-methoxychalcone
(0.5 g, 1.18 mmol) and styrene (1.88 mmol) in DMF (20 mL) were
added piperidine (1.18 mmol), Pd(PPh3)4 (0.035 mmol), and LiCl
(0.09 mmol). Then the mixture was refluxed for 14 h (until the
completion of reaction). The above mixture was cooled to room
temperature and filtered through Celite. The filtrate was poured into
water (100 mL, acidified with dilute HCl, pH 5−6) and extracted with
ethyl acetate (2 × 40 mL). The combined organic layer was washed
with water (2 × 15 mL), brine (1 × 10 mL), dried over Na2SO4, and
vacuum evaporated. The residue was subsequently purified by column
chromatography on silica gel (60−120 mesh size) using hexane/ethyl
acetate (9.3:0.7) to give product 1.
( 2 E ) - 1 - { 4 - [ ( E ) - 2 - P h e n y l e t h e n y l ] p h e n y l } - 3 - ( 4 -
methoxyphenyl)prop-2-en-1-one (1). Cream solid (48% yield);
mp 162−164 °C; 1H NMR (CDCl3, 300 MHz), δ (ppm) 8.06 (2H, d,
J = 8.3 Hz), 7.86 (1H, d, J = 15.6 Hz), 7.65 (4H, d, J = 8.3 Hz), 7.56
(2H, d, J = 7.3 Hz), 7.49 (1H, d, J = 15.6 Hz), 7.38 (1H, d, J = 7.6
Hz), 7.34 (1H, d, J = 7.3 Hz), 7.27 (3H, q), 6.98 (2H, d, J = 8.1 Hz),
3.88 (3H, s); 13C NMR (75.4 MHz, CDCl3), δ (ppm) 189.9, 162.1,
144.9, 142.0, 137.7, 137.2, 131.7, 130.7, 129.4, 129.2, 128.7, 128.1,
128.0, 127.2, 127.0, 120.0, 114.8, and 55.8. HRMS-ESI: m/z [M + H]+
for C24H20O2, calculated 341.1536; observed 341.1541.
Synthesis of Other Stilbene−Chalcone Hybrids 2−7
(Scheme 1 and Figure 3). The above procedure was followed.
(2E)-1-{4-[(E)-2-Phenylethenyl]phenyl}-3-(3, 4-
dimethoxyphenyl)prop-2-en-1-one (2). Yellow solid (44% yield);
mp 147−149 °C; 1H NMR (CDCl3, 300 MHz), δ (ppm) 8.06 (2H, d,
J = 8.1 Hz), 7.83 (1H, d, J = 15.6 Hz), 7.65 (2H, d, J = 8.1 Hz), 7.57
(2H, d, J = 7.6 Hz), 7.46 (1H, d, J = 15.6 Hz), 7.42 (2H, t, J = 7.4 Hz),
7.34 (1H, d, J = 7.6 Hz), 7.28−7.24 (3H, m), 6.93 (1H, d, J = 8.1 Hz),
4.19 (1H, s), 3.97 (3H, s), 3.96 (3H, s); 13C NMR (75.4 MHz,
CDCl3), δ (ppm) 189.9, 151.8, 149.6, 145.2, 142.0, 137.6, 137.1, 131.7,
129.6, 129.2, 128.9, 128.8, 128.7, 128.0, 127.2, 127.0, 123.6, 111.5,
110.5, and 56.4. HRMS-ESI: m/z [M + H]+ for C25H22O3, calculated
371.1641; observed 371.1622.
(2E)-1-{4-[(E)-2-Phenylethenyl]phenyl}-3-(2,4,5-
trimethoxyphenyl)prop-2-en-1-one (3). Yellow solid (52%
yield); mp 148−150 °C; 1H NMR (CDCl3, 300 MHz), δ (ppm)
8.16 (1H, d, J = 15.7 Hz), 8.06 (2H, d, J = 7.9 Hz), 7.66 (2H, d, J = 7.9
Hz), 7.59 (2H, t, J = 7.5 Hz), 7.48 (1H, d, J = 15.7 Hz), 7.41 (2H, d, J
= 7.5 Hz), 7.38 (1H, d, J = 7.1 Hz), 7.32 (1H, d, J = 7.5 Hz), 7.20 (2H,
d, J = 10.9 Hz), 6.55 (1H, s), 3.97 (3H, s), 3.93 (6H, s); 13C NMR
(75.4 MHz, CDCl3), δ (ppm) 190.6, 155.1, 152.9, 143.7, 141.7, 140.4,
138.1, 137.2, 131.5, 129.4, 129.2, 128.6, 128.1, 127.2, 126.8, 120.7,
116.0, 111.9, 97.3, 57.0, 56.8, and 56.5. HRMS-ESI: m/z [M + H]+ for
C26H24O4, calculated 401.1747; observed 401.1721.
(2E)-1-{4-[(E)-2-(4-Hydroxyphenyl)ethenyl]phenyl}-3-(2,4,5-
trimethoxyphenyl)prop-2-en-1-one (8). Reddish brown solid
1
(30% yield); mp 78−80 °C; H NMR (CD3COCD3, 300 MHz), δ
(ppm) 8.15 (1H, d, J = 15.7 Hz), 8.03 (2H, d, J = 8.1 Hz), 7.60 (2H, d,
J = 8.2 Hz), 7.53 (1H, d, J = 15.7 Hz), 7.44 (2H, d, J = 8.3 Hz), 7.15
(2H, s), 7.02 (1H, d, J = 16.3 Hz), 6.90 (2H, d, J = 8.3 Hz), 6.53 (1H,
s), 3.95 (3H, s), 3.92 (6H, s); 13C NMR (75.4 MHz, CDCl3), δ (ppm)
190.8, 157.3, 155.1, 152.9, 143.7, 142.4, 140.4, 137.5, 131.4, 129.5,
128.6, 126.5, 125.4, 120.6, 116.3, 116.0, 111.9, 97.3, 57.0, 56.8, and
56.5. HRMS-ESI: m/z [M + H]+ for C26H24O5, calculated 417.1696;
observed 417.1672.
(2E)-1-{4-[(E)-2-Phenylethenyl]phenyl}-3-(thiophen-2-yl)-
prop-2-en-1-one (4). Yellow solid (46% yield); mp 138−140 °C;
1H NMR (CDCl3, 300 MHz), δ (ppm) 8.06−7.99 (2H, m), 7.96 (1H,
Synthesis of Other Hydroxylated Stilbene−Chalcone Hy-
brids Including 9−13 (Scheme 1 and Figure 4). The above
procedure was followed.
s), 7.66 (2H, d, J = 8.9 Hz), 7.56 (2H, d, J = 7.2 Hz), 7.46 (1H, d, J =
7.2 Hz), 7.41−7.28 (5H, m), 7.25 (2H, d, J = 8.9 Hz), 7.14 (1H, q);
13C NMR (75.4 MHz, CDCl3), δ (ppm) 189.0, 163.8, 147.7, 143.9,
140.0, 135.7, 134.4, 131.6, 131.2, 130.7, 129.3, 129.0, 127.1, 126.4,
121.7, 114.2, and 104.0. HRMS-ESI: m/z [M + H]+ for C21H16OS,
calculated 317.0994; observed 317.0982.
(2E)-1-{4-[(E)-2-(4-Hydroxy-3-methoxyphenyl)ethenyl]-
phenyl}-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (9). Yel-
low solid (29% yield); mp 78−80 °C; 1H NMR (CDCl3, 300
MHz), δ (ppm) 8.15 (1H, d, J = 15.8 Hz), 8.03 (2H, d, J = 7.8 Hz),
7.60 (2H, d, J = 7.8 Hz), 7.52 (1H, d, J = 15.8 Hz), 7.19−7.01 (5H,
m), 6.96 (1H, t, J = 5.2 Hz), 6.52 (1H, s), 5.84 (1H, s), 3.95 (6H, s),
3.90 (6H, s); 13C NMR (75.4 MHz, CDCl3), δ (ppm) 190.6, 155.1,
152.9, 147.2, 146.6, 143.7, 142.1, 140.3, 137.6, 131.5, 129.9, 129.4,
126.6, 125.8, 121.4, 120.6, 116.0, 115.1, 111.9, 108.9, 97.3, 57.0 56.8,
56.5, and 56.3. HRMS-ESI: m/z [M + H]+ for C27H26O6, calculated
447.1802; observed 447.1816.
(2E)-1-{4-[(E)-2-(3,4-Dihydroxyphenyl)ethenyl]phenyl}-3-
(2,4,5-trimethoxyphenyl)prop-2-en-1-one (10). Light orange
solid (24% yield); mp 121−123 °C; 1H NMR (CD3COCD3, 300
MHz), δ (ppm) 8.21 (2H, d, J = 15.7 Hz), 8.12 (2H, d, J = 8.2 Hz),
8.03 (1H, s), 7.80 (1H, d, J = 15.7 Hz), 7.72 (2H, d, J = 8.2 Hz), 7.50
(1H, s), 7.33 (1H, d, J = 16.4 Hz), 7.13 (1H, d, J = 16.4 Hz), 7.03 (2H,
(2E)-1-{4-[(E)-2-Phenylethenyl]phenyl}-3-(furan-2-yl)prop-2-
1
en-1-one (5). Yellow solid (43% yield); mp 122−124 °C; H NMR
(CDCl3, 300 MHz), δ (ppm) 8.08 (2H, d, J = 7.8 Hz), 7.66 (1H, s),
7.63−7.54 (5H, m), 7.43−7.38 (3H, t, J = 7.2 Hz), 7.35−7.30 (2H,
m), 7.20 (1H, d, J = 16.3 Hz), 6.75 (1H, s), 6.55 (1H, s); 13C NMR
(75.4 MHz, CDCl3), δ (ppm) 189.3, 152.2, 145.3, 142.2, 137.4, 137.2,
131.8, 130.9, 129.4, 129.2, 128.7, 128.0, 127.2, 127.0, 119.6, 116.6, and
113.1. HRMS-ESI: m/z [M + H]+ for C21H16O2, calculated 301.3659;
observed 301.3650.
(2E)-1-{4-[(E)-2-Phenylethenyl]phenyl}-3-(5-chloro-1H-indol-
3-yl)prop-2-en-1-one (6). Yellow solid (47% yield); mp 248−250
306
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q), 6.88 (1H, d, J = 8.2 Hz), 6.81 (1H, s), 3.96 (3H, s), 3.94 (3H, s),
3.86 (3H, s); 13C NMR (75.4 MHz, CD3COCD3), δ (ppm) 189.6,
156.1, 154.8, 147.3, 146.7, 145.1, 143.5, 140.0, 138.5, 132.8, 130.8,
130.2, 127.5, 126.0, 121.1, 120.5, 116.8, 116.6, 114.6, 113.1, 98.8, 57.5,
57.3, and 56.8. HRMS-ESI: m/z [M + H]+ for C26H24O6, calculated
433.1645; observed 433.1621.
(2E)-1-{4-[(E)-2-(4-Hydroxy-3,5-dimethoxyphenyl)ethenyl]-
phenyl}-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (11).
Brownish yellow solid (33% yield); mp 68−70 °C; 1H NMR
(CDCl3, 300 MHz), δ (ppm) 8.15 (1H, d, J = 15.7 Hz), 8.04 (2H,
d, J = 7.9 Hz), 7.62 (2H, d, J = 7.9 Hz), 7.53 (1H, d, J = 15.7 Hz), 7.19
(2H, q, J = 15.8 Hz), 7.15 (1H, s), 6.80 (2H, s), 6.54 (1H, s), 5.69
(1H, s), 3.97 (9H, s), 3.92 (6H, s); 13C NMR (75.4 MHz, CDCl3), δ
(ppm) 190.5, 155.1, 152.9, 147.7, 143.7, 141.9, 140.3, 137.8, 135.8,
131.6, 129.4, 128.9, 126.6, 126.2, 120.6, 116.0, 112.0, 104.1, 97.3, 57.0,
56.7, and 56.5. HRMS-ESI: m/z [M + H]+ for C28H28O7, calculated
477.1907; observed 477.1902.
56.7, and 56.5. HRMS-ESI: m/z [M + H]+ for C28H28O7, calculated
477.1907; observed 477.1946.
Synthesis of Other Hydroxylated Stilbene−Chalcone Hy-
brids Including 15 and 16 (Scheme 2 and Figure 4). The above
procedure was followed.
(2E)-3-{4-[(E)-2-(4-Hydroxy-3,5-dimethoxyphenyl)ethenyl]-
phenyl}-1-(4-methoxyphenyl)prop-2-en-1-one (15). Dark yel-
low solid (29% yield); mp 189−191 °C; 1H NMR (CDCl3, 300
MHz), δ (ppm) 8.08 (3H, s), 7.80−7.57 (5H, m), 7.01(4H, s), 6.80
(2H, s), 5.67 (1H, s), 3.97 (9H, s); 13C NMR (75.4 MHz, CDCl3), δ
(ppm) 189.0, 163.8, 147.7, 143.9, 140.0, 135.7, 134.4, 131.6, 131.2,
130.7, 129.3, 129.0, 127.1, 126.4, 121.7, 114.2, 104.0, 56.7, and 55.9 .
HRMS-ESI: m/z [M + H]+ for C26H24O5, calculated 417.1696;
observed 417.1659.
(2E)-3-{4-[(E)-2-(4-Hydroxy-3-methoxyphenyl)ethenyl]-
phenyl}-1-(4-methoxyphenyl)prop-2-en-1-one (16). Brownish
1
yellow solid (28% yield); mp 148−150 °C; H NMR (CDCl3, 300
MHz), δ (ppm) 8.08 (2H, s), 7.85 (1H, s) 7.65−7.52 (6H, m), 7.16−
6.93 (6H, m), 5.61 (1H, s), 3.97 (3H, s), 3.91 (3H, s); 13C NMR (75.4
MHz, CDCl3), δ (ppm) 189.1, 163.8, 147.2, 146.4, 143.9, 140.2, 134.3,
131.6, 131.2, 130.5, 130.0, 129.3, 127.1, 126.0, 121.6, 121.2, 115.1,
114.2, 108.8, 56.3, and 53.8. HRMS-ESI: m/z [M + H]+ for C25H22O4,
calculated 387.1590; observed 387.1561.
Representative Procedure for the Methylation of 11 into
(2E)-1-{4-[(E)-2-(3,4,5-Trimethoxyphenyl)ethenyl]phenyl}-3-
(2,4,5-trimethoxyphenyl)prop-2-en-1-one (17) (Scheme 3). To
a stirred mixture of 11 (0.2 g, mmol) in acetone (8 mL) were added
K2CO3 (0.84 mmol) and dimethyl sulfate (0.84 mmol). The reaction
mixture was refluxed for 24 h. After the usual workup, the residue was
purified by column chromatography (hexane/ethyl acetate) to obtain
product 17.
(2E)-1-{4-[(E)-2-(3,4,5-Trimethoxyphenyl)ethenyl]phenyl}-3-
(2,4,5-trimethoxyphenyl)prop-2-en-1-one (17). Dark orange
semi solid (75% yield); 1H NMR (CDCl3, 300 MHz), δ (ppm)
8.13−7.91 (3H, m) 7.56−7.38 (3H, m), 7.11 (2H, t, J = 14.4 Hz), 6.76
(2H, s), 6.59−6.46 (2H, m), 3.87 (18H, s); 13C NMR (75.4 MHz,
CDCl3), δ (ppm) 190.2, 154.7, 154.0, 149.7, 143.8, 141.9, 138.4, 137.2,
132.0, 130.4, 129.2, 128.5, 127.1 126.4, 119.9, 113.6, 105.5, 103.9, 97.6,
60.9, 56.5, 56.3, and 56.1. HRMS-ESI: m/z [M + H]+ for C29H30O7,
calculated 491.2064; observed 491.2035.
Representative Procedure for the Acetylation of 11 into 2,6-
Dimethoxy-4-[(E)-2-{4-[(2E)-3-(2,4,5-trimethoxyphenyl)prop-2-
enoyl]phenyl}ethenyl]phenyl Acetate (18) (Scheme 3). To a
stirred mixture of 11 (0.2 g, 0.42 mmol) in DMF (8 mL) were added
acetic anhydride (1.7 mmol), sodium acetate (1.7 mmol), and a
catalytic amount of DMAP (dimethylaminopyridine). The reaction
mixture was allowed to stir for 3 h until completion of the reaction.
Then the reaction mixture was poured into water (50 mL) and
extracted with ethyl acetate (2 × 20 mL). The combined organic layer
was washed with water (2 × 10 mL), brine (1 × 10 mL), dried over
Na2SO4, and vacuum evaporated. The obtained residue was purified by
washing with hexane and ether to give product 18.
(2E)-1-{4-[(E)-2-(4-Hydroxy-3,5-dimethoxyphenyl)ethenyl]-
phenyl}-3-(2,3,4-trimethoxyphenyl)prop-2-en-1-one (12).
Brick red solid (25% yield); mp 100−102 °C; 1H NMR (CDCl3,
300 MHz), δ (ppm) 8.06 (3H, t, J = 8.5 Hz), 7.63 (3H, t, J = 8.5 Hz),
7.44 (1H, d, J = 8.7 Hz), 7.21 (2H, q, J = 16.2 Hz), 6.81 (2H, s), 6.76
(1H, d, J = 7.9 Hz), 5.68 (1H, s), 3.98 (9H, s), 3.92 (6H, s); 13C NMR
(75.4 MHz, CDCl3), δ (ppm) 190.4, 156.2, 154.2, 147.7, 142.1, 140.3,
137.5, 135.8, 131.8, 129.5, 128.8, 126.6, 126.1, 124.3, 122.5, 121.7,
117.0, 108.0, 104.1, 61.8, 61.3, 56.7, and 56.5. HRMS-ESI: m/z [M +
H]+ for C28H28O7, calculated 477.1907; observed 477.1919.
(2E)-1-{4-[(E)-2-(4-Hydroxy-3,5-dimethoxyphenyl)ethenyl]-
phenyl}-3-(2,4,6-trimethoxyphenyl)prop-2-en-1-one (13).
1
Dark yellow solid (32% yield); mp 150−152 °C; H NMR (CDCl3,
300 MHz), δ (ppm) 8.32 (1H, d, J = 15.8 Hz), 8.05 (2H, d, J = 8.2
Hz), 7.95 (1H, d, J = 15.8 Hz), 7.61 (2H, d, J = 8.2 Hz), 7.28 (1H, s),
7.19 (1H, q, J = 16.9 Hz), 6.80 (2H, s), 6.16 (2H, s), 5.67 (1H, s), 3.98
(6H, s), 3.93 (6H, s), 3.88 (3H, s); 13C NMR (75.4 MHz, CDCl3), δ
(ppm) 191.6, 163.5, 162.1, 147.7, 141.5, 138.3, 136.2, 135.7, 131.4,
129.4, 129.0, 126.5, 126.4, 122.4, 107.1, 104.1, 91.0, 56.7, 56.2, and
55.8. HRMS-ESI: m/z [M + H]+ for C28H28O7, calculated 477.1907;
observed 477.1934.
Representative Procedure for the One Pot Synthesis of (2E)-
3-{4-[(E)-2-(4-Hydroxy-3,5-dimethoxyphenyl)ethenyl]phenyl}-
1-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (14) via Knoeve-
nagel Condensation−Decarboxylation−Heck Sequence
(Scheme 2 and Figure 4). To a solution of 2,4,5-trimethoxyaceto-
phenone (3.2 mmol) in methanol (15 mL) were added 10% aqueous
NaOH (6 mmol) and 4-iodo benzaldehyde (3 mmol). The reaction
mixture was stirred until complete consumption of starting material.
The methanol was vacuum evaporated from the above mixture, and
the obtained precipitates were washed with dilute HCl, excess water,
dried in air, and finally recrystallized with methanol to obtain pure 1-
(2,4,5-trimethoxyphenyl)-3-(4-iodophenyl)prop-2-en-1-one (90%
yield).
In the second step, to the stirred mixture of malonic acid (8.4
mmol) and piperidine (10.60 mmol) in DMF (20 mL) were added 4-
hydroxy-3,5-dimethoxybenzaldehyde (2.12 mmol), Pd(PPh3)4 (0.035
mmol), LiCl (0.09 mmol), and the reaction mixture was refluxed for
14 h. The above mixture was cooled to room temperature and filtered
through Celite. The filtrate was poured into water (100 mL, acidified
with dilute HCl, pH 5−6) and extracted with ethyl acetate (2 × 40
mL). The combined organic layer was washed with water (2 × 15
mL), brine (1 × 10 mL), dried over Na2SO4, and vacuum evaporated.
The residue was subsequently purified by column chromatography on
silica gel (60−120 mesh size) using hexane/ethyl acetate (9.3:0.7) to
give product 14.
2,6-Dimethoxy-4-[(E)-2-{4-[(2E)-3-(2,4,5-trimethoxyphenyl)-
prop-2-enoyl]phenyl}ethenyl]phenyl Acetate (18). Orange solid
(78% yield); mp 170−172 °C; 1H NMR (CDCl3, 300 MHz), δ (ppm)
8.16 (1H, d, J = 15.7 Hz), 8.05 (2H, d, J = 8.1 Hz), 7.63 (2H, d, J = 8.1
Hz), 7.51 (1H, d, J = 15.7 Hz), 7.20−7.05 (3H, m), 6.79 (2H, s), 6.53
(1H, s), 3.96 (3H, s), 3.92 (6H, s), 3.89 (6H, s), 2.37 (3H, s); 13C
NMR (75.4 MHz, CDCl3), δ (ppm) 190.5, 169.1, 155.1, 153.0, 152.7,
143.7, 141.4, 140.4, 138.2, 137.2, 135.7, 131.2, 129.4, 129.2, 128.4,
126.9, 115.9, 111.9, 103.8, 97.3, 57.0, 56.8, 56.6, 56.5, and 20.9.
HRMS-ESI: m/z [M + H]+ for C30H30O8, calculated 519.2013;
observed 519.2026.
(2E)-3-{4-[(E)-2-(4-Hydroxy-3,5-dimethoxyphenyl)ethenyl]-
phenyl}-1-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (14). Or-
ange solid (31% yield); mp 95−97 °C; 1H NMR (CDCl3, 300
MHz), δ (ppm) 7.71 (2H, d, J = 11.8 Hz), 7.62 (4H, q, J = 8.0 Hz),
7.41 (1H, s), 7.13 (2H, q), 6.78 (2H, s), 6.56 (1H, s), 5.67 (1H, s),
3.98 (3H, s), 3.96 (9H, s), 3.95 (3H, s); 13C NMR (75.4 MHz,
CDCl3), δ (ppm) 190.2, 155.2, 154.0, 147.7, 143.8, 141.9, 139.6, 135.6,
134.9, 130.4, 129.2, 129.1, 127.0, 126.5, 120.9, 113.6, 103.9, 97.6, 57.3,
Representative Procedure for the Sulfonylation of 11 into
2,6-Dimethoxy-4-[(E)-2-{4-[(2E)-3-(2,4,5-trimethoxyphenyl)-
prop-2-enoyl]phenyl}ethenyl]phenyl4-methylbenzene-1-sul-
fonate (19) (Scheme 3). To a stirred mixture of 11 (0.2 g, 0.42
mmol) in DMF (8 mL) was added pyridine (1.2 mmol) at room
temperature followed by p-toluenesulfonyl chloride (0.42 mmol) and
then a catalytic amount of DMAP (dimethylaminopyridine). The
reaction mixture was allowed to stir for 2 h until completion of the
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reaction. The reaction mixture was poured into water (50 mL, acidified
with dilute HCl, pH 5−6) and extracted with ethyl acetate (2 × 20
mL). The combined organic layer was washed with water (2 × 10
mL), brine (1 × 10 mL), dried over Na2SO4, and vacuum evaporated.
The obtained residue was subsequently washed with hexane and
diethyl ether to obtain the product 19.
2,6-Dimethoxy-4-[(E)-2-{4-[(2E)-3-(2,4,5-trimethoxyphenyl)-
prop-2-enoyl]phenyl}ethenyl]phenyl4-methylbenzene-1-sul-
fonate (19). Yellow solid (64% yield); mp 90−92 °C; 1H NMR
(CDCl3, 300 MHz), δ (ppm) 8.15 (1H, d, J = 15.7 Hz), 8.03 (2H, d, J
= 8.0 Hz), 7.88 (2H, d, J = 8.0 Hz), 7.60 (2H, d, J = 8.1 Hz), 7.50 (1H,
d, J = 15.7 Hz), 7.35 (2H, d, J = 8.1 Hz), 7.13 (1H, s), 7.09 (2H, d, J =
4.7 Hz), 6.70 (2H, s), 6.51 (1H, s), 3.93 (3H, s), 3.90 (3H, s), 3. 89
(3H, s), 3.72 (6H, s), 2.46 (3H, s); 13C NMR (75.4 MHz, CDCl3), δ
(ppm) 190.4, 155.1, 153.8, 153.0, 144.9, 143.7, 141.2, 140.4, 138.3,
136.7, 135.3, 130.8, 129.6, 129.4, 129.1, 128.8, 128.4, 127.0, 120.3,
115.9, 111.8, 103.8, 97.2, 57.0, 56.7, 56.5, 56.4, and 20.1. HRMS-ESI:
m/z [M + H]+ for C35H34O9S, calculated 631.1996; observed
631.1978.
reaction mixture was stirred until complete consumption of starting
material. Methanol was vacuum evaporated from the above mixture,
and the precipitate obtained was washed with dilute HCl, excess water,
dried in air, and finally recrystallized with methanol to obtain the pure
product 23.
1-(4-Methoxyphenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-
1-one10e (23). Yellow solid (81% yield); mp 122−124 °C; 1H NMR
(CDCl3, 300 MHz,) δ (ppm) 8.11 (1H, d, J =15.7 Hz), 8.05 (2H, J =
7.0 Hz), 7.51 (1H, d, J = 15.7 Hz), 7.14 (1H, s), 7.00 (2H, d, J = 9.7
Hz), 6.53 (1H, s), 3.94 (3H, s), 3.91 (6H, s), 3.89 (3H, s); 13C NMR
(75.4 MHz, CDCl3) δ (ppm) 189.7, 163.5, 154.9, 152.7, 143.7, 139.7,
132.1, 131.1, 120.6, 116.2, 114.1, 112.1, 97.5, 57.3, 56.8, 56.5, and 55.8.
Synthesis of (2E)-1-{3-[(1E)-3-Oxo-3-(2, 4, 5-
trimethoxyphenyl)prop-1-en-1-yl]phenyl}-3-(2,4,5-
trimethoxyphenyl)prop-2-en-1-one (24) (Scheme 5). The
above procedure was followed except that the equivalents of the
respective benzaldehyde and NaOH were doubled.
(2E)-1-{3-[(1E)-3-Oxo-3-(2,4,5-trimethoxyphenyl)prop-1-en-
1-yl]phenyl}-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one(24).
1
Synthesis of Another Sulfonyl Derivative 20 (Scheme 3).
The same procedure was followed.
Yellow solid (78% yield); mp 152−154 °C; H NMR (CDCl3, 300
MHz), δ (ppm) 8.63 (1H, s), 8.22−8.19 (3H, m), 8.14 (1H, s), 7.66
(1H, t, J = 7.7 Hz), 7.55 (2H, d, J = 15.7 Hz), 7.16 (2H, s), 6.54 (2H,
s), 3.96 (9H, s), 3.92 (9H, s); 13C NMR (75.4 MHz, CDCl3), δ (ppm)
190.9, 155.3, 153.2, 143.8, 141.3, 139.5, 132.4, 129.2, 128.7, 120.3,
115.7, 111.8, 97.2, 57.0, 56.8, and 56.5. HRMS-ESI: m/z [M + H]+ for
C30H30O8, calculated 519.2013; observed 519.2051.
2,6-Dimethoxy-4-[(E)-2-{4-[(2E)-3-(2,4,5-trimethoxyphenyl)-
prop-2-enoyl]phenyl}ethenyl]phenyl 3,5-dichlorobenzene-1-
sulfonate (20). Yellow solid (63% yield); mp 217−218 °C; 1H
NMR (CDCl3, 300 MHz), δ (ppm) 8.16 (1H, d, J = 15.7 Hz), 8.11
(2H, d, J = 8.0 Hz), 7.92 (2H, s), 7.66 (3H, t, J = 6.8 Hz), 7.51 (1H, d,
J = 15.7 Hz), 7.15 (3H, t, J = 3.5 Hz), 6.75 (2H, s), 6.54 (1H, s), 3.97
(3H, s), 3.93 (6H, s), 3.82 (6H, s); 13C NMR (75.4 MHz, CDCl3), δ
(ppm) 190.5, 171.5, 155.2, 153.6, 153.0, 143.7, 141.0, 140.9, 140.5
138.5, 137.2, 135.9, 133.8, 130.6, 129.5, 128.1, 127.2, 127.0, 120.4,
115.9, 111.9, 103.7, 97.3, 60.7, 57.0, 56.8, and 56.4. HRMS-ESI: m/z
[M + H]+ for C34H30O9SCl2, calculated 685.1060; observed 685.1047.
Representative Procedure for the Synthesis of 4-Hydroxy-
3,4′,5-trimethoxystilbene (21) (Scheme 4). Malonic acid (6.15
mmol) was taken in a round-bottom flask, and piperidine (4.6 mmol)
was added gradually. The above mixture was stirred in DMF (15 mL)
for 2 min at room temperature. Thereafter, 4-hydroxy-3,5-dimethox-
ybenzaldehyde (1.51 mmol), p-methoxyiodoanisole (0.85 mmol),
Pd(PPh3)4 (0.025 mmol), piperidine (3.1 mmol), and LiCl (0.07
mmol) were added, and the reaction mixture was allowed to reflux for
14 h. The above mixture was cooled to room temperature and filtered
through Celite. The filtrate was poured into water (100 mL, acidified
with dilute HCl, pH 5−6) and extracted with ethyl acetate (2 × 40
mL). The combined organic layer was washed with water (1 × 30
mL), brine (1 × 10 mL), dried over Na2SO4, and vacuum evaporated.
The residue was subsequently purified by column chromatography on
silica gel (60−120 mesh size) using hexane/ethyl acetate to give a solid
which was further recrystallized in methanol to provide pure product
21.
Measurement of Inhibition of P. f alciparum Growth in
Culture. In this study chloroquine sensitive Pf3D7 and chloroquine
resistant Pf Dd2 and Pf INDO strains were used in in vitro culture.
Parasite strains were cultivated by the method of Trager and Jensen1a
with minor modifications. Cultures were maintained in fresh O+
human erythrocytes at 4% hematocrit in complete medium (RPMI
1640 with 0.2% sodium bicarbonate, 0.5% Albumax, 45 mg/L
hypoxanthine, and 50 mg/L gentamicin) at 37 °C under reduced O2
(gas mixture 5% O2, 5% CO2, and 90% N2). Stock solutions of
chloroquine (CQ) were prepared in water (Milli-Q grade), and test
compounds were dissolved in DMSO. All stocks were then diluted
with complete culture medium to achieve the required concentrations.
(In all cases the final concentration was 0.4% DMSO, which was found
to be nontoxic to the parasite.) CQ and test compounds were then
placed in triplicate wells of 96-well flat-bottom tissue culture grade
plates with drug concentrations ranging from 0 to 100 μM in a final
well volume of 100 μL for primary screening. Chloroquine was used as
a positive control in all experiments. Parasite culture was synchronized
at ring stage with 5% sorbitol. Synchronized culture was aliquoted to
drug containing 96-well plate at 2% hematocrit and 1% parasitemia.
After 48 h of incubation under standard culture conditions, plates were
harvested and read by the SYBR Green I fluorescence-based method13
using a 96-well fluorescence plate reader (Victor, Perkin-Elmer), with
excitation and emission wavelengths of 497 and 520 nm, respectively.
The fluorescence readings were plotted against drug concentration,
and IC50 values were obtained by visual matching of the drug
concentration giving 50% inhibition of growth.
Measurement of Cytotoxic Activity against Mammalian Cell
Lines in Culture. Animal cell lines (HeLa and fibroblast L929) were
used to determine drug toxicity by using MTT assay for mammalian
cell viability assay as described by Mosmann 198336 using HeLa and
fibroblast L929 cells cultured in complete RPMI (cRPMI) containing
10% fetal bovine serum, 0.2% sodium bicarbonate, 50 μg/mL
gentamycin. Briefly, cells (104 cells/200 μL/well) were seeded into
96-well flat-bottom tissue-culture plates in complete culture medium.
Drug solutions were added after overnight seeding and incubated for
24 h in a humidified atmosphere at 37 °C and 5% CO2. DMSO (final
concentration 10%) was added as +ve control. An aliquot of a stock
solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) (5 mg/mL in 1× phosphate-buffered saline) was added
at 20 μL per well and incubated for another 3 h. After the plate was
spun at 1500 rpm for 5 min, the supernatant was removed and 100 μL
of the stop agent DMSO was added to the well to dissolve the
formazan crystals. Formation of formazon, an index of growth, was
4-Hydroxy-3,4′,5-trimethoxystilbene11c (21). White solid
1
(52% yield); mp 96−98 °C; H NMR (CDCl3, 300 MHz), δ (ppm)
7.46 (2H, d, J = 6.9 Hz), 6.98−6.90 (4H, m), 6.75 (2H, s), 5.60 (1H,
s), 3.96 (6H, s), 3.85 (3H, s); 13C NMR (75.4 MHz, CDCl3), δ (ppm)
159.2, 147.3, 134.6, 130.3, 129.4, 127.5, 126.9, 126.5, 114.2, 103.2,
56.4, and 55.4.
Synthesis of ((E,E)-1,4-Bis(4-hydroxy-3,5-dimethoxy)-
styrylbenzene (22) (Scheme 4). The same procedure was followed
except the equivalents of the respective benzaldehyde, malonic acid,
piperidine, Pd catalyst, and LiCl were doubled.
((E,E)-1,4-Bis(4-hydroxy-3,5-dimethoxy)styrylbenzene11c-
1
(22). Greenish brown solid (65% yield); mp 246−250 °C; H NMR
(DMSO, 300 MHz), δ (ppm) 8.45 (2H, s), 7.46 (4H, s), 7.06 (2H, d, J
= 16.4 Hz), 7.05 (2H, d, J = 16.4 Hz), 6.82 (4H, s), 3.74 (12H, s); 13C
NMR (75.4 MHz, DMSO), δ (ppm) 148.6, 136.8, 136.3, 129.1, 128.1,
125.9, 124.6, 104.7, and 55.7. HRMS-ESI: m/z [M + H]+ for
C26H26O6, calculated 435.4984; observed 435.4967.
Representative Procedure for the Synthesis of 1-(4-
Methoxyphenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one
(23) (Scheme 5). To a solution of 4-methoxyacetophenone (0.42 g,
3.2 mmol) in methanol (15 mL) were added 10% aqueous NaOH (6
mmol) and 2,4,5-trimethoxybenzaldehyde (0.6 g, 3 mmol). The
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read at 570 nm by 96-well plate reader (Versa Max), and IC50 values
were determined by analysis of dose−response curves. The therapeutic
index was calculated as IC50(mammalian cell)/IC50(Pf 3D7).
cultures were treated with IC100 for 18 and 24 h. The treated and
untreated parasites were incubated with Hoechst 33342 stain for 10
min at room temperature. Then the parasites were washed with PBS
and thin smears were prepared. The slides were observed by using a
fluorescence microscope (Nikon 50i).
Fluorimetric Assay for Falcipain-2 Activity. Recombinant
falcipain-2 was purified as described by Shenai et al.37 with minor
modifications. The falcipain-2 activity was measured by 96-well plate
based high throughput fluorimetric method. Briefly the enzyme (2 μg)
was incubated for 10 min at room temperature in 100 mM sodium
acetate buffer, pH 5.5, and 10 mM DTT with or without test
compounds in triplicate format. The test compound stocks were
prepared in DMSO (1% final concentration of DMSO was maintained
in all wells). E64 was used as a positive control for falcipain-2. After
incubation, substrate benzyloxycarbonyl-Phe-Arg-7-amino-4-methyl-
coumarin hydrochloride (ZFR-AMC (Sigma), 1 mM stock in
DMSO, 2 μL) was added to a final concentration of 10 μM in the
same mixture. The enzyme cleaved the substrate and released
fluorescent 7-amino-4-methylcoumarin (AMC). The fluorescence
was monitored using a 96-well fluorescence plate reader (Victor,
Perkin-Elmer), with excitation and emission wavelengths of 355 and
460 nm, respectively. The fluorescence readings were plotted against
drug concentration, and IC50 values were obtained by visual matching
of the drug concentration giving 50% inhibition of enzyme activity.
In Vitro Stage-Specific Inhibition Assay. In order to find if
there was stage specificity in the action of the molecules under study,
highly synchronized P. falciparum (3D7) cultures at ring (R),
trophozoite (T), and schizont (S) stages were treated with the two
most potent compounds at their respective IC100 (11, 6 μM; 18, 10
μM). The assay was done in two sets of triplicates, one each for the
Giemsa stain and SYBR Green assay. Drug pressures were maintained
over 48, 24, and 8 h for ring, trophozoite, and schizont stages,
respectively, following which the drugs were removed by media wash
and the cultures were maintained in drug free complete RPMI medium
for 48 h. Percentage of stage-specific inhibition of each compound was
calculated in comparison to drug free control by microscopic counting
of 3000 cells for each stage and independently assessed for growth by
SYBR Green fluorescence assay as described earlier. Parasitized and
nonparasitized cells were counted by using Plasmodium auto count 0.1
software developed by Ma et al.38 The parasites with pycnotic
morphology were considered as nonviable cells.
Determination of P. f alciparum Mitochondrial Membrane
Potential (ΔΨm). Plasmodium falciparum mitochondrial membrane
potential (ΔΨm) was determined by using JC-1 dye. Cell-permeable
cationic carbocyanine dye JC-1 (Invitrogen), also known as 5,5′,6,6′-
tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodine,
emits green fluorescence (525 nm) in its monomeric form. However,
upon transfer to the membrane environment of a functionally active
mitochondrion, it exhibits an aggregation dependent orange-red
fluorescence (emission at 590 nm). Briefly, Plasmodium falciparum
cultures were treated with IC100 of test drugs for 24, 42, and 48 h (ring
stage culture) or for 18 and 24 h (trophozoite stage cultures). After
drug treatment the parasites were incubated with JC-1 dye for 20 min
at 37 °C. The wet mounts of stained cultures were observed by using a
fluorescence microscope (Nikon 50i).
ASSOCIATED CONTENT
■
S
* Supporting Information
Additional experimental details and NMR (1H and 13C) and
HRMS spectra of compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*For A.K.S.: phone, 01894-230426; fax, 91-1894-230433; e-
mail, aksinha08@rediffmail.com. For D.S.: phone, +91-11-
26742357; fax, +91-11-26742316; e-mail, dinkar@icgeb.res.in.
Present Address
§Department of Chemistry, Katholieke Universiteit Leuven,
Celestijnenlaan 200F, B-3001, Leuven, Belgium.
Author Contributions
∥These authors contributed equally.
Microscopic Evaluation of the Minimal Duration of 11/18−
P. f alciparum Ring/Trophozoite Exposure Necessary for
Causing Growth Inhibition. Plasmodium falciparum (3D7) ring
stage cultures were treated with IC100 for 18, 24, and 48 h. Similarly,
trophozoite stage cultures were treated with IC100 for 18 and 24 h.
After the respective incubation periods, drug pressures were removed
by media wash and the cultures were maintained under normal culture
conditions for a total of 96 h in each case. After this incubation period,
thin blood smears were prepared from control and treated wells and
processed for Giemsa staining. Percentage of stage-specific inhibition
by each compound was calculated in comparison to the corresponding
drug free control by counting 3000 cells for each stage as described
earlier.
Egress and Invasion Not Affected by 11 and 18. Highly
synchronized schizont stage Plasmodium falciparum (3D7) cultures
were treated with IC100 of 11 and 18 and incubated in normal culture
conditions for 8 h. The untreated parasites were maintained as control.
After incubation, drug pressure was removed by media wash and the
washed cultures were incubated for 48 h with or without drug
pressure. Thin smears were prepared from control and treated wells
and processed for Giemsa staining. The drug effects were monitored
microscopically and compared with control.
ACKNOWLEDGMENTS
■
We graciously acknowledge Department of Science and
Technology, New Delhi, India (Vide Grant No. SR/S1/OC-
16/2008) and the I.H.B.T., Palampur, India (Grant MLP 0025)
for financial support. The authors gratefully acknowledge the
Directors of I.H.B.T., C.S.I.R., Palampur, and I.C.G.E.B, New
Delhi, India, for their kind encouragement as well as providing
infrastructure for conducting the above work. N.S. and A.S. are
indebted to CSIR, New Delhi, for a Senior Research fellowship.
D.M. and D.S. thank MR4 who generously provided the
chloroquine resistant Dd2 and INDO strains used in the study.
Thanks are extended to David Walliker and X. Su who
deposited these strains with MR4. We thank Dr. Pawan
Malhotra and Sri Vidya Sundararaman for providing recombi-
nant falcipain-2 and Dr. R. L. Coppel and Charles Ma for
Plasmodium auto count 0.1 software. We are thankful to S.
Kumar for technical support regarding NMR spectra and
HRMS recordings (IHBT Communication No. 2252).
ABBREVIATIONS USED
P. falciparum DNA Staining by Hoechst 33342. Hoechst
33342 (2′-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5′-bis-1H-
benzimidazole trihydrochloride trihydrate, Invitrogen) was used to
detect chromatin condensation. DNA minor groove binding Hoechst
33342 is a cell permeable fluorescent dye that emits blue fluorescence
at 460 nm. It stains the condensed chromatin of apoptotic cells more
brightly than the relaxed chromatin of healthy cells. Briefly,
Plasmodium falciparum (3D7) ring stage cultures were treated with
IC100 of test drugs for 24, 42, and 48 h. Similarly, trophozoite stage
■
S-C, stilbene−chalcone; DNA, deoxyribonucleic acid; PCD,
programmed cell death; SAR, structure−activity relationship;
CQ, chloroquine; Pf, Plasmodium falciparum
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