Hydroxylated di- and tri-styrylbenzenes, a new class of antiplasmodial agents: Discovery and mechanism of action

Article abstract of DOI:10.1039/c6ra06059e

The first systematic evaluation of the antiplasmodial activity of the hydroxystilbene family of natural products and di/tristyrylbenzenes is described. A library of 27 diversely substituted hydroxy stilbenoids was rapidly synthesized using modified Knoevenagel-Perkin-decarboxylation-Heck sequences from readily available starting materials (i.e. hydroxybenzaldehyde-phenylacetic acid-arylhalide). These compounds were evaluated for in vitro antiplasmodial activity against three different strains of Plasmodium falciparum. Notably, 4,4′4′′-((1E,1′E,1′′E)-benzene-1,3,5-triyltris(ethene-2,1-diyl))tris(2,6-dimethoxyphenol) (27), an octupolar stilbenoid, showed IC₅₀ (μM) values of 0.6, 0.5 and 1.36 while a distyrylbenzene (11) showed IC₅₀ values of 0.9, 2.0 and 2.7 against 3D7 (chloroquine sensitive), Dd2 and Indo (chloroquine resistant) strains of Plasmodium falciparum respectively. Moreover, 27 and 11, which exhibited selectivity indices of 40 and >111 were also found to be nontoxic to the HeLa cell line. Microscopic studies revealed that the rings and trophozoites obtained from the 27 and 11 (an octupolar tristyrylbenzene and distyrylbenzene respectively) treated cultures were growth inhibited and morphologically deformed. These cultures also showed DNA fragmentation and loss of mitochondrial membrane potential (ΔΨ_m), suggestive of apoptotic death of the parasite. Together, these studies introduce di/tristyrylbenzenes as a new class of antimalarial agents.

Full text of DOI:10.1039/c6ra06059e

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PAPER
Hydroxylated di- and tri-styrylbenzenes, a new
class of antiplasmodial agents: discovery and
mechanism of action†
Cite this: RSC Adv., 2016, 6, 49348
Naina Sharma,ab Dinesh Mohanakrishnan,c Amit Shard,a Abhishek Sharma,ab
a
c
*
*
Arun K. Sinha‡ and Dinkar Sahal
The first systematic evaluation of the antiplasmodial activity of the hydroxystilbene family of natural
products and di/tristyrylbenzenes is described. A library of 27 diversely substituted hydroxy stilbenoids
was rapidly synthesized using modified Knoevenagel–Perkin-decarboxylation–Heck sequences from
readily available starting materials (i.e. hydroxybenzaldehyde–phenylacetic acid–arylhalide). These
compounds were evaluated for in vitro antiplasmodial activity against three different strains of
Plasmodium falciparum. Notably, 4,40400-((1E,10E,100E)-benzene-1,3,5-triyltris(ethene-2,1-diyl))tris(2,6-
dimethoxyphenol) (27), an octupolar stilbenoid, showed IC50 (mM) values of 0.6, 0.5 and 1.36 while
a distyrylbenzene (11) showed IC50 values of 0.9, 2.0 and 2.7 against 3D7 (chloroquine sensitive), Dd2
and Indo (chloroquine resistant) strains of Plasmodium falciparum respectively. Moreover, 27 and 11,
which exhibited selectivity indices of 40 and >111 were also found to be nontoxic to the HeLa cell line.
Microscopic studies revealed that the rings and trophozoites obtained from the 27 and 11 (an octupolar
tristyrylbenzene and distyrylbenzene respectively) treated cultures were growth inhibited and
morphologically deformed. These cultures also showed DNA fragmentation and loss of mitochondrial
membrane potential (DJm), suggestive of apoptotic death of the parasite. Together, these studies
introduce di/tristyrylbenzenes as a new class of antimalarial agents.
Received 7th March 2016
Accepted 27th April 2016
DOI: 10.1039/c6ra06059e
www.rsc.org/advances
describing the antimalarial potential of natural prenylated,4
glycosidic5 or benzamide6 containing stilbene derivatives
Introduction
(Fig. 1), however, the exploration of higher order stilbenoids
(dimeric/trimeric) against P. falciparum has not received much
attention. For instance, although distyrylbenzenes (DSBs) nd
important applications in the detection7 and treatment8 of
neurodegenerative disease, to date DSBs as well as tristyr-
ylbenzene have never been investigated as antimalarials.
Malaria is one of the most infectious diseases known to
mankind and it causes enormous mortality.1 Although diverse,
potent antimalarial agents including quinoline (e.g. chloro-
quine) and endoperoxides (e.g. artemisinin) are available, resis-
tance against antimalarial drugs has been increasing at an
alarming pace. Thus, the search for newer efficacious drugs as
well as new molecular scaffolds possessing antimalarial activity
remains a vital goal towards achieving control over malaria.2
Hydroxylated stilbenes represent an important class of
natural products having special therapeutic signicance owing
to their various pharmacological activities3 including anticancer
and anti-inammatory effects. Although there are few reports
aNatural Plant Products Division, CSIR-Institute of Himalaya Bioresource Technology,
Palampur, H.P.-176061, India. E-mail: aksinha08@rediffmail.com
bDepartment of Chemistry, University of Illinois at Urbana-Champaign, Urbana,
Illinois 61801, USA
cMalaria Drug Discovery Group, International Centre for Genetic Engineering and
Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India. E-mail: dinkar@
icgeb.res.in
† Electronic supplementary information (ESI) available: 1H NMR, 13C NMR data of
selected compounds. See DOI: 10.1039/c6ra06059e
‡ Present address: Medicinal and Process Chemistry, C.S.I.R-Central Drug
Research institute (C.D.R.I.), Lucknow 226031, India, Email: ak.sinha@cdri.res.in
Fig. 1 Example of some previously reported antimalarial stilbenoids.
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Pursuant to our interest in synthesis9–11 and biological activ- ring A, were synthesized by Perkin–Knoevenagel-condensation–
ities12 of hydroxylated stilbenoids it was envisaged to synthesize decarboxylation–Heck strategy under microwave irradiation.11
a library of monomeric, dimeric and trimeric hydroxylated stil-
In the course of our efforts to further enhance the diversity of
benoids and screen them as antimalarials. Herein we show that our panel of higher order stilbenoids, the oligophenyleneviny-
hydroxylated octupolar13 stilbenoid (27) and a distyrylbenzene lene (OPV, 27)9 with 4-hydroxy-3,5-dimethoxy substitution
(11) are highly potent antiplasmodial agents across chloroquine (Scheme 1) was prepared via Heck reaction of in situ formed 4-
sensitive and chloroquine resistant strains of P. falciparum. hydroxy-3,5-dimethoxystyrene with 1,3,5-tribromobenzene in
Mechanistic studies have indicated that these molecules cause DMF under reux conditions. In order to gauge the specic
morphological deformations, DNA fragmentation and loss of effect of octupolar moiety, an analogue14 of 27 possessing biaryl
mitochondrial membrane potential (DJm) in malaria parasite linkage (28, Scheme 2) was synthesized via palladium catalyzed
suggesting apoptotic cell death.
triple Suzuki coupling of 1,3,5-tribromobenzene with 3,4,5-
trimethoxy-phenylboronic acid under microwave in the pres-
ence of Pd(PPh3)4, K2CO3 in dioxane : water (5 : 1).
Results and discussion
Synthesis of hydroxylated stilbenoids
Amongst the various possible synthetic routes towards hydrox-
ystilbenoids (A–HC]CH–B), the modied Knoevenagel Heck9
and Perkin10 approaches were utilized, as these allowed a rapid
access to diversely functionalized stilbenoids using readily
available starting materials such as hydroxylated benzaldehyde,
phenyl acetic acid and halobenzene. A library comprising of 27
diversely substituted hydroxylated stilbenoids including stil-
benes, distyrylbenzenes and tristyrylbenzenes was synthesized.
Thus, hydroxylated stilbenes, 1 to 8 (Scheme 1) were obtained
via synthesis of hydroxy substituted styrenes (ring A) using
modied Knoevenagel Doebner-decarboxylation approach fol-
lowed by their in situ Heck coupling9 with diversely substituted
aryl halides (ring B).
Biological activity of monomeric/dimeric/trimeric stilbenes
The hydroxylated (1–8) and brominated (9–10) stilbenes were
subjected to microtiter plate high-throughput format SYBR
Green I uorescence based antiplasmodial screen.15 As shown
in Table 1, 4,40-dihydroxystilbene (1) displayed an IC50 of 92 mM
against the 3D7 strain of P. falciparum. Replacement of one of
the hydroxy groups with methoxy i.e. 4-hydroxy-40-methox-
ystilbene (2) caused a decrease in potency (IC50 > 100 mM).
On the other hand, 9 & 10 possessing bromo functional
group on ring B (Scheme 1) were synthesized via modied
Perkin condensation–decarboxylation10 reaction. Hydroxy
substituted symmetrical DSBs (11–19) with different substitu-
tion patterns on rings A and B (11–17) or with variations on ring
C (18 & 19) were synthesized via sequential Knoevenagel/
decarboxylation–double Heck reaction in one-pot.9
Scheme 2 General conditions: 3,4,5-trimethoxyphenylboronic acid,
1,3,5-tribromobenzene, K2CO3, Pd(PPh3)4, MW (250 W, 115 ꢀC) in
dioxane : water (5 : 1) for 35 min.
Unsymmetrical DSBs 20–26 (Scheme 1) having a 4-hydroxy
substitution at ring B and different electron releasing groups at
Scheme 1 Reagents and conditions: (a) substituted iodobenzene, CH2(COOH)2, Pd(PPh3)4, piperidine, LiCl, DMF, reflux 10 h (b) p-bromo-
phenylacetic acid, N-methylimidazole, piperidine, PEG-200, microwave (160 ꢀC) for 25 min (c) malonic acid, substituted p-hydrox-
ybenzaldehyde, Pd(PPh3)4, piperidine, LiCl, DMF, MW (150 ꢀC) for 45 min (d and e) CH2(COOH)2, piperidine, Pd(PPh3)4, LiCl, substituted 1,4-
diiodobenzene in condition (d) and 1,3,5-tribromobenzene in condition (e) were refluxed in DMF for 14.
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Table 1 Antiplasmodial activities of unsymmetrical and symmetrical Table 1 (Contd.)
stilbenoids against P. falciparum 3D7 strain
Compound no.
Structure
IC50 (mM)
Compound no.
Structure
IC50 (mM)
1
2
92
18
19
16
>100
3
4
>50
>100
>100
20
21
>100
1.9
5
6
47.5
>50
22
23
2.4
6.0
7
8
9
>100
88
99
24
1.4
10
11
63
25
26
15.0
0.9
>100
12
13
66
18
27
0.62
14
15
16
17
11
50
26
15
28
>100
Chloroquine
40 nM
Later on, 4-hydroxy-3-methoxy substitution on ring A was
kept constant and the effects of different electron releasing
(Table 1, 3–5) and electron withdrawing groups (7–9) on the ring
B were evaluated. Interestingly, 5 (IC50 47.5 mM) possessing
greater electron density on both rings showed slightly better
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antiplasmodial activity than 1–4 and 7–9 (IC50 > 50 mM). application in material science16 and non-linear optics17 is well
However none of these monomeric stilbenoids showed signi- documented. The dose dependent growth inhibition of malarial
cant antimalarial potential.
parasites by potent stilbenoids is shown in Fig. 2A. Since test
Hence, it was decided to extend the conjugation of the core molecules can interfere with SYBR green uorescence,15 all the test
stilbene monomers (Table 1, 1–10) and evaluate the anti- molecules were checked for (a) auto uorescence and (b)
plasmodial potency of resulting distyrylbenzenes (11). It is quenching effects in cultures of malaria parasites. Measurement of
evident from the screening results that a dimer of the inactive auto orescence helps avoid false negatives among the active
4,40-dihydroxy monomeric stilbene (1, IC50 92 mM) i.e. 4,40-dihy- molecules which may be uorescent. Evaluation of quenching
droxy substituted symmetrical distyrylbenzene (11) displayed effects helps to avoid false negatives from test molecules that may
promising activity (IC50 0.9 mM) against the 3D7 strain of P. fal- be quenchers of uorescence but may have no antiplasmodial
ciparum. An increase in hydroxy substitution on rings A and B of action. As shown in Fig. 2B, none of the 27 molecules studied by us
dimeric stilbene led to decreased antiplasmodial potential (12, (a) had intrinsic uorescence or (b) had the ability to quench the
IC50 66.0 mM) while introduction of methoxy substitution (13) uorescence due to SYBR Green. This gives validity to the results
resulted in comparatively better activity (IC5018 mM) than 12. obtained by the SYBR Green assay. The results of SYBR Green assay
Interestingly, a further increase in electron density (14) slightly were further corroborated by examining the time dependent
improved the activity (IC50 11 mM). On the other hand dimeric effects of 11 & 27 on the malaria parasite using microscopy on
stilbenes 15 (IC50 50 mM) and 16 (IC50 26 mM) having the same Giemsa stained smears of parasite cultures (Fig. 3).
substituents as 14, but having the electron withdrawing uoro
Subsequently, trimeric 27 (Table 1) and some potent DSBs
groups on ring C (15 & 16) showed decrease in antiplasmodial (IC50 < 2.5 mM) (Table 1, 11, 21, 22 & 24) were explored against
potency. Further, the replacement of central ring C with 2,5- two chloroquine resistant strains of P. falciparum (PfDd2 &
dimethoxybenzene (17) & anthracenyl (18) led to moderate anti- PfINDO) (Table 2). Importantly, 27 displayed a broad spectrum
plasmodial activity (17, IC50 15 mM & 18, IC50 16 mM), whereas the antiplasmodial potential with IC50 1.36 mM (Pf INDO) and 0.5
presence of a biaryl ring (19) diminished the activity (IC50 > 100 mM (Pf Dd2). Further, 27 also showed a selectivity index of 40
mM). It is evident from above discussion that the presence of against HeLa cell lines thereby indicating it to be non-toxic.
either 4-hydroxy or 4-hydroxy-3,5-dimethoxy substitution on
terminal rings A & C augments antiplasmodial activity.
Aer the antiplasmodial analysis of the symmetrical DSBs
(Table 1, 11–19), it was decided to evaluate the effect of
unsymmetrical substitution pattern of distyrylbenzenes (Table
1, 20–26). The antiplasmodial evaluation results for the
unsymmetrical DSBs (20–26) indicated that among the three
compounds (20–22), the DSB possessing 3,4-dihydroxy func-
tionality at ring A (20) (IC50 > 100 mM) was inactive (Table 1).
Surprisingly, slight variation of substituents i.e. having 4-
hydroxy-3-methoxy (21, IC50 1.9 mM) and 4-hydroxy-3,5-
dimethoxy (22, IC50 2.4 mM) substitutions at ring A, signi-
cantly enhanced the antiplasmodial activity (Table 1).
Interestingly, a further increase in electron density on ring B
(23 & 24) of dimeric stilbenes led to decreased activity in 23 (IC50
6 mM) while increase in potency was observed in case of 24 (IC50
1.4 mM). Further, replacement of a hydroxy group with methoxy
in ring B (25) decreased the activity (IC50 15 mM). Similarly, 26
(IC50 > 100 mM) having an unsubstituted ring B was also found to
be inactive. In view of above results, potent activity seems to be
associated with the presence of 4-hydroxy-3-methoxy (ring A) and
4-hydroxy (ring B) (21) or 4-hydroxy-3,5-dimethoxy i.e. syringol
Fig. 2 (A) Dose dependent antiplasmodial activities of potent stilbe-
noids estimated by SYBR green assay against P. falciparum 3D7.
(ring A) and 3,4-dihydroxy (ring B) (24). Thereaer, the tristyr- Compound numbers and the corresponding color codes are indicated
in the strip on the right. Standard deviation bars at each data point have
been calculated from triplicate observations. (B) Data validation.
Fluorescence intensity test of autofluorescent or quenching nature of
test molecules: untreated (UT) or drug (1–27) treated cultures were
ylbenzene 27 (Table 1) was also evaluated for antimalarial
activity. Interestingly it was found to be the most potent Pf3D7
inhibitor with IC50 0.62 mM. In contrast to octupolar stilbene (27,
IC50 0.62 mM), the triarylbenzene (28) with IC50 > 100 mM (Table
subjected to fluorescence intensity measurements using excitation/
1, Scheme 2) was found to be inactive suggesting a specic role emission of 480/535 nm (red bars). The nearly identical intensities
across control and test samples indicate that the test compounds
caused no interference due to autofluorescence. Control and test
molecule treated cultures were read for fluorescence intensities
following lysis by SYBR Green I containing lysis buffer (blue bars). The
of tristyrylbenzene moiety in P. falciparum inhibition.
It is worthwhile to mention that unlike DSBs (11–26) which
have been used for the detection/treatment of Alzheimer's
disease, such octupolar stilbenoids (27) have not yet been
nearly identical intensities across control and test samples indicate
explored as biologically active agents even though their absence of quenching effects.
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apoptotic programmed cell death in the parasite. Synchronized
parasite cultures of Pf3D7 were incubated with 27 and 11 at
their respective IC90 {1.3 mM (27) & 1.8 mM (11)} for 12, 24, 48 h
(rings) and 6, 12, 24 h (trophozoites). In contrast to the healthy
appearances of the untreated control cultures and their
sequential transition to the subsequent stages, the pycnotic
appearances of the rings and the trophozoites in the treated
cultures suggested that stress and probable death had pre-
vented the parasite to transit to the respective next stages of its
life cycle. It may be noted that cell shrinkage (crisis form) which
can be readily observed under the microscope, is one of the
major characteristic features of apoptotic cell death.18 The
shrunken, condensed and darkly stained nuclei of 27 and 11
treated rings and trophozoites stages as observed by Giemsa
staining (Fig. 3 and ESI Fig. S1a† for zoom) led us to look for
other indicators of apoptotic cell death.
DNA fragmentation and condensation are among the major
features of apoptosis.18 Minor groove binding DNA stain,
Hoechst 33342 was used to monitor DNA condensation in drug
treated parasite cultures. As shown (Fig. 3), the control rings
showed progressively greater staining as the rings matured to
become early trophozoites (12 h) and late trophozoites (24 h). In
contrast, 11 and 27 treated rings failed to become trophozoites
and appeared as highly condensed dot like bodies till 48 h.
Staining with Hoechst 33342 in control parasite cultures owes
its low intensity to the fact that highly compact and condensed
native chromatin exposes very few sites for the binding of dyes
like the Hoechst 33342. DNA fragmentation results in higher
degree of exposure and greater number of binding sites
resulting in brighter uorescence associated with cells under-
going apoptosis. The Hoechst 33342 staining of 11 and 27
treated trophozoites showed strong uorescence at 12 h. At 24 h
while the control culture showed dull staining in the form of
small dots corresponding to rings, the treated cultures showed
strong uorescence originating from arrested trophozoites
harboring fragmented chromatin spread over a large area (Fig. 3
and ESI Fig. S1b† for zoom). It is interesting to note that the
chromatin fragmentation observed here bears resemblance to
the pattern of chromatin fragmentation we have earlier
observed in trophozoite stage cultures treated with stilbene–
chalcone hybrids.12 While Hoechst 33342 staining based results
described above were suggestive of DNA fragmentation, a more
direct proof of DNA fragmentation was obtained from agarose
Fig. 3 Microscopic studies indicating apoptotic cell death in 11 and 27
treated malaria parasites. Ring and trophozoite stages of Pf3D7 were
treated with compounds 27 and 11 at their IC90 values for different
time points indicated against each panel. Giemsa staining showed
stressed and shrunken “crisis forms” in 27 & 11 treated cultures. Drug
treated ring and trophozoite stages lagged behind their respective
controls in terms of ring to trophozoite and trophozoite to schizont
transitions. Increased fluorescence of Hoechst 33342 in treated
trophozoites (24 h) suggests DNA condensation and fragmentation in
malaria parasites. JC-1 staining indicates loss of mitochondrial
membrane potential in 27 and 11 treated ring and trophozoites stages.
For zoom of these images please see ESI Fig. S1a–c.†
Probing the antiplasmodial action of DSB 11 and octupolar
stilbenoid 27
Microscopic examination of 11 and 27 treated parasite cultures gel electrophoretic analysis of genomic DNA obtained from
revealed stressed and shrunken “crisis forms” (Fig. 3). This led 11/27 treated vs. control parasite cultures (Fig. 4). While the
us to explore if these two lead compounds were triggering untreated culture showed the only intense band of genomic
Table 2 Antiplasmodial potential, resistance and selectivity indices for potent stilbenoids
SYBR green assay IC50 (mM)
Resistance index
IC50 Dd2/IC50 3D7
Selectivity index
Comp. no.
Pf3D7
Pf3D2
PfIndo
IC50 Indo/IC50 3D7
IC50 HeLa/IC50 3D7
IC50 L929/IC50 3D7
11
21
22
24
27
0.9
1.9
2.4
1.4
0.62
2.0
3.2
3.2
6.25
0.5
2.7
2.2
2.8
6.1
1.36
2.2
1.5
1.3
4.4
0.8
3
>111.11
15.8
4.7
18.6
40.3
>111.11
>52.6
>41.6
39.3
1.2
1.2
4.5
2.2
29.0
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However it is now becoming increasingly apparent that the
evolutionary origins of apoptosis may go back to unicellular
organisms like Leishmania, Plasmodium, yeast, bacteria, blas-
tocystis, Trypanosoma, and Trichomonas.22,23 Our observation of
apoptotic death in stilbenoid treated Plasmodium falciparum
represents a new avenue of targeting a sensitive niche of the
malaria parasite with a new pharmacophore. The high selec-
tivity with which 11 and 27 have been found to target the blood
stage malaria parasite but not the mammalian cells like the
HeLa and L929 suggests that the apoptotic machinery of Plas-
modium, a primitive protozoan, may be characteristically
different from the corresponding machinery in the highly
evolved mammalian cells. It is worth noting that CQ has also
been found to inict apoptotic death in malaria parasite.24,25
Moreover the report of a putative P. falciparum metacaspase (Pf
MCA-1)25,26 and apoptotic features in P. berghei ookinetes27,28
endorse the presence of apoptotic machinery in the malaria
parasite. Thus apoptosis machinery of the malaria parasite
appears to be a valid target worthy of attack by novel pharma-
cophores like the stilbenoids in the present study.
Fig. 4 Detection of DNA fragmentation in 11 and 27 treated PfIndo by
agarose gel electrophoresis: early trophozoite stage cultures were
treated with indicated concentrations of CQ (+ control), 11 and 27 for
12 and 24 h. The DNA isolated from parasites was subjected to elec-
trophoretic separation. Note the strong intensity of genomic DNA
band in untreated (UT) culture. Drug treated cultures band intensities
of genomic DNA is diminished with simultaneous appearance of low
MW DNA fragments.
Conclusions
In conclusion, the rst systematic antiplasmodial evaluation of
a library of hydroxy substituted monomeric and oligomeric
stilbenoids (stilbenes, distyrylbenzenes and tristyrylbenzene)
was conducted. Importantly, the above study led to the intro-
duction of distyrylbenzenes and tristyrylbenzene as a novel class
of potent antiplasmodial scaffolds. Compound 11, a dimeric
form of hydroxyl stilbene {IC50: Pf 3D7 0.9 mM, PfDd2 2.0 mM,
PfIndo 2.7 mM, selectivity index > 111 (HeLa and L929)}
remarkably shows high antiplasmodial potency and high
selectivity index. Likewise, compound 27: 4,40400-((1E,10E,100E)-
benzene-1,3,5-triyltris(ethene-2,1-diyl))tris(2,6-dimethox-
yphenol), displayed highly promising antiplasmodial activity
(IC50: Pf 3D7 0.62 mM, Pf Dd2 0.5 mM, Pf Indo 1.36 mM) as well as
good selectivity indices of 40.3 (HeLa) and 29 (L929). Further
mechanistic investigations have revealed that distyrylbenzene
(11) and octupolar stilbenoid (27) trigger selective apoptotic cell
death in malaria parasite.
band, the DNA obtained from 11/27 treated cultures showed
a diminished intensity of genomic DNA together with a streak of
low molecular weight DNA fragments. Interestingly, the inten-
sity of these fragmented pieces of genomic DNA was higher in
cultures from 12 h exposure than from analogous cultures
resulting from 24 h exposure to 11/27. This data suggests that 11
and 27 may induce DNA fragmentation at times earlier than 12
h and 24 h may be the time corresponding to extensive DNA
breakdown to sizes too small to be detected by this gel based
staining method. Indeed this time dependent phenomenon
observed with 11/27 found a good match with chloroquine
(used as positive control in this experiment) which also showed
a higher intensity of fragmented DNA bands at 12 h than at 24 h.
Loss of mitochondrial membrane potential (DJm), a hall-
mark of apoptosis19 was observed using JC-1 dye uorescence
microscopy in 27 and 11 treated malarial parasites. This dye is
known to acquire a red color upon aggregation in the ambience
of a high DJm. When DJm is abolished by drugs that trigger
apoptosis, the disaggregation of the dye causes its color to
change from red to green. Ring stage parasites treated with 27
and 11 showed complete loss of DJm (fully green) at 12 h and 24
h respectively (Fig. 3 and ESI Fig. S1c† for zoom). However, the
vulnerability of trophozoites to 11 and 27 appeared to be iden-
tical since 12 h treatments with each one of them resulted in
complete loss of DJm.
Experimental section
Materials & instruments
All the starting materials were reagent grade. The palladium
catalyst was purchased from Aldrich and used as such. The
substituted benzaldehydes, haloarenes, 4-bromophenylacetic
acid and all other reagents were obtained from commercial
sources (Merck and Aldrich). The solvents used for isolation/
purication of compounds were obtained from commercial
sources (Merck) and used without further purication. Column
chromatography was performed using silica gel (Merck, 60–120
mesh size). The chromatographic solvents are mentioned as
volume : volume ratios. 1H (300 MHz) and 13C (75.4 MHz) NMR
spectra were recorded on a Bruker Avance-300 spectrometer.
The following abbreviations have been used to designate
chemical shi multiplicities: s ¼ singlet, d ¼ doublet, t ¼
Apoptosis, a process of programmed cell death involving
features of cell shrinkage, DNA fragmentation, chromatin
condensation, formation of apoptotic bodies, translocation of
phosphatidyl serine from inner to the outer leaet of the plasma
membrane and the loss of mitochondrial membrane potential
has found vivid description in multicellular organisms.20,21
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triplet, m ¼ multiplet. The 13C NMR spectra are proton decou- MHz, CDCl3), 147.1, 146.2, 136.9, 132.1, 130.0, 129.7, 128.1,
pled. The melting points were determined on a digital Barnsted 125.6, 121.2, 121.0, 115.0, 108.6 and 56.3.
Electrothermal 9100 apparatus and are uncorrected. HRMS-ESI
The above procedure was also followed for synthesis of 4-
spectra were determined using micromass Q-TOF ultima spec- bromo-40-hydroxy-30,50-dimethoxystilbene (10, Table 1).
trometer and reported as m/z (relative intensity). A CEM Dis-
Representative procedure for the synthesis of 4,40-((1E,10E)-
1,4-phenylenebis(ethene-2,1-diyl))diphenol 11, via
Knoevenagel condensation–double decarboxylation–double
Heck coupling reaction (Table 1)
cover® focused microwave oven (2450 MHz, 300 W) was used for
reactions.
Representative procedure for the synthesis of (E)-4,40-(ethene-
1,2-diyl)diphenol (1) via Knoevenagel condensation–double
decarboxylation–Heck coupling reaction (Table 1)
Malonic acid (21.6 mmol) was taken in a round bottom ask
and piperidine (16.45 mmol) added gradually. The above
Malonic acid (0.64 g, 6.15 mmol) was taken in a round bottom mixture was stirred in DMF (25 mL), for 2 min at room
ask and piperidine (0.45 mL, 4.6 mmol) added gradually. The temperature. Thereaer, 4-hydroxybenzaldehyde (2.7 mmol),
above mixture was stirred in DMF (15 mL) for 2 min at room 1,4-diiodobenzene (0.25 g, 0.755 mmol), Pd(PPh3)4 (0.045
temperature. Thereaer, 4-hydroxybenzaldehyde (1a, 1.51 mmol), piperidine (10.55 mmol) and LiCl (0.12 mmol) were
mmol), 4-iodophenol (0.2 g, 0.90 mmol), Pd(PPh3)4 (0.025 added and the reaction mixture allowed to reux for 14 h. The
mmol), piperidine (3.1 mmol) and LiCl (0.07 mmol) were added, above mixture was cooled to room temperature and ltered
and the reaction mixture allowed to reux for 10 h. The above through celite. The ltrate was poured into water (250 mL,
mixture was cooled to room temperature and ltered through acidied with dil. HCl, pH ¼ 5) and extracted with ethyl acetate
celite and washed with ethyl acetate. The ltrate was poured (3 ꢁ 50 mL). The combined organic layer was washed with water
into water (100 mL, acidied with dil. HCl, pH ¼ 5–6) and (1 ꢁ 50 mL), brine (1 ꢁ 20 mL), dried over Na2SO4 and vacuum
extracted with ethyl acetate (2 ꢁ 40 mL). The combined organic evaporated. The resultant residue was subsequently puried by
layer was washed with water (1 ꢁ 30 mL), brine (1 ꢁ 10 mL), column chromatography on silica gel (60–120 mesh size) using
dried over Na2SO4 and vacuum evaporated. The obtained hexane : ethyl acetate (6 : 4) and obtained solid was recrystal-
residue was subsequently puried by column chromatography lized with methanol to provide pure product (11).
on silica gel (60–120 mesh size) using hexane : ethyl acetate
(9.5 : 0.5) to give a solid which was further recrystallized in Grey solid (61% yield), mp 362–366 ꢀC (lit. mp 310–312 ꢀC), 1H
4,40-((1E,10E)-1,4-Phenylenebis(ethene-2,1-diyl))diphenol (11).31
methanol to provide pure 4,40-dihydroxystilbene (1, Table 1).
NMR (DMSO, 300 MHz), d (ppm) 9.68 (2H, s), 7.51–7.45 (8H,
(E)-4,40-(Ethene-1,2-diyl)diphenol (1).29 White solid (33% m), 7.13–7.04 (4H, m), 6.78 (4H, s); 13C NMR (75.4 MHz,
1
ꢀ
yield), mp 200–202 C, H NMR d (CD3COCD3, 300 MHz), 8.54 DMSO), d (ppm) 157.7, 136.7, 128.6, 128.5, 128.3, 126.8, 125.3,
(2H, s), 7.39 (4H, d, J ¼ 8.7 Hz), 6.96 (2H, s), 6.82 (4H, d, J ¼ 8.7 116.0.
Hz); 13C NMR d (75.4 MHz, CD3COCD3), 157.3, 129.9, 127.8,
125.9 and 115.9.
The above procedure was also followed for synthesis of other
symmetrical distyrylbenzenes 12–19 (Table 1).
The same procedure was also followed for synthesis of other
hydroxylated stilbenes including 2–8 (Table 1).
Representative procedure for the synthesis of 4-((E)-4-((E)-4-
hydroxystyryl)styryl)benzene-1,2-diol (20, Table 1) from 3,4-
dihydroxy benzaldehyde
Representative procedure for the synthesis of (E)-4-(4-
bromostyryl)-2-methoxyphenol (9, Table 1) via modied
Perkin condensation–decarboxylation reaction
To a stirred mixture of 4-bromophenylacetic acid (1.5 g, 7.0
mmol), methylimidazole (0.46 mL, 5.8 mmol) and piperidine
A stirred mixture of 4-bromophenylacetic acid (3.6 mmol), (0.62 mL, 6.30 mmol) in PEG-200 (5 mL), 3,4-dihydrox-
methylimidazole (4.9 mmol), piperidine (4.9 mmol) and 4- ybenzaldehyde (3.94 mmol) was added and the reaction mixture
hydroxy-3-methoxybenzaldehyde (0.5 g, 3.28 mmol) in poly- irradiated under microwave (180 W, 150 ꢀC) for 20 min.
ethylene glycol-200 (3–4 mL) was irradiated under focused Thereaer, a mixture of malonic acid (4.08 g, 39.2 mmol),
monomode microwave (150 W, 160 ꢀC) tted with reux piperidine (3.34 mL, 39.2 mmol), 4-hydroxybenzaldehyde (1.2 g,
condenser for 25 min. Aer the completion of reaction, the 9.82 mmol), Pd(PPh3)4 (0.136 g, 0.12 mmol), K2CO3 (0.54 g, 3.9
reaction mixture was cooled and acidied with dil. HCl (pH ¼ mmol), LiCl (0.014 g, 0.32 mmol) in DMF (10 mL) was added to
ꢀ
5). Then the aqueous layer was extracted with ethyl acetate (2 ꢁ the above pot and irradiated under microwave (180 W, 150 C)
20 mL) and the organic layer was dried over sodium sulfate, for 45 min. The reaction mixture was cooled to room tempera-
vacuum distilled to obtain crude product which was further ture and ltered through celite and washed with little amount of
puried by column chromatography using silica-gel (60–120 ethylacetate. The ltrate was poured into water (150 mL), acid-
mesh size) with a 0.5 : 9.5 mixture of ethyl acetate : hexane to ied with dil. HCl, (pH ¼ 5–6) and extracted with ethyl acetate (2
give the pure stilbene (9).
ꢁ 40 mL). The combined organic layer was washed with water (2
(E)-4-(4-Bromostyryl)-2-methoxyphenol (9).30 Cream solid ꢁ 15 mL), brine (1 ꢁ 10 mL), dried over Na2SO4 and vacuum
(42% yield), mp 120–122 ꢀC, 1H NMR d (CDCl3, 300 MHz), 7.50 evaporated. The residue was subsequently puried by column
(2H, d, J ¼ 8.5 Hz), 7.38 (2H, d, J ¼ 8.5 Hz), 7.10–7.02 (3H, m), chromatography on silica gel (60–120 mesh size) using hex-
6.95 (2H, d, J ¼ 8.3 Hz), 5.75 (1H, s), 3.97 (3H, s); 13C NMR d (75.4 ane : ethylacetate (9.4 : 0.6) to give product (20).
49354 | RSC Adv., 2016, 6, 49348–49357
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4-((E)-4-((E)-4-Hydroxystyryl)styryl)benzene-1,2-diol
Green solid (40% yield), mp 293–295 C, H NMR (CD3COCD3 1,10:30,100-terphenyl (28). White solid (45% yield), mp 278–280
: DMSO-d6 (7 : 3) 300 MHz), d (ppm) 7.98 (4H, s), 7.91 (2H, d, J ꢀC, 1H NMR (CDCl3, 300 MHz), d (ppm) 7.68 (3H, s), 6.87 (6H, s),
¼ 8.3 Hz), 7.60 (2H, d, J ¼ 18.1 Hz), 7.54–7.53 (1H, m), 7.51 (2H, 3.96 (27H, s); 13C NMR (75.4 MHz, CDCl3), d (ppm) 154.0, 143.1,
d, J ¼ 18.1 Hz), 7.35–7.34 (1H, m), 7.30 (3H, d, J ¼ 8.7 Hz); 13C 138.4, 137.5, 125.7, 105.1, 61.4 and 56.7. HRMS-ESI: m/z [M +
NMR (75.4 MHz, CD3COCD3 : DMSO-d6 (7 : 3)), d (ppm) 158.3, H]+ for C33H36O9, calculated 577.2432; observed 577.2456.
146.5, 146.3, 137.2, 135.5, 129.7, 129.2, 129.0, 128.8, 128.4,
RSC Advances
(20).32
3,300,4,400,5,500-Hexamethoxy-50-(3,4,5-trimethoxyphenyl)-
1
ꢀ
127.0, 126.7, 125.5, 125.4, 119.3, 116.2, 115.7 and 113.9.
The above procedure was also followed for synthesis of other
unsymmetrical DSB's 21–26 (Table 1).
Measurement of inhibition of P. falciparum growth in culture
In this study, chloroquine sensitive 3D7 and chloroquine
resistant Dd2 and INDO strains of P. falciparum were cultivated
in vitro by the method of Trager and Jensen33 with minor
modications. 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 Lꢂ1
Representative procedure for the one pot synthesis of 4,40,400-
((1E,10E,100E)-benzene-1,3,5-triyltris(ethene-2,1-diyl))tris(2,6-
dimethoxyphenol) (27, Table 1)
Malonic acid (16.05 g, 154.2 mmol) was taken in a round bottom hypoxanthine and 50 mg Lꢂ1 gentamicin) at 37 ꢀC under
ask and piperidine (12.75 mL, 128.7 mmol) added gradually. reduced O2 (gas mixture 5% O2, 5% CO2, and 90% N2). Stock
The above mixture was stirred in DMF (30 mL) for 2 min at room solutions of chloroquine were prepared in water (Milli Q grade)
temperature. Thereaer, 4-hydroxy-3,5-dimethoxybenzaldehyde and test compounds were dissolved in DMSO. All stocks were
(2.36 g, 12.96 mmol), 1,3,5-tribomobenzene (0.75 g, 2.38 then diluted with culture medium to achieve the required
mmol), Pd(PPh3)4 (0.247 g, 0.21 mmol), piperidine (6.37 mL, 63.7 concentrations (in all cases the nal concentration contained
mmol) and LiCl (0.025 g, 0.058 mmol) were added, then reaction 0.4% DMSO, which was found to be non-toxic to the parasite).
mixture allowed to reux for 16 h. The above mixture was cooled Drugs and test compounds were then placed in 96-well at-
to room temperature and ltered through celite. The ltrate was bottom tissue culture grade plates to yield triplicate wells with
poured into water (250 mL, acidied with dil. HCl, pH ¼ 5) and drug concentrations ranging from 0 to 100 mM in a nal well
extracted with ethyl acetate (3 ꢁ 50 mL). The combined organic volume of 100 mL. Chloroquine was used as a positive control in
layer was washed with water (1 ꢁ 50 mL), brine (1 ꢁ 20 mL), experiments (100 nM with 3D7 and 1000 nM with the chloro-
dried over Na2SO4 and vacuum evaporated. The residue was quine resistant strains). Parasite culture was synchronized at
subsequently puried by column chromatography on silica gel ring stage with 5% sorbitol. Synchronized culture was aliquoted
(60–120 mesh size) using hexane : ethyl acetate (6 : 4) and ob- to a drug containing 96-well plates at 2% hematocrit and 1%
tained solid was washed with methanol to provide pure ((E,E,E)- parasitemia. Aer 48 h of incubation under standard culture
1,3,5-tris(4-hydroxy-3,5-dimethoxy)styrylbenzene) (27).
conditions, plates were harvested and read by the SYBR Green I
4,40,400-((1E,10E,100E)-Benzene-1,3,5-triyltris(ethene-2,1-diyl)) uorescence-based method15 using a 96-well uorescence plate
tris(2,6-dimethoxyphenol) (27).9 Yellow solid (30% yield), mp reader (Victor, Perkin Elmer), with excitation and emission
1
ꢀ
223–225 C, H NMR (CDCl3, 300 MHz), d (ppm) 7.53 (3H, s), wavelengths at 485 nm and 530 nm, respectively. The uores-
7.17 (3H, d, J ¼ 15.7 Hz), 7.04 (3H, d, J ¼ 15.7 Hz), 6.81 (6H, s), cence readings were plotted against drug concentration, and
5.62 (3H, s), 3.98 (18H, s); 13C NMR (75.4 MHz, CDCl3), d (ppm) IC50 values obtained by visual matching of the drug concen-
147.4, 138.3, 135.1, 129.4, 129.0, 126.7, 123.4, 103.6 and 56.5. tration giving 50% inhibition of growth. In view of the uores-
HRMS-ESI: m/z [M + H]+ for C36H36O9, calculated 613.2432; cence basis of the SYBR Green assay, it was important to assess
observed 613.2432.
artefacts due to autouorescence or quenching effects of each
test molecule. To measure the auto uorescence of test mole-
cules, the parasites were treated with 100 mM of all test mole-
cules and incubated for 1 h at 37 ꢀC following which the
cultures were lysed by lysis buffer {20 mM Tris; 5 mM EDTA;
0.008% (w/v) saponin; 0.08% (v/v) Triton X, pH 7.5} and read at
485/530 nm (excitation/emission). To determine possible
quenching effects, untreated parasite cultures or parasite
cultures treated with the test molecules (100 mM) were lysed by
1ꢁ SYBR Green I containing lysis buffer and read for their
uorescence values at 485/530 nm (excitation/emission).
Comparison of uorescence counts (+/ꢂ test molecule) was
used as a measure of quenching or lack of quenching.
Representative procedure for the synthesis of 3,300,4,400,5,500-
hexamethoxy-50-(3,4,5-trimethoxyphenyl)-1,10:30,100-terphenyl
(28, Table 1)
To a stirred mixture of 1,3,5-tribromobenzene (0.95 mmol) in
dioxane : water (5 : 1, 10 mL), 3,4,5-trimethoxyphenylboronic
acid (3.42 mmol), Pd(PPh3)4 (0.085 mmol), K2CO3 (1.4 mmol)
were added and the reaction mixture was irradiated under MW
ꢀ
(250 W, 115 C) for 35 min. The above mixture was cooled to
room temperature and was poured into water (250 mL, acidied
with dil. HCl, pH ¼ 5) and extracted with ethyl acetate (3 ꢁ 50
mL). The combined organic layer was washed with water (1 ꢁ 50
mL), brine (1 ꢁ 20 mL), dried over Na2SO4 and vacuum evapo-
rated. The resulting residue was subsequently puried by
column chromatography on silica gel (60–120 mesh size) using
Measurement of cytotoxic activity against mammalian cell
lines in culture
hexane : ethyl acetate (9 : 1) to provide pure 1,3,5-tris(3,4,5- Animal cell lines (HeLa and broblast L929) were used to
trimethoxyphenyl)benzene (28).
determine drug toxicity by using MTT assay for mammalian cell
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viability assay as described by Mosmann34 using HeLa and 1,10,3,30-tetraethylbenzimidazolylcarbocyanine iodine) (Molec-
broblast L929 cells cultured in complete RPMI containing 10% ular Probes, USA). JC-1 shows membrane potential dependent
fetal bovine serum, 0.2% sodium bicarbonate and 50 mg mLꢂ1 transition from a green (lmax emission 525 nm) monomeric form
gentamicin. Briey, cells (104 cells per 200 mL per well) were (at low transmembrane potential) to a red (lmax emission 590 nm)
seeded into 96-well at-bottom tissue-culture plates in complete aggregated oligomeric form (at higher trans-membrane poten-
culture medium. Drug solutions (in all cases the nal concen- tial). The assay was performed according to the manufacturer's
tration contained 0.4% DMSO) were added aer overnight instructions. Briey, synchronized ring and trophozoites stage
seeding and incubated for 24 h in a humidied atmosphere at cultures (1% parasitemia and 2% hematocrit) were incubated
37 ꢀC and 5% CO2. DMSO (nal concentration 10% v/v) was with IC90 of 27 (1.3 mM) and 11 (1.8 mM) for 12, 24, 48 (rings) and
added as +ve control. An aliquot of a stock solution of 3-(4,5- 6, 12, 24 h (trophozoites) in wells of 96 well plate. The cultures
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 were incubated with JC-1 dye for 20 min at 37 ꢀC and washed with
mg mLꢂ1 in 1ꢁ phosphate buffered saline) was added at 20 mL PBS. Wet mount slides were prepared and observed by using
per well, and incubated for another 4 h. Aer spinning the plate a uorescence microscope (Nikon 50i) using FITC lter.
at 1500 rpm for 5 min, supernatant was removed and 100 mL of
the stop agent DMSO was added to each well. Formation of
formazon, an index of growth, was read at 570 nm and IC50
values were determined by analysis of dose–response curves.
Detection of drug induced DNA fragmentation by agarose gel
electrophoresis
Drug induced DNA fragmentation was examined by agarose gel
electrophoresis. Early trophozoite stage cultures (4% para-
sitemia) of Plasmodium falciparum (Indo) were treated with IC90
of 27 (1.3 mM) and 11 (1.8 mM) for 12 and 24 h. CQ (2 mM) was
used as positive control. Aer treatment, the parasites were
isolated by the 0.05% saponin treatment procedure, genomic
DNA was isolated and samples were analysed by 1% agarose gel
electrophoresis method.35 The gels were photographed using
Alpha Imager EC.
Selectivity index was calculated as IC50 mammalian cell/IC50 Pf
3D7.
Microscopic evaluation of morphological changes in P.
falciparum
Morphological changes of P. falciparum were monitored by
microscopy based Giemsa staining method. Briey, synchro-
nized ring and trophozoites stage cultures (1% parasitemia, 2%
hematocrit) were incubated with IC90 of 27 (1.3 mM) and 11 (1.8
mM) for 12, 24, 48 (rings) and 6, 12, 24 h (trophozoites) in wells
of 96 well plate. Thin blood smears were prepared from treated
and untreated cultures, methanol xed, stained by Giemsa
(Sigma, India) and examined by bright eld optical microscopy
at 100ꢁ.
Acknowledgements
We graciously acknowledge Department of Science and Tech-
nology, New Delhi (Vide Grant No. SR/S1/OC-16/2008). The
authors gratefully acknowledge Director(s) of C.S.I.R.-IHBT,
I.C.G.E.B., Delhi and C.S.I.R.-C.D.R.I., Lucknow, for their kind
cooperation and encouragement. DM and DS thank MR4 who
generously provided the chloroquine resistant Dd2 and INDO
strains used in the study. Thanks to X. Su and late David
Walliker and who deposited these strains with MR4. IHBT
Communication No. 2331. DM thanks to ICMR, New Delhi for
senior research fellowship. CDRI communication no. 9230.
Detection of DNA fragmentation and chromatin condensation
by Hoechst 33342
DNA fragmentation and condensation were detected by
Hoechst 33342 (20-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-
2,50-bis-1H benzimidazoletrihydrochloridetrihydrate, Molecular
Probes, USA). Hoechst 33342 stains the condensed chromatin of
apoptotic cells far more brightly than is the case with native
chromatin of live healthy cells. The assay was performed
according to the manufacturer's instruction. Briey, synchro-
nized ring and trophozoite stage cultures (1% parasitemia and
2% hematocrit) were incubated with IC90 of 27 (1.3 mM) and 11
(1.8 mM) for 12, 24, 48 (rings) and 6, 12, 24 h (trophozoites) in
wells of 96 well microtiter plate. Cultures were incubated with
Hoechst 33342 stain (lmax emission 460 nm) for 20 min at ice
temperature. Following transfer of cells to microfuge tubes, the
cells were centrifugally washed (200 mL, twice) with PBS and wet
mount slides were prepared. The slides were observed by using
a uorescence microscope (Nikon 50i).
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