Divergent Synthesis of Styryl–Cinnamate Hybrid Analogues Inspired by the Natural Product Salvianolic Acid F as a Premise To Investigate Their Anticancer Activity and Its Metabolomic Profiling

Article abstract of DOI:10.1002/ejoc.201601104

Protecting-group-free synthesis of hydroxylated styryl–cinnamate hybrids (C₆–C₂–C₆–C₃unit), inspired by the natural product salvianolic acid F, was achieved by a step-economical route involving sequential double C–C bond formation in one pot. The method involved multiple reactions (Perkin condensation/decarboxylation–Heck cross-coupling reactions) by using simple precursors (i.e., hydroxylated benzaldehyde, arylacetic acid, and acrylic acid derivatives) in one pot and yielded the desired unnatural small hybrid molecules in varying yields of 35–65 % with E selectivity under microwave irradiation, whereas the reported conventional route for the synthesis of salvianolic acid F itself requires six steps with an overall yield of 10.0 % in addition to tedious separation of E/Z isomers that arise from a Wittig reaction. Apart from an economical synthesis and product diversity, we herein report the potential of some hybrid molecules with the catechol core to selectively inhibit glioma cells. The intrinsic mode of action of our lead molecule, involving caspase 6 and the quinonemethide pathway, is also reported on the basis of¹H NMR spectroscopy guided metabolomic profiling. We emphatically demonstrate the role of our hybrid molecules, analogues of salvianolic acid F, in forcing glioma cells towards apoptosis by specifically perturbing the concentration of glutathione along with that of caspase 6.

Full text of DOI:10.1002/ejoc.201601104

A Journal of
Accepted Article
Title: Natural Product Salvianolic acid F-Inspired Divergent Synthesis of Styryl-
Cinnamate Hybrid Analogues as a Premise to Investigate Anticancer Activity
and its Metabolomic Profiling
Authors: Amit Shard; Kiran Rawat; Arun Kumar Sinha; Yogendra Padwad; Dinesh
Kumar
This manuscript has been accepted after peer review and the authors have elected
to post their Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by using the Digital
Object Identifier (DOI) given below. The VoR will be published online in Early View as
soon as possible and may be different to this Accepted Article as a result of editing.
Readers should obtain the VoR from the journal website shown below when it is pu-
blished to ensure accuracy of information. The authors are responsible for the content
of this Accepted Article.
To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201601104
Link to VoR: http://dx.doi.org/10.1002/ejoc.201601104
Supported by

European Journal of Organic Chemistry
10.1002/ejoc.201601104
FULL PAPER
Natural Product Salvianolic acid F-Inspired Divergent Synthesis
of Styryl-Cinnamate Hybrid Analogues as a Premise to
Investigate Anticancer Activity and its Metabolomic Profiling
Amit Shard, [a,d]KiranRawat,[c,d] Arun K. Sinha,*[a,b,d] YogendraPadwad*[c,d] and Dinesh Kumar[a]
This article is dedicated with respect to Late Professor Richard F. Heck
Abstract: The natural product salvianolic acid F-inspired protecting-
group-free synthesis of hydroxylated styryl-cinnamate hybrids (C6-
C2-C6-C3 unit) has been achieved by step-economical route via
sequential double C-C bond formation in one pot. The present
made in its molecular pathogenesis; extensive efforts are
underway to evaluate novel chemotherapeutics against glioma,
but there has been limited progress in developing effective
therapies.[1a-b] The poor prognosis is due to high cellular and
molecular heterogeneity, altered signaling pathways and intrinsic
resistance.[1c] This has caught interest from various chemists
and biologists to synthesize better molecules with higher yields
and cytotoxic potential. Simultaneously, drug discovery invites
the development of new synthetic approaches to generate
method
involves
multiple
reactions
(Perkin-condensation/
decarboxylation-Heck cross-coupling reactions) using simple
precursors (i.e. hydroxylated benzaldehyde, arylacetic acid and
acrylic acid derivatives) in one pot which yields desired unnatural
small hybrid molecules (1-12) in varying yields of 35-65% with E-
selectivity under microwave irradiation whereas the reported
conventional route for synthesis of salvianolic acid F itself requires
six steps with an overall yield of 10.0% besides tedious separation of
E/Z isomers that arise from Wittig reaction. Apart from an
economical synthesis and products diversity, we herein report the
potential of some hybrid molecules (3,8,10 and 11) with the
catecholic core, to selectively inhibit glioma cells. The intrinsic mode
of action (MOA) for our lead molecule involving caspase 6 and
structurally diverse scaffolds.[1d-e]
It is well accepted that the unique ability of organic
chemists allows tailoring of many natural products,[2] which itself
is an excellent pharmacy.[2a-b] This provides an extraordinary
approach to unlock the full potential of an anticancer natural
product like podophyllotoxin[2c ]which eventually lead to the
synthesis of etoposide.[2d] Similarly, many natural hybrid
molecules and their synthetic analogues have shown
multifarious bioactivities including anticancer property.[2b] Hybrid
molecules[3] are of great therapeutic repertoire as they confer
superior biological profiles (Figure 1) than their parent molecule.
Among numerous hybrid molecules reported so far,
Salvianolic acid F (Figure 1), one of the natural tetrahydroxy-
substituted phenolic acids[4] isolated from Salvia multihoriza,[5]
has been subject of major scientific attention as this natural
hydroxylated stilbene-cinnamate scaffold possesses numerous
biological activities. [4a-b]
1
quinonemethide pathway is also reported based on HNMR-guided
metabolomic profiling. We emphatically demonstrate the role of our
hybrid molecules, an analogue of salvianolic acid F, in compelling
the glioma cells towards apoptosis by specifically perturbing the
concentration of glutathione along with caspase 6.
Introduction
Gliomas[1] are most common type of primary malignant brain
tumors with poor prognosis. Although a significant progress is
[a]
[b]
[c]
A. Shard, A. K. Sinha, D. Kumar
Natural Product Chemistry and Process Development Division,
CSIR-Institute of Himalayan Bioresource Technology, Palampur-
176061 (H.P.), India
Figure 1. Structure of biologically active natural and unnatural hybrid
molecules
A. K. Sinha*
To the best of our knowledge, salvianolic acid A and B[4b-c]
have shown significant activity against glioma,[4d] however, there
is no report for testing of Salvianoic acid F against it. This may
be due to the fact that despite its esteemed biological profile, the
synthesis of Salvianolic acid F has been an arduous task as it
involves multiple steps[4a,6] reactions via Wittig reaction with
requirements of additional deprotection with BBr3[4a] leading to
the formation of undesired side product. Moreover, this protocol
has some shortcomings, such as using inaccessible starting
materials, poor selectivity (E/Z),[4a] low yields (overall 10.0%)[4a]
(Scheme 1) or limited substrate scope and product diversity.
Thus, it is still necessary to develop efficient and protection
group-free synthesis of Salvianolic acid F and their structural
Medicinal and Process Chemistry, CSIR-Central Drug Research
Institute, Lucknow-226031(U.P.), India
E-mail: aksinha08@rediffmail.com
K.Rawat, Y.Padwad*
Food and Nutraceuticals Division, Pharmacology and Toxicology
Lab, CSIR-Institute of Himalayan Bioresource Technology,
Palampur-176061 (H.P.), India
E-mail: yogendra@ihbt.res.in
Academy of Scientific and Innovative Research (AcSIR), New Delhi,
India
[d]
A. Shard, K. Rawat contributed equally
Supporting Information for this article is available on the WWW under
http://dx.doi.org/10.1002/anie.201xxxxxx
This article is protected by copyright. All rights reserved

European Journal of Organic Chemistry
10.1002/ejoc.201601104
FULL PAPER
analogues (Figure 1) as many a times analogues synthesized are
found more potent than the parent scaffold besides it allows short
and concise synthesis of desired analogues in better yield with
possibilities to conduct many additional biological activity studies.
keeping this in mind, two esteemed name reactions Perkin
condensation (C6-C2-C6 unit) and Heck coupling (C3 unit) were
designed for the generation of hydroxylated styryl-cinnamate
(C6-C2-C6-C3)unit. The fundamental retro synthetic route (I or II)
adopted is shown for better clarity (Scheme 2).
Scheme 1. Comparison of multistep prevalent protocol for natural Salvianolic
acid F with our concise approach for unnatural stilbene-cinnamate in one pot.
With this notion in mind, we came across literature report[7]
which revealed that unnatural stilbene-cinnamate[7a] (C6-C2-C6-
C3 unit; Figure 1), an analogue of salvianloc acid F, has potent
protein tyrosine phosphatase 1b (PTP1b) inhibitory[7a] activity.
Interestingly, PTP1b was also reported to be a potent target for
glioma.[7b] Unfortunately, the available synthetic route for such
stilbene-cinnamate[7a] (Figure 1), also requires tedious seven-
Scheme 2. Retrosynthetic analysis of hydroxylated styrylcinnamic acids, an
analogue of Salvianolic acid F[4a] (Scheme 1).
In this communication, we report a rationally designed
protecting-group-free (PGF) synthesis of hydroxylated styryl-
cinnamate (S-C) hybrids (1-12) via sequential Perkin-Heck
reactions (condensation-decarboxylation/coupling reactions) in
one-pot that accomplished in shorter time under the influence of
microwave irradiation[15] using polyethylene glycol (PEG) as a
green solvent. The structurally diverse hybrid molecules (1-12)
that resemble natural “Salvianolic acid F” core, were screened
against C6 glioma cells in vitro with a conclusive report that their
mode of action involves caspase 6 activity and reduction in
Glutathione (GSH) concentration.
steps protocol via
Wittig-Horner reaction along with
requirements of additional protection-deprotection strategy to
provide
desired
3,4-dihydroxy-4'-(methoxy-carbonyl-1-
ethenyl)stilbene)[7a] (see SI).
We now thoroughly investigated the possible alternative
short routes for protection-group-free synthesis of such styryl-
cinnamate scaffold with (E)-selectivity and it was visualized that
above shortcomings can be fulfilled by the most acclaimed
carbon-carbon cross coupling reactions i.e. palladium catalyzed
Mizoroki-Heck reaction.[8] Then it was thought to join the planar
centers for the synthesis of (E)-styryl-cinnamate(C6-C2-C6-C3
unit) by double[9] Heck coupling of dihalo substituted benzene
(C6 unit) with appropriate hydroxylated styrene (C6-C2 unit) and
acrylate (C3 unit) in one pot in sequential manner at two different
temperatures[9a] using Pd nano-particle as a catalyst. However,
problem of chemoselectivity[9a] during synthesis of dihydroxylated
hybrid molecules (C6-C2-C6-C3 unit) surfaced due to possibility of
formation of various side products including phenylenediacrylate
or distyrylbenzene (DSB) (see SI). Another problem was
propensity of hydroxylated styrenes towards polymerization
apart from the fact that many styrenes such as 3,5-dimethoxy-4-
hydroxystyrene (i.e. canolol)[10] are not commercially available.
In seeking a more efficient route towards step-economic
synthesis of hydroxylated stilbene−cinnamate hybrids from
readily available substrates (hydroxylated benzaldehyde,
arylacetic acid and acrylic acid derivatives), it was equally
visualized that the halogenated stilbene intermediate (Scheme
2) can act as a synthetic handle to synthesize targeted
hydroxylated stilbene-cinnamates through cascade[11] Heck
coupling with acrylate, by avoiding protection-deprotection
strategy.[12] In this context, our group has been actively engaged
in designing such cascade protocols where privileged Heck
coupling[13]along with other highly useful name reactions like
Suzukui/Aldol/Knoevenagel[14] remains pivotal.[13b] Therefore,
Results and Discussion
Initially, to prove the concept of two different name
reactions functioning together in one-pot towards concise
synthesis of styryl-cinnamic acid 1 (Scheme 3, Method A), a
tandem Heck-Perkin strategy was planned. Accordingly, 4-
bromophenylacetic acid (1.2 equiv.), acrylic acid (1.2 equiv.) and
3,4-dihydroxy-benzaldehyde (0.050g) were mixed in one pot
with 1-methyl imidazole (MIM)/Piperidine as bases (1.5 equiv.
each) and Pd(OAc)2 (5 mol %) as catalyst using PEG-200 as
solvent. The mixture was then microwave (MW) irradiated (at
160oC) for 35 min but unfortunately the tandem reaction led to
the formation of multiple side products along with trace amount
of 1. Hence, a sequential Heck-Perkin strategy (Scheme 3,
Method B) was planned where 4-bromophenylacetic acid and
acrylic acid were MW irradiated in presence of MIM/piperidine,
Pd(OAc)2 and PEG-200 to obtain intermediate 3-(4-
carboxymethyl-phenyl)-acrylic acid. This was followed by
sequential addition of 3,4-dihydroxybenzaldehyde in the same
pot for Perkin reaction with simultaneous condensation-
decarboxylation[16] under MW irradiation (160oC, 35 min), yet the
This article is protected by copyright. All rights reserved

European Journal of Organic Chemistry
10.1002/ejoc.201601104
FULL PAPER
desired product 1 (Scheme 3) was obtained in 15% yield only
(S.I.).
molecule 5 (Table 2), a close analogue of bioactive salvianolic
acid F and salvianolic acid A, an ETAR (Endothelin-A receptor)
antagonist[4e] (Figure 1), was obtained in 51% yield (Table 2) in
two step instead of six steps[4a] required for synthesis of
salvianolic acid F (Scheme 1).
With remarkable success towards step-economic and
protection-group-free synthesis of hydroxylated hybrids S-C
molecules (1-7, Table 2), we screened them against C6 cells (rat
glioma). The preliminary screening[18] was focused on glioma[18a-
b]
because of two reasons. Firstly, PTP1b is one of its prime
oncotargets[7b] and secondly because of the presence of ETAR
Scheme 3. Tandem Heck-Perkin and Sequential Heck and Perkin reaction
towards synthesis of unnatural styryl cinnamic acids (1)
receptor on its membrane[4e] which indicates possibility of
receptor-mediated
endocytosis
of
our
molecules.
Antiproliferative screening of molecules 1-7 was performed by
sulforhodamine B based colorimetric assay[19] and only methyl
stilbene-cinnamate 3 (IC50 5.4 µm) came out to be significant as
compared to Vinblastine (IC50 9.9 µm; Figure 2). Encouraged by
the potential of 3 to target C6 that has stem cell like property;[20]
the panel was also screened against four different cancer cell
lines viz. lung (A549), colon (COLO205), cervix (HeLa) and
ovary (CHOK1), but none of the hybrids could induce significant
(>50%) antiproliferative activity (See S.I). However, 3,4-
dihydroxy substituted ethyl stilbene-cinnamte 8 came out to be
significantly active on C6 cells with an IC50 of 3.4 µm (Figure 2).
On the other side, a non hydroxylated hybrid molecule 9
(Scheme 4) synthesized by using our recent dehydrative-
Heck[13a] methodology could not exhibit potential activity on C6
cells.
After the failure of above tandem Heck-Perkin coupling
reaction and with limited success of sequential Heck-Perkin
(Scheme 3) strategies, our attention moved towards sequential
Perkin (condensation-decarboxylation)-Heck coupling reaction.
Here, the first step involves the modified Perkin reaction[16]
between 4-bromophenylacetic acid (1.2 equiv.) and 3,4-
dihydroxybenzaldehyde (0.050g) in the presence of
MIM/piperidine (1.5 equiv. each) and PEG-200 (4 mL) with
simultaneous condensation and decarboxylation of intermediate
α-phenylcinnamic acid into trans-4-bromo-3',4'-hydroxy stilbene.
This was followed by Heck coupling with acrylic acid in the
presence of Pd(OAc)2 (5 mol %) which led to the formation of 1
in 28% yield (Table 1, entry 1). Enthused by this, different
palladium catalysts were screened and amount of acrylic acid
was optimized (Table 1, entries 2-8). Delightedly, Pd(PPh3)4 and
acrylic acid (2 eq.) provided hydroxylated S-C hybrid 1 in 57 %
yield (Table 1, entry 7) in one pot in comparison to prevalent
multistep protocol.[7a]
Table 2. Perkin-Heck sequence led synthesis of various styryl-cinnamates.a
Table 1. Optimization of reaction conditions for SequentialPerkin-Heck sequence.a
a
CEM monomode microwave; General conditions. For step I reaction was
Microwave (MW) irradiated at 150W, 160oC, 35 min. For step II reaction was
MW irradiated at 110W, 110oC, for 45 min.
a CEM monomode microwave; General conditions. For step I, reaction mixture
was Microwave (MW) irradiated at 150W, 160oC for 35 min (Perkin
condensation). For step II, reaction mixture was MW irradiated at 110W,
110oC, for 45 min (Heck coupling).
b Isolated Yields.
With the optimized reaction conditions in hand, the
synthesis of stilbene-cinnamic acid 2 having 4 hydroxy 3,5-
dimethoxy styryl (syringol)[10] substitution was also achieved in
52% yield (Table 2). Further we planned to enhance the skeletal
diversity to see the effect of various substituent’s in SAR studies.
The methyl/ethyl esters[17] of hybrid cinnamic acids (1-2) were
prepared by following the above sequential Perkin-Heck reaction
just by replacing acrylic acid with methyl/ethyl acrylate and the
desired ester hybrids (3-8, Table 2) were obtained in good yield
upto 65% (4, Table 2). It is worth mentioning that hybrid
Scheme 4. Synthesis of styryl-cinnamate hybrid (S-C) using dhydrative-Heck
methodology.
From here onwards, we thought of concentrating on the
synthesis of 3,4-dihydroxy substituted styryl-cinnamate as out of
nine S-C hybrids molecules (1-9), only methyl or ethyl
substituted styryl-cinnamates (3 and 8) were found to be potent.
However, it is imperative to mention that propyl, isopropyl and
This article is protected by copyright. All rights reserved

European Journal of Organic Chemistry
10.1002/ejoc.201601104
FULL PAPER
butyl substituted acrylates are not commercially unavailable,
hence the synthesis of targeted hybrid esters (10-12) were
prepared sequentially in two steps as mentioned in scheme 5.
Thus molecule 1 was prepared in large amount following Perkin
and Heck protocol followed by reacting 1 with respective
alcohols (excess of propyl, isopropyl or butyl alcohol) in
presence of acetyl chloride with concomitant release of mineral
acid. This led us to prepare the three hybrid esters 10, 11 and
12 respectively in good yield up to 55% (Scheme 5). To our
delight molecules 10 and 11 (Figure 2) also came out to be
active against C6 cells (see SI) whereas n-butyl substituted
stilbene-cinnamate (12) was found inactive against C6 cells.
The plausible mechanism of the sequential Perkin-Heck protocol
is expected to be quinone methide species.[13] Further,
capitalizing on the inherent properties of 3,4-dihydroxy
substituted hybrid molecule[21] and their electrophilic ortho-
quinone methide[22] (QM) structure obtained under physiological
conditions, we also proposed a plausible mode of action (see
S.I). It was postulated that once the S-C hybrid molecule enter
the cells, widespread occurrence of esterases[22] inside cells
might cause hydrolysis of an ester bond and generate tantalizing
QM’s. Most importantly, QM’s being strong electrophiles are
vulnerable to the similar set of reactions usually committed by
other anticancer drugs.[22b] It’s well established that most of the
anticancer drugs like mitomycin-C or platinum complexes reacts
with N7 of guanine in DNA and also with other thiols containing
cellular nucleophiles like glutathione (GSH). So, we
hypothesized that our molecules produce QMs that on reacting
with GSH reduces its availability in the cells hinting towards the
activation of executioner proteins causing apoptosis.
Scheme 5. Synthesis of styryl-cinnamate (S-C) hybrids using Perkin-Heck
methodology followed by esterification.
(a)
(b)
(c)
(d)
(e)
80
60
40
20
0
POSITIVE
CONTROL
(Vinblastine)
IC50 9.9 µm
10µM 25µM 50µM 100µM
Vinblastine at various concentrations
80
60
40
20
0
CO2Me
HO
OH
IC50 5.4 µm
10µM
25µM
50µM
100µM
Molecule3
Molecule 3 at various concentrations
80
60
40
20
0
CO2Et
HO
OH
IC50 3.9 µm
10µM
25µM
50µM
100µM
Molecule8
Molecule 8 at various concentrations
80
O
O
60
40
20
0
HO
OH
IC50 6.4 µm
Molecule10
10µM
25µM
50µM
100µM
Molecule 10 at various concentrations
O
O
80
60
40
20
0
HO
OH
IC50 15.1 µm
Molecule11
10µM
25µM
50µM
100µM
Molecule 11 at various concentrations
Figure 2. Cytotoxicity profile of lead hybrid molecules (3, 8, 10 and 11) compared to positive control (Vinblastine). (a) Molecules under study. (b) Image of C6
cells using Phase-contrast microscopy after 48 h insult with molecules under study. (c) Toxicity induced by Vinblastine as well as molecules 3, 8, 10 and 11. C6
cells (2x104) were treated with different concentrations (10-100 µm) for 48 h before staining with 0.4% Sulforhodamine B dye. Data was analyzed by ANNOVA
using STATISTICA, version 7. Graph represents the percentage toxicity of molecules since SRB staining is indicative of number of alive cells. (d) After insult with
the molecules at IC50, cells (1x105) were subjected to Annexin V-FITC-PI double staining. Flow Cytometry results were obtained on BD calibur for 10,000
events/sample. X-axis represents Apoptosis while Y-axis depicts necrosis. e) Cells were treated for 12 h and harvested by mechanical dislodging, resuspended in
1X Caspase Buffer with final concentration of 5x106 cells/mL and proceed for staining to study Multicaspase profiling.
This article is protected by copyright. All rights reserved

European Journal of Organic Chemistry
10.1002/ejoc.201601104
FULL PAPER
Figure 3. Determining the Mechanism of Action for our lead molecule 8. (a) 1H NMR spectra (three independent repliecates) of aqueous metabolites from three
groups. The spectral regions from 0.0 to 4.5 ppm is shown (3-HIVA= 3-hydroxyisovaleric acid; La= Lactate; Ly= Lysine; Ac= Acetate; Glum= Glutamine; Glt=
Glutathiona; Tau= Taurine; Val= Valine; Leu= Leucine; Ileu= Isoleucine; Ala= Alanine; Lys= Lysine; Glm= Glutamate; Gly= Glycine; Ph= Phenylalanine;
Th=Threonine; Cr= Creatine; CG= Control Group; DG= Diseased Group; DTG= Drug Treated Group; TSP= 3-(Trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt;
For full spectra refer SI). (b) Matrix plot generated using Past 3.x - software to identify the differential quantitative variation in metabolites among three groups
(CG= Control Group; DG= Diseased Group; DTG= Drug Treated Group; refer SI) . (c) Representative Western Blots indicate the release of activated caspase 3
upstream of caspase 6 in cells (1x106) treated at IC50. Lane 1 represents untreated cells (control) while lane 2-5 indicate protein level at various time points for
which insult was given. (d) Effect on intracellular GSH levels after incubation of C6 cells with 3.9 µm of molecule for 36 h, as measured by Ellman’s assay.
Results are presented as the mean of three independent experiments, with bars depicting the standard deviations.
In light of the cytotoxic ability (Figure 2a-c) of our lead
molecules and the plausible hypothesis generated thereby, we
analyzed the ability of these hybrid molecules to interfere in
phosphatidylserine translocation[23] along with DNA damage.
Accordingly, data generated by Propidium iodide(PI)-AnnexinV-
FITC double staining[23a] indicated that all lead hybrid molecules
(3, 8; Table 2; 10 and 11; Scheme 5) were able to trigger
apoptosis (Figure 2d) within 24 h of incubation while molecule 8
displayed remarkable potential even at low concentration (3.9
µm; Figure 2b).
Further multicaspase study to understand if the cell death
is caspase dependant or not (Figure 2e) was carried out and
selectivity index (SI Index; Table 3) was calculated to
understand the specificity of our lead moieties against cancer
cells opposed to normal glial cells.[23b] Molecule 8 depicted
highest Selectivity Index of 22 enabling us to appreciate its
unique ability to specifically target cancerous cells while being
less harmful to normal brain cells. This encouraged us to uptake
the analysis of activated caspases by immunoblotting through 8.
It was found that apoptosis triggered by it was through
caspase 6 (Figure 3). It is well established that the proapoptotic
protein, Bax, localizes to the mitochondria in response to
numerous apoptotic stimuli and leads to the release of
cytochrome c, which further activates the caspase cascade.[23c]
Therefore, the expression level of Bax was evaluated and it was
also found to increase with time confirming upstream signalling
of caspase 6 pathway. Since Poly (ADP-ribose) polymerase
(PARP) is involved in repair of damaged DNA, the blot obtained
for cleaved PARP indicated that 8 caused irreparable and
irreversible DNA damage (Figure 3c).
Table 3. Selectivity Index (SI) of our lead molecule against normal glial cells.
It was calculated as IC50 against normal glial cells/IC50 against C6 cells
Molecule
under study
IC50 against
Normal glial
cells
IC50
against C6
Selectivity
Index (SI)
Molecule 3
Molecule 8
Molecule 10
Molecule 11
142
86
103
103
15.4
3.9
10
9
22
10
3
39
In continuation to this, we tried to understand DNA damage
through QM formation for which a NMR based metabolomic
This article is protected by copyright. All rights reserved

European Journal of Organic Chemistry
10.1002/ejoc.201601104
FULL PAPER
study of 8 was undertaken which has become an important tool
now-a-days.[24] The comparative NMR spectra (aqueous phase)
acquired for 3 groups[24a] (CG= Control Group; DG= Diseased
Group; DTG= Drug Treated Group) is shown in Figure 3a. We
found that these metabolic profiles were highly reproducible. It
was observed that the accumulation of metabolites was high in
C6 cells and was significantly altered by 8. Concentration of
Glutathione (GSH; δ 2.908 ppm, m) was markedly reduced by
molecule 8 after 36 h of insult, clearly indicating its role in
modulating QM formation. Encouragingly, it had a pronounced
effect on other metabolites as well (Figure 3b), where the
concentration of Glycine (δ 3.54 ppm, s) and Lysine (δ 1.764
ppm, m) were considerably reduced. Studies have established
that Lysine has significant role in histone modification at DNA
level while glycine is well known marker for grade IV glioma.[24b-e]
Eventually, Ellman’s assay[22b] was performed and GSH
reduction was validated (figure 3d). Further, due to the success
of molecule 8 against C6, it was also screened against human
glioma U87MG[4d] cells but a moderate cytotoxicity of 45.4%
(See SI) was observed. This warrants further chemical
manipulation to target human glioma.
Finally, our initial presumption is consistent with our
biological results that the hybrid stilbene-cinnamates (Figure 1)
which targets PTP1b enzyme as anti-diabetic agent[7a] might be
potent target for glioma.[7b] It will although be premature to state
whether the cluster of our bioactive hybrid molecules (3, 8, 10
and 11) would safely cross the “Valley of death[25]” or will move
from “Table to be a tablet”, yet the important lessons learned
from this odyssey of small unnatural hybrid molecues may
reinvigorate interest for future studies to tackle these recalcitrant
cells for anticancer drug development.
metabolites and proteins as well as their role at the
transcriptomic level will provide further insight into the mode of
action. For the same, proteomic as well as transcriptomic
studies are in progress. Further the study will be also expedited
towards modification of hybrid 8, enabling it suitable against
human glioma.
Experimental Section
General Experiments. From our preliminary screening (1-7) we
found that 3 could not significantly inhibit other cancer types
than glioma. Since our results came positive for molecule 3
alone towards glioma (C6) cell line of rat, further designed hybrid
molecules (8-12) were screened on this cell line only. All the cell
lines were purchased from National Center for Cell Science
(NCCS, Pune) and were cultured in respective media as
suggested by NCCS. Media was purchased from Gibco,
Invitrogen and cells were grown in 10% FBS (Gibco, Invitrogen)
along with 1% Antibiotic-antimytotic (Gibco, Invitrogen).
Cultures were incubated at 37°C with 5% CO2 and 95% humidity
till required number of cells were obtained. In order to test the
efficacy of our lead molecules on primary glial[23b] cells, we
isolated the cells employing standard protocol from rat pup (P0-
P1). This protocol was approved by the Institutional Animal
Ethical Committee (IAEC) at CSIR-IHBT in compliance with
relevant State and Federal Regulations (IHBT/P2 March, 2015).
Anticancer screening. All the S-C hybrid analogues (1-
12) were assayed for antiproliferative activity using
Sulphorodamine B (SRB) assay. Linearity over a 20-fold range
of cell number, sensitivity, non-destructive as well as indefinitely
stable colorimetric end point and lower signal-to-noise ratio
made SRB an assay of choice in our study of drug-induced
cytotoxicity screening. For the same, cell monolayer in its log
phase was trypsinized and final suspension of 2x105/mL was
prepared using DMEM. 0.1 mL (20,000cells) of this suspension
was added to require number of wells of 96-well microtiter plates
and incubated at 37ºC with 5% CO2 in 95% humidified
atmosphere. Insult at required concentrations was given for 48 h
in triplicate following which cell monolayer was fixed with 50%
Trichloroacetic acid (w/v) and subjected to colorimetric study
where cells were stained with 0.1 mL of anionic/aminoxanthine
0.4 % SRB dye. Allowed 30 min staining to ensure its optimum
electrostatic binding where it forms a complex with basic amino
acids on live cell membrane. Thereafter, unbound dye was
washed with 1% acetic acid and protein-bound dye was
dissolved in 10mM Tris base [tris(hydroxymethyl)aminomethane]
solution. OD540 was determined using microplate reader [BioTek
Synergy H1 Hybrid Reader] and Percentage inhibition was
calculated using following formula.
Conclusions
In summary,
a protecting-group-free, eco-sustainable and
potentially economic synthesis of the hybrid styryl-cinnamates
(1-12) via sequential Perkin-Heck strategies is unambiguously
established wherein carbon dioxide and water are released
during the course of condenstion-decarboxylation-coupling
reactions in one pot. Our protocol concurrently addresses issues
of simplicity and unprecedented utility by generation of S-C
hybrids, which show close structural analogy to naturally
occurring hybrid Salvianolic acid F. Some of these hybrid
molecules (3, 8, 10 and 11) served as potent chemical warheads
against glioma. The preliminary bio-activity guided syntheses of
variable small S-C hybrids have inhibited the growth of C6 cells
selectively without perturbing the physiology of normal glial cells.
Damaging effects of these antagonists against glioma cells in
vitro are manifested by superior and selective antitumor potential
without causing significant distress in healthy cells. Thus our
study strongly emphasizes the value of small hybrid orthologue-
specific antagonists with catecholic nucleus conjugated to an
ester group. The hybrid stilbene-cinnamates may act as
cornerstones in future to compel the biological terrorists (glioma
cells) towards apoptosis thus offering an effective method of
chemotherapy. The effect of these hybrid molecules on the other
%
Inhibition = [(C-S)/C]*100 where C= OD540 of Control and
S=OD540 of Sample
Three-factor factorial ANNOVA was applied on this data
employing STATISTICA (Version 7).
Flow Cytometry analysis. To quantify apoptosis and
necrosis induced by our small-stillbene conjugates, Propidium
iodide (PI) -Annexin V-FITC double staining was employed.
Viable, early apoptotic as well as necrotic or late apoptotic
cells were distinguished owing to differences in their plasma
membrane integrity and permeability due to phosphatidylserine
translocation. After treatment was given to cultured cells
(1x106/mL) in 6-well plate for 36 h, the adherent cells were
This article is protected by copyright. All rights reserved

European Journal of Organic Chemistry
10.1002/ejoc.201601104
FULL PAPER
suspended in PBS and subjected to Annexin V/Propidium iodide
staining using Annexin V-FITC Apoptosis Detection Kit II
(Becton Dickinson-BD; Biosciences Products) according to the
manufacturer’s protocol. Parallel to drug-treated group we also
µl of chloroform as well as 200 µL of distilled water was added to
it. The lysates were centrifuged at 15,000g for 20min at 4°C.
After centrifugation, polar layer was carefully transferred to a
new micro-centrifuge tube. The samples were dried and
aqueous residue was dissolved in 600 µl of NMR buffer (2mM
Na2HPO4, 5mM NaH2PO4, 0.025% TSP in D2O). For NMR
analysis an aliquot of 600 μl of sample was transferred to the 5-
mm NMR tube. Proton NMR (1H NMR) spectra were recorded
(at 300 K) on 600 MHz Bruker AVANCE III-600 spectrometer
(Bruker, Karlsruhe, Germany) operating at a proton NMR
maintained vehicle control cells.
The resultant green
fluorescence in FL-1 channel and red fluorescence in FL-2
channel was measured by FACS Calibur flow cytometer (Becton
Dickinson) thorough 10,000events/sample. Accordingly, dual
negative cells in lower left quadrant show live cells while dual
positive in upper right indicate mid-apoptotic stage. Single
positive cells in lower right and upper left indicate early apoptotic
and necrotic cells respectively. Data was collected at 36 h.
1
frequency of 600.13 MHz. Each H NMR spectrum consisted of
128 scans requiring 13 min and 16 s acquisition time with the
following parameters: 0.11 Hz/point and relaxation delay (RD)=5
s. Pre-saturation sequence was employed to suppress residual
H2O/D2O signal with low power selective irradiation at the H2O
frequency during the recycle delay. FIDs were Fourier
transformed with LB=0 Hz. The resulting spectra were manually
phased and baseline corrected, and calibrated at 0.0 ppm, using
Topspin (version 3.2, Bruker). As signal intensity is absolutely
proportional to molar concentration of metabolites13, the
quantification of the selected metabolites was performed by
using ratio method. A known internal standard (in this case TSP)
was used to determine the concentration of the targeted
metabolites by using the following equation:
Multicaspase profiling.
MuseTM Multicaspase Kit
(MCH100109; Milipore, USA) was exercised for fluorescence-
based facile, rapid and quantitative measurements. As directed
by manufacturer, treated cells were harvested by mechanical
dislodging, resuspended in 1X Caspase Buffer with final
concentration of 5x106cells/mL and proceeded for staining. Data
Acquisition and analysis was performed on the MuseTM Cell
Analyzer (version 1.3.0.0). Assay involves use of derivatized
VAD peptide having Fluoromethylketone (FMK) and fluorescent
moiety which is permeable and non-toxic to cells. This peptide
binds to activated caspases with resulting fluorescent signal
proportional to number of active caspases in cells which appears
as increased signal (lower right quadrant) in the Caspase axis.
On the other hand, a dead cell marker dye (7-AAD) shows
increased fluorescence on Viability axis (upper left quadrant).
Dual positive and dual negative results appear in upper right and
lower left quadrant respectively. Protocol by Promega (USA)
employing use of Apo-ONE® Homogeneous Caspase 3/7 Kit
was followed on 2x104cells/well of 96-well microtiter plate.
Higher Florescence resulting by higher activity of caspase 3/7 on
profluorescent substrate rhodamine 110 (bis-(N-CBZ-L-aspartyl-
L-glutamyl-L-valyl-L-aspartic acid amide; Z-DEVD-R110) was
measured at 499nm using microplate reader [BioTek Synergy
H1 Hybrid Reader]. Emission maxima were obtained at 521nm
and Relative Florescence Unit was calculated.1x105 cells were
subjected to Pan Caspase study employing Muse analyzer and
Multicaspase Kit and analyzed after 12 h of treatment.
mX = mST x (AX / AST) x (MWX / MWST) x (NST / NX)
where,
mX – mass of target analyte
mST – mass of TSP
AX – Integral area of selected signal
AST- Integral area of TSP
NX- number of protons generating integral signals
NST- Number of protons generating integral signal of
TSP
MWX- Molecular weight of target analyte
MWST- Molecular weight of TSP
In order to validate the lead obtained from NMR metabolomic
profiling, total glutathione quantification was undertaken
employing Ellman’s Assay using standard protocol from Sigma
Aldrich (Cat. # CS0260).
Western Blotting.
Control and treated cells were
collected after 0 h, 12 h, 24 h, 36 h and 48 h. These were lysed
with RIPA buffer (Sigma-aldrich, USA) along with protease
inhibitry cocktail (PIC; Sigma-aldrich, USA) in the of ratio 9:1 at
4°C for 1 h. Lysates were spun at 12000 r.p.m. for 20 min to
remove cellular debris. Protein concentration was determined
with Bradford reagent (Sigma-aldrich, USA). Equal amounts of
protein (60 µg) were subjected to electrophoresis on SDS-
polyacrylamide gels and transferred to nitrocellulose membranes
(Sigma) by electro-blotting. The blots were blocked with 5% Non
Fat Dried milk for an hour and probed overnight at 4°C with the
respective primary antibody (1:1000 dilution). Blots were then
washed with PBST and probed further with secondary anti-
mouse (1:15,000 dilution) or anti-rabbit antibody (1:10,000
General Procedure for the Preparation of hybrid compounds
(1-8)
via
sequencial
Perkin-Heck
reaction:
4-
Bromophenylacetic acid (1.3 eq., 0.093g, 0.434mmol) was
stirred in the presence of 1-methylimidazole (1.5 eq.) and
piperidine (1.5 eq.) in the solvent PEG-200 (4 mL) to generate
the carbanion. Next, the 3,4-dihydroxybenzaldehyde (1eq.,
0.050g, 0.362 mmol) was added to the formerly stirred reaction
mixture and the reaction mixture were microwave irradiated (MW,
CEM Discover© focused) for 30-35 min (150W, 160oC) or till
completion of the reaction. To the obtained intermediate 4-
bromo-3',4'-hydroxy stilbene, a mixture of acrylic acid or acrylic
ester (2 eq.) and Pd(PPh3)4 (5 mol%) were added in the same
pot for Heck coupling reaction. Finally, the reaction mixture was
again MW irradiated (110 W, 110oC, 40 min) to yield 1. The
crude reaction mixture after column purification (silica gel,
hexanes:ethyl acetate) was further recrystallized in methanol to
obtain the desired pure product (1-8). The compound 9 was
prepared following the previously developed protocol
dilution)
conjugated
with
horseradish
peroxidase.
Chemiluminescence was performed with ECL substrate (Biorad,
USA). The following monoclonal primary antibodies were used:
caspase-3, PARP, caspase 6, Bax and β-tubulin, that were
purchased from cell signalling technology (USA).
Metabolomic profiling probed by NMR. A standard
extraction protocol (See SI) was followed to obtain aqueous
extract. Accordingly 1x107 cell pellet (normal glial cells from rat
pup, C6 control and C6 cells treated with 8 for 36 h at 3.9 µM)
was resuspended with 400 µL of methanol and 200 µL of
chloroform. These 3 samples were sonicated for 15min and 200
General Procedure for the Preparation of hybrid esters (10-
12): The hybrid esters (10-12) were prepared by reacting 1 with
excess of corresponding alcohols (propyl, isopropyl or butyl
This article is protected by copyright. All rights reserved

European Journal of Organic Chemistry
10.1002/ejoc.201601104
FULL PAPER
alcohol) at cooled temp. in the presence of hydrochloric acid gas
as generated by reaction of acetyl chloride with respective
alcohol). The NMR and HRMS spectra of compounds 1-12 are
given below:
8.10 (s, 1H), 6.88-6.51 (m, 5H), 5.95-5.90 (d, J = 15.0 Hz, 1H),
5.86-5.83 (m, 4 H), 5.22-5.20 (m, 2H), 4.01 (s,3H); 13C NMR
(75.4 MHz, (CDCl3+DMSO-d6 (3:1): (ppm) 170.0, 163.0, 148.0,
144.4, 138.8, 136.1, 135.2, 134.1, 129.1, 128.2, 120.6, 119.6,
118.7, 56.6. HRMS-ESI: m/z [M+H]+ calcd for C18H16O3,
281.1308; found. 281.1326.
Compound 1: (2E)-3-{4-[(E)-2-(3,4-dihydroxyphenyl)-
ethenyl]phenyl}prop-2-enoic acid
Yield: 57%;mp: 246-248°C, Rf=0.2 (hexanes:ethyl acetate, 50:50
v/v with 1-2 drops of glacial acetic acid); 1H NMR (DMSO-d6,
300MHz): (ppm) 7.58 (d, J = 8.5 Hz, 2H), 7.52 (t, J = 9.3 Hz,
3H), 7.11 (d, J = 16.5 Hz,1H), 6.94 (d, J = 15.1 Hz, 1H), 6.83 (d,
J = 8.7 Hz, 2H), 6.67 (d, J = 7.6 Hz, 1H), 6.46 (d, J = 16.5 Hz,
1H); 13C NMR (75.4 MHz, DMSO-d6): (ppm) 168.2, 146.4,
145.9, 144.1, 140.0, 133.1, 130.7, 129.1, 128.9, 126.9, 124.7,
119.4, 118.7, 116.2, 113.9. HRMS-ESI: m/z [M+H]+ calcd for
C17H14O4, 283.0964; found 283.0956.
Compound 7: 3-{4-[2-(2-Hydroxy-phenyl)-vinyl]-phenyl}-
acrylic acid methyl ester
Yield: 40%;mp: 202-205°C;Rf= 0.4 (hexanes:ethyl acetate, 85:15
v/v); 1H NMR (CDCl3 + DMSO-d6, 600 MHz): (ppm) 8.71 (s, 1H),
7.83-7.43 (m, 3H), 7.35- 6.80 (m, 4), 6.44 (d, J = 15.6Hz, 1H),
6.41 (d, J = 15.6Hz, 1H), 3.75 (s, 3H); 13C NMR (150 MHz,
CDCl3 + DMSO-d6): (ppm) 167.5, 160.2, 155.0, 153.5, 144.5,
134.7, 131.7, 128.9, 128.0, 126.6, 125.6, 124.6, 119.7, 119.4,
118.5, 116.8, 116.4, 51.7. HRMS-ESI: m/z [M+H]+ calcd for
C18H16O3, 281.1308; found. 281.1332.
Compound 2: (2E)-3-{4-[(E)-2-(4-hydroxy-3,5-dimethoxy-
phenyl)ethenyl]phenyl}prop-2-enoic acid
Compound 8: 3-{4-[2-(3,4-Dihydroxy-phenyl)-vinyl]-phenyl}-
acrylic acid ethyl ester Yield: 57%;mp: 182-184 °C;Rf = 0.7
(hexanes:ethyl acetate, 85:15 v/v); 1H NMR (CD3COCD3,
300MHz): (ppm) 8.09 (s, 2H), 7.69-7.59 (m, 5H), 7.22 (d, J
=16.2 Hz, 1H), 7.16-7.15 (m, 2H), 7.03 (d, J =16.2 Hz, 1H), 6.97-
6.50 (m, 2H), 4.22 (q, 2H), 1.29 (t, 3H); 13C NMR (75.4 MHz,
CD3COCD3): (ppm) 167.5, 146.9, 146.6, 145.1, 141.4, 134.3,
131.5, 130.8, 129.8, 127.8, 126.0, 120.8, 118.7, 116.7, 114.4,
61.1, 15.0. HRMS-ESI: m/z [M+H]+ calcd for C19H18O4,
311.1458; found. 311.0770.
Yield: 52%;mp: 202-205°C;Rf=0.3 (hexanes:ethyl acetate, 50:50
v/v with 1-2 drops of glacial acetic acid); 1H NMR (DMSO-d6,
300MHz): (ppm) 8.65 (s, 1H), 7.68 (d, J = 8.5 Hz, 2H), 7.60 (t, J
= 8.5 Hz, 3H), 7.28 (q, J = 16.5 Hz, 2H), 6.92 (s, 2H), 6.55 (d, J
= 15.9 Hz, 1H), 3.81 (s, 6H); 13C NMR (75.4 MHz, DMSO-d6):
(ppm) 168.2, 148.5, 144.0, 140.0, 136.5, 133.2, 130.9, 129.2,
127.8, 126.9, 125.4, 118.8, 104.8, 56.4. HRMS-ESI: m/z [M+H]+
calcd for C19H18O5, 327.1227; found 327.1216.
Compound 3: Methyl (2E)-3-{4-[(E)-2-(3,4-dihydroxyphenyl)-
ethenyl]phenyl}prop-2-enoate
Compound 9: Ethyl (2E)-3-{4-[(E)-2-(naphthalen-2-yl)-
ethenyl]phenyl}prop-2-enoate
Yield: 54%; mp: 208-210°C;Rf = 0.6 (hexanes:ethyl acetate,
85:15 v/v); 1H NMR (DMSO-d6, 300MHz): (ppm) 9.32 (s,1H),
9.11 (s, 1H), 7.72 (d, J = 7.9 Hz, 3H), 7.65 (t, J =10.5 Hz, 3H),
7.24 (d, J = 15.6 Hz, 1H), 7.18 (d, J = 15.6 Hz, 1H), 6.96 (d, J =
8.7 Hz, 1H), 6.82 (d, J = 9.1 Hz, 1H), 6.67 (d, J = 16.7 Hz, 1H),
3.75 (s, 3H); 13C NMR (75.4 MHz, DMSO-d6): (ppm) 167.7,
146.9, 146.3, 145.1, 140.7, 133.3, 131.3, 129.6, 129.3, 127.3,
125.0, 119.9, 117.7, 116.6, 114.4, 52.3.HRMS-ESI: m/z [M+H]+
calcd for C18H16O4, 297.1121; found. 297.1103.
Yield: 52%;mp: 172-174°C; Rf = 0.8 (hexanes:ethyl acetate,
98:2 v/v); 1H NMR (300 MHz, CDCl3): (ppm) 7.89-7.77 (m, 5H),
7.74 (d, J = 16.0 Hz, 1H), 7.60-7.46 (m, 6H), 7.38 (d, J = 16.3 Hz,
1H), 7.26 (d, J = 16.3 Hz, 1H), 6.50 (d, J = 16.0 Hz, 1Hz), 4.34
(q, J = 7.1 Hz, 2H), 1.40 (t, J = 7.1 Hz, 3H); 13C NMR(75.4 MHz,
CDCl3): (ppm) 167.5, 144.5, 139.7, 134.8, 134.1, 133.6, 130.5,
128.9, 128.8, 128.5, 128.1, 127.4, 127.3, 126.8, 126.5, 123.8,
118.3. 60.9,14.7. HRMS-ESI: m/z [M+H]+ calcd for C23H20O2,
329.1626; found. 329.1646. IR (KBr) υ 1714 cm.-1 (ester).
Compound 4: 3-{4-[2-(4-Hydroxy-3,5-dimethoxy-phenyl)-
vinyl]-phenyl}-acrylic acid methyl ester
Compound
10:
3-{4-[2-(3,4-Dihydroxy-phenyl)-vinyl]-
Yield: 65%;mp: 190-192°C;Rf= 0.8 (hexanes:ethyl acetate, 85:15
v/v); 1H NMR (CDCl3, 300MHz): (ppm) 7.46 (d, J = 15.9 Hz, 1H),
7.51- 6.78 (m, 8H), 6.45 (d, J = 15.9 Hz, 1H), 5.66 (s, 1H), 3.96
(s, 6H), 3.82 (s,3H); 13C NMR (75.4 MHz, CDCl3): (ppm) 167.9,
147.6, 144.7, 139.9, 135.6, 133.6, 130.6, 128.9, 127.0, 126.2,
117.5, 103.9, 56.7, 52.1.HRMS-ESI: m/z [M+H]+ calcd for
C20H20O5, 341.1608; found. 341.1644.
phenyl}-acrylic acid propyl ester
Yield: 50%;mp: 146-148°C;Rf=0.8 (hexanes:ethyl acetate, 85:15
1
v/v); H NMR (CD3COCD3, 300MHz): (ppm) 7.91 (s, 1H), 7.90
(s, 1H), 7.77-7.51 (m, 3H), 7.34-7.28 (d, J =18.0 Hz, 1H), 7.11-
6.71 (m, 6H), 6.61-6.55 (d, J = 18.0 Hz, 1H), 4.15 (t, 2H), 1.8 (m,
2H), 0.96 (t, 3H); 13C NMR (75.4 MHz, CD3COCD3, 300MHz):
(ppm) 169.1, 147.2, 146.7, 145.9, 141.8, 134.3, 131.7, 130.9,
129.8, 127.7, 125.9, 120.8, 118.1, 116.6, 114.2, 67.4, 23.3, 10.9.
HRMS-ESI: m/z [M+H]+ calcd for C20H20O4, 325.1614; found.
325.1645.
Compound 5: 3-{2-[2-(4-Hydroxy-3-methoxy-phenyl)-vinyl]-
4,5-dimethoxy-phenyl}-acrylic acid methyl ester
Yield: 51%;mp: 172-174°C;Rf= 0.8 (hexanes:ethyl acetate, 85:15
v/v);1H NMR (CDCl3, 600MHz): (ppm) 7.73 (s,1H), 7.28 (s,1H),
6.94- 6.91 (d, J = 18.0 Hz, 1H), 6.84-6.81 ( m, 5H) , 6.51 (s,1H),
3.92 (s, 3H), 3.89 (s,3H), 3.82 (s,3H), 3.61 (s,3H);13C NMR (150
MHz, CDCl3): (ppm) 168.9, 149.7, 149.0, 146.8, 141.0, 136.4,
130.1, 129.2, 126.1, 122.6, 113.4, 113.2, 112.0, 108.3, 56.4,
56.3, 52.7. HRMS-ESI: m/z [M+H]+ calcd for C21H22O6,
371.1758; found. 371.1752.
Compound
11:
3-{4-[2-(3,4-Dihydroxy-phenyl)-vinyl]-
phenyl}-acrylic acid isopropyl ester
Yield: 55%;mp: 150-152 °C;Rf= 0.7 (hexanes:ethyl acetate,
85:15 v/v); 1H NMR (CD3COCD3, 300 MHz),  (ppm) 8.17 (s,1H),
8.00 (s,1H): 7.68-7.59 (m, 4H), 7.28-7.16 (m, 3H), 7.03 (d, J =
16.0 Hz, 1H), 6.97-6.84 (m, 2H), 6.50 (d, J = 16.01 Hz, 1H),
5.08 (m, 1H), 1.29 (d, 6H); 13C NMR (75.4 MHz, CD3COCD3):
(ppm) 167.3, 147.2, 146.8, 145.1, 141.6, 134.6, 131.8, 131.1,
130.0, 128.1, 126.3, 121.0, 119.4, 116.9, 114.6, 68.6, 22.7.
HRMS-ESI: m/z [M+H]+ calcd for C20H20O4, 325.1614; found.
325.1645.
Compound 6: 3-{4-[2-(4-Hydroxy-phenyl)-vinyl]-phenyl}-
acrylic acid methyl ester
Yield: 60%;mp: 182-184 °C, Rf= 0.7 (hexanes:ethyl acetate,
90:10 v/v); 1H NMR (CDCl3+DMSO-d6 (3:1), 300MHz): (ppm)
This article is protected by copyright. All rights reserved

European Journal of Organic Chemistry
10.1002/ejoc.201601104
FULL PAPER
[8]
[9]
a) V. Calo, A. Nacci, A. Monopoli, P. Cotugno, Angew. Chem. Int. Ed.
2009, 48, 6101-6103; b) J. Petrignet, A. Boudhar, G. L. Blond, J. Suffert,
Angew. Chem. Int. Ed. 2011, 50, 3285-3289; c) D. H. Wang, K. M.
Engle, B. F. Shi, J. Q. Yu., Science. 2010, 327, 315-319.
a) X. Zhang, A. Liu, W. Chen, Org. Lett. 2008, 10, 3849-3852; b) V.
Calo, A. Nacci, A. Monopoli, P. Cotugno, Angew. Chem. Int. Ed. 2009,
121, 6217-6219.
Compound
phenyl}-acrylic acid butyl ester
12:
3-{4-[2-(3,4-Dihydroxy-phenyl)-vinyl]-
Yield: 35%; mp: 188-190°C;Rf = 0.9 (hexanes:ethyl acetate,
1
85:15 v/v); H NMR (CD3COCD3, 300MHz): (ppm) 8.16 (s, 1H),
8.00 (s, 1H), 7.70-7.59 (m,5H), 7.22 (d, J = 16.3 Hz, 1H), 7.16-
6.97 (m, 3 H), 6.85 (d, J = 8.15 Hz, 1H) ,6.55 (d, J = 16.3 Hz,
1H), 4.15 (t, 2H), 1.46 (m, 2H), 1.29 (s, 2H), 0.96 (t, 3H); 13C
NMR (75.4 MHz, CD3COCD3): (ppm) 167.7, 147.0, 146.7,
145.2, 141.5, 134.5, 131.7, 130.9, 129.9, 127.9, 126.2, 120.8,
118.8, 116.7, 114.5, 65.0, 32.0, 20.3, 14.4. HRMS-ESI: m/z
[M+H]+ calcd for C21H22O4, 339.1781; found. 339.1794.
[10] H. Kuwahara, A. Kanazawa, D. Wakamatu, S. Morimura, K. Kida, T.
Akaike, H. Maeda, J. Agric. Food. Chem. 2004, 52, 4380-4387.
[11] K. C. Nicolaou, D. J. Edmonds, P. G. Bulger, Angew. Chem. Int. Ed.
2006, 45, 7134-7186.
[12] a) S. L. Schreiber,Proc. Natl. Acad. Sci. USA 2011, 108, 6699-6702; b)
I. S. Young, P.S. Baran, Nat. Chem. 2009, 1, 193-205.
[13] a) R. Kumar, A. Shard, R. Bharti, Y. Thopate, A. K. Sinha, Angew.
Chem. Int. Ed. 2012, 51, 2636-2639; b) A. Shard, N. Sharma, R. Bharti,
S. Dadhwal, R. Kumar, A. K. Sinha, Angew. Chem. Int. Ed. 2012, 51,
12250-12253; c) N. Sharma, A. Sharma, A. Shard, R. Kumar, Saima,
and A. K. Sinha Chem. Eur. J. 2011, 17, 10350-10356.
Acknowledgements
Amit Shard and Kiran Rawat would like to thank CSIR and DST
INSPIRE for SRF award respectively. The authors gratefully
acknowledge AcSIR, Director(s) CDRI, Lucknow and IHBT,
Palampur for their kind cooperation and encouragement. We
also thank Sh. Shiv Kumar of IHBT and SAIF of CDRI for
providing spectroscopic data. C.D.R.I./ I.H.B.T. Communication
No. /3538.
[14] N. Kumar, Richa, N. H. Andhare, A. Shard, A. K. Sinha, Chem. Eur. J.
2013, 19, 14798-14803.
[15] a) X. Zhang, A. Liu, W. Chen, Org. Lett. 2008, 10, 3849-3852; b) T. N.
Glasnov, W. Stadlbauer, C.O. Kappe, J. Org. Chem. 2005, 13, 3864-
3870.
[16] a) A. K. Sinha, V. Kumar, A. Sharma, A. Sharma, R. Kumar,
Tetrahedron. 2007,63, 11070-11077; b) A. K. Sinha, B. P. Joshi, A.
Sharma. U.S. Patent 6,989,467 (2006).
Keywords: C-C coupling • glioma • hybrid styryl-cinnamates •
Sequential Perkin-Heck reactions • metabolomic NMR
[17] a) R. B. Greenwald, C. W. Gilbert, A. Pendri, C. D. Conover, J. Xia, A.
Martinez, J. Med. Chem. 1996, 39, 424-431; b) A. de Jong, J. Plat, R.
P. Mensink, The Journal of Nutritional Biochemistry. 2003, 14, 362-369.
[18] a) D. Kumar, M. Sukapaka, G. D. K. Babu, Y. Padwad, Plos One. 2015,
e0133823; b) A. Acharya, K. Rawat, K. A. Bhat, V. Patial, Y. Padwad,
Mater. Res. Express. 2015, 2, 115401-115406
[1]
a) P.Y. Wen, S. Kesari, N Engl J Med. 2008, 359, 492-507; b) A.
Omuro, L. M. DeAngelis, JAMA. 2013, 310, 1842-1850; c) T. F.
Cloughesy, W. K. Cavenee, P. S. Mischel, Ann Rev Pathol. 2014, 9, 1-
25; d) M. Garcia-castro et al., Nat. Comm. 2015, 6, 6516-6518; e) T.
Cloughesy et al., Science Translational Medicine. 2016, 8, 341ra75.
DOI:10.1126/scitranslmed.aad9784.
[19] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMohan, D. Vistica,
J. T. Warren, H. Bokesch, S. Kenney, M. R. Boyd, J. Natl. Cancer Inst.
1990, 82, 1107-1112.
[2]
[3]
a) E. A. Crane, K. Grademan, Angew. Chem. Int. Ed. 2016, 55, 3882-
3902; b) M. Decker, Curr. Med. Chem. 2011, 18, 1464-1475; c) Y. Wu,
H. Zhang, Y. Zhao, J. Zhao, J. Chen, L. Li, Org. Lett. 2007, 9, 1199-
1202; d) T. G. Gantchev, D. J. Hunting, Mol. Pharmacology. 1998, 53,
422-428.
[20] X. Zheng, G. Shen, X. Yang, W. Liu., Cancer Res.2007, 67, 3691-3697.
[21] a) K. M. Huttunen, J. Rautio, Curr. Top. Med. Chem. 2011, 11, 2265-
2287; b) A. Y. Loo, K. Jain, I. Darah, Food Chem. 2008, 107, 1151-
1160; c) J. B. Harborne, P. M. Dey, Methods in plant biochemistry: A
handbook of plant phenolics Volume 1 (Academic press Inc., CA,
1989); d) M. Koufaki, E. Theodorou, D. Galaris, L. Nousis, E. S.
Katsanou, M. N. Alexis., J. Med. Chem. 2006, 49, 300-306.
a) N. Sharma, D. Mohanakrishnan, A. Shard, A. Sharma, Saima, A. K.
Sinha, D. Sahal, J. Med. Chem. 2012, 55, 297-311; b)L. F. Tietze, H.P.
Bell, S. Chandrasekhar, Angew. Chem. Int. Ed. 2003, 42, 3996-4028; c)
G. Mehta., V. Sing, Chem. Soc. Rev. 2002, 31, 324–334;d) B. Meunier,
Acc. Chem. Res. 2008, 41, 69-77; e) M. N. Aminakeet al., Bioorg. Med.
Chem. 2012, 20, 5277-5289; (f) P. G. Baraldi, A. N. Zaid, D. Preti, F.
Fruttarolo, M.A. Tabrizi, A. Iaconinoto, M. G. Pavani, M. D. Carrion, C.
L. Cara, R. Romagnoli, Arkivoc 2006, (vii) 20-34.
[22] a) F. Dufrasne, M. Gelbcke, J. Neve, R. Kiss , J. L. Kraus, Curr. Med.
Chem. 2011, 18, 3995-4011; b) N. Hulsman, J. P. Medema, C. Boss, A.
Jongejan, R. Leurs, M. J. Smit, I. J. de Esch, D. Richel, M. Wijtmans, J.
Med. Chem. 2007, 50, 2424-2431.
[23] a) V. A. Fadok, D. R. Voelker, P. A. Campbell, J. J. Cohen, D. L. Bratton,
P. M. Henson, J. Immunol. 1992,148, 2207-2216; b) M. Mecha, M. L.
Paula, M. Leyre, H. Miriam, B. Jose, G. Carmen, Nature Protocol
Exchange,2014 (www.nature.com/protocolexchange/ protocols/2051#/
reagents); c) J. C. Reed, Oncogene. 1998, 17, 3225-3236.
[4]
a) V. Dalla, P. Cotelle, Tetrahedron. 1999, 55, 6923-6930; b) J. H. C.
Ho, C. Y. Hong, J. Biomed. Sci. 2011, 18, 30-34; c) P. Hu, Q.L. Liang,
G.A. Luo, Z.Z. Zhao, Z. H.Jiang, Chem. Pharm. Bull. (Tokyo) 2005,
53,677-683; d) Z. Wang, P. Luo, S. Dai, Z. Liu, X. Zheng, T. Chen, Cell
Mol Neurobiol. 2013, 33, 921-928; e) Q. Zhang, S. Wang, Y. Yu, S. Sun,
Y. Zhang, Yu. Zhang, W. Yang, S. Li, Y. Qiao, Int. J. of Mol. Sci. 2016,
17, 1244-1259 .
[24] a) S. J. Hur, H. W. Lee, A. H. Shin, S. J. Park, Journal of the Korean
Magnetic Resonance Society. 2014, 18, 10-15R. b) Hamamoto, V.
Saloura, Y. Nakamura, Nat. Rev. Can. 2015, 15, 110-124; c) A. A.
Maudsley, R. K. Gupata, R. Stoyanova, N. A. Parra, B. Roy, S. Sheriff,
N. Hussain, S. Behari, Am J Neuroradiol. 2014, 35, S31-36; d) I. Amelio,
F. Cutruzzola, A. Antonov, M. Agostini, G. Melino, Trends in
Biochemical Sciences. 2014, 39, 191-198; e) M. Jain, R. Nilsson, S.
Sharma, N. Madhusudhan, T. Kitami, A. L. Souza, R. Kafri, M. W.
Kirschner, C. B. Clish, V. K. Mootha, Science. 2012, 336, 1040-1044;
e) H. Wen , Y. J. An , W. J. Xu , K. W. Kang, S. Park, Angew. Chem. Int.
Ed. Engl. 2015, 127, 1-5.
[5]
S. multihoriza, commonly known as "Danshen" in China, rich in
polyphenolic acids including Salvianolic acid F, A and B, is reported for
a large number of human health benefits. This patented Chinese
traditional herbal plant has successfully completed Phase II clinical
trials in the United States and will soon begin Phase III investigations.
V. Dalla, P. Cotelle, Tet. Lett. 1998, 39, 8285-8287.
[6]
[7]
a) Jung et al., Bioorg. Med. Chem. Lett. 2007, 17, 4481-4483; b) A. C.
Navis, M. V. Eijnden, J. T. G. Schepens, R. H. V. Huijsduijnen, P.
Wesseling, W. J. A. J. Hendriks, Acta Neuropathol. 2010, 119, 157-175;
c) Belley et al., Bioorg. Med. Chem. Lett. 2005, 15, 527-530.
[25] S. Hoelder, P.A. Clarke, P. Workman, Mol. Oncol. 2012, 6, 155-176.
This article is protected by copyright. All rights reserved

European Journal of Organic Chemistry
FULL PAPER
10.1002/ejoc.201601104
Entry for the Table of Contents
FULL PAPER
Key Topic*
The specifically tailored catecholic
Apoptosis
styryl-cinnamates
(C6-C2-C6-C3
Amit Shard, Kiran Rawat, Arun K.
Sinha,* Yogendra Padwad* and
Dinesh Kumar
unit), an analogue of natural
salvianolic acid F designed by a
novel and sustainable Perkin-Heck
sequence in one pot, have
enriched the diversity on ever
O
OR
Quinone
methide
Glioma
Cells
expansive
molecules
antagonistic profiles against glioma
C6 cell line that corresponds to one
of the most lethal and malignant
canvas
with
of
hybrid
superior
HO
Page No. – Page No.
Title: The Natural Product
R= Me, Et etc
OH
Salvianolic
acid
F-Inspired
Divergent Synthesis of Styryl-
Cinnamate Hybrid Analogues as
form
of
brain
tumors. NMR
a
Premise
to
Investigate
metabolomic studies of the potent
hybrid molecule received after
phenotypic screening revealed that
these novel variants exhibited
remarkable reduction in glutathione
levels and also increased cleaved
caspase-3 and -6 having definite
role in cancer cell progression. In
Anticancer Activity and its
Metabolomic Profiling
addition,
of necrotic machinery
the
activation
by our
catecholic molecules in C6 cells is
an added advantage thus helping
to nip the evil in the bud and
strongly
obviates
about
its
*one or two words that highlight the emphasis of the paper or the field of the study: Cross-coupling, Anticancer
This article is protected by copyright. All rights reserved