Methoxy-enriched cationic stilbenes as anticancer therapeutics

Article abstract of DOI:10.1016/j.bioorg.2020.103719

Stilbene-based compounds are largely described for their antioxidant activity. But their use as anticancer chemotherapeutics is hampered by poor pharmacokinetic properties and non-selectivity towards cancer and non-cancer potency. To overcome these drawbacks, twin chain cationic lipid conjugated, methoxy-enriched stilbene derivatives were designed, synthesized and evaluated for their anticancer potency. Our findings reveal that HMSC16, a molecule with the highest number of methoxy groups and with C16-twin chain lipid, is the most potent as well as the most selective anticancer agent when compared to the other synthesized derivatives and commercially available stilbene-based drug, tamoxifen, and resveratrol. To justify these results, we have conducted a series of mechanistic experiments where we found that HMSC16 induced ROS generation, apoptosis, and autophagy by affecting the mitochondrial, lysosomal and nuclear pathways. Further cell cycle analysis data reveals that HMSC16 not only induces cell death but is also involved in the arrest of the cell cycle at the sub-G1 phase. Moreover, HMSC16 showed self-aggregation property owing to a possibly favorable hydrophilic-lipophilic balance. The self-aggregation property of HMSC16 allowed it to entrap hydrophobic drugs, withaferin. With entrapped withaferin, HMSC16 showed additive if not synergistic cell killing effect in HeLa cells. From the above results, we concluded that HMSC16 can be used not just as a drug but also as a drug delivery agent.

Full text of DOI:10.1016/j.bioorg.2020.103719

Journal Pre-proofs
Methoxy-enriched cationic stilbenes as anticancer therapeutics
Md Yousuf, Sudhakar Jinka, Susanta Adhikari, Rajkumar Banerjee
PII:
S0045-2068(19)32269-2
DOI:
https://doi.org/10.1016/j.bioorg.2020.103719
YBIOO 103719
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
31 December 2019
8 February 2020
1 March 2020
Please cite this article as: M. Yousuf, S. Jinka, S. Adhikari, R. Banerjee, Methoxy-enriched cationic stilbenes as
anticancer therapeutics, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103719
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Inc.

Methoxy-enriched cationic stilbenes as anticancer
therapeutics
MdYousufa,b,1,*, SudhakarJinkaa,c,1, SusantaAdhikarid, RajkumarBanerjeea,c,*
aApplied Biology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007,
India.
bSERB-National Postdoctoral Fellow, DST-New Delhi, India.
cAcademy of Scientific and Innovation Research (AcSIR), CSIR-HRDC campus, Ghaziabad
201002, India.
dDepartment of Chemistry, University of Calcutta, Kolkata 700073, India
*Corresponding authors: E-mail: banerjee@iict.res.in; usuf.chem@gmail.com
1 These authors contributed equally to this work.
Highlights
 Developed a series of cationic amphiphilic molecules based on stilbene.
 Nine methoxy enriched substituted stilbenes with varying hydrophobic twin chains have
been synthesized.
 All compounds tested in both cancer and non-cancer cell lines for their anticancer potency
and selectivity.
 Compound with the highest number of methoxy groups and with C16-twin chain lipid
(HMSC16), proved to be the most potent as well as the most selective anticancer agent.
 HMSC16 induced ROS generation, apoptosis, and autophagy, cell cycle arrest.
 HMSC16 showed better self-aggregation property and subsequently allowed to entrap
drugs; it can be used as a drug delivery agent.
1

Abstract
Stilbene-based compounds are largely described for their antioxidant activity. But their use as
anticancer chemotherapeutics is hampered by poor pharmacokinetic properties and non-selectivity
towards cancer and non-cancer potency. To overcome these drawbacks, twin chain cationic lipid
conjugated, methoxy-enriched stilbene derivatives were designed, synthesized and evaluated for
their anticancer potency. Our findings reveal that HMSC16, a molecule with the highest number
of methoxy groups and with C16-twin chain lipid, is the most potent as well as the most selective
anticancer agent when compared to the other synthesized derivatives and commercially available
stilbene-based drug, tamoxifen, and resveratrol. To justify these results, we have conducted a series
of mechanistic experiments where we found that HMSC16 induced ROS generation, apoptosis,
and autophagy by affecting the mitochondrial, lysosomal and nuclear pathways. Further cell cycle
analysis data reveals that HMSC16 not only induces cell death but is also involved in the arrest of
the cell cycle at the sub-G1 phase. Moreover, HMSC16 showed self-aggregation property owing
to a possibly favorable hydrophilic-lipophilic balance. The self-aggregation property of HMSC16
allowed it to entrap hydrophobic drugs, withaferin. With entrapped withaferin, HMSC16 showed
additive if not synergistic cell killing effect in HeLa cells. From the above results, we concluded
that HMSC16 can be used not just as a drug but also as a drug delivery agent.
Keywords: Stilbene, a cationic lipid, anticancer drug, ROS generation, apoptosis, autophagy,
self-aggregation, combination treatment
2

Graphical Abstract
3

1. Introduction
Cancer is one of the foremost causes of death throughout the world population for decades.
Cervical cancer stands in fourth place in terms of the incidence of female cancer deaths worldwide
[1]. Cervical cancer is caused majorly by human papillomavirus apart from lifestyle risk factors.
Of course, many interventional strategies for the treatment of cervical cancers are available [2].
Targeted chemotherapy is widely adopted now-a-days and plays a vital role in the treatment of this
kind of disorders. But, the application of chemotherapeutics is limited primarily owing to their
non-selectivity towards non-cancer cells [3]. Because of this reason many studies were envisioned
in this direction of designing innovative compounds, such as mimicking natural compounds with
anticancer capabilities but with negligible collateral damages to normal cells. Natural and synthetic
stilbene derivatives are widely used for the prevention of various human diseases, including
cardiovascular and neurodegenerative, immune regulation, and cancer [4].
Regardless of the multifaceted anticancer potential of resveratrol (a molecule with stilbene
backbone), it has shown poor pharmacokinetic properties due to its poor bioavailability and
extensive phase-II conjugation reactions in vivo [5]. The IC50 for resveratrol as reported in the
literature varied between 40 and 200µM, while most of the studies did not compare the activity
difference in non-cancer and cancer cells [6].
To address these concerns one promising strategy is to modify resveratrol analogs with methoxy
groups, which has been an emerging area of exploration for the synthesis of new drugs with
improved therapeutic index [7]. The wholly methoxylated resveratrol derivatives, (E)-3,5,4-
trimethoxystilbene, shown to have great activity against angiogenesis [8]. Likewise, (E)-3,4,5,4-
tetra methoxy stilbene, has been shown to possess higher activity against cancer concerning
apoptosis induction than resveratrol [9][10].
During the last decade, Reddy et al. developed some synthetic stilbene derivatives based on type
and position of substituent on the stilbene backbone, where almost all compounds demonstrated
significant anti-proliferative activity against only the four cancer cell lines tested but without any
data on normal cells [11][12].
Very recently, Chillemiet al. developed amphiphilic resveratrol lipo-conjugates to improve
stability, uptake and bioavailability and targeted release. Regardless of the encouraging outcomes,
4

still novel approaches are needed to accomplish optimal pharmacological response with improved
resveratrol bioavailability in targeted cells [13].
In this study, our primary objective of developing a novel class of molecule i.e., conjugate of
cationic lipid with aliphatic chains to an established or unknown molecule/moiety for imparting
selective anticancer property (if any), was based on earlier observations. SAR studies with
molecules (such as 17β-estradiol [14][15], haloperidol [16], emodin [17], anisamide [18],
dexamethasone [19]) conjugated with various chain lengths of cationic lipid moiety exhibited
reduced anticancer activity of those molecules that contain mostly C8-C10 aliphatic chain length.
Others, while conjugating anisamide with C2-C18 aliphatic chain lengths also showed that C8-
C12 as the optimum chain length exhibiting selective anticancer property against various cancer
cells [18]. Among longer chain length derivatives, we additionally showed that only
hydrocortisone with C16 chain length showed selective anticancer property [20].
Br
Cl
R
N
R
Cl
H
CH3
C8H17
C8H17
OH
Cl
OH
O
O
O
O
N
C8H17
C8H17
N
N
H
O
O
H
CH3
N
CH3
H3C
OH
H
HO
Emodin
Haloperidol
Estradiol
O
Cl
N
R
O
R
R
Cl
F
OH
O
HO
OH
I
R
HO
N
N
C
10H21
O
NH
H
H
CH3
N
H
CH3
N
C10H21
H
H
H
CH3
F
H
Dexamethasone
MeO
O
O
Benzamide
Hydrocortisone
Figure1. Developed cationic lipid conjugated small molecules as anticancer agents
Keeping in mind, the serious concern of resveratrol bioavailability and the advantage of lipidation
strategy, in our design, we sought to modify the pharmacokinetic properties of the stilbene
backbone and adopted two structural manipulations. First, we synthesized stilbene derivatives by
varying the position and number of methoxy groups to find out the best one. Secondly, taking in
cue the lipid conjugation strategy used in the synthesis of ESC8 and others [14-19] we have
conjugated cationic lipid moiety, but with maximum C16 aliphatic chain length, to this methoxy
5

enriched stilbene analogues via amide bond formation (Figure 2), to impart cancer selectivity and
potency.
Cl
N
MeO
MeO
OH
H
OMe
MeO
H
H
Cationic lipid-anchored estradiol
(e.g., ESC8)
HO
Methoxy enriched resveratrol
OMe
MeO
MeO
Cl
N
H
N
O
n
`
R1=R3=H, R2=OMe
TMSC8
n=1,
R1==R2=R3=OMe
HMSC8
n=1,
R1=R2=OMe, R2=H
PMSC8
n=1,
R3
R1
R2
TMSC10
TMSC16
HMSC10
HMSC16
n=3;
n=9;
n=3;
n=9;
PMSC10
PMSC16
n=3;
n=9;
Figure 2. Structure of resveratrol derivative and design strategy as adopted in the present
study
In this work, we show that by grafting this cationic twin-chain lipid moiety through an amide
linkage onto the ortho position of methoxy enriched stilbene, the anticancer activity against several
cancer cell lines can be significantly enhanced with a certain degree of selectively. Among all,
HMSC16 showed specificity towards cervical cancer cells without affecting normal cells even at
higher concentrations. Further, we have evaluated the self-aggregation property of the most
selective, twin chain containing HMSC16 molecule and linked it with its selective toxicity. With
its efficient anticancer activity, cellular uptake potential, and also lipophilic drug-carrying ability,
HMSC16 can be projected as a potent drug delivery vehicle that can induce a combination of
anticancer effects of both the drugs with potential synergism.
2. Results
2.1. Chemistry
6

Cationic twin chain-conjugated stilbene derivatives were synthesized by several steps as
highlighted in Scheme I-III. Initially, we undertook the synthesis of methoxy-enriched stilbene
skeleton (3a-c) with ortho functionalization by varying the number and position of methoxy groups.
For this, we synthesized ethyl 2-iodo-3, 4, 5-trimethoxybenzoate (2), by the iodination of ethyl
1,2,3-trimethoxy benzoate (1) ester using molecular iodine and silver trifluoroacetate at room
temperature. This iodo-derivative (2) was subjected to Heck coupling with various
methoxyphenylstyrenes (4a-c), which was synthesized by Wittig reaction with methyl
triphenylphosphonium bromide and butyl lithium at -78 °C. Here, we optimized the reaction
condition, where among different base and solvent, sodium acetate as a base and dry DMF as a
solvent worked best and gave good to an excellent yield of (E)-stilbene (5a-c) without any
isomerization (E/Z). These methoxy enriched stilbenic ester (5a-c) derivatives were further de-
esterified by LiOH at 78 °C temperature for 3 h. Next, we synthesized various hydrophobic (C8,
C10, and C16) twin-chain substituted ethylenediamine derivatives (11a-c). For this, we used
commercially available ethylene diamine and Boc-anhydride to get mono-Boc protected ethylene
diamine. This protected amine reacted with various alkyl halide in presence of K2CO3 under the
refluxing condition to give twin aliphatic chain attached, protected amine derivatives (9a-c). These
ethylene diamine derivatives were further quaternized with methyl iodide. This was passed through
Cl-ion exchanging Amberlite IRA-400Cl resin to give the Boc-protected derivatives (10a-c)
(Scheme II). Trifluoroacetic acid (TFA) mediated deprotection of the Boc group of these
derivatives provided the ultimate amine product with a twin aliphatic chain quaternary center.
Finally, EDC-mediated amide coupling between stilbene acid (6a-c) and ethylenediamine
derivatives (11a-c) in dry DCM at room temperature generated the respective target lipids. Three
series of lipid molecules, e.g., tetramethoxy stilbene lipid (TMSC8, TMSC10 &TMSC16),
pentamethoxy derivatives (PMSC8, PMSC10 & PMSC16) and hexamethoxy derivatives (HMSC8,
HMSC10 & HMSC16) were isolated with moderate to good yields (Scheme III). Thus, by the use
of simple synthons and reagents, such as mono-, di- or trimethoxy benzaldehyde and using a
common methodology we made the present stilbene analogues with tetra-, penta- and
hexamethoxy moieties respectively. The identity and purity of each of these compounds were
confirmed by thin-layer chromatography, NMR and HRMS spectroscopy.
7

O
O
OEt
MeO
MeO
O
OEt
I
OEt
4a 4b 4c
i
/
/
R3
R2
ii
OMe
MeO
OMe
MeO
OMe
OMe
R1
5a
1
OMe
1
3
2
2
; R = R = H; R = OMe
iii
1
2
3
5b
; R = R = OMe; R = H
1
2
3
5c
; R = R = R = OMe
O
CHO
iv
MeO
MeO
OH
R3
R2
R1
R3
R1
R3
R2
OMe
R2
1
3
2
1
3
2
1
3
2
R1
6a
; R = R = H; R = OMe
3a
4a
4b
; R = R = H; R = OMe
; R = R = H; R = OMe
1
2
3
1
2
3
1
2
3
6b
; R = R = OMe; R = H
3b
; R = R = OMe; R = H
; R = R = OMe; R = H
1
2
3
1
2
3
1
2
3
6c
; R = R = R = OMe
3c
; R = R = R = OMe
4c
; R = R = R = OMe
The scheme I: Synthesis of various methoxy enriched (E)-stilbenes. Reagents and condition:(i)
AgO2CF3, I2, CHCl3, 99%; (ii) Pd(OAc)2, TBAB, NaOAc, dry DMF, 90 °C, 6 h, 70-78%;(iii)
LiOH, THF:MeOH:H2O (2:1:1), 78°C, 3 h; (iv) n-BuLi, methyltriphenylphosphonium bromide, -
78 °C to r.t., 4 h.
O
R4
N
O
i
ii
iii
H2N
H2N
NH2
R4
N
H
8
O
R4
N
O
H
7
4
9a
; R = -C8H17
;
4
O
9b
; R = -C10H21
;
R4
4
N
iv
9c
; R = -C16H33
;
R4
N
4
O
11a
11b
11c
; R = -C8H17
;
N
R4
Cl
NH3
CF3COO
H
4
4
Cl
; R = -C10H21
;
10a
10b
10c
; R = -C8H17
;
4
4
; R = -C16H33
;
; R = -C10H21
;
4
; R = -C16H33
;
Scheme II: Synthesis of various hydrophobic twin chain containing ethylenediamine precursors.
Reagents and condition: (i) (Boc)2O, rt., overnight; (ii) RBr, K2CO3, ethyl acetate, 48 h reflux,74-
82%; (iii) CH3I, K2CO3, 12 h, rt., followed by chloride ion exchange by Amberlite IRA 400Cl,
methanol; (iv) TFA/DCM (1: 2), 4 h, r.t.
8

O
O
R4
R4
Cl
MeO
MeO
11a 11b 11c
/
/
MeO
MeO
N
OH
4
12a
12b
12c
TMSC8
; R = -C8H17;
N
4
H
EDC, HOBt, DIPEA,
DCM,12 h, rt.
47-63%
TMSC10
; R = -C10H21;
4
TMSC16
; R = -C16H33;
OMe
OMe
OMe
OMe
OMe
6a
O
O
R4
Cl
11a 11b 11c
/
/
MeO
MeO
MeO
MeO
N
4
N
R4
13a
13b
13c
PMSC8
; R = -C8H17;
OH
EDC, HOBt, DIPEA,
DCM,12 h, rt.
42-57%
4
H
PMSC10
PMSC16
; R = -C10H21;
4
; R = -C16H33;
OMe
OMe
OMe
6b
OMe
OMe
O
O
R4
Cl
N
11a 11b 11c
/
/
MeO
MeO
MeO
MeO
4
14a
14b
14c
HMSC8
; R = -C8H17;
OH
N
R4
EDC, HOBt, DIPEA,
DCM,12 h, rt.
49-59%
4
H
HMSC10
HMSC16
; R = -C10H21;
OMe
OMe
OMe
4
; R = -C16H33;
OMe
OMe
OMe
6c
OMe
OMe
Scheme III: Synthesis of various hydrophobic twin chain conjugated-methoxy enriched stilbenic
lipid derivatives.
2.2. Biology
2.2.1. In vitro selective toxicity of HMSC16 against cervical cancer cells
The in vitro anticancer capabilities for all of the synthesized, tetramethoxy, pentamethoxy and
hexamethoxy C8, C10, C16 long-chain derivatives of stilbene molecules were assessed against
cancer cell lines such as HeLa, MCF-7 (an Estrogen receptor-positive breast cancer cell line),
MDA-MB- 231 (an Estrogen receptor-negative cell line) and non-cancer cell lines such as HEK-
293 (Human embryonic kidney), CHO (Chinese hamster ovary) and NRK-49F (normal rat kidney
fibroblast) cell lines. The results are shown in Table1. Even though almost all molecules exhibited
anticancer activities, but some of them also exhibited toxicity against normal cells also. Among all
the compounds tested, HMSC8, HMSC10 and HMSC16 showed promising results in terms of
selectivity. Even though HMSC8, HMSC10 has shown broader specificity towards cancer cells,
HMSC16 is the only molecule shown to hold good beneficial effects among all tested molecules
for cervical cancer cells with an IC50 of 4.61µM. As is evident, with an increasing number of
methoxy groups and long-chain lengths the selectivity got increased with lesser IC50. Noticeably,
9

the anticancer potencies of these derivatives are even 5-10 folds more than tamoxifen and
resveratrol. When we evaluated cell viability among non-cancer and cancer cells, HMSC16 did
not affect the non-cancer cells even at 40µM concentration but showed specificity towards cervical
cancer cells with increasing concentrations from 5 to 40µM. (Figure 3). Interestingly, when we
compared the cytotoxicities of non-cationic stilbenes with cationic lipid grafted stilbenes, we find
that non-cationic stilbenes (i.e., the stilbenes without the charge and aliphatic chains), either in
acid form (acid analogues, compounds 6, Scheme I) or ethyl ester form (ester analogues,
compounds 5, Scheme I) are non-toxic to all cancer cells tested as well as most non-cancer cells.
Exceptionally, ester of hexa-methoxy compound exhibited high toxicity against non-cancer cells
CHO, indicating a high level of non-selectivity prevailing in non-cationic lipid analogues (Table
S1). Clearly, our strategy of including aliphatic long chain lipids with cationic charge imparted
increased cytotoxicity and selectivity against cancer cells so far tested.
Table.1. IC50values of various stilbene derived cationic amphiphiles on different cancer and
non-cancer cell lines
IC50 [µM]
Cancerous cell line
Non-cancerous cell line
Compounds
n= HeLa
MDA-
MCF-7
HEK-
293
NRK-
49F
CHO
MB-231
OMe
1
3.55±0.05 3.44±0.085
16.38±
0.82
4.16±0.26
8.01±0.13
4.91±0.04
MeO
MeO
Cl
H
N
N
CH3
CH3
3
9
6.17±0.85
>40
2.66±0.46
9.48±1.55
6.19±1.05
>40
6.18±0.56
>40
6.07±0.33
>40
n
O
n
31.61±0.70 >40
OMe
OMe
1
3
9
18.45±0.08 4.43±1.34 31.23±4.72 8.47±0.47 27.31±0.36 11.07±3.34
MeO
MeO
Cl
H
21.58±2.58
>40
4.41±2.5
>40
17.73±0.96 11.55±3.65 10.92±1.53 9.97±1.10
18.58±1.56 >40 27.18±4.88 >40
N
N
CH3
CH3
n
O
n
MeO
OMe
10

OMe
1
3
9
19.59±2.94 3.33±0.09
>40
>40
>40
13.59±1.23
>40
21.73±0.90 24.20±4.31
MeO
MeO
Cl
N
H
N
15.17±1.70
4.61±0.37
3.76±0.7
>40
>40
>40
5.79±2.11
>40
CH3
CH3
n
O
17.67±0.60
n
MeO
OMe
OMe
Tamoxifen
Resveratrol
35.0±2.73 38.27±3.57 23.34±2.94 36.43±3.26 15.15±0.44 21.20±2.17
>40
>40
>40
>40
>40
>40
Figure 3. Cell viability studies of non-cancer and cancer cells of HMSC16 by MTT assay: A)
Cell viability studies in non-cancer cells (CHO, HEK-293, and NRK-49F) at 5, 10, 20 and 40µM
concentrations of HMSC16 for 48 hours. B) Cell viability studies in cancer cell lines (HeLa,
MCF-7 and MDA-MB231) for 48 hours with 5, 10, 20, 40µM of HMSC16.
2.2.2. HMSC16 induces apoptosis in HeLa cells
In a cell, among cell death pathways, apoptosis is very important to maintain cellular homeostasis.
However, upon severe and irreparable cellular stress all cells including cancer cells undergo
apoptosis. To confirm if there is any induction of apoptosis by the cytotoxic molecule HMSC16,
11

we treated HeLa and CHO cells with 5µM, 10µM of the molecule for 24 hours. The Annexin V/PI
binding profile confirmed by flow cytometry shows that HMSC16 induced 37% apoptosis [25.4
% early apoptotic + 11.71 % late apoptotic cells] with 5µM treatment and 50% apoptosis [24.02
% early apoptotic + 26.48 % late apoptotic cells] with 10µM treatment of molecule among HeLa
cells with respect to cells in untreated (UT) condition. It did not induce any significant apoptotic
effect on CHO cells (Figure 4).
Figure 4. HMSC16 induced apoptosis selectively in HeLa cells. A) annexin V-FITC/PI binding
profile in HeLa cells either left untreated or treated with 5µM and 10µM of HMSC16 respectively
for 24 hours. B) Annexin V-FITC/PI binding profile in CHO cells either left untreated or treated
with 5µM and 10µM of HMSC16 respectively. Among the quadrants, upper left represents
necrotic cells, upper right represents late apoptotic cells, lower right represents: early apoptotic
cells and the lower-left represents: normal live cells.
12

2.2.3. HMSC16 induce autophagy in HeLa cells
Due to dual behavior i.e., cell death and cell survival, the autophagic pathway is considered as a
double-edged sword [21]. To check whether autophagic cell death is induced or not by HMSC16
treatment, we used dansylcadaverine (DC) staining. DC stains autophagic vesicles. Using this
staining process, it is very clear that HMSC16 induces autophagic cell death in HeLa cells, when
cells are treated with increasing concentrations (2.5, 5 & 10 µM) of HMSC16 for 24 hours. Up to
5 M concentration one can observe the prominent increase in the number of DC stained cells
indicating a higher number of autophagic vesicles (Figure 5). Moreover, at higher concentrations
(10 M) as well as lower concentrations of HMSC16, degradation of nuclear and the cell
membrane is also evident, due to which scattered vesicles and small nuclear sizes are also
observed. Among the cells in the untreated group, the number of vesicles is either low or absent.
From this and cytotoxicity data in HeLa, it is obvious that HMSC16-induced autophagy is not
involved in cell survival-associated recycling process, but is involved in cell death.
Figure 5. HMSC16 induces autophagy in HeLa cells: A. Dansylcadaverine staining results
observed under a microscope with 100x objective lens after treatment of HeLa cells with 2.5, 5,
13

and 10µM of HMSC16 for 24 hours. B. zoom scan (2.5 times) of 100x objective lens results of the
same image observed in A. Here with increasing concentrations, an increased quantity of vesicles
and nuclear membrane destabilization was observed. In this figure BF denotes bright field, DC
denotes dansylcadaverine and PC denotes phase-contrast images. All the images shown here are
the representations of 3 different regions of the same image under respective treatment conditions.
2.2.4. HMSC16 arrest cell cycle at sub G0 population
The cell cycle is firmly structured in normal cells [22] but becomes anomalous in abnormal cells.
We wanted to recognize whether HMSC16-induced HeLa cells killing is due to arrest of certain
cell cycle phase. To confirm this, we stained HMSC16-treated HeLa cells with propidium iodide
and cell cycle phase distribution was analyzed by flow cytometry. The flow cytometry analyses of
untreated and treated cells revealed the following: With increasing concentration of HMSC16, the
sub-G0 population was increased. The respective percent of population (P1, P2, P3, P4) for
untreated cells (4.4, 47.6, 7.9, 40.1) gradually changed when cells were treated respectively with
2.5µM (7.9, 38.2, 12.4, 41.5), 5µM (12.2, 36.4, 11.7, 39.8), and 10µM (31.4, 28.6, 11.0, 29.0) of
HMSC16. The data overall reveals that in HeLa cells, HMSC16-treatment arrests cell cycle in the
sub-G0 phase, thereby arresting further process of mitotic cell cycle (Figure 6).
14

Figure 6. Cell cycle analysis by FACS in HeLa cells after 24h treatment with HMSC16: HeLa
cells were treated with HMSC16 for 24 hours with 2.5, 5, 10µM concentrations or are left untreated.
In these figures, P1 represents the sub-G0 phase population, P2 represents the G0/G1 phase
population, P3 represents the S phase population and P4 represents the G2/M phase population.
2.2.5. HMSC16 stimulates ROS production in HeLa cells.
Levels of reactive oxygen species (ROS), up to some extent, are pro-survival for cancer cells, but
if the concentration of ROS increases, it leads to its cell death [23]. Earlier reports mentioned that
cationic lipids and related derivatives induce ROS production that leads to changes in
mitochondrial membrane potential, ultimately leading the cells to apoptosis [24]. As our
compound HMSC16 is also a cationic lipid derivative, we wanted to check if it could indeed
induce ROS production in HeLa cells. Using DCFDA staining, flow cytometric studies reveal that
with increasing concentration of HMSC16, there was a limited but gradual increase in ROS
content in cells (Figure 7).
Figure7: ROS induction by HMSC16: Flow cytometry analyses of HeLa cells promptly followed
either by the treatment of HMSC16 [2.5 µM (red) and 5 µM (blue)] for 24h or are left untreated
(UT, black).
15

2.2.6. HMSC16-treatment directs a change in the morphology and distribution of organelles
As the above results illustrated, HMSC16-treatment leads to the induction of apoptosis, a process
where mitochondrial and nuclear morphological changes happen. Hence, we checked the
morphology and distribution pattern of mitochondria and nucleus following HMSC16-treatment.
Under the confocal microscope, the morphology of HMSC16-treated HeLa cells indeed changed
and the cell nuclei became very small, due to the significant leakage of nuclear content in the
cytoplasm. This is evident as DAPI (blue) stained contents of nucleus are scattered on cytoplasm
in treated cells (Figure 8A). Additionally, drastic changes in the distribution pattern of
mitochondria compared to untreated cells are also observed (Figure 8B). The mitotracker green
(MG) stains mitochondria mass. MG stained cells reveal that all the mitochondria in treated cells,
unlike in untreated cells, tend to concentrate in one area rather than evenly distribute throughout
the cytoplasm. As the previous result indicates that HMSC16 induces autophagy, we wanted to
check the distribution pattern and levels of lysosomes, which are crucially involved and
upregulated during autophagy. Lysotracker Red (LR) stains lysosomes in cells. LR stains in
HMSC16-treated cells indicate an increased quantity of lysosomes and are concentrated in an area
where mitochondria (and possibly other organelles) are concentrated, indicating eventual
autophagic destruction of vital cellular organelles such as mitochondria, thereby leading to
eventual cell death.
16

Figure 8: HMSC16 induces a change in the distribution pattern of organelles. A) HeLa cells treated
with 10 µM of HMSC16 or left untreated for 24h and observed under confocal microscopy with a
100x objective lens with a zoom scan of 1.5. B) HeLa cells treated with 10 µM of HMSC16 or
left untreated for 24h, followed by treatment of Mitotracker Green (MG) and Lysotracker Red
(LR). Cells were then fixed and counterstained by DAPI (blue) and observed under confocal
microscopy with a 100x objective lens. Here BF denotes a bright field and PC denotes phase-
contrast image.
2.2.7. HMSC16 can form self-aggregates which efficiently entrap rhodamine-PE
Molecules maintaining hydrophobic-hydrophilic balance (HLB) tend to form self-aggregations in
aqueous media. The molecules so designed and synthesized in this study carry a cationic charge
and hydrophobic moieties. Among these, we chose to check the self-aggregation property of
hexamethoxy derivatives only, as these series exhibited some level of selectivity and anticancer
efficiency. All the three molecules of hexamethoxy derivatives carry a single positive charge as
hydrophilic moiety and predominantly bear all hydrophobic moieties including aliphatic chains.
Using the ethanolic injection procedure, these molecules readily formed self-aggregates and
possessed distinct sizes and surface charges in nanometric ranges with HMSC16 exhibiting lowest
size and highest surface charge. As a proof of formation of self-aggregates, the hydrodynamic
diameter (size), polydispersity index (PDI) and zeta potential (charge) as obtained through
dynamic light scattering (DLS) experiments are represented in Table 2 and the representative
histograms obtained from DLS are depicted in Figure 9.
17

Table 2. DLS data of HMSC8, HMSC10, and HMSC16 compound loaded with rhodamine-
PE.
Self-
Size
PDI (%)
Charge
(mV)
aggregates
(nm)
HMSC8
HMSC10
HMSC16
356±37.59
22.9±3.28
26.06±0.56
138.61 ± 4.9
17.7 ± 1.89 30.86±0.20
137.04 ± 2.50 11.5 ± 7.62 47.30 ±1.21
Figure 9: Representative histograms depicting size and charge data obtained from DLS.
Hydrodynamic sizes and surface charges of HMSC8, HMSC10, HMSC16 self-aggregates with
rhodamine-PE in water.
2.2.8. HMSC16 aggregate encapsulating rhodamine-PE is efficiently taken up by HeLa cells
18

After establishing the self-aggregating nature of these molecules, we checked the uptake of these
self-aggregates in HeLa cells. The entrapment property of aggregates depends on the lipophilic or
hydrophilic property of the cargo. Rhodamine-PE, a red fluorescent dye-conjugated lipid having
distinct ability to associate with lipid-based aggregates, could be efficiently entrapped in
hydrophobic/lipophilic core of the newly formed aggregates by simple anchoring of lipophilic
moiety of rhodamine-PE in the lipophilic core of the aggregate thereby making the aggregate
fluorescently labeled. HeLa cells were treated respectively with 10 µM of HMSC8, HMSC10, and
HMSC16 self-aggregates carrying rhodamine-PE for 6 hours. Microscopic study confirms that the
self-aggregate of HMSC16 is taken up in a higher amount than the uptake of HMSC8, HMSC10
self-aggregates in HeLa cells (Figure 10). Thus, it is evident that the killing effect of HMSC16 in
HeLa cells could be because of its higher uptake than other molecules. This higher uptake could
be because of higher positive surface charge of HMSC16, thus enabling better interaction with cell
surface which is known to be of the negatively charged surface.
Clearly, if HMSC16 aggregate can entrap rhodamine-PE, it can hence also entrap any other
hydrophobic cancer drug within its self-aggregating milieu. This can potentially be used to induce
the double anticancer effect of HMSC16 and drug-cargo.
19

Figure 10: Rhodamine uptake studies in HeLa cells: HeLa cells were treated with 10µM of nano-
liposomes or self-aggregates of HMSC16loaded with rhodamine-PE (0.05µM) for 4 hours, fixed
and DAPI used as a counterstain and observed under confocal microscopy with 100x objective
lens.
2.2.9. HMSC16 can be used as a drug delivery agent
Further, we checked for the drug delivery capability of HMSC16. For this, initially, we made
withaferin-entrapped HMSC16 nanosized self-aggregates by maintaining a 1:0.25 mole ratio of
HMSC16 and withaferin using the ethanolic solution method as described before. The drug-
carrying nano-aggregate was characterized for size (147.49 ± 5.56), PDI (23.06 ± 0.93%) and
charge (35.26 ± 2.83 mV) by DLS method. Then we treated HeLa cells with free withaferin (W),
HMSC16 aggregate (H) and HMSC16-entrapped withaferin (WH) and assessed their viability
using MTT assay. We found that the viability of cells treated with WH is significantly less
compared to a native drug, W in all concentrations tested. Herein, HMSC16 self-aggregate alone
showed IC50 at 4.03µM and free withaferin (W) showed IC50 greater than 5µM (data not shown).
20

However, IC50 for WH is obtained at a combination of 1.25µM HMSC16 and 0.31µM of withaferin.
The data clearly indicates that in the nano-aggregate combination of WH, the killing effect of
HMSC16 could be increased more than 3-folds and withaferin more than 17-folds. Figure 11
reveals that instead of using HMSC16-self-aggregate and withaferin individually, if one uses
withaferin-entrapped HMSC16, cell killing will be enhanced, if not synergistic.
Figure 11: Withaferin entrapped HMSC16 nanosized self-aggregates enhance the cancer cell
killing: HeLa cells were treated with free withaferin (W), HMSC16 self-aggregate (H) and
withaferin-entrapped HMSC16 (WH) for 48 hours at different concentrations of either W or H,
and their cell viabilities were assessed by MTT assay. Here comparison made between W or H
with respect to WH for assessing the significance. Here * indicates p < 0.05, ** indicates p < 0.01,
*** indicates p < 0.001, **** indicates p < 0.0001.
3. Discussion
Numerous studies have indicated that methoxy group-priming in place of hydroxyl groups in
resveratrol structure enhances potency and enhance the anti-proliferative activity by inducing
apoptosis and cell cycle inhibition. Our lab has previously reported an anti-cancer molecule ESC8,
a cationic lipid derivative of β-estradiol that induced apoptosis and autophagy selectively in cancer
cells while demonstrating negligible effects in non-cancer cells [14]. In an effort to show similar
cancer-cell selectivity in terms of apoptosis and autophagy induction and to enhance the anti-
cancer potential of resveratrol, we synthesized tetra-, penta- and hexa-methoxy enriched stilbene
21

derivatives with hydrophobic twin chains. From cytotoxicity data one can observe that for tetra-
methoxy (TMS) analogs, molecules with lower aliphatic chains (i.e., C8, C10) exhibit potent, yet
non-selective killing of both cancer and non-cancer cells. The same trend of non-selectivity albeit
with a lower level of cytotoxicity is also observed for penta-methoxy (PMS) analogues with lower
aliphatic chains (C8, C10). For both TMS and PMS analogues, molecules with C16 aliphatic
chains have neither selectivity nor cytotoxicity. However, for hexa-methoxy (HMS) analogues, C8
molecule (HMSC8) showed the better selective killing of MDA-MB-231 cells only. But, the C16
analog (HMSC16) showed the best selective killing of HeLa cancer cells.
To find the primary cause of selective killing, we decided to see if these TMS, PMS and HMS
analogues have any selective uptake in HeLa cells. Aided by the solvent (here, ethanol) HMS
molecules assumed aggregative structures above its critical micellar (or aggregate) concentration
(CMC) in aqueous solution with definitive hydrodynamic size and surface charge. With more and
more inclusion of these cationic lipid-conjugated molecules in water, the entropy loss becomes
higher, thus favorably leading to the assembly of non-polar tails to make a lipophilic core in
aggregation and polar hydrophilic head on the surface, which is in touch with aqueous milieu. The
data shows that as the aliphatic chain length increases from C8 to C10 or C16, the hydrodynamic
sizes of the respective aggregates become smaller and gets saturated at ~140 nm. This indicates
better compaction of aggregates with bigger aliphatic chains. This trend is also reflected in terms
of higher positive surface charges, which is more prominent in the case of HMSC16. As to our
knowledge, this is the first stilbene-based molecule that showed its self-aggregating property,
which is propelled by the inclusion of cationic lipid moieties in the chemical structure.
The cationic surface charge of synthesized lipids, logically imparts a nuclear and mitochondrial
DNA binding ability to the molecules besides their potent interaction with negatively charged cell
surface. However, only a few of these shows selective killing, thus underpinning the importance
of a balanced aggregative behavior of the molecules carrying increased hydrophobicity either in
the form of increased methoxy, which also imparts more polarity to the molecule, or in the form
of longer aliphatic chain or both. We believe that with this increased hydrophobicity as well as
some other yet unclear but favorable factors helped HMS molecules to gain aggregative property.
The most selectivity in killing of cancer cell (here, HeLa) is obtained for the most compacted
aggregate (formed by HMSC16), hence with lower hydrodynamic size but with highest positive
surface charge. The compaction, aided with high surface charge, possibly led to higher uptake and
22

hence more killing of HeLa cells. But no such killing was observed among non-cancer cells, clearly
indicating the selective action of the aggregate-forming HMSC16 molecule on the HeLa cell
surface, followed by the instigation of specific cell killing pathways in those cells.
Mitochondria remain the chief source and target of reactive oxygen species which plays a vital
role in diverse metabolic signaling pathways. According to previous reports, it is evident that ROS
production leads to apoptosis due to reduced redox balance and caspases activation, and JNK
signaling pathway action. Some drugs cause mitochondrial DNA damage which further leads to
ROS production and apoptosis induction [25]. Autophagy, a key metabolic pathway in cancer so
induced by HMSC16 did not trigger the pro-survival mechanism as mitochondria and nucleus
were damaged with increasing concentrations of treatment. Co-induction of apoptosis and
autophagy may be due to the mTOR pathway downregulation, which is well observed in MDA-
MB-231 breast cancer cells with ESC8 treatment [14]. So, further studies are warranted to see if
mTOR pathway is also a possible target for this stilbene-based molecule.
Tubulin is indeed a predominant molecular target for most stilbene-based anticancer drugs
developed so far. Notably, tubulin plays a major role during the G2/M phase of growth [3].
However, HMSC16 led to the arrest of HeLa cells at the G0/G1 phase and that many stilbenes
described before us are also known to arrest cells in the G0/G1 phase, indicating that the effect of
those stilbenes on tubulin could be minimal. Along with tubulins, stilbenes also target CYP1A1,
CYP1A2, CYP1B1, COX-2, PPAR-α, and DNA minor grooves, etc. The key molecule exhibiting
self-aggregative behavior with a net high positive surface charge shows the selectivity of
anticancer action on only HeLa cancer cells but has no action on cancer and non-cancer cells
(although all having tubulin) so far tested. This is very interesting but, the anticancer action of
these stilbenes is more complex than conventionally thought and need not be solely based on
tubulin inhibition. The exact mechanism of action of these stilbenes is certainly a futuristic study.
The benefit of delivering dual drugs for combination anticancer effect is well known. Herein,
HMSC16 acted both as a drug as well as a delivery vehicle for carrying other drugs, thereby
imparting a combination effect of both the drugs and reducing the overall dosage for effective
treatment. The molecule hence can be projected as a tool to deliver drugs and induce possible
23

synergistic combinatorial effects against cancer cells. Further research to check the feasibility of
tumor models in vivo is currently underway.
4. Conclusion
Here we describe a series of nine structurally diverse cationic amphiphilic molecules based on
stilbene which are synthesized and evaluated for its anticancer potential in the panel of cancer and
normal cell lines. Among them, HMSC16 carrying a novel blend of methoxy enrichment and
cationic long-chain attachment, becomes the most proficient anti-cervical cancer agent with self-
aggregation property. Hence, we can say that to the best of our knowledge this is a unique small
molecule exhibiting apoptosis, autophagy and cell cycle arrest in cervical cancer cells as well as
self-aggregation property. We hope this strategy will open up new windows for cervical cancer
treatment.
5. Material and methods:
5.1. Chemistry
Materials and Methods: All chemicals were purchased from Sigma-Aldrich, Avra chemical and
Spectrochem, India, and used as received. Silica gel (100-200 mesh) was used for column
chromatography, and thin-layer chromatography was performed on Merck-precoated silica gel 60-
F254 plates. All other chemicals and solvents were obtained from commercial sources and purified
using standard methods. The 1H and 13C NMR spectra were recorded on a Bruker-Avance 300
MHz /400 MHz/ 500 Mhz (see the Supporting Information). Chemical shifts (δ) are reported in
parts per million, using TMS (δ = 0) as an internal standard in CDCl3.Data for1H NMR is reported
as follows: chemical shift (δ in ppm), multiplicity (s-singlet, br. s-broad singlet, d-doublet, t-triplet,
q-quadruplet, m-multiplet), coupling constant (Hz), integration. ESI mass spectra were recorded
on a Finnigan LCQ Advantage max spectrometer. All products reported showed 1H NMR and 13C
NMR spectra in agreement with the assigned structures. All tested compounds yielded data
consistent with a purity of at least 95% compared with the theoretical values.
5.1.1. Synthesis of methyl 2-iodo-3,4,5-trimethoxybenzoate (2)[26]
24

To a solution of methyl 3,4,5-trimethoxybenzoate (1.0 g, 4.42 mmol) and silver trifluoroacetate
(1.07 g, 4.86 mmol) in CHCl3 (20 mL) iodine (1.23 g, 4.86 mmol) was added in several portions.
Three hours after completion, the reaction mixture was filtered to remove the silver salt byproduct.
The purple organic layer was washed twice with a 20% Na2S2O3 solution to remove any remaining
iodine. The organic layers were combined and concentrated in rotary evaporator give crude
product. Purification by flash column chromatography (Silica gel 100-200 mesh, 10-20% ethyl
acetate/ petroleum ether) afforded the desired product as gummy liquid. (1.4 g, 90%): Rf = 0.45
(30% EtOAc in hexanes)
1H NMR (400 MHz, CDCl3) δ 7.18 (s, 1H), 3.93 (s, 3H), 3.92 (s, 3H), 3.89 (s, 3H), 3.87 (s, 3H);
13C NMR (75 MHz, CDCl3) δ 166.95, 153.88, 153.38, 152.92, 144.88, 131.05, 125.14, 110.46,
106.78, 83.89, 61.07, 60.86, 56.28, 52.53, 52.25.
5.1.2. General procedure for styrene preparation
To a stirred suspension of methyl triphenylphosphonium bromide (1.5 equiv.) in THF at -78 °C
under argon atmosphere, n-butyllithium (1.5 equiv. 1.6 M in THF) was added drop wise. The
yellow solution was allowed to stir for 15 min before addition of the aldehyde (1 eq.) in THF, upon
its addition, the mixture turned white or pale yellow. After 2h (or observed completion of the
reaction by TLC), saturated ammonium chloride was added and the mixture was extracted with
CH2Cl2. The combined organic extracts were then dried (Na2SO4), and concentrated in vacuo.
Evaporation of the solvent under reduced pressure gave the crude product. Purification by flash
column chromatography (Silica gel 230-400 mesh, 1-10% ethyl acetate/ petroleum ether) afforded
the desired styrene as a colorless liquid.
5.1.2.1. Synthesis of 1-Methoxy-4-vinylbenzene (4a) [27]
Compound 4a starting with 4-methoxybenzaldehyde (3c) (1 g, 7.34 mmol), was synthesized by
general procedure of Styrene preparation, with similar workup and purified by flash
chromatography on (230-400 mesh) silica. [Yield: 91% (895 mg, colorless oil (Rf ≈ 0.7, PET/
EtOAc (9/1)].
1H NMR: (500 MHz, CDCl3) δ (ppm) 3.82 (s, 3H), 5.13 (d, J = 11.2 Hz, 1H), 5.61 (d, J = 17.6
Hz, 1H), 6.67(dd, J1=10.8 Hz, J2=17.6 Hz, 1H), 6.87 (dd, J1 = 2 Hz, J2 = 9 Hz, 2H), 7.35 (d, J =
8.5 Hz, 2H).
25

5.1.2.2. Synthesis of 1,2-Dimethoxy-4-vinylbenzene (4b) [28]
Compound 4b starting with 1,2-dimethoxybenzaldehyde (3b) (1 g, 6.00 mmol), was synthesized
by general procedure of styrene preparation, with similar workup and purified by flash
chromatography on (230-400 mesh) silica gel. [Yield: 90% (885 mg, colourless oil (Rf ≈ 0.6, PET/
EtOAc (9/1)].
1H NMR: (500 MHz, CDCl3) δ (ppm) 3.87 (s, 3H), 3.90 (s, 3H), 5.14 (d, J = 11 Hz, 1H), 5.61 (d,
J = 17.5 Hz, 1H), 6.65(dd, J1 = 10.5 Hz, J2 = 17.5 Hz, 1H), 6.81 (d, J = 8 Hz,1H), 6.93-6.96 (m,
13
2H); C NMR: (125 MHz, CDCl3) δ (ppm) 55.9, 56.0, 108.8, 11.2, 111.9, 119.6, 130.9, 136.6,
149.1, 149.2.
5.1.2.3. Synthesis of 1,2,3-Trimethoxy-5-vinylbenzene (4c) [27]
Compound 4c starting with 1,2,3-Trimethoxybenzaldehyde (3c) (1.5 mg, 7.64 mmol), was
synthesized by general procedure of Styrene preparation, with similar workup and purified by flash
chromatography on (230-400 mesh) silica. [Yield: 86% (1.3 g, colourless semi-solid (Rf ≈ 0.5,
PET/ EtOAc (9/1)].
1H NMR: (500 MHz, CDCl3) δ (ppm) 3.85 (s, 3H), 3.88 (s, 6H), 5.21 (d, J = 10.5 Hz, 1H), 5.66
(d, J = 17.5 Hz, 1H), 6.63(m, 3H); 13C NMR: (125 MHz, CDCl3) δ (ppm) 56.2, 61.0, 103.5, 113.3,
133.4, 136.9, 138.2, 153.4
5.1.3. Representative procedure of Heck coupling
A solution of compound 2 (1 equiv.), appropriate styrene (1.2 equiv.), Pd(OAc)2 (5 mol%),
triphenylphosphine ligand (10 mol%), terabutylammonium bromide (TBAB) (3 equiv.), and
NaOAc (3 equiv.) was stirred in dry DMF under argon atmosphere at 90 °C for 5-6 h (depending
on the substrate). After completion of reaction (monitored by TLC), the reaction mixture was
cooled to r.t. and diluted with ethylacetate. The organic layer was successively washed with cold
water and brine and dried over anhydrous Na2SO4. The solvent was removed under reduced
pressure and the residue was purified by flash chromatography on silica gel (100-200 mesh) with
20-30% ethyl acetate in petroleum ether.
5.1.3.1. Synthesis of (E)-ethyl 3,4,5-trimethoxy-2-(4-methoxystyryl) benzoate (5a)
Compound (5a) was synthesized by general procedure of Heck coupling between compound (2)
(1.00 g, 2.83 mmol) and styrene 4a (447.48 mg, 3.39 mmol), with similar workup, and purified by
26

flash chromatography on (100-200 mesh) silica gel with petroleum ether/EtOAc (4/1) as eluent as
eluent, which gave compound (5a). [Yield: 78% (790 mg, gummy liquid); Rf ≈ 0.45, petroleum
ether/EtOAc (3/1)].
1H NMR (400 MHz, CDCl3) δ 7.44 (d, J = 8.6 Hz, 2H), 7.28 (d, J = 16.5 Hz, 2H), 7.13 (s, 1H),
6.96 (d, J = 16.5 Hz, 1H), 6.89 (d, J = 8.8 Hz, 1H), 4.32 (q, J = 7.1 Hz, 2H), 3.95 (s, 3H), 3.90 (s,
3H), 3.82 (s, 3H), 3.81 (s, 3H), 1.31 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 168.35,
159.27, 152.19, 151.75, 145.42, 132.86, 130.94, 127.68, 126.31, 126.12, 120.42, 114.07, 109.22,
61.28, 61.06, 60.62, 56.12, 55.34, 14.35; ESI-MS: m/z [M+H]+ calcd for C21H25O6 :373.1, found:
373.1.
5.1.3.2. Synthesis of (E)-Methyl 2-(3,4-dimethoxystyryl)-3,4,5-trimethoxybenzoate (5b)
Compound (5b) was synthesized by general procedure of Heck coupling between compound (2)
(1.00 g, 2.83 mmol) and styrene 4b (549 mg, 3.39 mmol), with similar workup, and purified by
flash chromatography on (100-200 mesh) silica gel with petroleum ether/EtOAc (4/1) as eluent as
eluent to give compound (5b). [Yield: 70% (768 mg, gummy liquid); Rf ≈ 0.45, petroleum
ether/EtOAc (3/1)].
1H NMR (500 MHz, CDCl3) δ 7.35 – 7.29 (m, 1H), 7.16 (s, 1H), 7.10 – 7.03 (m, 2H), 6.98 (d, J =
16.4 Hz, 1H), 6.86 (d, J = 8.0 Hz, 1H), 3.96 (d, J = 6.0 Hz, 3H), 3.94 (s, 3H), 3.91 (s, 3H), 3.90 (s,
13
3H), 3.86 (s, 3H), 3.82 (s, 3H); C NMR (75 MHz, CDCl3) δ 168.48, 152.22, 151.72, 149.10,
148.91, 145.61, 133.18, 131.34, 126.48, 125.49, 120.62, 119.70, 111.25, 109.34, 109.04, 61.06,
60.64, 56.10, 55.99, 55.90, 52.28, 29.71; ESI-MS: m/z [M+H]+ calcd for C21H25O7 :389.1, found:
389.2.
5.1.3.3. Synthesis of (E)-Methyl 3,4,5-trimethoxy-2-(3,4,5-trimethoxystyryl) benzoate (5c)
Compound (5c) was synthesized by general procedure of Heck coupling between compound (2)
(1.00 g, 2.83 mmol) and styrene 4c (447.48 mg, 3.39 mmol), with similar workup, and purified by
flash chromatography on (100-200 mesh) silica gel with petroleum ether/EtOAc (4/1) as eluent as
eluent to give compound (5c). [Yield: 75% (887 mg, gummy liquid); Rf ≈ 0.4, petroleum
ether/EtOAc (3/1)].
1H NMR (500 MHz, CDCl3) δ 7.35 (d, J = 16.2 Hz, 1H), 7.17 (s, 1H), 6.95 (d, J = 16.3 Hz, 1H),
13
6.73 (s, 2H), 3.95 (s, 3H), 3.90 (s, 9H), 3.86 (6H), 3.81 (s, 3H); C NMR (126 MHz, CDCl3) δ
27

168.44, 153.50, 152.39, 152.03, 145.79, 134.06, 133.46, 126.35, 125.67, 122.16, 109.54, 103.79,
61.15, 61.05, 60.79, 56.27, 56.21, 52.38; ESI-MS: m/z [M+H]+ calcd for C55H95N2O6 :419.1,
found: 419.2.
5.1.4. General Procedure of Ester Hydrolysis
To a stirred solution of appropriate stilbene ester derivative (1 mmol) in THF: MeOH: H2O (4:1:1)
LiOH was added. The reaction mixture was heated to 75-80 °C for 3 h. Then the solvent was
removed under reduced pressure and diluted with EtOAc. The organic layer was washed with brine
(3 × 20 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure to give crude
product as pale yellow or white solid. This product was used directly for further reaction.
5.1.4.1. Synthesis of (E)-3,4,5-trimethoxy-2-(4-methoxystyryl) benzoic acid (6a)
1H NMR (400 MHz, CDCl3) δ 7.46 (d, J = 6.9 Hz, 2H), 7.40 (d, J = 16.5 Hz, 1H), 7.37 (s, 1H),
7.11 (d, J = 16.5 Hz, 1H), 6.89 (d, J = 8.8 Hz, 2H), 3.98 (s, 3H), 3.92 (s, 3H), 3.82 (s, 3H), 3.80
(s, 3H); 13C NMR (101 MHz, CDCl3) δ 172.82, 159.32, 152.99, 152.31, 151.49, 146.76, 133.46,
131.01, 128.05, 127.86, 123.63, 120.29, 114.09, 110.38, 107.37, 61.15, 60.55, 56.28, 56.12, 55.36;
ESI-MS: m/z [M+H]+ calcd for C19H21O4 :345.1, found: 345.1.
5.1.4.2. Synthesis of (E)-2-(3,4-dimethoxystyryl)-3,4,5-trimethoxybenzoic acid(6b)
1H NMR (400 MHz, CDCl3) δ 7.41 (d, J = 16.4 Hz, 1H), 7.35 (s, 1H), 7.13 – 6.99 (m, 3H), 6.84
(d, J = 8.2 Hz, 1H), 3.98 (s, 3H), 3.91 (d, J = 0.9 Hz, 6H), 3.89 (s, 3H), 3.81 (s, 3H); 13C NMR (75
MHz, CDCl3) δ 172.54, 152.31, 151.55, 149.08, 148.92, 146.74, 133.57, 131.39, 127.87, 123.65,
120.67, 119.79, 111.24, 110.36, 109.09, 61.10, 60.57, 56.09, 55.97, 55.82; ESI-MS: m/z [M+H]+
calcd for C20H23O7 :375.1, found: 375.1.
5.1.4.3. Synthesis of (E)-3,4,5-trimethoxy-2-(3,4,5-trimethoxystyryl) benzoic acid (6c)
1H NMR (400 MHz, CDCl3) δ 7.45 (d, J = 16.4 Hz, 1H), 7.36 (s, 1H), 7.04 (s, 1H), 6.75 (s, 2H),
3.98 (s, 3H), 3.92 (s, 3H), 3.89 (s, 6H), 3.85 (s, 3H), 3.81 (s, 3H); 13C NMR (126 MHz, CDCl3) δ
172.23, 153.50, 152.47, 151.88, 146.91, 143.29, 138.11, 134.16, 133.78, 127.73, 123.78, 122.27,
110.53, 103.88, 61.21, 61.06, 60.75, 56.22, 29.54, 0.10; ESI-MS: m/z [M+H]+ calcd for C21H25O8
:405.1, found: 405.2.
5.1.5. Representative procedure for the synthesis of tert-butyl (2-(dihexadecylamino) ethyl)
carbamate (9c)[20]
28

To a stirring solution of tert-butyl (2-aminoethyl) carbamate 8 (1 g, 6.25 mmol) in dry ethyl acetate
(20 ml), 1-bromohexadecane (4.41 g, 18.75 mmol) and K2CO3 (2.58 g, 18.75 mmol) were added.
The resulting reaction mixture was refluxed for 24 h reaction under N2 atmosphere. After
completion of reaction, K2CO3 was filtered out and the residue was washed with ethyl acetate.
Combined filtrate further diluted with ethyl acetate (100 ml), washed successively with water (2 ×
100 ml), and saturated brine (2×100 ml. It was then dried over anhydrous Na2SO4. Solvent was
evaporated under reduced pressure which afforded crude product. It was purified by column
chromatography using 10% ethyl acetate/hexene (v/v) as the eluent to afford the tertiary amine
(9c, 2.89 g) as colourless viscous oil, [76% yield, Rf 0.55 (10% ethyl acetate/hexene)].
Remaining derivatives (9a & 9b) were synthesized following this and our earlier reported
procedure with analytical data well matched with previously synthesised products [20].
1H NMR (400 MHz, CDCl3) δ 3.14 (d, J = 5.2 Hz, 2H), 2.49 (t, J = 5.8 Hz, 2H), 2.42 – 2.33 (m,
4H), 1.59 (s, 4H), 1.45 (s, 9H), 1.42 – 1.35 (m, 4H), 1.26 (s, 48H), 0.88 (t, J = 6.9 Hz, 6H); 13C
NMR (126 MHz, CDCl3) δ 58.69, 54.03, 53.19, 38.28, 31.96, 29.73, 29.69, 29.66, 29.40, 28.48,
27.55, 27.05, 22.72, 14.15; ESI-MS: m/z [M+H]+ calcd for C39H82N2O2 :609.6, found: 609.6.
5.1.6. Representative quaternization procedure: Synthesis of N-(2-((tert-butoxycarbonyl) amino)
ethyl)-N-hexadecyl-N-methylhexadecan-1-aminium chloride (10c)[20]
Tertiary amine tert-butyl 2-(dihexadecylamino)ethylcarbamate (9c) (1 g, 1.64 mmol) was
dissolved in chloroform (15 ml) and add methyl iodide (excess) and K2CO3 (452.6 mg, 3.28 mmol).
The resulting suspension was left stirred at room temperature for 12 h. The reaction mixture was
filtered and the solvent was evaporated on a rotary evaporator to afford the crude residue that was
subsequently purified by column chromatography using 2% MeOH/CHCl3 (v/v) as the eluent to
yield the compound 10c as a colourless liquid 941 mg (87% yield).
Remaining derivatives (10a &10b) were synthesized following this and our earlier reported
procedure [20].
1H NMR (400 MHz, CDCl3) δ 6.17 (s, 1H), 3.70 (s, 4H), 3.53 – 3.36 (m, 4H), 3.31 (s, 3H), 1.79-
1.71 (m, Hz, 4H), 1.43 (s, 9H), 1.36 (d, J = 2.3 Hz, 4H), 1.30 (s, 44H), 0.88 (t, J = 6.8 Hz, 6H);
13C NMR (101 MHz, CDCl3) δ62.51, 60.69, 49.44, 35.07, 31.95, 29.72, 29.69, 29.63, 29.50, 29.42,
29