Potent Antiglioblastoma Agents by Hybridizing the Onium-Alkyloxy-Stilbene Based Structures of an α7-nAChR, α9-nAChR Antagonist and of a Pro-Oxidant Mitocan

Article abstract of DOI:10.1021/acs.jmedchem.8b01052

Adenocarcinoma and glioblastoma cell lines express α7- and α9α10-containing nicotinic acetylcholine receptors (nAChRs), whose activation promotes tumor cell growth. On these cells, the triethylammoniumethyl ether of 4-stilbenol MG624, a known selective antagonist of α7 and α9α10 nAChRs, has antiproliferative activity. The structural analogy of MG624 with the mitocan RDM-4′BTPI, triphenylphosphoniumbutyl ether of pterostilbene, suggested us that molecular hybridization among their three substructures (stilbenoxy residue, alkylene linker, and terminal onium) and elongation of the alkylene linker might result in novel antitumor agents with higher potency and selectivity. We found that lengthening the ethylene bridge in the triethylammonium derivatives results in more potent and selective toxicity toward adenocarcinoma and glioblastoma cells, which was paralleled by increased α7 and α9α10 nAChR antagonism and improved ability of reducing mitochondrial ATP production. Elongation of the alkylene linker was advantageous also for the triphenylphosphonium derivatives resulting in a generalized enhancement of antitumor activity, associated with increased mitotoxicity.

Full text of DOI:10.1021/acs.jmedchem.8b01052

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Article
Potent anti-glioblastoma agents by hybridizing the
onium-alkyloxy-stilbene based structures of an alpha7-,
alpha9-nAChR antagonist and of a pro-oxidant mitocan
francesco bavo, susanna pucci, francesca fasoli, Carmen Lammi, milena moretti, vanessa mucchietto,
donatella lattuada, paola viani, clara de palma, Roberta Budriesi, irene corradini, Cheryl Dowell,
J. Michael McIntosh, francesco clementi, Cristiano Bolchi, cecilia gotti, and Marco Pallavicini
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01052 • Publication Date (Web): 07 Nov 2018
Downloaded from http://pubs.acs.org on November 9, 2018
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Potent anti-glioblastoma agents by hybridizing the
onium-alkyloxy-stilbene based structures of an 7-,
9-nAChR antagonist and of a pro-oxidant mitocan
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°
°
°
Francesco Bavo1 , Susanna Pucci2 , Francesca Fasoli2 , Carmen Lammi1, Milena Moretti2,3,
Vanessa Mucchietto2, Donatella Lattuada3, Paola Viani3, Clara De Palma4, Roberta Budriesi5,
Irene Corradini2, Cheryl Dowell6, J. Michael McIntosh6,7,8, Francesco Clementi2,3, Cristiano
Bolchi1* , Cecilia Gotti2,3*, and Marco Pallavicini1
1Dipartimento di Scienze Farmaceutiche , Universitàꢀdegli Studi di Milano, Via Mangiagalli 25, I-
20133, Milano, Italy
2CNR, Institute of Neuroscience, Via Vanvitelli 32, I-20129, Milano, Italy
3Department of Medical Biotechnology and Translational Medicine, Università degli Studi di
Milano, Via Vanvitelli 32, I-20129, Milano, Italy
4Unit of Clinical Pharmacology, University Hospital “Luigi Sacco”-ASST Fatebenefratelli Sacco,
Via G. B. Grassi 74, I-20157, Milano, Italy
5Dipartimento di Farmacia e Biotecnologie, Università degli Studi di Bologna, Via Belmeloro 6,
I-40126, Bologna, Italy
6 Department of Biology University of Utah, 257S.1400 East, Salt Lake City, UT 84112, USA.
7George E. Wahlen Veterans Affairs Medical Center, 500 Foothill Drive, Salt Lake City, UT 84148,
USA.
8Department of Psychiatry, University of Utah, 501 Chipeta Way, Salt Lake City, Ut 84108, USA
° Co-first authors
*Co-corresponding authors
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Abstract
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Adenocarcinoma and glioblastoma cell lines express 7 and 9-10-containing nAChRs, whose
activation promotes tumour cells growth. On these cells, the triethylammoniumethyl ether of 4-
stilbenol MG624, a known selective antagonist of 7 and 9-10 nAChRs, has antiproliferative
activity. The structural analogy of MG624 with the mitocan RDM-4’BTPI,
triphenylphosphoniumbutyl ether of pterostilbene, suggested us that molecular hybridisation among
their three substructures (stilbenoxy residue, alkylene linker and terminal onium) and elongation of
the alkylene linker might result in novel antitumour agents with higher potency and selectivity. We
found that lengthening the ethylene bridge in the triethylammonium derivatives results in more potent
and selective toxicity towards adenocarcinoma and glioblastoma cells, which was paralleled by
increased 7 and 9-10 nAChR antagonism and improved ability of reducing mitochondrial ATP
production. Elongation of the alkylene linker was advantageous also for the triphenylphosphonium
derivatives resulting in a generalized enhancement of antitumor activity, associated with increased
mitotoxicity.
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INTRODUCTION
Since 2000, a considerable number of reports have suggested that cancer cells, compared to their
non-tumoral counterparts, are characterized by altered redox homeostasis and hyperpolarized inner
mitochondrial membrane (IMM). 1,2 Permanently enhanced production of Reactive Oxygen Species
(ROS) by mitochondria may predispose to carcinogenesis, tumor growth and metastasis, but, on the
other hand, sudden, notable and targeted ROS enhancement can activate pro-apoptotic signaling and
3
induce Mitochondrial Permeability Transition (MPT) resulting in cell death. These observations
have suggested that the antitumor potential of substances increasing ROS production, such as
resveratrol at high doses, quercetin and α-tocopheryl succinate, can be maximized if the compounds
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are targeted to mitochondrial matrix, with preference for the hyperpolarized mitochondria of cancer
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cells, by coupling to
a
membrane-permeable lipophilic cation.4 Connection to
a
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triphenylphosphonium head, a charged but lipophilic moiety, through an alkylene linker is the most
widely used strategy to make these molecules ‘mitochondriotropic’. MitoVES,5 Q-7BTPI6 and, more
recently, the 3’,5’-dimethoxy-4-stilbenol (pterostilbene) derivative RDM-4’BTPI from now on
defined PtP-4 7 are successful examples of mitochondria-targeted cytotoxic pro-oxidants, classified
within the so-called ‘mitocans’ (Chart 1).
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O
O
-
O
+
PPh3
O
O
MitoVES
O
O
OH
+
O
PPh3
-
HO
(E)
MeO
+
I
(E)
HO
HO
PPh3
O
-
I
OMe
+
Q-7BTPI
RDM-4'BTPI (PtP-4)
O
-
I
NEt3
(E)
MG624 (StN-2)
Chart 1. Cytotoxic pro-oxidants targeted to mitochondria by triphenylphosphonium head and the
stilbenoxyethylammonium α7 and α9 nAChR antagonist MG624.
Our attention was drawn to RDM-4’BTPI and to some analogies between its structure and that of
MG624 from now on defined StN-2 , an antagonist of the α7 and α9 neuronal nicotinic acetylcholine
receptors (nAChRs) that we developed in 1998 (Chart 1).8 nAChRs are ligand-gated ion channels
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widely distributed throughout the central and peripheral nervous systems, that also have an
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extraneuronal localisation. In particular, the subtypes containing the α7 or the α9 subunits are
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present in endothelial cells, glia, immune cells, lung epithelia and cancer cells, where they regulate
cell differentiation, proliferation and inflammatory responses. 11,12 Recently, we characterized StN-2
for its ability to increase ROS production and to block the α7 and α9 nAChR mediated pro-
proliferative effect of nicotine in pulmonary adenocarcinoma cells. 13 The two molecules, PtP-4 and
StN-2, share a stilbene scaffold with a para positioned quaternary ω-onium alkyloxy residue, four
and two carbons long respectively, and both behave as cytotoxic pro-oxidants. Furthermore, SAR
studies on regioisomeric resveratrol 4-triphenylphosphoniumbutyl ethers as mitocans proved that
methylation of the two remaining free hydroxyls, both meta positioned on the same phenyl (PtP-4)
or meta and para separately positioned on the two phenyls, resulted in highly increased cytotoxicity
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due to ROS generation. This indicated that, at variance with mitochondriotropic derivatives of
quercetin, free hydroxyls are not essential for the activity of mitochondriotropic resveratrol
derivatives and suggested that the two methoxy functions could be removed by switching to the
unsubstituted stilbene scaffold.
Based on these premises, we planned a systematic hybridization between the three constitutive
portions of PtP-4 (pterostilbene residue (Pt), triphenylphosphonium head (P) and 4C linker) and of
StN-2 (stylbenoxy residue (St), triethylammonium head (N) and 2C linker) and a biological
characterization of the resultant series of eight phosphonium and ammonium iodides. These are
shown in Chart 2 and identified with a code composed of the initials of the name of the aromatic
scaffold (Pt for pterostilbene and St for stilbene), the onium head hetero atom (P or N), followed by
the number of the linker carbons. In particular, we wished to test their anti-proliferative activity on
different tumor cells expressing α7 and /or α9 nAChRs and to analyze the underlying mechanisms.
In this way, we hoped to understand the role played by the three substructures (onium head, stilbene
scaffold and interposed alkylene linker) in addressing the activity towards one or more biological
targets and to find advantageous synergies between different mechanisms.
Furthermore, we were particularly interested in the elongation of the alkylene linker, a structural
modification expected to be relevant to both nicotinic antagonism, for the increase of steric
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encumbrance, and cytotoxic pro-oxidant activity. Useful information would have been gained from
α7 and α9 nAChR antagonism comparison between PtN-2 and its two-carbon-elongated analogue
PtN-4 and between StN-2 and the elongated StN-4, StN-6 and StN-8 analogues, while a further
elongation of the butylene linker of PtP-4 was strongly suggested by evidences from the literature.
In fact, PtP-4 has been suggested to act as mitoVES, whose activity has been reported to be
diminished by shortening its eleven-carbon long linker. 14 Consistently with such a trend, elongation
of the short 4 C linker of StP-4, the stilbene analogue of PtP-4, could be beneficial. Therefore, we
extended the series of compounds to be investigated over the stilbenoxyalkyl triphenylphosphonium
iodides PtP-3 and PtP-5-PtP-10 having alkylene linkers of increasing length.
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Here, we report the synthesis of the compounds shown in Chart 2, except for the previously
described StN-2 (MG624) and PtP-4 (RDM-4’BTPI), and discuss their pharmacological activity and
those of MG624 and RDM-4’BTPI comparing their profiles as antiproliferative agents against
glioblastoma, other tumor cells and normal glial cells (astrocytes) and analysing their behaviour as
α7 and α9 nAChR ligands and cytotoxic pro-oxidants. Unfortunately, compounds with two-carbon
linker and phosphonium head, PtPt-2 and StP-2, could not be enclosed into this investigation because
ready decomposition to pterostilbene and stilbenol, respectively, and vinyl-triphenylphosphonium
precluded their isolation and characterization.15
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Chart 2. Target phosphonium and ammonium compounds resulting from hybridization between
MG624 (StN-2) and RDM-4’BTPI (PtP-4) and from alkylene linker elongation.
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MG624/RDM-4'BTPI hybridization
+
+
O
O
PPh3
NEt3
-
-
MeO
MeO
I
I
(E)
(E)
PtP-2
PtN-2
OMe
OMe
+
PPh3
+
O
O
NEt3
-
-
I
I
(E)
(E)
StP-2
StN-2 (MG624)
+
+
O
O
NEt3
-
PPh3
-
MeO
MeO
I
I
(E)
(E)
PtN-4
PtP-4 (RDM-4'BTPI)
OMe
OMe
+
NEt3
+
PPh3
O
O
-
-
I
I
(E)
(E)
StP-4
StN-4
Alkylene linker elongation
+
+
O
O
PPh3
NEt3
( )n
( )n
-
-
I
I
(E)
(E)
n = 3 StP-3
= 5 StP-5
= 6 StP-6
= 7 StP-7
= 8 StP-8
= 9 StP-9
n = 6 StN-6
= 8 StN-8
= 10 StP-10
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RESULTS
Chemistry
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The pterostilbene ammoniumalkyl ethers PtN-2 and PtN-4 were synthesized from commercial
resveratrol, while the stilbenol ammoniumalkyl ethers StN-4, StN-6 and StN-8 and the eight stilbenol
phosphoniumalkyl ethers StP-3 – StP-10 from commercial 4-(E)-stilbenol (Scheme 1). Alkylation of
resveratrol with 1-bromo-2-choloroethane and 1-bromo-4-chlorobutane afforded, respectively, the
intermediates 1a and 1c, whose free phenolic functions were methylated to obtain the O-chloroalkyl
pterostilbene intermediates 2a and 2c. These were converted into the corresponding iodoalkyl
analogues 3a and 3c by Finkelstein reaction and then into the final triethylammonium derivatives
PtN-2 and PtN-4 by treatment with triethylamine. The ethers with stilbene scaffold were synthesized
by the same strategy but using ,-dibromoalkanes to O-alkylate 4-(E)-stilbenol. The resulting -
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bromoalkyl ethers 4b-i were converted into the corresponding -iodoalkyl ethers 5b-i. Reaction of
5c, 5e and 5g with triethylamine afforded the triethylammonium derivatives StN-4, StN-6 and StN-
8, while reaction of 5b-i with triphenylphosphine the corresponding triphenylphosphonium
derivatives StP-3-10. RDM-4’BTPI (PtP-4) and StN-2 (MG624) were synthesized as previously
reported. 7,8
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Scheme 1
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O
X
n
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n= 2
n= 3
n= 4
a
b
c
n= 5
n= 6
n= 7
d
e
f
n= 8
n= 9
n= 10 i
g
h
R
(E)
R
a
2a
3a
PtN-2
1a
+ -
d
e
c
resveratrol
X=Cl R=OMe
X=I R=OMe
X=NEt I R=OMe
X=Cl R=OH
3
b
2c
3c
1c
PtN-4
e
StN-4,StN-6,StN-8
+ -
5c,e,g
X=I R=H
X=NEt I R=H
3
g
StP-4,StP-6,StP-8
X=PPh I R=H
+ -
f
d
3
stilbenol
4b-i
X= Br R=H
g
5b,d,f,h,i
X=I R=H
StP-3,StP-5,StP-7,StP-9,StP-10
+ -
X=PPh I R=H
3
Reagents and conditions: (a) 1-bromo-2-chloroethane, K2CO3, DMF, 60 °C, overnight; (b) 1-
bromo-4-chlorobutane, K2CO3, DMF, 60 °C, overnight; (c) MeI, CsCO3, DMF, rt, overnight; (d) NaI
sat. in acetone, reflux, overnight; (e) NEt3, toluene, reflux, 5 h; (f) 1,3-dibromopropane (4b) or 1,4-
dibromobutane (4c) or 1,5-dibromopentane (4d) or 1,6-dibromohexane (4e) or 1,7-dibromoheptane
(4f) or 1,8-dibromooctane (4g) or 1,9-dibromononane (4h) or 1,10-dibromodecane (4i), NaOH,
DMSO, rt, 3h; (g) PPh3, neat, 100 °C, 3 h.
Biology
Binding studies
We tested the affinity of the synthesized compounds for the human α42 or α7 subtypes expressed in
heterologous cells respectively labeled by [3H]-Epibatidine or [125I]-Bungarotoxin. Compounds
PtN-2, StN-2, StN-4, and StN-8 had very similar α7 affinities ranging from 100 to 200 nM Ki,
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whereas PtN-4 and StN-6 had slightly lower affinities (305 nM and 465 nM). All of the phosphonium
compounds had low α7 affinities that were approximately 6-10 times lower than that of StN-2 (see
Tables 1 and 2). When tested on the α4β2 subtypes, all of the ammonium compounds showed very
similar α4β2 low affinities (Ki > 3 M) (data not shown).
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In vitro functional activity on α7 and α9α10 nAChR subtypes
We have previously shown that StN-2 is a selective antagonist of the human α7 and α9α10 nicotinic
subtypes expressed in oocytes13, and so we tested the new compounds using the same expression
system and subtypes. Table 1 shows the potency of all of the ammonium compounds in inhibiting
10 µM or 200 µM ACh-induced currents in oocytes expressing the human α9α10 and α7 subtypes.
Their IC50 values were all <100 nM. Increasing the linker length of StN-2 (compounds StN-4 and
StN-6) increased antagonist potency against both subtypes, whereas StN-8 was more active on
α9α10. We also tested the possible antagonist activity of PtP-4 on the α7 and α9α10 subtypes and
found that the IC50 was > than 10 µM.
Effect of ammonium and phosphonium compounds on tumoral cells and normal astrocyte
viability
The cytotoxic/cytostatic action of nine compounds (the six ammoniums PtN-2, StN-2, PtN-4 ,
StN-4, StN-6 and StN-8 and the three phosphoniums PtP-4, StP-4 and StP-10) was tested using
MTS assays on U87MG glioblastoma, A549 adenocarcinoma, SH-SY5Y neuroblastoma cell lines
and wild-type mouse astrocytes. All of the tumor cells and control astrocytes were incubated with
increasing concentrations of the compounds for 72 h, and their activity was tested taking the
absorbance of the control cells (not treated with the compounds) as 100%. The dose–response
curves were fitted by means of non-linear regression analysis, and the concentration that blocked
50% of the viability (IC50) was determined.
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Table 1. Pharmacological and biological characterisation of the ammonium and phosphonium compounds
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Cpd
Affinitya
7
receptors
Ki, nM
(CI)
Oocyte-
Oocyte-
Viabilityc Viabilityc
Viabilityc
IC50 ,M
SH-SY5Y
72h
Viabilityc
IC50 ,M
mouse
astrocytes
72h
expressedb expressedb
IC50 ,M
U87MG
72h
IC50 M
A549
72h
7
IC50 ,nM
(CI)
910
IC50 ,nM
(CI)
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StN-2
104
41
(20-72)
47
10
(8-13.4)
17.2
4.4
5.3
4.7
12
>100
>100
11
47
MG624
(54-202)
189
PtN-2
23
22
20
5.8
2
(96-373)
305
(30-74)
24.3
(7-41)
11.9
PtN-4
StN-4
StN-6
StN-8
0.57
1.8
3.3
(69-1356)
186
(15-39)
6.5
(7.3-19.4)
5
3.5
31
(80-433)
465
(4.5-9.4)
12.3
(3.8-6.4)
4.8
3.9
3.6
30
(168-1291)
164
(7.7-19.5)
92
(3.9-5.8)
6.6
0.11
0.86
0.64
0.06
0.49
0.26
0.42
0.10
10
(44-619)
1826
(67-125)
>10000
(5.2-8.6)
>10000
PtP-4
RDM-4’BTPI
StP-4
0.55
3.0
1
(830-4017)
1638
Not done
Not done
Not done
Not done
0.4
0.6
(617-4342)
1930
StP-10
0.21
(587-6336)
a The affinity (Ki) for the human 7 subtype ammoniums MG624, PtN-2, PtN-4 , StN-4, StN-6
and StN-8 and the phosphoniums RDM-4’BTPI, StP-4 and StP-10 was tested in 3-4 independent
competition binding experiments using [125I]- Bungarotoxin and the transfected 7 subtype.
CI=confidence interval of the Ki.
b All of the ammonium compounds and RDM-4’BTPI were functionally tested. In order to obtain
concentration response data concerning their inhibitory effects, concentrations between 0.01 nM
and 100 M were co-applied with 10 µM ACh (α9α10 nAChR) or 200 µM ACh (α7 nAChR) and
the peak current responses were normalized to the peak of the responses elicited by ACh alone;
the normalized data were then fitted to the Hill equation using non-linear regression. Shown are
the IC50 values and the confidence interval (CI) of the IC50.
c The cytotoxic/cytostatic action of the nine compounds was tested using the MTS assays of three
tumoral cell lines (U87MG glioblastoma, A549 adenocarcinoma and SH-SY5Y neuroblastoma)
and wild type mice astrocytes. All of the cells were incubated with increasing concentrations for
72 h. The IC50 values were determined from the dose–response curves fitted using non- linear
regression analysis, taking the absorbance of the control cells (not treated with compounds) as
100%.
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As shown in Table 1, all the ammonium compounds, other than StN-8 , were less potent (higher
IC50 values) than the phosphonium compounds, which were similarly potent against all of the
tumoral and non-tumoral cells. Although less potent, the ammonium compounds were efficacious
on U87MG and A549 cells, but not on SH-SY5Y cells or control astrocytes. In both ammonium
and phosphonium series, the maximum potencies corresponded to the maximum linker length, eigth
(StN-8) and ten methylenes (PtP-10) respectively, resulting in similar ≤ 100 nM IC50 values
against U87MG glioblastoma and A549 adenocarcinoma. Table 2 shows the cytotoxic/cytostatic
action of StP-3 to StP-10 on U87MG glioblastoma and A549 cell lines, the potency of which
increased with the increasing length of the alkylene linker.
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ATP production
We determined the ability of the nine compounds listed in Table 1 to interfere with ATP production
by incubating them with U87MG cells for one or 72 hours. The tested concentrations were those
determined by the MTS assay, which were in the range of the IC50 values. StN-4, StN-6, PtP-4,
StP-4 and StP-10 significantly reduced ATP production (Fig 1A) after just one hour of incubation,
whereas all of the compounds significantly decreased ATP production after 72 h (Fig 1B).
Compound StP-10 was the most potent one as it significantly reduced ATP production at both one
and 72 hours at a concentration that was 10 times lower than that of PtP-4 at 1 and 72 h, whereas
compound StP-4 affected ATP production only at a concentration that it was 2.5 times greater
than its IC50. We also measured mitochondrial membrane potential using the tetramethylrhodamine
methyl ester (TMRM), a voltage sensitive dye that accumulates inside intact mitochondria, and
found that one hour’s treatment with the compounds had no effect, but 72 hours’ treatment greatly
reduced it (not shown).
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Table 2. Pharmacological and biological characterization of the phosphonium compounds
8
9
Cpd
Affinitya
7 receptors
Ki, nM
Viabilityb
IC50 , M
U87MG
72h
Viabilityb
IC50 , M
A549
% ROSc
production
over 0.05
mM
Antimycin A
16.3
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60
(CI)
72h
PtP-4
1826
0.86
0.26
RDM-4’BTPI
(830-4017)
StP-3
StP-4
StP-5
StP-6
StP-7
StP-8
StP-9
StP-10
984
1.4
1.8
21
(276-3600)
1638
0.64
0.67
0.38
0.36
0.07
0.09
0.06
0.42
0.32
0.15
0.12
0.26
0.11
0.10
1.5
(617-4342)
2800
20
(500-4280)
559
27.3
39.5
46.3
56.3
58.2
(31-9900)
617
(102-3700)
909
(477-1732)
1288
(614-2699)
1930
(587-6336)
a
b
c
See note (a) Table 1. See note (c) Table 1. The FACS experiments were performed using the
fluorescence indicator, a costant concentration of 500 nM of each compound and taking the ROS
production induced by 50 M antimycin as 100%.
ROS production
The quaternary onium group should target the molecules to the mitochondrial matrix, which is held
at a negative relative voltage, where their pro-oxidant effect is demonstrated by increased ROS
production. We therefore verified the effects of 1 h exposure to the phosphonium and ammonium
compounds on the ROS production of U87MG cells as monitored by FACS experiments. Treatment
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with the ammonium compounds did not induce the production of mitochondrial ROS, whereas the
phosphoniums PtP-4, StP-4 and StP-10 increased ROS production in a dose- dependent manner and
with an increasing potency (StP-10 > PtP-4 > StP-4) (Fig 1C).
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We also tested whether there was a correlation between the length of the linker and the ability of the
phosphonium compounds to produce ROS. The FACS experiments were performed using the
fluorescence indicator and a costant compound concentration of 500 nM, taking the ROS production
induced by 50 M antimycin (the positive control) as 100%. As shown in Table 2, increasing the
length of the linker increases the capability of phosphonium compounds to produce mitochondrial
ROS.
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in U87MG cells.
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The figure shows the decrease in the level
7
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9
of mitocondrial ATP after one (Fig 1A) or
72 hours (Fig 1B) incubation of U87MG
cells with the indicated compounds. The
data shown were obtained using the
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concentrations
of the ammonium
compounds MG624 (StN-2), PtN-2, PtN-
4 , StN-4, StN-6 and StN-8 of respectively
4, 4, 0.5, 2, 4 and 0.25 M and
concentrations of
the phosphonium
compounds RDM-4’BTPI (PtP-4), StP-4
and StP-10 of respectively 1, 2 and 0.1 M.
For almost all cases, these concentrations
correspond to the IC50 concentrations
determined in the viability assays of
U87MG cells. The data from at least five
separate experiments are expressed by
setting the basal level of control cells as 1
and were analysed by means of one way
ANOVA followed by Dunn's post hoc test
*P<0.05, ** P<0.01, *** P<0.001, ****
P<0.0001 versus basal control. (Fig. 1 C)
Relative ROS production at increasing
compound concentrations. Data are
expressed by setting the ROS production of
the positive control 50 M antimycin A as
100%.
Figure 1. Effects of onium compounds on the
mitochondrial ATP and ROS production
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HepG2 cells permeation
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8
9
The ability of StN-2 to permeate the HepG2 cells was investigated exploiting the natural behavior
of StN-2 to emit fluorescence at 350 nm after excitation at 260 nm using a fluorescent plate reader.
Briefly, HepG2 cells were treated with 1.0, 10.0, 50.0, and 100.0 μM StN-2 or vehicle (DMSO,
blank samples) for 6 and 24 h, respectively. Fluorescence signals emitted at 350 nm of each
compound at intracellular level have been normalized for the total amount of cells (fluorescence
emission of the nuclei after their staining with DAPI) at 620 nm obtaining, therefore, the Relative
Fluorescence Unit (RFU) 350/620 nm after 6 and 24 h of incubation. The RFU 350/620 nm of the
blank samples, which represents the cellular background, was subtracted from each normalized
fluorescence signal (RFU 350/620 nm). Our findings clearly indicated that StN-2 is taken up by
HepG2 cells with a concentration and time response trend. In particular, StN-2 is able to permeate
the human hepatic cells by 4.75, 6.82, 7.67, and 9.43 RFU at 1.0, 10.0, 50.0, and 100.0 μM,
respectively after 6h. After 24 h, the fluorescence signal deriving from the intracellular level
reaches 5.42, 7.18, 9.13, and 14.35 RFU, after the treatment with the compound at 1.0, 10.0, 50.0,
and 100.0 μM, respectively (Figure 2).
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Cells treated with 100 µM StN-2 for 24 h were lysed and the cell lysates were analyzed by mass
spectrometry. The MS/MS fragmentation pattern and the observed product ions are completely
assigned to the product ions generated during the fragmentation. In the MS/MS spectrum the base
peak at m/z 222.9 corresponds to the loss of the styrene moiety.
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Figure 2. Fluorescence signals (bars normalized using the DAPI staining of the nuclei) of HepG2
internalized StN-2 after 6h (black bars) and 24 h (grey bars) at different concentrations. StN-2
naturally emits fluorescence at 350 nm after excitation at 260 nm.
We investigated the ability to permeate the HepG2 also of compounds StN-4, StN-8 and StP-8 at
1 M concentration, after ascertaining that their fluorescence properties (emission at 350 nM upon
irradiation at 260 nM) were just like those of StN-2. Figure 3 shows the intracellular fluorescence
signals reached after 1, 6 and 24 hours treatment with the three compounds in comparison with StN-2.
All the four compounds are able to permeate, the two longest ones, having eight-carbon linker and
phosphonium or ammonium head, a little more rapidly. Overall, these experiments suggest that cell
permeation should be prejudiced neither by polymethylene linker length or onium head nature and
thus allowed to all our compounds.
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Figure 3. Fluorescence signals (bars normalized using the DAPI staining of the nuclei) of HepG2
internalized StN-2, StN-4, StN-8 and StP-8 after 1 h, 6 h and 24 h at 1 M concentration.
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DISCUSSION
The hybridisation planning. The two pillars of our investigation were RDM-4’BTPI (PtP-4) and
MG624 (StN-2): the former, the mitochondriotropic triphenylphosphoniumbutyl ether of
pterostilbene (3’,5’-dimethoxy-4-stilbenol), owes its antiproliferative activity to its known cytotoxic
pro-oxidant action7; the latter, the triethylammoniumethyl ether of 4-stilbenol, selectively
antagonises ACh at 7- and 9-nAChR and, as recently found, has an antiproliferative activity, that
is related to its interaction with 7- and 9-nAChRs.13 The two molecules were hybridised with the
idea that recombining their similar constituents (onium head, alkyl linker and stilbene scaffold) may
not only clarify the contribution of the three substructures to the various components of the
antiproliferative activity, but also lead to a sort of hybridisation of the biological profiles of RDM-
4’BTPI and MG624 by creating synergies between different mechanisms of action.
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Nicotinic binding and nicotinic antagonist affinity of the ammonium hybrids. The synthesis of
the three structural hybrids with triethylammonium head, StN-4, PtN-2 and PtN-4, was not
problematic. We could prepare the StN-2 analogue with a four-carbon linker, StN-4, and the two
pterostilbene analogues PtN-2 and PtN-4 with respectively two- and four-carbon linkers. As single
modifications, the linker elongation (compound StN-4) and the 3’,5’-dimethoxy substitution
(compound PtN-2) only slightly diminished the 7 nicotinic affinity of StN-2 (0.104 M Ki ), but,
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together, they led to a greater decrease (compound PtN-4. 0.3 M Ki). Therefore, further linker
elongation to six and eight carbons was accomplished in the StN series rather than in the PtN series.
StN-6 showed a decreased 7 nicotinic affinity (0.465 M Ki ), but its eight-carbon analogue StN-8
had an 7 affinity (0.164 M Ki ) similar to that of StN-2. Like StN-2, all the five new ammonium
derivatives had modest micromolar 4β2 affinity. On the basis of these findings, we tested
functionally their ability to antagonise the ACh activation of the 7 and the 910 nicotinic subtypes
heterologously expressed in oocytes. We found that the pterostilbene analogue of StN-2, namely PtN-
2, was as potent as StN-2 (IC50 47 vs 41 nM at the 7 subtype and IC50 17 vs 10 nM at the 910
subtype) thus indicating that the 3’,5’-dimethoxy substitution is irrelevant in this respect. Consistently
with these results, elongation to four carbons was beneficial for both of them: StN-4 had IC50 6.5 nM
at 7 and IC50 5 nM at 910, while PtN-4 IC50 24.3 nM at 7 and IC50 11.9 nM at 910. Further
elongation in the StN series to six and eight carbons confirmed the 910 sub-10 nM potency
achieved by StN-4 and, though not ameliorative, the considerable sub-100 nM potency of StN-2 at
the 7 subtype. Overall, as shown in Table 1, elongation of the ethylene linker in the ammonium
compounds resulted in generally higher antagonist activities at the two nicotinic receptors with non-
negligible 910 vs 7 selectivity in the case of StN-8.
Anti-adenocarcinoma activity of the phosphonium hybrids. On the other hand, of the three
structural hybrids with phosphonium head, only StP-4, the stilbene analogue of PtP-4 (RDM-
4’BTPI), could be obtained as the phosphonium ethyloxy derivatives PtP-2 and StP-2 were highly
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unstable. PtP-4 has been characterised in detail as a mitocan by its inventors, who reported that it
induces the necrotic death of CT-26 mouse colon tumor cells in the low-M range and that it is
respectively 5-fold and 10-100-fold more potent than its demethylated analogue and resveratrol,
which has all three phenolic hydroxyls free and is therefore not mitochondriotropic7. Its cytotoxicity
is due to the production of ROS, particularly of H2O2, upon accumulation in mitochondria and there
is evidence that, like mitoVES, it induces the generation of ROS by interacting with respiratory chain
complexes anchored in the mitochondrial inner membrane16.
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Supported by these data, we decided to test the antiproliferative activity of the two phosphonium
derivatives, PtP-4 and StP-4, on the A549 adenocarcinoma cell line derived from lung tumors, the
same cell line as that previously used to investigate the antiproliferative properties of StN-2. These
cells express 7 and 9 nAChRs, whose activation promotes lung carcinoma cell growth, and StN-2
not only blocks the 7- and 9-mediated pro-proliferative effects of nicotine on A549 cells, but also
has dose-dependent A549 cytotoxicity.13 We found that both PtP-4 and StP-4 have sub-micromolar
cytotoxicity (0.26 and 0.42 M IC50) on A549 cells that is comparable with their low-M cytotoxicity
for CT-26 mouse colon tumor cells.7 Importantly, these data indicate that replacing 3’,5’-
dimethoxystilbene with stilbene does not negatively affect antitumor activity and allowed us to
undertake a systematic comparison among our six triethylammonium and triphenylphosphonium
hybrids.
Comparison of the antitumoral activity of ammonium and phosphonium hybrids. In addition
to the A549 human lung carcinoma line, we also used the human SH-SY5Y neuroblastoma and
U87MG glioblastoma cell lines. It has been proved that glioblastoma cells express both 7 and 9
mRNAs,17 while neuroblastoma cells express 7 mRNA and protein.18 As shown in Table 1, the four
triethylammonium derivatives StN-2, PtN-2, PtN-4 and StN-4 had less A549 cytotoxicity than the
two triphenylphosphonium compounds, PtP-4 and StP-4; their IC50 values are in the 3-12 M range,
whereas those of PtP-4 and StP-4 are in the 0.2-0.5 M range. In particular, the antiproliferative
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activity of StN-2 (4.4 M IC50) confirmed the extent of the previously observed decrease in A549
cell proliferation.13 Furthermore, compounds StN-4 and PtN-4, with four-carbon O-N+ linker,
showed slightly greater A549 cytotoxicity (respectively 3.5 and 3.3 M IC50). Viability assays of the
neuroblastoma and glioblastoma cells followed similar trends but with some noteworthy divergences.
When tested on neuroblastoma cells the two phosphonium compounds PtP-4 and StP-4 were more
potent (respectively 0.55 and 3.0 M IC50) than the elongated ammonium compounds PtN-4 and
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StN-4 (11 and 31 M IC50), in turn much more potent than their shorter analogues PtN-2 and StN-2.
On the other hand, compounds PtN-4 and StN-4 were as potent as the two phosphoniums against
glioblastoma, thus showing pronounced glioblastoma vs neuroblastoma selectivity, and, notably, they
were much less toxic than the two phosphoniums towards normal glial cells.
Mechanisms of action. After investigating their antiproliferative activity on various tumor cells, we
investigated whether the six hybrids mechanisms of action underlying their antitumor activity were
similar by considering two intracellular parameters: the increase in mitochondrial ROS generation
and the decrease in ATP production in U87MG cells. We first ascertained that the
triethylammoniumalkyl derivatives can permeate cell membranes, as reported for RDM-4’BTPI and
other molecules linked to triphenylphosphonium through an aliphatic carbon chain. Initially, we
considered StN-2 and StN-4. The ability to permeate the HepG2 cells was investigated using two
different approaches. The former permitted to detect StN-2 and StN-4 directly in intact cells
exploiting their natural ability to emit fluorescence at 350 nm after excitation at 260 nm, whereas the
latter, applied only to StN-2, was based on the detection of StN-2 in the cell lysates by LC-MS/MS.
Both approaches suggest that the two ammoniums are bioavailable at intracellular levels. In
particular, already after 1 h, HepG2 cells were able to uptake them from the extracellular environment
with a concentration-response trend, which was highly improved after 24 h of treatment (Figure 2
and 3). In agreement with this result MS analysis unequivocally confirmed the presence of StN-2 in
the HepG2 cell lysates after 24 h treatment. As shown in Figure 1C, mitochondrial ROS production
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was induced in U87MG glioblastoma cells by PtP-4 (RDM-4’BTPI) and StP-4, but not by the four
ammonium derivatives, StN-2, StN-4, PtN-2 and PtN-4. Phosphonium StP-4 was about 10-fold less
potent than RDM-4’BTPI, whose response in glioblastoma cells at a concentration of 1M, after one
hour incubation, was almost identical to that reported in Jurkat cells at the same concentration and
after the same incubation time.7
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The second intracellular parameter we examined was the decrease in ATP production, which is
indicative of dysregulated mitochondrial function and antitumor activity.19 Tumor cells have to up-
regulate their inefficient glycolytic ATP production in order to satisfy their energy requirements and
preventing mitochondrial ATP production has a highly toxic and antiproliferative effect. There was
a reduction in the ATP production in glioblastoma cells, as early as one hour after the administration
of PtP-4, StP-4 and StN-4, whereas ATP was significantly reduced only after 72 hours in the case of
the other three ammonium derivatives, StN-2, PtN-2 and PtN-4.
In order to complete the picture symmetrically, we had to establish whether the two phosphonium
hybrids, PtP-4 and StP-4, have 7 nicotinic affinity and, if so, whether they have antagonist activity
at 7 nAChRs. The data shown in Table1 indicate that PtP-4 and StP-4 have very modest
supramicromolar binding affinity for 7 nAChRs and that the IC50 of PtP-4 for Ach activated 7
and 910 nAChRs is greater than 10 µM.
Overall, the results of these experiments indicate that the mechanism of action underlying the
antitumor activity of the triphenylphosphonium derivatives is the profound impairment of
mitochondrial functions, as revealed by the immediate and conspicuous increase in ROS production
and decrease in ATP production and by their quite indistinct cytotoxicity. On the other hand, the
antitumor activity of the triethylammonium derivatives seems to be mainly connected to their
interactions with 7 and 910 nAChRs. However it cannot be excluded that, after entering cells,
the triethylammonium derivatives, interfere with mitochondria function albeit less potently than the
phosphonium analogues possibly due to a delayed membrane crossing and a lower level of
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mitochondriotropism. This may be why, with the exception of StN-4, our triethylammonium
derivatives decreased ATP production, only after 72 hours, and induced no immediate increase in
ROS production.
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Multi-carbon linker elongation in ammonium and phosphonium compounds. As stated in the
introduction, alkylene elongation was expected to be the most promising modification of StN-2
resulting from its hybridisation with the PtP-4 substructures, and StN-4 proved to be a more potent
7 and 910 nAChR antagonist than StN-2 and PtN-2 and, together with PtN-4, had greater
antitumor activity, particularly against glioblastoma cells. Such a trend was substantially confirmed
by the further elongation of the linker to six and eight methylenes. Compared with StN-4, compound
StN-8 showed a 16-fold more potent anti-glioblastoma activity, characterized by higher selectivity
over the other tumor and non-tumor cells. Interestingly, such a profile was associated with
maintaining the highly potent 910 nAChR antagonism reached by StN-4 and improvement of ATP
production impairment.
With regard to linker length in the phosphoniums, shortening the four-carbon bridge of PtP-4 by
two carbons was not only hampered by the high chemical instability of the
triphenylphosphoniumethyloxy residue, but was also discouraged by the finding that PtP-4 and StP-4
have modest nicotinic affinity and no antinicotinic properties, probably due to the unsuitable triphenyl
substitution at P+ regardless of the length of the alkylene linker. Furthermore, SAR studies have
recently been made on the mitochondrially targeted derivative of vitamin E (VE) analogus -
tocopheryl succinate, MitoVES (or MitoVE11S), which has an eleven-carbon linker between its
triphenylphosphonium head and chromane nucleus.14 MitoVE11S inhibits mitochondrial respiratory
complex II (CII) leading to ROS generation and cell death induction with IC50 values for apoptosis
in various malignant cell lines mainly ranging between 0.5 and 3 M. 20 A similar mechanism of
action, preferentially involving CI and CIII, has been invoked also in support to PtP-4 proapoptotic
activity, 16 for which we determined slightly higher cytotoxicity values (0.86, 0.55 and 0.26 M IC50
on U87MG, SH-SY5Y and A549 cell lines respectively). These SAR studies on MitoVE11S have
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highlighted that a key role is played by the suitable length of the linker between the targeting group
(P+Ph3) and the group responsible for the interaction with the binding site of the mitochondrial
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complex. Short-linker analogs, such as MitoVE9S, MitoVE7S and MitoVE5S, show decreasing
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effects with the number of carbons of the linker suggesting that their bioactive group cannot reach
the binding site.
On the basis of these observations, we wondered whether increasing the four-carbon distance between
the stilbenoxy group and the triphenylphosphonium head of StP-4 would lead to a parallel increase
in activity. The answer was yes. Though with some slight incongruities, anti-glioblastoma and anti-
adenocarcinoma activities gradually increase with the number of carbons (compounds StP-5→StP-
9). Compound StP-10, whose linker is ten-carbon long, was 10-fold (vs U87MG) and four-fold (vs
A549) more potent than compound StP-4 and also more potent than its long-bridged analogue
MitoVE11S . Consistently, compound StP-3, which has a shortened three-carbon linker, was 2-fold
less cytotoxic for glioblastoma cells than StP-4 and 4-fold less cytotoxic for adenocarcinoma cells.
Linker elongation gradually increased also ROS generation: compound StP-10 was almost four-fold
more potent than PtP-4 and 39-fold more potent than StP-4. Furthermore, the decrease in ATP
production was observable as early as one hour after the administration of StP-10.
In view of these findings, the greater antitumor activity of the elongated ammonium derivatives in
comparison with StN-2 and PtN-2 may also be due to the wider spacing between the onium head and
stilbene portion, a modification beneficial for the interaction at mitochondrial level.
Fluorescence experiments with HepG2 cells demonstrated that elongated ammonium and
phosphonium compunds (StN-8 and StP-8) are able to rapidly permeate cell membrane.
CONCLUSIONS
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Adenocarcinoma, neuroblastoma and glioblastoma cell lines express 7- and, excepting for
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neuroblastoma, 9-containing nAChRs, whose activation has been proved to increase cell
proliferation. A specific antagonist of these receptors, StN-2 (MG624), reduces the viability of such
tumor cells in a dose-dependent manner. Its structure, a quaternary ammonium head linked through
an alkylenoxy chain to the stilbene para position, resembles that of the known mitocan RDM-4’BTPI
(PtP-4), the triphenylphosphoniumbutyl ether of pterostilbene, which we found to be cytotoxic for
the same tumors impairing mitochondrial functions. The hybridisation of these two lead compounds
by recombining their three structural elements (onium head, alkyleneoxy linker and stilbene or 3’,5’-
dimethoxystilbene scaffold) and lengthening the polyalkylene chain proved to be a profitable design
approach, as summarized in Table 3, which lists the magnitude of the action mechanisms and of the
cytotoxicities.
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Table 3. Mechanisms of action of compounds reported in Table 1
MITOCHONDRIAL
OXPHOS
ATP
DECREASE
CELL VIABILITY /
PROLIFERATION
DECREASE AT
nAChR
ACTION ON
ROS
CPD
ANTAGONISM MITOCHONDRIA
PRODUCTION
-
(O2 )
1h
72h
StN-2
(MG624)
INDIRECT (72h)
[M]
++
-
-

INDIRECT (72h)
PtN-2
PtN-4
StN-4
StN-6
StN-8
[M]
[nM]
[M]
[M]
[nM]
++
++
++
++
-
-
-
-
-
-
-


-





INDIRECT (72h)
DIRECT (1h)
DIRECT (1h)
INDIRECT (72h)
++
PtP-4
(RDM-
4’BTPI)
DIRECT (1h)
[M]
-





DIRECT (1h)
StP-4
[nM]
[nM]
-
-


DIRECT (1h)
StP-10
 
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The two ammonium hybrids StN-4 and PtN-4 with linker elongated to butylene were more toxic
against cancer cells, particularly glioblastoma cells, and their potency was similar to the
submicromolar potency of PtP-4, but they were more selective and less toxic against non-tumor cells
than PtP-4. Notably, the enhanced anti-glioblastoma activity of compound StN-4 coincided with
greater antagonism against 7 and 9 nAChRs and StN-4 was the only ammonium hybrid capable
of reducing mitochondrial ATP production after 1h. Such a positive trend was confirmed by further
elongation to six and eight carbons (StN-6 and StN-8).
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Hybridisation of the phosphonium derivatives was limited to replacing pterostilbene with stilbene
for feasibility reasons. This modification of PtP-4 (compound StP-4) did not prejudice its ability to
impair mitochondrial function, but it did not confer, as expectable, those antinicotinic properties of
which, as we demonstrated, PtP4 is devoid. Nevertheless, the approach was effective in suggesting
that further elongation of the linker would also benefit the pro-oxidant cytotoxic activity of
triphenylphosphonium derivatives. The analogue of StP-4 with a ten-carbon linker (compound StP-
10) was a much more potent mitocan than both PtP-4 and StP-4, which have butylene linkers.
In conclusion, we developed more potent antitumor triphenylphosphonium analogues of RDM-
4’BTPI, and we have also demonstrated that the less lipophilic triethylammoniumalkyloxystilbenes,
hitherto considered as acting at membrane 7 and 9 nAChRs, cross membranes and interfere with
mitochondrial functionality. Interestingly, we found that enhancement of such a capacity, associated
with more potent 7 and 9 antagonism, as in ammonium compounds StN-4, StN-6 and, above all,
StN-8, improves the moderate antitumor activity of the quaternary ammonium StN-2 without
impairing its selectivity.
EXPERIMENTAL SECTION
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Chemistry. All chemicals and solvents were used as received from commercial sources or prepared
as described in the literature. Flash chromatography purifications were performed using KP-Sil 32-
63 m 60 Å cartridges. TLC analyses were carried out on alumina sheets precoated with silica gel 60
F254 and visualized with UV light; Rf values are given for guidance. 1H and 13C NMR spectra were
recorded at 300 and 75 MHz using an FT-NMR spectrometer. Chemical shifts are reported in ppm
relative to residual solvent (CHCl3, MeOH or DMSO) as internal standard. Melting points were
determined by Buchi Melting Point B-540 apparatus. Elemental analyses (CHN) are within  0.40%
of theoretical values. The results of elemental analyses indicated that the purity of all tested
compounds was higher than 95%. In each described preparation, the moles of reagents are given for
one mmole of substrate.
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METHOD A: general procedure for the preparation of 1a and 1c.
The intermediates 1a and 1c were synthesized according to a modified literature procedure. 21 K2CO3
(1.1 mmol) was added to a solution of resveratrol (1 mmol) in 1.2 ml of anhydrous DMF. After stirring
for 20 minutes, the appropriate bromo-chloro-alkane (1.5 mmol, 1-bromo-2-cloroethane for 1a and
1-bromo-4-chlorobutane for 1c) was added dropwise and the reaction mixture was stirred overnight
at 60 °C. After HCl/AcOEt extraction, the organic phase was dried over NaSO4, filtered and
evaporated under vacuum. The crude residue was then purified by flash column chromatography,
affording the desired intermediates as brownish solids.
METHOD B: general procedure for the preparation of 4b-i
The intermediates 4b-i were synthesized according to a modified literature procedure. 22 A solution
of trans-4-hydroxy-stilbene (1 mmol) in DMSO (1.4 ml) was added to a stirred suspension of 85 mg
of finely ground NaOH (2.1 mmol) in DMSO (5.6 ml). The mixture was stirred at room temperature
for 30 minutes and then the appropriate dibromoalkane (2 mmol) was added dropwise. After 3 hours,
the reaction mixture was filtered and 1M aqueous solution of HCl was added dropwise to the filtrate
to reach pH 3. The crude product was precipitated by slowly adding cold water. The precipitate was
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isolated by filtration, dried under vacuum and recrystallized from ethanol affording the desired
intermediates as white solids. In the only case of the bromopropyl ether 4b, purification was
accomplished by chromatography.
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METHOD C: general procedure for the preparation of 2a and 2c
The intermediates 2a and 2c were prepared according to a modified literature procedure. 21 Cs2CO3
(2.2 mmol) was added to a stirred solution of the appropriate precursor (1 mmol of 1a and 1c
respectively) in anhydrous DMF (1.5 ml). The resultant mixture was stirred for 20 minutes.
Afterward, MeI (2.2 mmol) was added dropwise and the reaction mixture was stirred overnight at
room temperature. After HCl/EtOAc extraction, the organic phase was dried over NaSO4, filtered and
concentrated under vacuum. The crude residue was then purified by flash column chromatography,
affording the desired intermediates as solids.
METHOD D: general procedure for the preparation of 3a, 3c and 5b-i
The intermediates 3a, 3c and 5b-i were obtained by dissolving 2a, 2c and 4b-i (1 mmol), respectively,
in a saturated solution of NaI in acetone (10 ml), which was then refluxed overnight. After
concentrating under vacuum, the residue was diluted with diethyl ether and subsequently washed with
10% aqueous Na2S2O5 and then with brine. The organic phase was dried over Na2SO4, filtered, and
the solvent was evaporated under vacuum to give the desired intermediates as solids.
METHOD E: general procedure for the preparation of PtN-2, PtN-4, StN-4, StN-6, StN-8
The compounds PtN-2, PtN-4, StN-4, StN-6 and StN-8 were obtained by stirring 3a, 3c, 5c, 5e and
5g (1 mmol), respectively, triethylamine (3 ml – 21.6 mmol) and toluene (3 ml) at reflux temperature
for 5 hours. Upon concentration under vacuum, the residue was diluted with diethyl ether and the
obtained suspension was filtered, affording the desired product as a solid.
METHOD F: general procedure for the preparation of compounds StP-3-StP-10
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The compounds StP-3-StP-10 were obtained by heating a solid mixture of PPh3 (20 mmol) and the
respective iodinated precursor 5b-i (1 mmol) to 100 °C so as to obtain a solution of the starting
material in molten PPh3. The reaction mixture was slowly stirred at 100 °C for 3 hours and then
cooled to room temperature without stirring any further. The re-solidified mixture was dissolved in
the smallest possible volume of dichloromethane and diethyl ether was added dropwise under
vigorous stirring. Afterwards, the solid precipitate was isolated by filtration affording the desired
products.
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(E)-3’,5’-Dihydroxy-4-(2-chloroethyloxy)stilbene (1a)
Obtained from resveratrol according to METHOD A, as a brown solid in 32% yield after purification
through silica gel chromatography (gradient dichloromethane/ethyl acetate from 9:1 to 8:2). Rf
(dichloromethane/ethyl acetate 9:1) = 0.22. M.p.= 161 °C. 1H NMR (300 MHz, DMSO) δ 9.20 – 9.13
(s, 2H, exchange with water), 7.49 (d, J = 8.8 Hz, 2H), 7.02 – 6.81 (m, 4H), 6.38 (d, J = 2.2 Hz, 2H),
6.10 (t, J = 2.2 Hz, 1H), 4.29 – 4.20 (m, 2H), 3.97 – 3.87 (m, 2H).
(E)-3’,5’-Dihydroxy-4-(4-chlorobutyloxy)stilbene (1c)
Obtained from resveratrol according to METHOD A, as a light brown solid in 30% yield after
purification through silica gel chromatography (gradient from dichloromethane to
dichloromethane/methanol 9:1). Rf (dichloromethane/methanol 9:1) = 0.54. M.p.= 148 °C. 1H NMR
(300 MHz, DMSO) δ 9.18 (s, 2H, exchange with water), 7.48 (d, J = 8.7 Hz, 2H), 6.91 (m, 4H), 6.38
(d, J = 2.0 Hz, 2H), 6.10 (t, J = 2.0 Hz, 1H), 4.00 (t, J = 5.9 Hz, 2H), 3.70 (t, J = 5.9 Hz, 2H), 1.97 –
1.70 (m, 4H).
(E)-3’,5’-Dimethoxy-4-(2-chloroethyloxy)stilbene (2a)
Obtained from 1a according to METHOD C as a salmon solid in 90% yield after purification by silica
gel chromatography (cyclohexane/ethyl acetate 9:1). Rf (cyclohexane/ethyl acetate 9:1) = 0.37. M.p.=
86-88 °C. 1H NMR (300 MHz, CDCl3) δ 7.44 (d, J = 8.7 Hz, 2H), 7.03 (d, J = 16.3 Hz, 1H), 6.96 –
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6.85 (m, 3H), 6.64 (d, J = 2.2 Hz, 2H), 6.37 (t, J = 2.2 Hz, 1H), 4.25 (t, J = 5.9 Hz, 2H), 3.85 – 3.79
(m, 8H).
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Obtained from 1c according to METHOD C as a pale yellow solid in 63% yield after purification by
silica gel chromatography (cyclohexane/ethyl acetate 95:5). Rf (cyclohexane/ethyl acetate 95:5) =
0.22. M.p.= 77 °C. 1H NMR (300 MHz, CDCl3) δ 7.43 (d, J = 8.6 Hz, 2H), 7.03 (d, J = 16.2 Hz, 1H),
6.95 – 6.81 (m, 3H), 6.65 (d, J = 1.9 Hz, 2H), 6.37 (t, J = 1.9 Hz, 1H), 4.03 (d, J = 5.8 Hz, 2H), 3.83
(s, 6H), 3.63 (t, J = 5.8 Hz, 2H), 1.97 (m, 4H).
(E)-3’,5’-Dimethoxy-4-(2-iodoethyloxy)stilbene (3a)
Obtained from 2a according to METHOD D. Yield: 97%. M.p.: 82-84 °C. Rf (cyclohexane/ethyl
acetate 9:1) = 0.43. 1H NMR (300 MHz, CDCl3) δ 7.44 (d, J = 8.8 Hz, 2H), 7.03 (d, J = 16.3 Hz, 1H),
6.91 (m 3H), 6.64 (d, J = 2.2 Hz, 2H), 6.38 (t, J = 2.2 Hz, 1H), 4.27 (t, J = 6.9 Hz, 2H), 3.82 (s, 6H),
3.45 – 3.39 (t, J = 6.9 Hz, 2H).
(E)-3’,5’-Dimethoxy-4-(4-iodobutyloxy)stilbene (3c)
Obtained from 2c according to METHOD D. Yield: 96%. M.p.: 87°C. Rf (cyclohexane/ethyl acetate
9:1) = 0.36. 1H NMR (300 MHz, CDCl3) δ 7.43 (d, J = 8.7 Hz, 2H), 7.03 (d, J = 16.3 Hz, 1H), 6.96
– 6.76 (m, 3H), 6.65 (d, J = 2.2 Hz, 2H), 6.38 (t, J = 2.2 Hz, 1H), 4.01 (t, J = 6.0 Hz, 2H), 3.83 (s,
6H), 3.27 (t, J = 6.8 Hz, 2H), 2.14 – 1.85 (m, 4H).
(E)-4-(3-Bromopropyloxy)stilbene (4b)
Obtained from 4-stilbenol synthesized according to METHOD B by reaction with 1,3-
dibromopropane. The crude product was purified by silica gel chromatography (gradient from hexane
to hexane/acetonitrile 9:1). Yield: 52%. M.p.: 109 °C. Rf (hexane/acetonitrile 9:1) = 0.24. 1H NMR
(300 MHz, CDCl3) δ 7.54 – 7.41 (m, 4H), 7.35 (t, J = 6.7 Hz, 2H), 7.26 – 7.19 (m, 1H), 7.08 (d, J =
15.5 Hz, 1H), 7.03 – 6.87 (m, 3H), 4.14 (t, J = 6.4 Hz, 2H), 3.62 (t, J = 7.0 Hz, 2H), 2.34 (m, 2H).
(E)-4-(4-Bromobutyloxy)stilbene (4c)
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