Development of mitochondria-targeted derivatives of resveratrol

Article abstract of DOI:10.1016/j.bmcl.2008.08.100

To target natural polyphenols to the subcellular site where their redox properties might be exploited at best, that is, mitochondria, we have synthesised new proof-of-principle derivatives by linking resveratrol (3,4′,5-trihydroxy-trans-stilbene) to the membrane-permeable lipophilic triphenylphosphonium cation. The new compounds, (4-triphenylphosphoniumbutyl)-4′-O-resveratrol iodide and its bis-acetylated derivative, the latter intended to provide transient protection against metabolic conjugation, accumulate into energized mitochondria as expected and are cytotoxic for fast-growing but not for slower-growing cells. They provide a powerful potential tool to intervene on mitochondrial and cellular redox processes of pathophysiological relevance.

Full text of DOI:10.1016/j.bmcl.2008.08.100

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5594–5597
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of mitochondria-targeted derivatives of resveratrol
Lucia Biasutto a,b, , Andrea Mattarei a, , Ester Marotta a, Alice Bradaschia b,
a
Nicola Sassi b, Spiridione Garbisa b, Mario Zoratti b,c, , Cristina Paradisi
*
a Department of Chemical Sciences, Università di Padova, via Marzolo 1, 35131 Padova, Italy
Department of Biomedical Sciences, Università di Padova, viale G. Colombo 3, 35131 Padova, Italy
b
c CNR Institute of Neuroscience, viale G. Colombo 3, 35131 Padova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
To target natural polyphenols to the subcellular site where their redox properties might be exploited at
best, that is, mitochondria, we have synthesised new proof-of-principle derivatives by linking resveratrol
(3,40,5-trihydroxy-trans-stilbene) to the membrane-permeable lipophilic triphenylphosphonium cation.
The new compounds, (4-triphenylphosphoniumbutyl)-40-O-resveratrol iodide and its bis-acetylated
derivative, the latter intended to provide transient protection against metabolic conjugation, accumulate
into energized mitochondria as expected and are cytotoxic for fast-growing but not for slower-growing
cells. They provide a powerful potential tool to intervene on mitochondrial and cellular redox processes
of pathophysiological relevance.
Received 7 August 2008
Revised 27 August 2008
Accepted 28 August 2008
Available online 31 August 2008
Keywords:
Resveratrol
Mitochondria
Triphenylphosphonium
Anti-oxidants
Ó 2008 Elsevier Ltd. All rights reserved.
Pro-oxidants
Reactive oxygen species
Plant polyphenols are the object of intense interest because
they display, at least in vitro, properties and effects of relevance
for physiopathological conditions ranging from aging to cancer.
These effects are ascribed to their redox properties and to interac-
tions with signaling proteins. Polyphenols can act either as anti- or
pro-oxidants, that is, inhibitors or enhancers of oxidative and rad-
ical chain processes.1 Whether an anti- or a pro-oxidant effect pre-
dominates depends, besides the redox potential of the polyphenol,
(MPT),11 promoting in both cases cell death. Cancer cells are con-
stitutively under oxidative stress4 and an intensification of this
stress may lead to their selective elimination. Resveratrol, in addi-
tion to other important activities,12 reportedly exerts anti-prolifer-
ative and pro-apoptotic effects on various tumor-derived cells13,14
and antagonizes growth of xenografts and mutagen-induced can-
cers.15 It has been recently shown that resveratrol-induced death
of cultured colorectal carcinoma cells involves generation of super-
oxide anion, that is, pro-oxidant action, at mitochondria.14 The IC50
on the abundance of metal ions sustaining a redox cycle (Fe2+/3+
,
Cu+/2+) and/or of oxidizing enzymes, on the ion-chelating proper-
ties of the molecule, on pH, on the concentration of the polyphenol,
and on the subcellular compartment. Either pro-oxidant or
anti-oxidant activity may lead to useful oncological applications.
Reactive Oxygen Species (ROS) are thought to be a major factor
in cancerogenesis.2 In particular, ROS production by mitochon-
dria3,4 is emerging as a key factor. The metastatic potential of cell
lines has been convincingly related to this parameter.5 Mitochon-
drial ROS are involved in the activation of Hypoxia Inducible Factor
(HIF),6,7 which influences angiogenesis and other aspects of tumor
development.7,8 Thus, an anti-oxidant action may limit metastasis
and tumor growth. Indeed resveratrol, the representative polyphe-
nol selected for this work, inhibits cell shedding from primary
tumors.9 On the other hand, ROS play fundamental roles in apopto-
sis10 and can induce the Mitochondrial Permeability Transition
for death induction was found to be in the hundreds of lM range, a
concentration which cannot be reached in vivo due to the poor bio-
availability of polyphenols.16
We are interested in exploiting the potential of polyphenols
through chemical modifications designed to serve specific purposes.
Thus, an increase in solubility was achieved via esterification with
aminoacids.17 The present work aims at targeting polyphenols to
thesubcellularcompartment wheretheyare expectedto bestrealize
their anti-cancer potential (as well as other functions), that is, mito-
chondria. We report here the synthesis and properties of new mitoc-
hondriotropic derivatives of resveratrol obtained by coupling it to
the membrane-permeable lipophilic cation triphenylphosphonium
(TPP+)18 which drives accumulation in compartments held at nega-
tive relative voltage, such as the mitochondrial matrix, according
to Nernst’s law. Since the mitochondria of cancer cells maintain a
higher-then-normal transmembrane potential,19 mitochondria-tar-
geted drugs may be cancer-selective.
* Corresponding author. Tel.: +39 049 8276054; fax: +39 049 8276049.
E-mail address: zoratti@bio.unipd.it (M. Zoratti).
The first two authors contributed equally to the work.
The target derivative 4 was synthesised starting from resvera-
trol (1) in three steps as outlined in Scheme 1.20 Briefly, O-alkyl-
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.100

L. Biasutto et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5594–5597
5595
ation introduces a chlorobutyl group which is then converted to
160
140
120
100
80
Intens.
the desired TPP+ derivative via two consecutive nucleophilic sub-
stitution steps: –Cl ? –I ? – TPP+Iꢀ. Direct substitution of chloride
by triphenylphosphine was unsatisfactory because it required high
temperatures which led to some decomposition. The assignment of
the site of O-alkylation in 2 is based on 1H NMR data: a unique sig-
nal is found for H-2 and H-6 indicating that these protons are
equivalent. The acetylated derivative 6 was also prepared (Scheme
1), so as to compare it with 4 and assess the importance of the free
hydroxyl groups for the behavior of these new mitochondriotropic
molecules.
545.2
600
x107
4
[M+H]+
4
2
0
200
800
400
m/z
*
60
*
40
6
4
20
The solubility in water of the resveratrol derivatives is
(3.12 0.20) ꢁ 10ꢀ5 mol Lꢀ1 and (9.7 0.6) ꢁ 10ꢀ5 mol Lꢀ1 for 4
and 6, respectively. These solubilities are significantly higher (15-
and 45-fold, respectively) than that of resveratrol. Both new com-
pounds are essentially stable in aqueous media: 4 for at least one
week both in deionised water and in Hank’s Balanced Saline Solu-
tion (HBSS) with 10% CH3CN (added to insure solubility of hypo-
thetical reaction products); 6 for at least 24 h in deionised water,
while in HBSS acetyl groups were slowly hydrolised (about 7% con-
version to the monoacetylated derivative in 6 h). There were no
detectable metabolic modifications of either 4 or 6 by cultured Hu-
man Colon Tumor (HCT) 116 cells20 over 6 h (Fig. 1) or whole
freshly drawn rat blood over 75 min (not shown), except for the
hydrolysis of the acetyl ester groups of 6 in both cases.
Two methods were used to verify accumulation of the new
compounds into mitochondria.21 First, their uptake by isolated,
respiring Rat Liver Mitochondria (RLM) was monitored using a
TPP+-sensitive electrode. A representative experiment with 6 is
shown in Figure 2. The introduction of mitochondria causes a de-
crease (upward deflection of the signal) of 6 in the medium, due
to uptake into the mitochondrial matrix. After addition of excess
Ca2+, which induces the MPT, or of uncouplers (not shown), 6 is
partially released. The release is incomplete presumably due to
binding of the resveratrol derivative to mitochondrial constituents.
Analogous results were obtained with 4 (not shown).
0
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30
Minutes
Figure 1. HPLC chromatograms recorded at 320 nm of the extracts obtained after
incubation of 4 (lower trace) and 6 (upper trace) with HCT116 cells for 6 h. Inset:
positive ESI-MS spectrum of 4. For clarity, the upper trace was shifted slightly to the
right along the time axis: the retention time and the mass spectrum of the major
peak in this chromatogram match perfectly those of 4. Peaks marked with ꢂ are due
to residual traces of acetone from the sample work-up.
tured cells by monitoring of their fluorescence upon excitation at
340 nm. Images from one such experiment are shown in Figure
3. After addition of 6 to the medium, intracellular structures be-
come progressively fluorescent due to accumulation of the resvera-
trol derivative (panel B). Addition of a transmembrane potential
(Dwm)-dissipating protonophore (carbonyl cyanide p-trif-
luoromethoxyphenylhydrazone (FCCP)) causes a loss of fluores-
cence due to efflux of the polyphenol (panel C). Some 6 remains
in the cytoplasm of the cells due to the plasma membrane poten-
tial maintained by K+ diffusion. Compound 4 behaved analogously
(not shown).
As a first test of potential anti-cancer activity, we verified the ef-
fects of 4 and 6, and of control compounds, on cultured cells
(Fig. 4). Controls consisted of the parent polyphenol resveratrol,
of the phosphonium salt TriPhenylMethylPhosphonium Iodide
(TPMP) and of resveratrol plus this latter compound. We used
In the second approach we exploited the spectral properties of 6
and 4, similar to those of resveratrol itself (Supporting Data, Fig. S1
and S2), to follow their accumulation in the mitochondria of cul-
HO
HO
HO
3
Cl
i
O
OH
5
4'
HO
1
2
I
O
O
HO
HO
I
O
O
iii
ii
O
O
3
5
iv
iv
I
I
PPh3
PPh3
HO
HO
O
O
O
O
O
O
4
6
Scheme 1. Synthesis of mitochondriotropic derivatives 4 and 6. Reagents and conditions: (i) 1-Bromo-4-chlorobutane (1.5 equiv), K2CO3 (1.1 equiv), DMF, Ar, rt, 20 h, yield
33%; (ii) NaI, acetone, reflux, 20 h, yield 89%; (iii) CH3C(@O)Cl (20 equiv), pyr, CH2Cl2, yield 73%; (iv) PPh3 (5 equiv), toluene, 100 °C, 6 h, yield 78%.

5596
L. Biasutto et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5594–5597
A
Control
TPMP 5 μM
1, 1 μM
1, 5 μM
1+TPMP, 5 μM
4 , 1 μM
4 , 5 μM
6 , 1 μM
6 , 5 μM
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
B
Control
TPMP 5 μM
1, 1 μM
1, 5 μM
1+TPMP, 5 μM
4 , 1 μM
4 , 5 μM
6 , 1 μM
6 , 5 μM
Figure 2. TPP+-selective electrode response to additions of 6 (thick gray arrows; 0.5
l
M each), Rat Liver Mitochondria (RLM) (1 mg prot. mLꢀ1) and CaCl2 (50
lM) at 20 °C.
0.0 0.2 0.4 0.6 0.8 1.0 1.2
C
Control
TPMP 5 μM
1, 1 μM
1, 5 μM
1+TPMP, 5 μM
4 , 1 μM
4 , 5 μM
6 , 1 μM
6 , 5 μM
0.0
0.1
0.2
0.3
0.4
Figure 4. Effect of the mitochondriotropic resveratrol derivatives and control
compounds on cell proliferation. Cells were allowed to grow for 3 days in the
presence of the specified compounds and assayed using the tetrazolium salt
reduction assay (see Supplementary data for details). All measurements were
performed in quadruplicate. Averages s.d. are given. (A) C-26 mouse colon tumor
cells. (B) Fast-growing Mouse Embryonic Fibroblasts (MEF). (C) Slow-growing MEF
(note different scale).
the murine colon cancer cell line C-26 and, as controls, fast- and
slow-growing non-tumoral mouse embryonic fibroblast (MEF)
lines. Cell growth and viability was quantified using the tetrazo-
lium salt reduction (MTT) assay.21
The various compounds had little effect on cell proliferation at
the 1 lM level. At 5 lM, 4 and 6 displayed a marked cytotoxic ef-
fect on the two rapid-growth cell types, that is, C-26 (Fig. 4A) and
non-tumoral ‘fast’ MEFs (Fig. 4B), but not on the slow-growth MEFs
(Fig. 4C). Resveratrol, TPMP, and their combination had little effect
also at 5 lM and with all three cell types, showing that the activity
of the resveratrol-TPP conjugates is not just the sum of the activity
of the two components. Selective cytotoxicity for fast-growing cells
is characteristic of many chemotherapeutic drugs.
In conclusion we have produced mitochondriotropic resveratrol
derivatives with good solubility and stability in aqueous media,
which accumulate as expected in regions at negative potential. A
class of natural compounds with useful properties can now be tar-
geted to subcellular compartments where they ought to realize their
biomedical potential in full. The results of initial cytotoxicity assess-
ments encourage further experimentation in vivo to determine
absorption, pharmacokinetics and possible anti-tumoral action.
Acknowledgments
Figure 3. Fluorescence microscope images of cultured HCT116 cells: (A) just before
the addition of 10 M 6, (B) 25 min after the addition of 6 and just before the
addition of 2 M FCCP, and (C) 10 min after the addition of FCCP.
We thank P. Bernardi, V. Petronilli, and A. Toninello for access to
instrumentation, V. Petronilli, A. Angelin, M.E. Soriano, and D. Dal
l
l

L. Biasutto et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5594–5597
5597
319, [M+H]+; HRMS (ESI-TOF): m/z 319.1092; Calcd for C18H19ClO3ꢃH+
319.1095. Anal.: Calcd for C18H19ClO3 C 67.81, H 6.01; found: C 67.78, H 6.02.
40-(4-O-iodobutyl) resveratrol (3). Compound 2 (500 mg, 1.57 mmol, 1 equiv)
was added to a saturated solution of NaI in dry acetone (10 mL) and heated at
reflux for 20 h. After cooling, the resulting mixture was diluted in EtOAc
(100 mL), filtered and washed with water (3ꢁ 30 mL). The organic layer was
dried over MgSO4 and filtered. The solvent was evaporated under reduced
pressure and the residue was purified by flash chromatography using CH2Cl2/
EtOAc 9:1 as eluent to afford 0.570 g of 3 (89%). 1H NMR (250 MHz, DMSO-d6) d
(ppm): 1.71–2.00 (m, 4H, CH2), 3.35 (t, 2H, CH2), 4.01 (t, 2H, CH2), 6.12 (t, 1H,
H-4, J = 2.0 Hz), 6.40 (d, 2H, H-2, H-6, J = 2.0 Hz), 6.82–7.04 (m, 4H, @CH, H-20,
H-60), 7.50 (d, 2H, H-30, H-50, J = 8.75 Hz), 9.21 (s, 2H, 3-OH, 5-OH); 13C NMR
(62.9 MHz, DMSO-d6) d (ppm): 8.54 (CH2I), 29.66 (CH2), 29.76 (CH2), 66.39
(OCH2), 104.38, 114.62, 126.61, 127.45, 127.76, 129.62, 139.07, 158.16, 158.49;
ESI-MS (ion trap): m/z 411, [M+H]+. Anal.: Calcd for C18H19IO3 C 52.70, H 4.66;
found: C 52.75, H 4.66.
Zoppo for operational instructions, B. Vogelstein, L. Scorrano, and
W.J. Craigen for cells. This work was supported in part by grants
of the Italian Association for Cancer Research (AIRC) (M.Z. and
S.G.) and the Italian Foundation for Basic Research (FIRB) (M.Z.)
and by a fellowship of the Fondazione Cassa di Risparmio di Padova
e Rovigo (L.B.).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.08.100.
40-(4-O-triphenylphosphoniumbutyl) resveratrol iodide (4).
A mixture of 3
References and notes
(500 mg, 1.22 mmol) and triphenylphosphine (1.60 g, 6.09 mmol, 5 equiv) in
toluene (15 mL) was heated at 100 °C under argon. After 6 h, the solvent was
eliminated at reduced pressure and the resulting white solid was dissolved in
the minimum volume of acetone (3 mL) and precipitated with diethyl ether
(100 mL). The solvents were decanted and the procedure repeated 4 more
times. The precipitate was then filtered to afford 600 mg of 7 (73%). 1H-NMR
(250 MHz, DMSO-d6) d (ppm): 1.73 (quintet, 2H, CH2), 1.92 (quintet, 2H, CH2),
3.67 (t, 2H, CH2), 4.06 (t, 2H, CH2), 6.13 (t, 1H, H-4, J = 1.9 Hz), 6.40 (d, 2H, H-2,
H-6, J = 1.75 Hz), 6.83–7.04 (m, 4H, @CH, H-20, H-60), 7.50 (d, 2H, H-30, H-50,
J = 8.75 Hz), 7.71–7.95 (m, 15H, aromatic-H), 9.22 (s, 2H, 3-OH, 5-OH). 13C NMR
(62.9 MHz, DMSO-d6) d (ppm): 18.48 (CH2), 20.07 (CH2), 29.15 (CH2), 65.93
(OCH2), 104.38, 114.66, 118.47 (Ph, J(13C/31P) = 85.6 Hz), 126.67, 127.41,
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127.73,
129.72,
130.25
(Ph,
J(13C/31P) = 12.4 Hz),
133.59
(Ph,
J(13C/31P) = 10.1 Hz), 134.93 (Ph, J(13C/31P) = 2.9 Hz), 139.03, 158.00, 158.49;
ESI-MS (ion trap): m/z 545, M+; HRMS (ESI-TOF): m/z 545.2236; Calcd for
C36H34O3P+ 545.2240.
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3,5-diacetyl-40-(4-O-iodobutyl) resveratrol (5).
A
solution of acetyl chloride
(1.1 mL, 15 mmol, 20 equiv) in CH2Cl2 (20 mL) was added dropwise and under
continuous stirring to mixture of (300 mg, 0.73 mmol, equiv) and
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a
3
1
anhydrous pyridine (0.85 mL, 10.5 mmol, 15 equiv) in CH2Cl2 (20 mL) cooled in
dry ice/acetone. The reaction mixture was then allowed to slowly warm up to
room temperature. CH2Cl2 (50 mL) was added and the organic layer was
washed with 1 N HCl (3ꢁ 50 mL), dried over MgSO4 and filtered. The solvent
was evaporated under reduced pressure and the residue was purified by flash
chromatography using CH2Cl2:n-hexane 4:1 as eluent to afford 280 mg of 5
(78%). 1H NMR (250 MHz, DMSO-d6) d (ppm): 1.73–2.02 (m, 4H, CH2), 2.29 (s,
6H, OAc), 3.38 (t, 2H, CH2), 4.02 (t, 2H, CH2), 6.86 (t, 1H, H-4, J = 1.9 Hz), 6.96 (d,
2H, H-20, H-60, J = 8.5 Hz), 7.08 (d, 1H, =CH, J = 16.5 Hz), 7.21–7.33 (m, 3H, H-2,
H-6, @CH), 7.53 (d, 2H, H-30, H-50, J = 8.75 Hz); 13C NMR (62.9 MHz, DMSO-d6) d
(ppm): 8.49 (CH2I), 20.83 (CH3), 29.62 (CH2), 29.75 (CH2), 66.43 (OCH2), 114.74,
116.79, 124.25, 128.08, 129.10, 130.09, 139.81, 151.15, 158.61, 168.99; ESI-MS
(ion trap): m/z 495, [M+H]+; HRMS (ESI-TOF): m/z 495.0664; Calcd for
C22H23O5IꢃH+ 495.0663.
17. Biasutto, L.; Marotta, E.; De Marchi, U.; Zoratti, M.; Paradisi, C. J. Med. Chem.
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3,5-diacetyl-40-(4-O-triphenylphosphoniumbutyl) resveratrol iodide (6). A mixture
of 5 (200 mg, 0.40 mmol) and triphenylphosphine (525 mg, 2.00 mmol, 5 equiv)
in toluene (10 mL) was heated at 100 °C under argon. After 6 h, the solvent was
eliminated underreducedpressure andtheresultingwhitesolidwas dissolvedin
the minimum volume of acetone (3 mL) and precipitated with diethyl ether
(100 mL) five times. The solvents were decanted after each precipitation. The
precipitate was than filtered to afford 210 mg of 6 of 96–98% purity (69% yield).
1H NMR (250 MHz, DMSO-d6) d (ppm): 1.73 (quintet, 2H, CH2), 1.93 (quintet, 2H,
CH2), 2.29 (s, 6H, OAc), 3.67 (t, 2H, CH2), 4.07 (t, 2H, CH2), 6.86 (t, 1H, H-4,
J = 2.2 Hz), 6.91 (d, 2H, H-20, H-60, J = 8.75 Hz), 7.08 (d, 1H, @CH, J = 16.25 Hz),
7.21–7.33 (m, 3H, H-2, H-6, @CH), 7.53 (d, 2H, H-30, H-50, J = 8.5 Hz), 7.72–7.96
(m, 15H, aromatic-H). 13C NMR (62.9 MHz, DMSO-d6) d(ppm): 19.25(CH2), 20.84
(CH2), 29.25 (CH2), 65.98 (OCH2), 114.79, 116.81, 118.46 (Ph, J(13C/31P) =
85.9 Hz), 124.31, 128.06, 129.19, 130.05, 130.25 (Ph, J(13C/31P) = 12.4 Hz),
133.59 (Ph, J(13C/31P) = 10.1 Hz), 134.93 (Ph, J(13C/31P) = 2.9 Hz), 139.76,
151.16, 158.46, 169.01; ESI-MS (ion trap): m/z 629, M+; HRMS (ESI-TOF): m/z
629.2453; Calcd for C40H38O5P+ 629.2451.
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20. Synthetic procedures and characterization.
40-(4-O-chlorobutyl) resveratrol (2). K2CO3 (1.33 g, 9.64 mmol, 1.1 equiv) and 1-
bromo-4-chlorobutane (2.25 g, 13.14 mmol, 1.5 equiv) were added under
argon to a solution of resveratrol (1) (2.00 g, 8.76 mmol) in DMF (10 mL). After
stirring overnight, the mixture was diluted in EtOAc (100 mL) and washed with
1 N HCl (3 ꢁ 50 mL). The organic layer was dried over MgSO4 and filtered. The
solvent was evaporated under reduced pressure and the residue was purified
by flash chromatography using CH2Cl2/EtOAc 85:15 as eluent to afford 0.930 g
of 2 (33%). 1H NMR (250 MHz, DMSO-d6) d (ppm): 1.76–1.98 (m, 4H, CH2), 3.72
(t, 2H, CH2), 4.02 (t, 2H, CH2), 6.12 (t, 1H, H-4, J = 2.0 Hz), 6.40 (d, 2H, J = 2.0 Hz,
H-2, H-6), 6.82–7.04 (m, 4H, @CH, H-20, H-60), 7.50 (d, 2H, H-30, H-50,
J = 8.75 Hz), 9.21 (s, 2H, 3-OH, 5-OH); 13C NMR (62.9 MHz, DMSO-d6)
d
21. For materials, instrumentation, and experimental details please see Supporting
Data.
(ppm): 26.14 (CH2), 28.90 (CH2), 45.19 (CH2Cl), 66.72 (OCH2), 104.38, 114.62,
126.62, 127.45, 127.76, 129.62, 139.07, 158.16, 158.49; ESI-MS (ion trap): m/z