Synthesis of 4′-ester analogs of resveratrol and their evaluation in malignant melanoma and pancreatic cell lines

Article abstract of DOI:10.1016/j.bmcl.2009.12.006

4′-Ester analogs of the disease preventative agent resveratrol were synthesized and evaluated for their potential as anti-melanoma and pancreatic cancer agents. A decarbonylative Heck coupling was used to assemble the protected stilbene core structure. Th

Full text of DOI:10.1016/j.bmcl.2009.12.006

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1198–1201
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of 40-ester analogs of resveratrol and their evaluation in
malignant melanoma and pancreatic cell lines
Yong Wong a, Gregory Osmond b, Kenneth I. Brewer a, Douglas S. Tyler b, Merritt B. Andrus a,
*
a Department of Chemistry and Biochemistry, Brigham Young University, C100 BNSN, Provo, UT 84602, USA
b Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA
a r t i c l e i n f o
a b s t r a c t
40-Ester analogs of the disease preventative agent resveratrol were synthesized and evaluated for their
potential as anti-melanoma and pancreatic cancer agents. A decarbonylative Heck coupling was used
to assemble the protected stilbene core structure. The 40-acetate and the palmitoate analogs demon-
strated selective activity with DM443 and DM738 cells over normal NHDF cells.
Ó 2009 Elsevier Ltd. All rights reserved.
Article history:
Received 3 August 2009
Revised 25 November 2009
Accepted 2 December 2009
Available online 5 December 2009
Keywords:
Heck coupling
Resveratrol
Stilbene
Anticancer activity
Melanoma
Gemcitabine
Malignant melanoma is among the most lethal and chemo-
resistant forms of cancer.1 Resveratrol 1, a dietary trihydroxy stil-
bene isolated from grapes and other sources known to possess sig-
nificant disease preventative and general health promoting
of the key decarbonylative Heck coupling reaction. The production
of pro-drug analogs of this important dietary agent offers the pos-
sibility of new applications with compounds that possess en-
hanced stability and lipophilicity (Scheme 1).
The synthesis involves the original reported acid chloride inter-
mediate with significant modifications and improvements to the
key coupling conditions (Scheme 2). Inexpensive resorcylic acid 4
(3,5-dihydroxy benzoic acid) was converted to the bis-MOM
(methoxymethyl ether) protected acid 5 now using potassium
carbonate and MOMCl (3 equiv), prepared conveniently from
bis-methoxymethane and hexanonyl chloride.8 Previously sodium
hydride was employed as base for this step.4 The MOM-ester inter-
mediate is hydrolyzed with aqueous NaOH followed by dilute HCl
activity,2 was found to bind and inhibit APE/Ref-I (12
l
M, IC50),3
a protein involved in DNA repair that is up-regulated in melanoma
cells. Previously we reported the synthesis of various 3, 5, and 40-
acetate ester analogs and fluoro variants of resveratrol together
with their anticancer activity using HL-60 cell assays and longevity
studies using yeast.4 In this study the 40-acetate analog 2 was
found to be most potent at 17 lM compared to 22 lM for resvera-
trol. In addition, this analog was found to be more resistant to
decomposition than resveratrol, remaining as a stable, pure white
solid over extended time.5 Longevity studies found that the 40-ace-
tate analog possesses enhanced metabolic stability with compara-
ble activity to resveratrol by significantly extending the lifespan of
yeast with reduced dosing required.6 Resveratrol, as a polar triol
stilbene has a short in vivo half life of 30 min and is rapidly cleared
(30 min).7 Based on previous findings, the synthesis and testing of
extended 40-ester variations 3 were pursued in an effort to discover
new agents tailored to specific therapeutic applications including
new types of cancer. A new set of 40-resveratrol esters were pro-
duced and evaluated using DM443 and DM738 (Duke Melanoma)
cell assays. This investigation also allowed for further exploration
OH
HO
OAc
HO
2
1
HO
HO
R
O
HO
O
3
HO
Scheme 1. Resveratrol and 40-ester analogs.
* Corresponding author. Tel.: +1 801 422 8171; fax: +1 801 422 0153.
E-mail address: mbandrus@chem.byu.edu (M.B. Andrus).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.006

Y. Wong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1198–1201
1199
MOMO
MOMCl (3 eq.)
2) NaOH / HCl
CO2H
R
1) NaOH
/ H+ 91%
2) RCOCl
pyr.
O
HO
CO2H 1) K2CO3,
HCl,
O
MOMO
THF
3
9
5
MOMO
4
HO
73-82%
10
88%
88-91%
MOMO
OAc
1.2 eq.
7
MOMO
COCl
SOCl2
benzotriazole
Pd(OAc)2 1%
O
Cl-
MOMO
6
8
R
N+
O
HO
N
R
O
O
O
NEt
, xyl. 120 ºC
3a
HO
OH
73%
OAc
82%
3b
MOMO
MOMO
R=adamantyl 88%
2,6-di-i-PrPh 72%
OH
(2 steps)
9
O
O
HO
Scheme 2. Decarbonylative Heck coupling.
74%
3c
OH
OH
O
to access the neutral product 5. Thionyl chloride with added ben-
zotriazole was used to access acid chloride 6, which was used with-
out purification in the decarbonylative Heck coupling step with 4-
acetoxystyrene 7. Palladium acetate (1 mol %) with an N-heterocy-
clic carbene (NHC) ligand 8 (1 mol %) were employed with N-ethyl-
morpholine as base in xylenes at 120 °C. The acid chloride 6 and
acetoxystyrene 7 were used at 1:1.25 stoichiometry. Previous re-
sults using N,N-bis-2,6-diisopropylphenyl-NHC (IPr) gave the prod-
uct stilbene 9 in 72% overall yield.4 The bulky commercially
available N,N-bis-adamantyl-NHC ligand 8 (R = adamantyl) now
produced the protected product in 88% yield for the two step
sequence. Steric constraints of NHC ligands have been previously
reported that improve related palladium catalyzed coupling
reactions.9
O
HO
OH
76%
3d
OH
O
O
O
HO
O
45%
3e
OH
Scheme 3. Synthesis of 40-resveratrol esters.
The acetate 9 was removed with base and the resulting 40-phe-
nol was acylated with various acid chlorides (Scheme 3). Sodium
hydroxide followed by acidification gave the phenol in 91% yield
and the acylations were efficiently performed individually with
the corresponding acid chlorides with added pyridine to access
the 40-esters 10 (88–91%). Finally, the desired 40-resveratrol esters
3a–e were obtained by deprotecting the 3,5-bis-MOM 10 interme-
diates using anhydrous HCl (2.0 M) in ether.10 Previous conditions
for selective bis-MOM removal employed trimethylsilyl iodide,
generated in situ from TMSCl and NaI.4 These conditions were
found to be problematic for larger scale production of 40-acetate
2 and with the new 40-esters. The new HCl approach for MOM re-
moval requires extended time (36 h), however the product is now
produced in reliable yield. Only the adipate dimer ester 3e was ob-
tained in lower yield, 45%. All the others, the n-butyrate, i-butyrate,
n-hexanoate, and palmitoyl products were produced in moderate
to good yield (73–82%).
F
OAc
HO
AcO
11
F
12
OAc
AcO
OH
OH
AcO
HO
13
AcO
14
Scheme 4. Additional resveratrol-analogs.
In addition to the new 40-esters, four other resveratrol analogs
were included in the melanoma cell assays.4 3,40-Difluoro-resvera-
trol 11, triacetate 12, 3,5-diacetate 13, and 3-acetate-resveratrol
14, reported previously (Scheme 4), were included to provide for
a broad structure–activity profile in the cancer cell inhibition
investigations.
trol or its analogs were then added and cells were incubated for 24,
48, or 72 h. Cell viability was then quantified using a colorimetric
assay. Cell lines were treated with resveratrol and ten analogs of
resveratrol at 10, 25, and 50
lM and evaluated at multiple time
With the set of new resveratrol analogs in hand, cell assays
were performed to assess in vivo potential. DM443 and DM738,
two previously characterized melanoma cell lines derived from hu-
man tumor tissue, were chosen for this study due to their chemo-
resistance properties to melphalan (LPAM) and temozolomide
(TMZ) respectively.11 DM443 and DM738 were plated and resvera-
points. In comparing efficacies at the 50
l
M dose, analogs 3b, 3c,
3a, and 11 are shown to be significantly more cytotoxic than resve-
ratrol at all time points in both cell lines (all p’s <0.025), analog 3d
does not show any effect at any time point in either cell line
(0.34 < p < 0.83), and analogs 2, 3e, 12, 13 and 14 have varying effi-
cacies between these two groups (Fig. 1). Analog 2 is shown to be

1200
Y. Wong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1198–1201
Figure 1. DM738 and DM443 treated with resveratrol and analogs for 24 (A and B) and 72 h (C and D) respectively. Data are expressed as mean SEM, n = 6.
equally effective as native resveratrol at every time point and dose
in both cell lines.
Normal human dermal fibroblast (NHDF) cells were treated
with resveratrol and analogs for 72 h at 50 lM to evaluate if the
analogs were also selectively cytotoxic to malignant cells. We
found that analogs 2 and 3e relatively spared NHDF cells compared
to their cytotoxic effect in melanoma cell lines (both p’s <0.0001)
(Fig. 2).
In order to evaluate whether the cytotoxic effects observed in
melanoma cell lines would translate to other cancer types, a malig-
nant line of pancreatic cells (Panc-1) was treated with either resve-
ratrol, analog 2 or analog 12 at 50
standard treatment of advanced pancreatic cancer,12 was also
added at 0, 1, or 10 g/mL. Resveratrol and both analogs mani-
lM for 72 h. Gemcitabine, a
l
fested significant cytotoxicity compared to both vehicle alone
(p <0.0001) and to treatment with gemcitabine alone at all doses
tested (p’s <0.0001) (Fig. 3). We also found that the addition of
gemcitabine did not enhance the cytotoxic response of resveratrol
or its analogs (all p’s > 0.05).
In summary, various 40-resveratrol esters have been produced
using a decarbonylative Heck coupling and a structure–activity
profile has been produced from melanoma cell assays. Further
studies with additional cells lines and in vivo investigations are
needed to further establish the potential of compounds of this
type.
Figure 3. (A) Panc-1 treated with resveratrol or analog for 72 h. (B) Panc-1 treated
with resveratrol or analog gemcitabine, or gemcitabine alone for 72 h. Data are
expressed as mean SEM, n = 6.
Acknowledgments
We are grateful for support provided by the Brigham Young
University Cancer Research Center. This work was also supported
by a Merit Review Grant to DST.
Supplementary data
Supplementary data (experimental procedures, characteriza-
tion, NMR spectra, and cell assay methods) associated with this
article can be found, in the online version, at doi:10.1016/
j.bmcl.2009.12.006.
Figure 2. Cell lines DM738, DM443, and NHDF were treated with resveratrol or
analog at 50 lM for 72 h. Data are expressed as mean SEM; n = 6.

Y. Wong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1198–1201
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