Rapid synthesis of triazole-modified resveratrol analogues via click chemistry

Article abstract of DOI:10.1021/jm051118z

Resveratrol is a phytoalexin able to display an array of biological activities. We decided to replace the double bond with a triazole ring using the archetypical click reaction: the Huisgen [3 + 2] cycloaddition. Seventy-two triazole derivatives were synt

Full text of DOI:10.1021/jm051118z

J. Med. Chem. 2006, 49, 467-470
467
Scheme 1. Huisgen [3 + 2] Cycloaddition
Rapid Synthesis of Triazole-Modified Resveratrol
Analogues via Click Chemistry
Francesca Pagliai,§ Tracey Pirali,§ Erika Del Grosso,
Riccardo Di Brisco, Gian Cesare Tron,* Giovanni Sorba, and
Armando A. Genazzani*
interesting pathways, but its low potency and the plethora of
actions limit its applications.
Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e
Farmacologiche and Drug and Food Biotechnology Center,
UniVersita` degli Studi del Piemonte Orientale “A. AVogadro”,
Via BoVio 6, 28100 NoVara, Italy
The concept of click chemistry is experiencing a growing
popularity.13 This term, coined by Barry K. Sharpless,14 now
refers to reactions yielding the product in high yield without
the need for further purification, generating inoffensive byprod-
ucts, and operating in a benign solvent, usually water. In this
way, it is possible to generate a plethora of new compounds
reliably and thereby accelerate the process of drug discovery.
In the present paper, we have synthesized a series of trans-
resveratrol analogues replacing the double bond with a triazole
ring. The substitution with a triazole ring would have the great
advantage of making these analogues suitable for combinatorial
chemistry. Indeed, we employed the archetypical “click” reac-
tion: the Huisgen [3 + 2] cycloaddition between alkynes and
azides catalyzed by copper(I) salts14 (Scheme 1). The presence
of the copper(I) salts at room temperature generates 1,4
regioselective products,15 which contrasts with the thermal
reaction, which demands high temperatures and gives equimo-
lecular quantities of the two regioisomers, which are often
difficult to separate. Indeed, out of 88 parallel reactions
attempted, 74 yielded precipitates, and 72 of these were
confirmed as the expected products. As a proof of principle,
we also have evaluated the cytotoxicity of these compounds,
and the preliminary biological analysis suggests that this
procedure is capable of generating active triazole-substituted
resveratrol analogues.
ReceiVed NoVember 7, 2005
Abstract: Resveratrol is a phytoalexin able to display an array of
biological activities. We decided to replace the double bond with a
triazole ring using the archetypical click reaction: the Huisgen [3 +
2] cycloaddition. Seventy-two triazole derivatives were synthesized via
a parallel combinatorial approach. Preliminary data suggest that this
procedure can lead to the synthesis of compounds that display some,
but not all, of resveratrol’s actions with increased potency.
The increasing knowledge of molecular cell biology and the
advances in biomedical research have increased exponentially
the known targets for therapeutic intervention. Such an increase
has been paralleled by an increased interest in natural com-
pounds as lead compounds for novel pharmacological screen-
ings. In this respect, there has been great interest in resveratrol
(1). The relatively high concentrations in wine and the original
To establish whether the double bond of resveratrol is
amenable to bioisosteric substitution with the triazole moiety,
compounds 2-6 were synthesized (Figure 1) using the Huisgen
[3 + 2] cycloaddition via the Sharpless protocol.14,15 As a
preliminary screening, these compounds were tested in a
cytotoxicity/proliferation assay in neuroblastoma (SH-SY5Y),
breast cancer (MDA-MB-231), basophilic leukemia (RBL 2H3),
and human pancreatic carcinoma (FG2) cell lines. We employed
a colorimetric method (MTT; based on mitochondrial activity),
which does not distinguish between decreased proliferation and
increased cell death.16 In all the cell lines, resveratrol was
capable of inducing a reduction of cell growth after 48 h
incubation. Triazole-derivatives were not uniform in their
behavior with some compounds not eliciting any effect and
observations of its cardioprotective role (which formed the basis
of the French paradox)1 brought this compound to notoriety.
Most of initial work performed on this molecule was indeed
on the cardiovascular system, including effects on lipid me-
tabolism and platelet function,2 but reports on cancer protection
opened new avenues.3-5 The main problem with resveratrol
research is that this molecule exerts most of its effects at high
micromolar concentrations,4 and it is therefore difficult to
ascertain which are the targets responsible for the single effects.
Among the potential beneficial uses of resveratrol, it has been
proposed that it might act as a cardioprotective and neuropro-
tective agent, as an antiinflammatory drug and in cancer
chemoprotection.4 The potential candidate targets are even
greater: alongside the inhibition of lipid peroxidation, inhibition
of nitric oxide synthase, inhibition of cyclooxygenases, and
accumulation of ceramide,4,6,7 there is growing evidence that a
number of proteins involved in signal transduction, in the cell
cycle, in regulating transcription, and in ionic conductance are
modulated by resveratrol.4,8-11 In this context, it has also been
shown that resveratrol can mimic estrogens in activating ERR
and â receptors.4,12 In summary, therefore, resveratrol possesses
the potential to intervene in a number of therapeutically
* To whom correspondence should be addressed. Tel: +39-0321-375857.
Fax: +39-0321-375821. E-mail addresses: tron@pharm.unipmn.it; genazzani@
pharm.unipmn.it.
§ These authors equally contributed to the present work.
Figure 1. Structure of triazole-modified resveratrol analogues.
10.1021/jm051118z CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/21/2005

468 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Letters
Table 1. Effect of Resveratrol and Triazole-Substituted Analogues on Viability in a Panel of Cell Linesa
1
2
3
4
5
6
SH-SY5Y
MDA-MB-231
RBL 2H3
FG2
33.5 ( 3.7
67.9 ( 0.9
52.1 ( 2.3
42.2 ( 1.6
93.4 ( 4.7
97.0 ( 2.4
110 ( 2.7
93.7 ( 2.7
86.6 ( 8.5
95.5 ( 0.8
114 ( 4.6
91.9 ( 2.8
85.9 ( 1.0
99.3 ( 5.8
113 ( 3.1
78.6 ( 4.3
93.2 ( 2.1
116 ( 1.4
88.2 ( 2.3
62.2 ( 2.1
72.2 ( 2.0
99.6 ( 5.0
83.7 ( 2.6
100.8 ( 4.6
a Values represent the % viability, as measured by the MTT assay, of control or treated cells at 48 h and are mean ( SEM of four determinations. All
compounds were used at a concentration of 100 µM.
others appearing efficacious at 100 µM (Table 1). Yet, differ-
ences in the extent of resveratrol action were observable among
the cell lines, as were differences in compounds 2-6. For
example, 6 did not have any effect on RBL 2H3 cells, it
paralleled the effect of 1 in MDA-MB-231 cells, and it had a
lower effect compared to 1 in the other two cell lines. To further
understand these differences, a time-course (measuring cell
number) up to 96 h was performed with compounds 1, 4, and
6 in MDA-MB-231 and SH-SY5Y cells. Indeed, major differ-
ences were observed in proliferation rates between control and
treated cells with the three compounds, and these differences
were more evident at 96 h treatment. Under these conditions,
100 µM resveratrol was capable of inhibiting completely cell
proliferation, as was compound 6. The per-methylated 4 was
unable to affect MDA-MB-231 proliferation, while it reduced
SH-SY5Y proliferation by 50% (data not shown). It has already
been shown that methylated or per-methylated analogues of
trans-resveratrol display biological activities, at times increased
over the parent compound,17 and therefore it was not surprising
that 6 and 4 unmasked an activity that was not significant in
the demethylated compounds (2,3). These preliminary data
suggested that a combinatorial approach could be beneficial
toward finding resveratrol analogues that maintained some, but
not all, properties of the parent compound.
As a proof of principle that click chemistry could be feasible
for rapid parallel combinatorial synthesis of resveratrol ana-
logues, we used 8 alkynes and 11 azides. The feasibility of this
approach is given by the precipitation of the resultant product.
The product can then be purified by filtration and ether washes.
This procedure resulted in 74 of the expected 88 compounds
being formed, as detected by precipitation. All precipitated
compounds were submitted to MS analysis to verify their
authenticity. Indeed, 67/74 (91%) were confirmed as the
expected product. Two of the compounds yielded no peak at
the predicted mass. Compounds Ia, Ic, Id, If, and Ig displayed
a mass fragmentation compatible with a triazole product yet
failed the original mass verification, as we observed a base peak
at m/z [M - H]+ in positive mode and a [M - 3H]- in negative
mode. Originally, we intended to use 2,3,5,6-tetrafluorophenyl
azide synthesized from 2,3,5,6-tetrafluoroaniline. Yet, the mass
suggested the presence of a hydroxyl group instead of a fluorine.
Indeed, 13C NMR, 1H NMR, and MS analysis (see Supporting
Information) of the azide I revealed that this compound
displayed a hydroxyl group in the ortho position instead of the
expected fluorine. This suggests that the original amine under-
went an aromatic nucleophilic substitution of one of the four
fluorine groups with water during the azidation protocol.18 In
conclusion, therefore, the procedure resulted in 72 triazole
products.
cytotoxic effect of 1 was modest in these cells (Table 2), this
compound can also induce a morphological change in these cells
that can be observed in phase contrast microscopy19 (see
Supporting Information). All compounds were tested at decreas-
ing concentrations in 96-well plates to establish an automated
procedure. Cells treated with 1 (100 µM) displayed 80.2% (
2.7% viability of control (n ) 47), while at 10 µM, they
displayed 106% ( 7.2% viability of control (n ) 33). Out of
the 72 compounds tested, 54 were unable to reduce viability (a
cutoff of 70% of control was arbitrarily chosen) at a concentra-
tion of 10 µM. It is interesting that some of these compounds
(Ic, Hc, and Mc) induced a significant proliferation of cells
(154% ( 12.8%, 123% ( 4.7%, and 174% ( 11.1% compared
to control, respectively; n ) 5). Since 1 has been shown to
possess estrogenic activity, it would be appealing to speculate
that these compounds retain this activity, while they do not retain
the antiproliferative activity.12,20 Of the remaining compounds,
nine possessed an approximate IC50 between 1 and 10 µM, eight
between 100 nM and 1 µM, and one between 10 nM and 100
nM (Fc). As a confirmation of the protocol, the lead compounds
arising from this screening (Aa, Ca, Da, La, Ac, Fc, Ad, and
Ed) were submitted to HPLC analysis to confirm purity.
Compounds with a purity lower than 95% (Aa, Ca, Da, and
La; all over 85%) were resynthesized, reverified, and reevalu-
ated (Aa bis, Ca bis, Da bis, La bis). The new compounds
possessed identical biological activity (data not shown).
It has been previously shown that MDA-MB-231 cells change
morphological characteristics upon resveratrol addition.19 This
change, which might or might not be correlated to the decreased
proliferation/increased cell death, is represented by an increased
filopodia extension.19 When cells were incubated for 48 h with
resveratrol this phenomenon could be reproduced at concentra-
tions over 50 µM (see Supporting Information). We then
attempted to establish whether the lead compounds were able
to reproduce this effect at low concentrations. We therefore used,
for each of the lead compounds, concentrations in the range of
the approximate IC50 established in the cytotoxicity assay.
Indeed, compounds Ac and Ad revealed similar processes
(although not to the same extent compared to high concentra-
tions of 1), while no morphological changes appeared evident
with Aa, Ca, Da, Ed, Fc, and La (see Supporting Information).
Among other reports on resveratrol is its ability to induce a
modest S-phase arrest of the cell cycle.21 Indeed, in MDA-MB-
231 cells, 31.5% ( 2.1% of control cells were in S-phase, while
this number increased to 49.5% ( 5.6% in resveratrol-treated
cells (10 µM). Of the lead compounds tested, only Aa (10 nM)
and Ad (10 nM) induced an increase in the S-phase percentage
(39% ( 4.2% and 41.5% ( 0.7%). Cells treated with the other
lead compounds did not show any significant change in the cell
cycle characteristics (S-phase Ca 100 nM 29% ( 1.4%; Da
100 nM 32.5% ( 6.4%; La 100 nM 32% ( 4.2%; Ac 10 nM
32% ( 8.5%; Fc 100 nM 35.5% ( 5.0%). It is interesting to
note that compound Ad shared with 1 both characteristics
investigated (morphology and cell cycle), while Ac and Aa
shared just one. This would suggest that our aim of generating
As mentioned above, resveratrol possesses numerous potential
molecular targets resulting in multiple potential cellular out-
comes. It is therefore possible that all the compounds synthe-
sized share with the original molecule at least one of its actions.
To evaluate a potential biological effect of these triazole-
substituted compounds, we decided to investigate cytotoxic
effects in MDA-MB-231 cells. Although the antiproliferative/

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 469
Table 2. Cytotoxic/Antiproliferative Effect of Click Chemistry Generated Resveratrol Analoguesa
a Experiments were performed at log intervals, and values represent the approximate IC50 values expressed as nM (n ) 4-6 for log unit performed);
np ) not precipitated; mf ) mass spectrometry failed to reveal the expected product.
potent analogues that did not display all the mechanisms of
resveratrol was accomplished.
analogues with estrogenic activity. On the other hand, this
preliminary screening should be extended to more sophisticated
and aimed biological assays to unravel structure-activity
relationships of other actions of resveratrol.
In conclusion, we have employed click chemistry to generate
triazole-substituted resveratrol analogues. At high concentra-
tions, resveratrol possesses numerous actions with a therapeutic
potential, and therefore the rapid triazole synthesis of resveratrol
analogues described here could be used to generate an enormous
chemical database. In the present paper, as a proof of principle,
we have generated a preliminary screening for analogues with
an antitumoral potential. We have found that some of the
compounds screened were more potent than resveratrol as
It would be appealing to draw a structure-activity relationship
with the compounds synthesized and screened. Indeed, a few
indications can be extrapolated or postulated. For example,
compounds with a para-methoxy group on either ring appear
to be cytotoxic. Yet, the cytotoxicity of these compounds is
unlikely to resemble that of combretastatin A4,22 because
compounds Hf and Db, the closest analogues of this molecule,
were devoid of cytotoxic activity. Furthermore, compounds with
a Grimm’s isosteric substitution (i.e., OH-NH2 in the para
position, Ic, Hc, and Mc) displayed a proliferative effect, and
these might therefore be good lead compounds for resveratrol

470 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Letters
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actions of resveratrol, it is difficult to ascertain whether the
effects observed are indeed correlated with a resveratrol-like
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did display similarities compared to resveratrol. This protocol
will allow us to generate agents that possess less promiscuity
compared to resveratrol and therefore might prove therapeuti-
cally useful.
Acknowledgment. Financial support from Universita` del
Piemonte Orientale and Regione Piemonte is gratefully ac-
knowledged.
Supporting Information Available: Synthesis of alkyne and
azide building blocks, characterization (MS and 1H and 13C NMR
data), and HPLC purities of the selected compounds, raw data
relative to Table 2, and morphological changes induced by the lead
compounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
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chemical function from a few good reactions. Angew. Chem., Int.
Ed. 2001, 40, 2004-2021.
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stepwise Huisgen cycloaddition process: copper (I) catalyzed regio-
selective “ligation” of azide and terminal alkynes. Angew. Chem.,
Int. Ed. 2002, 41, 2596-2599. (b) Tornøe, C. W.; Christensen, C.;
Meldal, M. Peptidotriazoles on solid phase: [1,2,3]-Triazole by
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