Synthesis and preliminary in vitro biological evaluation of 4-[(4-hydroxyphenyl)sulfanyl]but-3-en-2-one, a 4-mercaptophenol derivative designed as a novel bifunctional antimelanoma agent

Article abstract of DOI:10.1021/jm900642j

We report the synthesis and preliminary in vitro biological evaluations of 4-[(4-hydroxyphenyl) sulfanyl]but-3-en-2-one, a compound designed as a potential bifunctional antimelanoma agent, bearing both a tyrosinase-activatable phenolic moiety and a GSH-re

Full text of DOI:10.1021/jm900642j

J. Med. Chem. 2009, 52, 4973–4976 4973
DOI: 10.1021/jm900642j
Synthesis and Preliminary in Vitro Biological Evaluation of 4-[(4-Hydroxyphenyl)sulfanyl]but-3-
en-2-one, a 4-Mercaptophenol Derivative Designed As a Novel Bifunctional Antimelanoma Agent
Paolo Ruzza,† Antonio Rosato,‡,§ Alberto Nassi, Maria Rondina,‡ Matteo Zorzin,† Carlo Riccardo Rossi,‡,§
Maura Floreani, ,^ and Luigi Quintieri*, ,^
†Istituto di Chimica Biomolecolare del CNR, Sezione di Padova, via Marzolo 1, Padova, Italia, ‡Dipartimento di Scienze Oncologiche
e Chirurgiche, Universitaꢀ di Padova, Padova, Italia, §Istituto Oncologico Veneto (IOV), IRCCS, via Gattamelata 64, Padova,
Italia, and Dipartimento di Farmacologia e Anestesiologia, Universitaꢀ di Padova, L.go Meneghetti 2, Padova, Italia. ^ These authors
contributed equally to this work.
Received May 26, 2009
We report the synthesis and preliminary in vitro biological evaluations of 4-[(4-hydroxyphe-
nyl)sulfanyl]but-3-en-2-one, a compound designed as a potential bifunctional antimelanoma agent,
bearing both a tyrosinase-activatable phenolic moiety and a GSH-reactive R,β-unsaturated carbonyl
group. Both the E (1) and Z (2) isomers of the synthesized compound proved to be very good substrates
of mushroom tyrosinase, reacted quickly with GSH at physiological pH, and showed a significant
cytotoxic activity against B16F1 murine melanoma cells.
Introduction
R,β-unsaturated carbonyl group, this latter conferring reac-
tivity toward GSH.9 Three further compounds with different
substitutions at the 4-mercapto group and lacking the R,
β-unsatured moiety (3-5, Figure 1) were also synthesized
for the purpose of comparison. All the synthesized com-
pounds were tested in vitro for their rate of oxidation by
mushroom tyrosinase, their spontaneous and GST-catalyzed
reaction with GSH, and their cytotoxic activity against B16F1
murine melanoma cells.
Malignant melanoma is an extremely aggressive tumor of
melanocytic origin. Like normal melanocytes, melanoma
cells synthesize melanin pigments through a complex pathway
in which a crucial role is played by tyrosinase (EC 1.14.18.1).
This enzyme catalyzes both the rate-limiting oxidation of
L-tyrosine to L-DOPA and the subsequent conversion of
L-DOPA to L-DOPA quinone, a highly reactive o-quinone
yielding melanins through a series of enzymatic and none-
nzymatic reactions.1 Because tyrosinase is frequently upregu-
lated in melanoma cells, phenolic and catecholic compounds
have been investigated as tyrosinase-activated antimelanoma
prodrugs, ultimately producing alkylating cytotoxic o-qui-
nones. Examples are 4-hydroxyanisole (4-HAa, Figure 1),
4-S-cysteaminylphenol, and 4-S-cysteaminylcatechol, all hav-
ing shown antimelanoma activity both in in vitro and in in vivo
studies.2-5 Because a byproduct of melanogenesis is hydrogen
peroxide, which can easily cross cell membranes and that, if not
appropriately processed, can generate cytotoxic reactive oxy-
gen species, GSH and cytosolic GSTs (EC 2.5.1.18), are
critically important to the maintenance of cells of melanocytic
origin. Melanomas, as well as other tumors, often exhibit
upregulation of both GSH and some GSTs, in particular
GSTP1-1,6 these events likely contributing to tumor resistance
to some anticancer agents.7 In this regard, a novel therapeutic
strategy based on GST-activated anticancer prodrugs has been
recently proposed.8-11
Results and Discussion
Tyrosinase/O2-Mediated Oxidation of 1-5 and 4-HA. The
time-course of tyrosinase/O2-mediated oxidation of 1-5 and
4-HA, a prototypical tyrosinase-oxidizable phenol,2 was
analyzed by a UV-vis spectroscopy method. In the absence
of tyrosinase, all compounds showed a maximum absor-
bance peak in the 250-320 nm range (see Figure 2 in
Supporting Information). Upon the addition of tyrosinase
to 1-4 and 4-HA, the absorbance of the respective maxi-
mum absorbance peak decreased and a characteristic peak
appeared at 460 nm, thus indicating the formation of an
o-quinone.12 Surprisingly, 5 did not exhibit these modifica-
tions of its UV-vis spectrum, suggesting that methylation of
the carboxyl group prevents oxidation of 4 by tyrosinase.
Kinetic analyses were carried out by measuring the initial
rate of tyrosinase-catalyzed o-quinone formation in the pre-
sence of increasing concentrations of 1-4 and 4-HA. Com-
pounds 1-4 exhibited similar affinity for the enzyme, their
Km values (ranging from 20 to 36 μM) being lower than that
of 4-HA (about 47 μM). By contrast, significant differences
were found in Vmax and kcat values of the compounds, the
rank order of these parameters being 2>1.3>4>4-HA.
Overall, mean kinetic parameters for the oxidationof1-4 and
4-HA by tyrosinase (summarized in Table 2 in Supporting
Information) indicate that 1 and 2 are the best among the
herein studied tyrosinase substrates.
In this work, we report the synthesis of a novel bifunctional
antimelanoma agent, 4-[(4-hydroxyphenyl)sulfanyl]but-3-en-
2-one, obtained as E and Z isomers (1 and 2, respectively,
Figure 1), characterized by the simultaneous presence of a
tyrosinase-activatable phenolic moiety and a GSH-reactive
*To whom correspondence should be addressed. Phone: (þ39) 049
827 5083. Fax: (þ39) 049 827 5093. E-mail: luigi.quintieri@unipd.it.
a Abbreviations: GSH, glutathione; GST, glutathione S-transferase;
4-HA, 4-hydroxyanisole.
r
2009 American Chemical Society
Published on Web 07/14/2009
pubs.acs.org/jmc

4974 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Ruzza et al.
Scheme 1. Proposed Scheme for the GSH-Dependent Metabo-
lism of Compound 2
Figure 1. Chemical structures of 4-HA and compounds 1-5.
Reactivity toward GSH. The rate of the nucleophilic attack
of GSH on the β-carbon of R,β-unsaturated carbonyl com-
pounds is known to be pH-dependent.10 Thus, the reactivity
of each synthesized compound toward GSH was evaluated
by incubation with GSH for increasing times (0-120 min) at
different pH values (6.0, 7.4, and 8.0). Compounds 3-5,
which lack the R,β-unsatured carbonyl moiety, did not show
any detectable reactivity toward GSH (data not shown),
whereas 1 and 2 reacted with GSH in a pH-dependent
manner (Figure 3 in Supporting Information). While at
pH 6.0 the rate of the reaction of 1 and 2 with GSH was
negligible, at pH 7.4 and 8.0, the reaction proceeded at
a much higher rate and followed pseudo-first-order kinetics.
t1/2 values for 1 were 20.5 and 6.1 min at pH 7.4 and
8.0, respectively, and t1/2 values for 2 were 38.6 and 18.4
min at pH 7.4 and 8.0, respectively.
The interaction of 1 and 2 with GSH was further investi-
gated at pH 7.4 either in the presence or in the absence of rat
liver cytosol, as a source of GSTs. Rat liver cytosol markedly
increased the reactivity of both compounds toward GSH
(data not shown), and t1/2 values of 2.2 and 7.24 min were
obtained for 1 and 2, respectively.
The reaction of 1 or 2 with GSH yielded two major
products, which were separated by HPLC (Figure 4 in
Supporting Information) and then identified by ESI-MS.
A first product was identified as the expected GSH-bute-
none adduct (GS-A in Scheme 1), originating from the
addition of GSH to the activated double bond. The second
product was the geometrical isomer of the parent compound
(Scheme 1), the conversion of the Z isomer (2) into the
E isomer (1) being more rapid and occurring at a greater
extent than the reverse reaction. This reaction is very efficient
for the R,β-unsaturated compounds13 and proceeds via
addition-elimination of GSH to the double bond.14 Accord-
ing to the proposed Scheme 1, GS-A formation should
occur via elimination of the 4-mercaptophenol moiety.
However, in our experimental conditions, this species
was never found as a product of the reaction. By contrast,
the incubation mixtures of 1 or 2 with GSH and rat liver
cytosol contained a new compound with retention time and
m/z value identical to that of 4,40-dihydroxydiphenyl disul-
fide (6, Scheme 1), the product of spontaneous oxidation of
4-mercaptophenol.
Thereafter, we synthesized 6 and evaluated some of its
biochemical properties. Compound 6 was a good substrate
for mushroom tyrosinase, being provided with the lowest
Km value (16.44 ( 2.29 μM) among the prepared com-
pounds. Its Vmax value (0.234(0.012 μM s-1), which was
significantly higher than that obtained with 3, 4, and 4-HA,
as well as its kcat and kcat/Km values (1.52 s-1 and 32683,
respectively), indicated that tyrosinase catalytic efficiency
for 6 was quite similar to that for 2. Compound 6 was even
more reactive than 1 and 2 toward GSH, its t1/2 value being
less than 1.0 min upon incubation at pH 7.4 in the presence of
GSH and rat liver cytosol (data not shown).
Cytotoxic Effects of the Novel Compounds. The last set of
experiments evaluated the cytotoxic effects of the synthe-
sized compounds and 4-HA toward murine melanoma
B16F1 cells. The results in Table 1 show that 1, 2, and
6 were effective cytotoxic agents provided with similar
IC50 values (17.4, 19.2, and 21.2 μM for 1, 2, and 6,
respectively). They were more active and more potent than
4-HA, 3, and 4. Compound 5, which is not a substrate for
tyrosinase and does not react with GSH, was devoid of
cytotoxic effects. Moreover, N-acetyl-L-cysteine (NAC), a
cysteine donor for GSH synthesis,15 did not influence the
effect of 4-HA on B16F1 cell viability while it significantly
decreased the cytotoxicity of 1, 2, and 6 (Table 1). By
contrast, 1-phenyl-2-thiourea (PTU), an inhibitor of tyrosi-
nase activity,16 did not alter the cytotoxicity of 1, 2, and 6,
while slightly decreased the effect of 4-HA. These results

Brief Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4975
Table 1. Cytotoxic Effects of 1-6 and 4-HA on Murine Melanoma
B16F1 Cells: Effects of NAC and PTU
3-[(4-Hydroxyphenyl)sulfanyl]propanoic Acid (3). Rf: 0.45I,
0.55II. 1H NMR (DMSO-d6): H2 δ 2.40 (2H, t), H3 δ 2.91
(2H, t), H6,10 δ 7.21 (2H, d), H7,9 δ 6.72 (2H, d), H OH δ 9.57
(1H, s). [M - H]-=197.03 m/z, calcd for C9H10O3S 198.04 Da.
[(4-Hydroxyphenyl)sulfanyl]acetic Acid (4). Rf: 0.31I, 0.45II.
1H NMR (DMSO-d6): H2 δ 3.54 (2H, s), H5,9 δ 7.23 (2H, d),
H6,8 δ 6.70 (2H, d), H OH δ 9.55 (1H, s). [M - H]-=183.01 m/z,
calcd for C8H8O3S 184.02 Da.
cell viability at 100 μM (% of control)
IC50 value (μM)
-
þPTU (0.2 mM) þNAC (5 mM)
4-HA
nd
61.3 ( 7.1
1.3 ( 1.2
3.1 ( 1.3
77.4 ( 12.5
69.4 ( 9.3
101.4 ( 3.5
0.2 ( 0.1
78.7 ( 5.0
0.0
0.0
60.0 ( 3.5
41.4 ( 5.2
43.8 ( 4.5
1
2
3
4
5
6
17.4 ( 2.3
19.2 ( 1.9
nd
Methyl [(4-Hydroxyphenyl)sulfanyl]acetate (5). Rf: 0.73I,
0.75II. 1H NMR (DMSO-d6): H1 δ 3.56 (3H, s), H3 δ 3.62 (2H,
s), H6,10 δ 7.23 (2H, d), H7,9 δ 6.71 (2H, d), H OH δ 9.63 (1H, s).
[M - H]-=197.03 m/z, calcd for C9H10O3S 198.04 Da.
4,40-Dihydroxydiphenyl Disulfide (6). The compound was
synthesized by dissolving 4-mercaptophenol in 1:1 (v/v) H2O-
acetonitrile in a test tube at room temperature. The reaction was
allowed to proceed 12 h at room temperature, after which the
reaction mixture was lyophilized and the crude product was
purified by recrystallization from ethyl acetate/petroleum ether.
HPLC analysis showed that purity of the final product was
greater than 96%.
nd
nd
21.2 ( 5.6
0.0
22.5 ( 3.0
The results are means ( SE from three experiments carried
out in quadruplicate; nd, not determined.
allow to conclude that: (a) the mechanism responsible for
the cytotoxic activity of 1, 2, and 6 is different from that of
4-HA, (b) although the cytotoxicity of 4-HA could be
dependent on its tyrosinase-mediated metabolism, the cyto-
toxic effects of 1, 2, and 6 are mainly due to their high
reactivity toward GSH, (c) 6, which is a secondary product
of GSH reaction with 1 or 2, could play a role in the
biological effects of these compound because its IC50 value
was quite similar to those of the two parent compounds and
it was highly reactive toward GSH.
1
Rf: 0.65I; 0.60II. H NMR (DMSO-d6): H5,50,3,30 δ 7.25
(2H, d), H6,60,2,20 δ 6.73 (2H, d), H OH δ 9.81 (1H, s).
[M-H]-=249.00 m/z, calcd for C12H10O2S2 250.34 Da.
Acknowledgment. This work was supported by a grant
from “Associazione Piccoli Punti”;ONLUS, Padova, Italy,
and was partially supported by a grant (CPDA064182) from
University of Padova, Italy.
Conclusions
Supporting Information Available: Full experimental details
for TLC, RP-HPLC, mass spectrometry, and NMR; for bio-
chemical assays, and for the in vitro cytotoxicity assays. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Overall, our results indicate that 1 and 2 are very interesting
and promising lead compounds for future development of
potential agents targeting tumors containing a high concen-
tration of GSH and/or expressing high levels of GST.
Experimental Section
References
(E)-, (Z)-4-[(4-Hydroxyphenyl)sulfanyl]but-3-en-2-one (1, 2).
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3 equiv of 3-butyn-2-one were added to the solution. The
reaction was allowed to proceed for 30 min, and thereafter the
reaction mixture was lyophilized and the isomers were separated
and purified by preparative RP-HPLC (see Supporting In-
formation). Fractions containing either Z or E isomer were
collected, combined, and lyophilized. Purity of the final pro-
ducts was greater than 96% as determined by HPLC. Under the
reaction conditions used, the ratio of E to Z isomers formed was
approximately 1:1.
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