Selenium-containing chrysin and quercetin derivatives: Attractive scaffolds for cancer therapy

Article abstract of DOI:10.1021/acs.jmedchem.5b00230

Selenium-containing chrysin (SeChry) and 3,7,3′,4′-tetramethylquercetin (SePQue) derivatives were synthesized by a microwave-based methodology. In addition to their improvement in terms of DPPH scavenging and potential GPx-like activities, when tested in a panel of cancer cell lines both selenium-derivatives revealed consistently to be more cytotoxic when compared with their oxo and thio-analogues, evidencing the key role of selenocabonyl moiety for these activities. In particular, SeChry elicited a noteworthy cytotoxic activity with mean IC₅₀ values 18- and 3-fold lower than those observed for chrysin and cisplatin, respectively. Additionally, these seleno-derivatives evidenced an ability to overcome cisplatin and multidrug resistance. Notably, a differential behavior toward malignant and nonmalignant cells was observed for SeChry and SePQue, exhibiting higher selectivity indexes when compared with the chalcogen-derivatives and cisplatin. Our preliminary investigation on the mechanism of cytotoxicity of SeChry and SePQue in MCF-7 human mammary cancer cells demonstrated their capacity to efficiently suppress the clonal expansion along with their ability to hamper TrxR activity leading to apoptotic cell death.

Full text of DOI:10.1021/acs.jmedchem.5b00230

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Article
Selenium-containing Chrysin and Quercetin
Derivatives: Attractive scaffolds for cancer therapy.
Inês L. Martins, Catarina Charneira, Valentina Gandin, João L. Ferreira da Silva, Gonçalo C.
Justino, João P. Telo, Abel J.S.C. Vieira, Cristina Marzano, and Alexandra M. M. Antunes
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00230 • Publication Date (Web): 23 Apr 2015
Downloaded from http://pubs.acs.org on April 25, 2015
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Journal of Medicinal Chemistry is published by the American Chemical Society.
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Selenium-containing Chrysin and Quercetin Derivatives: Attractive scaffolds for cancer
therapy.
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ⱡ**
ⱡ**
‡*
ⱡ
Inês L. Martins , Catarina Charneira , Valentina Gandin , João L. Ferreira da Silva,
ⱡ
ⱡ
┤
‡
Gonçalo C. Justino, João P. Telo, Abel J.S.C. Vieira , Cristina Marzano , Alexandra
M. M. Antunesⱡ*
ⱡ
Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa,
Lisboa, 1049-001, Portugal
‡Dipartimento di Scienze del Farmaco, Università di Padova, via Marzolo 5, 35131
Padova, Italy
┤
LAQV, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia,
Universidade Nova de Lisboa, 2829-516 Caparica, Portugal
*
*
Corresponding authors.
* These authors contributed equally for this work.
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ABSTRACT
Selenium-containing chrysin (SeChry) and 3,7,3’,4’-tetramethylquercetin (SePQue)
derivatives were synthesized by a microwave-based methodology. In addition to their
improvement in terms of DPPH scavenging and potential GPx-like activities, when
tested in a panel of cancer cell lines both selenium-derivatives revealed consistently to
be more cytotoxic when compared with their oxo and thio-analogues, evidencing the
key role of selenocabonyl moiety for these activities. In particular, SeChry elicited a
noteworthy cytotoxic activity with mean IC50 values 18- and 3-fold lower than those
observed for chrysin and cisplatin, respectively. Additionally, these seleno-derivatives
evidenced an ability to overcome cisplatin and multidrug resistance. Notably, a
differential behavior towards malignant and non-malignant cells was observed for
SeChry and SePQue, exhibiting higher selectivity indexes when compared with the
chalcogen-derivatives and cisplatin. Our preliminary investigation on the mechanism of
cytotoxicity of SeChry and SePQue in MCF-7 human mammary cancer cells
demonstrated their capacity to efficiently suppress the clonal expansion along with their
ability to hamper TrxR activity leading to apoptotic cell death.
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.
INTRODUCTION
The key role of sulphur and selenium-containing small molecules and enzymes in a
wide range of essential biological functions1-2 led researchers to focus intensely on
developing new seleno and thio-compounds to treat or prevent diseases. In particular,
the considerable number of reports of organoselenium compounds presenting
antineoplastic effects have markedly increased the interest in this class of
compounds.3-6 Indeed, organoselenium compounds are promising candidates for
cancer therapy due to their ability to modulate multiple physiological functions
implicated in cancer development presenting either antioxidant, chemopreventive8-10 or
apoptotic activities.11
7
Flavonoids are a common group of plant polyphenols that have been extensively
studied in relation to their health promoting properties and potential pharmacological
applications, namely in cancer therapy and prevention.12,13 Representative examples
are the natural flavonol quercetin, (1, Que, Figure 1) and the flavone chrysin (2, Chry).
Whereas structurally distinct, Que (1) and Chry (2) have shown to induce growth
inhibition and apoptosis in multiple cancer cell lines.14-22 Additionally, it has been
suggested that 2 is an effective chemopreventive agent in a rat model-promoted renal
cancer23 and that this flavone efficiently modulates the toxic effects of the
chemotherapeutic drugs cisplatin 24 and doxorubicine.25 Recent studies in rat models
have also shown the ability of 1 to prevent liver carcinogenesis cell proliferation26 and
enhancing apoptosis along with its chemopreventive effect on prostate cancer.27
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Indeed, as a multitarget agent and being associated with a variety of anticancer
mechanisms, Que (1) has been proposed as a potential modulator of cancer MultiDrug
Resistance (MDR).28 Nonetheless, the pharmacological use of Que (1) is still
controversial mostly due to questions regarding its potential toxicity in vivo. Indeed, its
pro-oxidant properties and/or the possibility of being metabolically converted into
potentially toxic quinones have been amply discussed.29 In contrast, the lack of
catechol moiety in ring B of Chry (2) renders this flavone as a poor antioxidant with
negligible prooxidant character and considerable less expected toxic outcomes. The
fact that a water-soluble pro-drug of 1, QC12, has entered in phase-I clinical studies.30
have motivated researchers to develop several Que (1) and Chry (2) derivatives and
explore their anticancer properties. Representative examples of this strategy are: 1) the
methylated derivatives of 1 that have shown promising modulating activity of ABC-drug
transporters on multidrug resistance tumour cells;31 2) the di-methylated analogue of 2
reduced cell proliferation in acute lymphoblastic leukemia32 and significantly
suppressed the development of preneoplastic lesions induced in rats;33 3) the C-
isoprenylated hydrophobic derivatives of Chry (2) that are potential P-glycoprotein
(Pgp) modulators in tumor cells;34 and 4) the nitro derivatives of 2 that were found to
have strong activities against both human gastric adenocarcinoma cell line (SGC-7901)
and colorectal adenocarcinoma (HT-29) cells.35 However, and despite the bioisosteric
replacement of the oxygen atom by sulfur and/or selenium in known bioactive
compounds has been consistently reported as a successfully strategy for the
development of putative new anticancer agents,4,36 the potential anticancer properties
of selenium and sulphur-containing Que (1) and Chry (2) derivatives has yet to be
presented. In particular, the potential therapeutic properties of selenocarbonyl-
containing compounds remain almost unexplored. Therefore, the key strategy of the
current study was oriented towards exploring the modulation of important properties for
cancer chemoprevention and therapy, upon replacement of the oxygen atom of the
carbonyl moiety of 1 and 2 by a selenium atom and by its chalcogen neighbor sulphur.
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■
RESULTS AND DISCUSSION
Chemistry
Preparation of selenium and sulphur-containing flavonoids. The preparation of
sulphur and selenium-containing derivatives of 1, involved initial protection of hydroxyl
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groups by methylation with dimethylsulfate followed by chalcogenation of C4 position
Scheme 1), while 2 was directly chalcogenated (Scheme 2). The 3,7,3’,4’-
tetramethylquercetin (PQue (3), Scheme 1) was thionated upon reaction with
(
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phosphorous pentasulphide for 3 days at room temperature in THF, affording 4 in
6% yield. Following reaction with a large excess of boron tribromide in
dichloromethane at room temperature, 4 was efficiently deprotected giving SQue (5) in
6% yield. The sulphur-containing derivative SChry (6) was obtained from Chry (1) by
2
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the methodology described by Elisei et al.39 using an equimolar amount of
phosphorours pentasulphide in refluxing acetonitrile (Scheme 2).
For the preparation of selenium-derivatives we have used a microwave (MW)-based
synthetic methodology developed in our research group40 that allows an efficient
conversion of carbonyl groups into selenocarbonyl groups, using Woollins’ reagent
(WR) as selenium source (Scheme 1 and Scheme 2). Indeed, the successful
selenation of caffeine and uracil by a MW-based synthetic methodology mediated by
40
WR along with the efficient MW-accelerated thionation of flavonoids with Lawesson’s
reagent,41 motivated us to test the selenation of PQue (3) combining the use of WR
and MW irradiation. To select the most effective experimental conditions, screening
reactions were conducted using different solvents and WR quantities; the effects of
temperature, power and times of microwave irradiation were also screened. Under the
optimized conditions, consisting on 5 min. of microwave irradiation at 175 W (150 ºC)
of an acetonitrile WR and flavonoid solution on a sealed pyrex microwave vial, SePQue
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(
7) was prepared in 83%. However, despite we succeeded on the deprotection of the
sulphur derivative SQue, the seleno-derivative 5 revealed always to be unstable under
the several deprotection conditions used. Direct selenation of Chry (2) (Scheme 2)
was achieved under similar experimental conditions affording 8 in 63% yield. Whereas
we have not succeeded on the preparation of the derivative SeQue, we were
encouraged to use the tetramethylated derivative 7 for our subsequent biological
studies based on three main facts: 1) the reported promising antineoplastic properties
for methylated derivatives of 1;31 2) the protection of hydroxyl groups is expected to
conduct to increased bioavailability, due to the inability of methylated products of being
metabolized to excretable sulphate and glucuronide conjugates; and 3) reduced toxicity
is expected to arise from the protected catechol moiety due to the inability of forming
potentially toxic quinoid metabolites.29 In fact, the formation of metabolic quinoid
derivatives is one of the factors that may contribute to the toxic events induced by
catechol-containing flavonoids.
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Spectroscopic characterization. Evidence for the formation of selenium-containing
derivatives was first observed by mass spectrometry. Regardless of the ionization
method used, the mass spectra showed indistinctly five signals correspondent to
protonated molecules containing selenium isotopes. Further crucial evidence for
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selenocarbonyl existence was provided by Se NMR, where signals at 815.2 and
7
68.3 ppm were obtained for SeCry (8) and SePQue (7), respectively, which are in
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literature. Moreover, despite the use of different solvents, it is noticeable that
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NMR signals observed for C-4 position of 7 and 8 present the expected downfield shift
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as compared with the parent oxo-derivatives. Furthermore, definitive proof of
SePQue (7) formation was obtained by X-ray diffraction. This compound crystallizes in
acetone in a monoclinic P2
1
/c space group: a = 13.8279(19) Å; b = 15.465(2) Å ; c =
3
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.9179(11) Å;  = 90;  = 90.154(7); = 90; V = 1693.2(4) Å ; Z = 4. After refinement
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the residual factor, R
1
, has a value of 0.0452. The molecule is composed by two fused
6-membered rings linked by a C-C bond whose torsion angle [-174.7(4)º] is nearly
planar (Figure 2). Bond lengths and angles (cf. Supplementary Material) are well within
the expected values, as judged from extensive analysis of the values included in the
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CSD.
The steric hindrance caused by dimethoxyphenyl substituent group can be clearly
inferred from bond angles involving C2 atom and, to a less extent, from bond angles
involving C1' atom. Whereas the methoxyl group on position C3 (O3-C9) is
perpendicular to ring plane, as it can be observed from the torsion angle C(9)-O(3)-
C(3)-C(4) (-86.4º), the remaining three methoxyl groups are nearly on ring plane
[
torsion angles -7.3(5), -14.4(5) and -175.5º(3)]. This particularity of methoxyl group on
position C3 (O3-C9) can be explained by the influence of both the dimethoxyphenyl
group and the selenium atom. The proximity between the hydroxyl group and selenium
atom will also influence the angle values around position C4a, particularly C(5)-C(4a)-
C(4) [127.4(3)].
The hydrogen atom of the hydroxyl group on position C5, H5, is involved in a weak
intramolecular hydrogen bond with Se (Se1-O5 = 3.018 Å, Se1-H5 = 2.261 Å, Se1-H5-
1
O5 = 153.8). The existence of this hydrogen bonding is also in accordance with the H
NMR chemical shift obtained for this hydroxyl proton (OH-5, 13.84 ppm). This
1
13
resonance was unambiguously assigned to this position due to the H- C two and
three bond correlations, observed in the HMBC spectra (cf. Supplementary Material),
between this proton and carbons C4a (118.6 ppm), C5 (162.9 ppm) and C6 (100.7
ppm). Although we were unable to obtain crystals of SeChry (8) suitable for X-ray
diffraction analysis, all spectroscopic data are in accordance with assigned structures.
The SChry and SPQue derivatives were already prepared by other authors.41,48
Evidence for the formation of the new deprotected thio-quercetin, SQue, was obtained
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by mass spectrometry, where the spectrum obtained by field ionization presented a
molecular ion at m/z 318, compatible with the assigned structure. Further structural
evidence were obtained by H and 13C NMR where the absence of signals compatible
with methoxyl groups revealed effective deprotection.
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DPPH Scavenging Activity. The cancer chemopreventive properties of flavonoids,
seleno- and thio-compounds, are considered to be, at least partly, due to their
antioxidant activity, which can be mediated by their ability to scavenge free radicals
and/or by their capacity of affecting key redox enzymes and chelating metal ions.49 In
particular, the free radical scavenging activity of flavonoids is based on their ability to
act as H atom donors and is highly dependent on structural features and sensitive to
50
the electronic contribution of ring substituents. As a starting point to understand if the
chalcogenation of PQue and Chry could underlie the modulation of chemical properties
with potential biological significance to cancer chemoprotection and therapy, we have
performed a preliminary evaluation of the free radical scavenging activity of the thio
and seleno-derivatives prepared (Figure 3), as compared with their parent oxo
compounds, using the DPPH assay (Table 1).
Chry (2), is a flavone with two hydroxyl groups at the positions 7 and 5 of A ring, where
the 5-OH group does not participate in radical quenching due to its hydrogen bonding
with the neighbor carbonyl group.50 These structural features are responsible for the
low radical scavenging activity of 2, and, as expected, this flavone exhibited low
percentage of inhibition of DPPH radical absorbance. Indeed, DPPH IC50 value
exceeded largely its solubility range, at the tested conditions. However, the
chalcogenation of C4 position of 2 is clearly accompanied with an improvement of the
capacity to scavenge the DPPH radical. Whereas an increment of this activity was
already observed with sulphur-derivative 6, the most noticeable improvement was
exhibited with the selenium derivative SeChry (8).
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The structural features of Que (1) that underlie the high free radical scavenger activity
include the presence of a catechol group (3’,4’-dihydroxyl) in the B ring, that stabilizes
the B ring-centered radicals by H bonding, the presence of a hydroxyl group at position
3
of C ring and a network of conjugated double bonds that allows spin delocalization
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throughout the whole molecule, favoring radical stabilization.50 Therefore, the
protection of these hydroxyl groups is expected to abrogate the ability to trap radicals,
as observed by the low % of DPPH radical scavenging activity exhibited by PQue (3).
However, similarly to what was observed with chrysin derivatives, a dramatic increase
of the DPPH radical scavenging activity occurred upon selenation of 3. Accordingly,
using 1mM of tested compound the % of DPPH radical scavenging activity followed
the order: 95.6 ± 2.9 (Que) >90.4 ± 0.6 (SePQue) >18.3 ± 4.9 (SPQue) >2.5 ± 0.8
(
PQue). The selenium-derivative SePQue (7) displayed an IC50 value only 2.6 times
lower than the powerful radical scavenger Que (1). These results attest the dramatic
impact of the presence of the selenocarbonyl group adjacent to the aromatic ring on
the homolytic hydroxyl bond dissociation enthalpy (BDE) of these flavonoids, as
predicted by the DFT BDE calculations of the OH bonds (Table 1). Whereas the H
atom most prone to abstraction in Chry (2) is the OH-7 one, as attested by its lower
BDE, the bioisosteric replacement of the C4 oxygen atom by sulfur results in a
considerable decrease in the BDE of OH-5. Consequently in SChry (6) both OH
groups are nearly equally prone to abstraction. This effect is even more evident in the
selenium derivative SeChry (8), where the OH-5 becomes the proton more prone to
abstraction, with a BDE value very similar to the one exhibited by the OH-3 of Que (1).
Regarding the protected derivatives of Que, the same effect is observed. In fact, the
presence of the weak hydrogen bonding between the hydroxyl group on position C5
and the selenium carbonyl group of SePQue (7), attested by X-ray diffraction, does not
hamper its ability as radical scavenger, similarly to what is observed with other
compounds.51 Accordingly, this seleno-derivative exhibits a higher scavenging ability
when compared with phenol.52
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Catalytic properties as GPx mimics in a model system. A considerable number of
organoselenium compounds exhibit antioxidant properties mediated by the ability of
mimicking the catalytic activity of the antioxidant selenoenzyme glutathione peroxidase
(GPx).54-61 Thus, the GPx-like activity, that is characterized by the capacity of catalyzing
the reduction of harmful hydroperoxides with thiol cofactors, has attracted the interest
on this class of compounds as potential therapeutic agents. Towards a preliminary
evaluation of the potential catalytic properties of SeChry (8) and SePQue (7) as GPx
mimics, we have used an amply used H NMR-based methodology57-61 that consists in
monitoring the oxidation of reduced dithiothreitol (DTTred) into oxidized dithiothreitol
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(DTTox) mediated by H
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compound, as model system.
The results obtained (Figure 4) clearly show that from the tested compounds only the
selenium derivatives, SeChry (8) and SePQue (7), exhibit potential GP-x like activity;
attesting that the selenation of Chry and PQue induces a modulation of this activity. To
evaluate the relative efficiency of these selenium-derivatives we have estimated the
time required to oxidize 50% of the DTTred (t1/2) by linear regression. This allowed to
conclude that SeChry (8) (t1/2 =3.62 min) is more efficient in reducing the H
2
O in the
2
model system used, when compared with SePQue (7) (t1/2 =4.34 min). Whereas, these
values are higher than those exhibited by other selenocarbonyl-containing derivatives
such as a naphthyl selenourea (t1/2 = 2.0 min) and a sugar-derived selenoureas (t1/2
=
2
.9 min) reported by Merino-Montiel et al. 59 this constitutes an prospective important
finding since, to our knowledge, this is the first report of a selone substrate with
potential GPx-like activity. Nonetheless, a selone intermediary was recently reported to
be generated during the GPx cycle of a diselenide substract.56 The formation of this
selone is proposed to proceed via the formation of a selenenic acid intermediate
(
RSeOH) which is capable of undergoing reaction with thiols yielding a selenenyl
sulfide (RSeSR) derivative. The nucleophilic addition of a second thiol moiety allows
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the concomitant recovery of the selone moiety with the oxidation of the thiol. Whereas
a detailed mechanistic study at this context was considered to be beyond the scope of
the present paper, a similar mechanism can be expected to occur with 7 and 8.
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Biological tests
Cell viability assays. The cytotoxic properties of 6-8 (Figure 3) were evaluated in nine
human cancer cell lines of multiple origins: A375 (melanoma cells) HCT-15 (colorectal
adenocarcinoma cells), BxPC3 (pancreatic adenocarcinoma cells), MCF-7 (breast
adenocarcinoma cells), MCF-7-ADR (Multi Drug Resistant breast adenocarcinoma
cells), A431 (cervical adenocarcinoma cells), A431/Pt (cisplatin-resistant cervical
adenocarcinoma cells), 2008 (ovarian adenocarcinoma cells) and C13* (cisplatin-
resistant ovarian adenocarcinoma cells). The IC50 values, calculated from the dose-
survival curves after 72 h of incubation with the drugs are summarized in Table 2. For
comparison purposes, the cytotoxicities of Chry (2), Que (1), PQue (3) as well as of
cisplatin, one of the most widely used anticancer drugs, were evaluated in the same
experimental conditions. The sulphur-containing quercetin derivatives, SPQue (4) and
SQue (5), elicited no reproducible results mostly due to their low stability in solution.
The results obtained in malignant cells clearly show that the protection of the hydroxyl
groups of quercetin induces a drastic decrease of the cytotoxic activity. Indeed, 1 was
3-fold more cytotoxic than its protected derivative PQue. Nonetheless, whereas the
derivative 3 showed negligible cytotoxicity in cancer cells, the replacement of the
oxygen of position C4 by selenium, resulted in a dramatic increase of this activity. In
fact, SePQue (7) was on the average 3-fold more cytotoxic than cisplatin and about 9-
fold more cytotoxic than Que (1). This suggests that the structural feature that affects
the killing potential of 7 is not the flavonoid-like structure. Similarly, among the chrysin
derivatives, whereas the replacement of the oxygen of the carbonyl group of Chry (2)
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by sulphur did not change the cytotoxic profile of the flavonoid, replacing it with
selenium resulted in a marked increase of the cell-killing effect. Actually, SeChry (8)
elicited a noteworthy cytotoxic activity with mean IC50 values 18- and 3-fold lower than
those calculated with Chry (2) and cisplatin, respectively. This further corroborates the
key role of the selenocarbonyl group on the observed cytotoxicity. Coherently, the
cytotoxic activity of both seleno-derivatives was very similar.
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The panel of cell lines also included two cell line pairs that have been selected for their
sensitivity and resistance to cisplatin: A431 and A431/Pt, cisplatin-sensitive and -
resistant cervical adenocarcinoma cells, and 2008 and C13*, cisplatin-sensitive and -
resistant ovarian adenocarcinoma cells. Although cisplatin resistance is multifactorial,
the main molecular mechanisms involved in Pt resistance of C13* and A431/Pt have
been identified. In particular, in C13* cells resistance is correlated to reduced cellular
drug uptake, high cellular glutathione and thioredoxin reductase (TrxR) levels, and
62
enhanced repair of DNA damage. In A431/Pt cells resistance is due to defect in drug
uptake and to decreased levels of proteins involved in DNA mismatch repair (MSH2),
which causes an increased tolerance to cisplatin-induced DNA damage.63 By
comparing the RF values (where RF is the resistance factor and is defined as the ratio
between IC50 values calculated for the resistant cells and those obtained with the
sensitive ones), it appeared that all tested compounds possessed a quite similar
cytotoxic potency both on cisplatin-sensitive and on cisplatin-resistant cell lines, a clear
evidence of their ability to overcome cisplatin-resistance. Additionally, cytotoxicity
experiments gave information concerning flavonoid’s ability to overcome MDR
phenomenon. It is well known that acquired MDR, whereby cells become refractory to
multiple drugs, represents a major barrier for the chemotherapy success. MDR of
breast MCF-7 ADR cancer cells is associated to an overexpression of different species
of specific ATP-binding cassette transporters and membrane proteins.64, 65 When tested
on the MCF-7/MCF-7 ADR cell line pair all tested derivatives allowed the calculation of
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an RF value up to 18 times lower than that obtained with doxorubicin, clearly
suggesting that they are not potential MDR substrates. Among all compounds, once
again SePQue (7) and SeChry (8) were found to be the most potent derivatives, with
IC50 values ranging from 1.9 to 3.3 μM.
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Importantly, when tested in the non-transformed HEK293 cells, the flavonoid
derivatives exhibited a distinct cytotoxic activity when compared with malignant cells
(
Figure 5). In particular, the selenoderivatives 7 and 8 exhibited higher selectivity
indexes (S.I.) when compared with their parent compounds and with cisplatin. This
result is of the outmost importance when facing the potential application of these
derivatives as antineoplastic agents.
Clonogenic ability. By using a clonogenic assay on MCF-7 human breast cancer cells
treated for 6 h with increasing concentrations of the two seleno-derivatives 7 and 8, it
has been proved that they efficiently abrogated the clonogenic ability of breast cancer
cells in a dose-dependent manner (Figure 6). This constitutes an interesting result,
taking into consideration that suppression of the clonal expansion of transformed cells
is an attractive mechanism for cancer chemotherapy.
On the basis of the above data, SeChry (8) and SePQue (7) were selected for further
biological evaluations in order to obtain insight into the mechanisms by which they
promote antiproliferative effects. The investigations have been focused on MCF-7
human breast cancer taking into consideration the role of organoselenium compounds
9
on mammary cancer chemoprevention along with the sensitivity of this cell line to
quercetin and chrysin and their derivatives.66, 67
Cellular uptake. The study of the cellular accumulation, that represents the balance at
a given time between cellular influx and efflux, is an important factor influencing drug
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efficacy. In an attempt to correlate cytotoxic activity with cellular uptake, the selenium
content was evaluated in MCF-7 cells treated for 12 and 24 h with 12.5 or 25 μM
concentrations of SeChry (8) and SePQue (7). The intracellular selenium amount was
quantified by means of GF-AAS analysis, and the results, expressed as μg metal/L/mg
of cellular proteins, are shown in Figure 7. Treatment of MCF-7 cells resulted in a
marked intracellular time- and dose-dependent accumulation of both seleno-
derivatives, albeit for SePQue (7) the accumulations at 12 h were almost twice of those
detected with SeChry (8). The higher cellular uptake rate at short-time exposure might
be due to the predicted slightly higher lipophilic character of quercetin derivative (2.96
and 2.56, ClogP values calculated with the on-line software ALOGPS2 for 7 and 8,
respectively) which allows a faster passive diffusion through cell membrane.
Nevertheless, after 24 h treatment, intracellular selenium contents were similar for both
seleno-derivatives.
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Modulation of thioredoxin reductase (TrxR) activity. TrxR is a selenium-containing
flavoprotein that catalyzes the NADPH-dependent reduction of thioredoxin (Trx) and a
wide spectrum of other oxidized dithiols, being fundamental for maintaining a suitable
redox balance in cells. The fact that TrxR is upregulated in many malignant tumors
suggests that its inhibition could prevent the tumor initiation and progression, rendering
TrxR as a promising target for cancer therapy.68 This, along with the fact that the
anticancer and chemopreventive properties of flavonoids and organoselenium
derivatives have been attributed to their ability to inhibit/modulate the TrxR activity,69, 70
led us to evaluate the TrxR activity in MCF-7 cells upon incubation with 7 and 8 .
Figure 8 shows the results obtained measuring TrxR activity by means of standard
procedures in MCF-7 cells treated for 24 h with increasing concentrations of seleno-
derivatives 7 and 8. The TrxR activity of treated cells was expressed as percentage of
control cell enzyme activity. Both compounds were able to decrease cellular TrxR
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activity, even if the chrysin derivative 8 was more efficient. Actually, with a 20 μM of 8
treatment, a reduction of TrxR activity by approximately 75% with respect to untreated
cells was achieved. Of note is the fact the activity of TrxR was slightly enhanced at
concentrations up to 1.25 μM of the chalcogenated flavonoids. Indeed, the fact that
reduction of TrxR activity is only achieved with doses closer to IC50 suggests the
critical role of this redox system on the cytotoxicity mechanism of these seleno-
derivatives. Coherently, the observed increase in viability of human non tumor HEK293
cells upon treatment with low doses (ranging from 0.0625 to 0.625 μM) of SeChry and
SePQue was accompanied by an increase of the TrxR activity of up to 35%, compared
to untreated control cells (cf. Supplementary Material Figure S29). Interestingly, the
oxygen and sulphur chalcogen derivatives did not significantly alter the TrxR activity in
MCF-7 cells. These results further support that TrxR plays a key role in the cytotoxic
effect of SeChry (8) and PSeQue (7).
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Interestingly, both compounds did not affect the selenoenzyme glutathione peroxidase
(
GPx) and the flavoenzyme glutathione reductase (GR) (Figure 9, panel A and B),
thus attesting a specificity against the Trx redox system. Whereas mammalian TrxR
possess an N-terminal dithiol/disulfide redox motif with high homology to GR, TrxR
possess a unique and conserved cysteine–selenocysteine (Cys–Sec) redox pair at the
C-terminal active site.71 Therefore, the specificity of 7 and 8 to Trx-TrxR-NADPH redox
system suggests to be mainly based on their interaction with Sec residues at the C-
terminal.
The TrxR inhibitory effect of Que (1) is proposed to involve the modification of this
protein upon oxidation of the catechol moiety of 1.72 In addition, the presence of a
hydroxyl group at the position 3 of C ring of flavonoids has been found to be
determinant for their TrxR activity. Therefore, taking into consideration the absence of
these structural features in the selenoderivatives 7 and 8, it is reasonable to assume
that their TrxR inhibitory effect stems from the presence in both selenoderivatives of
the selenocarbonyl group. The fact that the oxygen and sulphur derivatives of chrysin
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did not significantly altered the TrxR activity in HEK293 cells, further corroborate the
key role of selenocarbonyl group for the TrxR inhibition. Whereas many
organoselenium compounds are known to be both substrates and inhibitors of
TrxR,3,69,70 to our knowledge, this is the first report of selone-containing derivatives with
TrxR inhibitory activity. Nonetheless, considering the wide range of substrate specificity
of these redox protein, it is plausible that the selenocarbonyl (selone) derivatives, 7 and
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8, can act as substrates for TrxR yielding selenol derivatives capable of modifying the
C-terminal of this protein (upon formation of Se-Se and S-Se bonds), thereby inhibiting
its activity, similarly to what happens during the catalytic cycle of TrxR, which involves
the formation of a selenenyl sulfide intermediate that undergoes redox reaction with
additional cysteine residues. Therefore, the higher TrxR activity of SeChry (8) when
compared with SePQue (7), can be explained on basis of its putative more suitable
redox properties and/or on the less electropositive character of its selenium atom (in its
selenol form) that favors the formation of disulphide products.
Measurement of total thiols and GSH redox state. Reduced Trx and TrxR are
fundamental for maintaining a suitable redox balance in cells and an inhibition of the
TrxR system can lead to a substantial decrease of total cellular sulfhydryls. A similar
effect can also arise from GPx-like activity. Therefore, the redox condition of
glutathione and total sulfhydryl groups were explored in MCF-7 cells treated for 24 or
48 h with IC50 doses of seleno-derivatives 7 and 8 (Figure 10). The results clearly
showed that after treatment with both seleno-flavonoids total glutathione concentration
(
GSH + GSSG) decreased (Figure 10, panel A). Following 48 h, 8 induced a 60%
reduction of total glutathione and a concomitant increase (about 4-fold higher than
control) of oxidized glutathione wherein a significant decline in reduced glutathione
accompanied by an increase in oxidized glutathione (Figure 10, panel A, inset a).
Coherently, total sulfhydryl groups markedly decreased after seleno-derivatives
treatment (about 60% with respect to the controls) (Figure 10, panel B). All these
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results suggest that the tested seleno-derivatives, and in particular the chrysin
derivative 8, are able to induce an oxidative shift in the redox status of MCF-7 cells.
Apoptosis studies. The development of cancer chemotherapeutic candidates (or as
adjuncts to existing therapy) that target the Trx system has attracted attention due to
the possibility of stimulating apoptosis of cancer cells.73 This can occur due to an
accumulation of ROS and alteration of the intracellular redox state and/or by
deregulation of pro-apoptotic pathways such as inhibition of procaspase-3
nitrosylation.74 As such, in order to characterize cell death pathways activated in
response to treatment with seleno-derivatives 7 and 8, we examined their effects in
terms of induction of apoptosis in MCF-7 cells. Figure 11, panel A shows the results
obtained upon monitoring cellular morphological changes of MCF-7 cells treated for 48
h with IC50 doses of 7 or 8 using Hoechst 33258/PI fluorescent staining analysis.
Compared with control cells, treated cells presented brightly stained nuclei and typical
apoptosis morphological features, such as chromatin condensation and fragmentation.
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Testing the ability of seleno-derivatives to activate caspase-3, a well-known executor
enzyme in the apoptotic pathway, it was found that they markedly stimulated caspase-3
activity (Figure 11, panel B). In particular, following a 48 h treatment with IC50 doses of
7
or 8, the protease activity was enhanced by factor of about 4 over untreated control
cells. These results are in agreement with those obtained by measuring the
mitochondrial energization of treated breast cancer cells in terms of retention of the
cationic fluorescent probe tetramethyl rhodamine methyl ester (TMRM) (Figure 11,
panel C). Actually, it is well known that mitochondrial membrane potential (MMP)
75
depletion generally precedes apoptotic cell death. By treating MCF-7 cells with IC50 of
7
or 8 for 24 and 48 h, TMRM fluorescence intensity markedly decreased by increasing
exposure times reflecting a dramatic alteration of MMP. In particular, after 48h, MMP
reduced by up to four times compared to control cells (Figure 11C).
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Oxidant production. There is experimental evidence that MMP is an important
regulator of efflux and influx of reactive oxygen species (ROS) from and into
mitochondria and mitochondrial ROS production is a very early event prior to the
collapse of MMP, the release of the pro-apoptotic factors and the activation of cell
death.76 Additionally, it has been previously shown that TrxR inhibition alters cell
conditions, as the increase of hydrogen peroxide concentration due to the prevention of
its removal causes an imbalance in cell redox conditions, thus leading to mitochondrial
membrane permeabilization and swelling.77 Moreover, the observed ability of 7 and 8
to promote an increase of oxidized glutathione could lead to increased ROS
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production. On these bases, we thought of interest to measure the hydrogen peroxide
basal production in MCF-7 cells treated with increasing concentrations of seleno-
derivatives by using the fluorescent probe CM-DCFDA to measure the oxidant
production (Figure 12). Antimycin, a classic inhibitor of the mitochondrial respiratory
chain at the level of complex III was used as a positive control. Both seleno-derivatives
were able to increase the oxidant basal production, even if 8 was more effective. The
enhancement of oxidant production in cells treated with 25 μM of 8 was nearly
superimposable to that obtained with 3 μM antymicin. These results suggest that the
TrxR inhibitory effect of seleno-derivatives overcomes their potential GPx-like activity.
This evidence the key role of the TrxR inhibitory effect for the apoptotic MCF-7 cell
death induced by 7 and 8.
■ CONCLUSIONS
We have synthesized selenium and sulphur-containing Chry and PQuerc derivatives.
For the synthesis of selenium-containing compounds a new MW-based synthetic
methodology was developed. The free radical scavenging activity of these compounds
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was preliminary evaluated by the DPPH assay, demonstrating that a considerable
modulation of this activity has occurred upon selenation. Indeed, whereas the oxygen
and sulphur-containing derivatives presented negligible DPPH radical scavenging
activity. The potential GPx-like ability of these two selenium derivatives was suggested
upon in vitro monitoring of the conversion of reduced dithiothreitol (DTTred) into oxidized
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dithiothreitol (DTTox), mediated by H
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2
tested compounds. Whereas these results constitute only initial evidence, per se
constitute important findings as the first report of selone derivatives with catalytic ability
to scavenge H
2
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2
flavonoid core is accompanied by the modulation of DPPH radical scavenging activity
and potential GPx-like ability. Taking into consideration the potential importance of
these properties for cancer progression, the synthesized selones constitute attractive
scaffolds for the preparation of new derivatives with enhanced antioxidant properties.
The activity of all chalcogen derivatives was tested against a panel of human cancer
cell lines of multiple origins: A375 (melanoma cells), HCT-15 (colorectal
adenocarcinoma cells), BxPC3 (pancreatic adenocarcinoma cells), MCF-7 (breast
adenocarcinoma cells), MCF-7-ADR (Multi Drug Resistant breast adenocarcinoma
cells), A431 (cervical adenocarcinoma cells), A431/Pt (cisplatin-resistant cervical
adenocarcinoma cells), 2008 (ovarian adenocarcinoma cells) and C13* (cisplatin-
resistant ovarian adenocarcinoma cells). The two seleno-derivatives 7 and 8 were
consistently more cytotoxic than their chalcogen-derivatives, fully demonstrating the
key role the selenocarbonyl group for their cytotoxic activity. All tested compounds
possessed a quite similar cytotoxic potency both on cisplatin-sensitive and on cisplatin-
resistant cell lines, as a clear evidence of their ability to overcome cisplatin-resistance.
Importantly, the two seleno-derivatives 7 and 8 exhibited a differential behavior in
tumor and non-tumor cell, with SI values higher than those exhibited by cisplatin and
their chalcogen derivatives. Of note is the fact that SeChry was more cytotoxic than
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SePQue in MCF-7 cells and when tested on the MCF-7/MCF-7 ADR cell line pair,
SeChry exhibited a RF value 18 times lower than that obtained with doxorubicin. Both
selenoderivatives exhibit similar ability to abrogate the clonogenic growth of these cells
and similar intracellular selenium levels. A preliminary evaluation of the mechanisms
underlying the cytotoxicity of these two seleno-derivatives in MCF-7 cells demonstrated
their ability to hamper the TrxR activity, contrasting with their lack of efficacy to affect
the GPx/GR redox system. The higher TrxR inhibitory effect of SeChry (8) was
accompanied with a higher decrease of the total sulfhydryl groups, a higher capacity to
induce apoptosis via caspase 3 inhibition, a more dramatic decrease of the MMP and a
higher ability to increase the hydrogen peroxide basal production, when compared with
SePQue (7). Taken together, our results suggest that selenium-containing falavonoids
and in particular SePQue (7) and SeChry (8) constitute attractive scaffolds for the
development of potential new candidates for cancer chemotherapy.
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■
EXPERIMENTAL SECTION
Materials and Methods. Woollins’ reagent (WR) was prepared as described in
Wood et al.78 involving the initial formation of the pentamer (PhP)
. All other
5
commercially available reagents were acquired from Sigma-Aldrich Quimica, S.A.
(Madrid, Spain), unless specified otherwise, and were used as received. PQue was
prepared in 52% yield by adaptation of published methodology,37 involving initial Que
treatment with potassium carbonate followed by methylation with dimethylsulphate in
refluxing acetone. PSQue was prepared from PQue in 26% yield according to
38
published procedure, involving reaction with P
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S in THF at RT for 3 days. Whenever
2
79
necessary, solvents were purified by standard methods. Microwave syntheses were
performed on a CEM reactor (Discover Benchmate). Experiments were performed in
sealed Pyrex microwave vials (300 W maximum power) using temperature control
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mode. Melting temperatures were measured in a Leica Galen III hot stage apparatus
and are uncorrected. Low resolution Mass spectra were recorded either on a Varian
500-MS LC Ion Trap mass spectrometer, operated in the electrospray ionization (ESI)
mode or on a API 4000 (Applied Biosystems) spectrometer operated in atmospheric
pressure chemical ionization (APCI) mode, where samples were delivered by direct
probe. High Resolution Mass Spectra were recorded on a Autospec (Micromass, UK
10
11
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1
Ltd) sprectrometer operated in the electronic impact ionization mode. H NMR spectra
were recorded on Bruker Avance III 500 spectrometer (Bruker BioSpin GmbH,
Rheinstetten, Germany) operating at 500 MHz. 13C NMR spectra were recorded on the
same instrument, operating at 125.77 MHz. Chemical shifts are reported in ppm
downfield from tetramethylsilane, and coupling constants are reported in Hz. The
presence of labile protons was confirmed by chemical exchange with D
spectra were recorded on a Bruker Avance II 500 operating at 95.4 MHz, using Me
as external reference. Resonance and structural assignments were based on the
2
O. 77Se NMR
2
Se
13
1
analysis of coupling patterns, including the C- H coupling profiles obtained in
bidimensional heteronuclear multiple bond correlation (HMBC) and heteronuclear
single quantum coherence (HSQC) experiments, performed with standard pulse
programs.
X-ray crystallographic data were collected from crystals using an area detector
diffractometer (Bruker AXS-KAPPA APEX II) equipped with an Oxford Cryosystem
open flow nitrogen cryostat at 150 K and graphite-monochromated MoKa ( = 0.711 Å)
radiation. Cell parameters were retrieved using Bruker SMART software and refined
with Bruker SAINT80 on all observed reflections. Absorption corrections were applied
using SADABS.81 The structures were solved by direct methods using SIR 97. and
82
83
refined with full-matrix least-squares refinement against F2 using SHELXL-97. All the
programs are included in the WINGX package (version 1.70.01).84 All non-hydrogen
atoms were refined anisotropically, and the hydrogen atoms were inserted in idealized
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1
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1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
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5
6
positions, riding on the parent C atom, except for the methyl hydrogens, whose
orientation was refined from electron density, allowing the refinement of both C–C
torsion angles and C–H distances, which were found directly in the density map.
Drawings were made with ORTEP3 for Windows.46 Intermolecular interactions were
analysed using Mercury 1.4.2 (Build 2). Relevant details of the X-ray data analysis are
displayed in Table S1. Crystallographic data for SePQue (7), were deposited with the
Cambridge Crystallographic Data Centre (CCDC 848449) and can be obtained free of
charge from: CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (Fax: +44-1223-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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5
6
7
8
9
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1
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
336033; e-mail: deposit@ccdc.cam.ac.uk; http://www.ccdc.cam.ac.uk/deposit).
HPLC analysis was performed on an Ultimate 3000 Dionex system (Dionex Co.,
Sunnyvale, CA) using a Luna C18 (2) column (250 mm ×4.6 mm; 5 μm; Phenomenex,
Torrance, CA) column. A 30-min linear gradient from 5 to 70% acetonitrile in 0.1%
aqueous formic acid, followed by a 2-min linear gradient to 100% acetonitrile and an 8-
min isocratic elution with acetonitrile, with flow rate of 1.0 mL/min and UV-detection
254/335 nm, was used in all instances. All key compounds were proven by this method
to show ≥95% purity.
Synthesis
Seleno-derivatives
Synthesis of 2-(3’,4’-dimethoxyphenyl)-5-hydroxy-3,7-dimethoxy-4H-
chromen-4-selenone (SePQue, 7). A solution of PQue (3) (50 mg, 140

mol) in acetonitrile (3 mL) was prepared in a microwave reaction vial.
WR (30 mg, 56 mol) was then added. The suspension was heated by
microwave irradiation at 150 ºC (MW power 175 W) for 5 minutes. The
solvent was distilled under reduced pressure and the residue obtained
was purified by silica gel flash chromatography (dichloromet hane)
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affording 34 mg (83%) as a red solid. MP 168-170ºC. IR (KBr) max
.
3
441 (OH), 1627 (C=C). 1H-NMR (acetone-d
6
) δ 13.84 (1H, exchanges
with D
2
O, s, OH-5), 7.99 (1H, dd, J = 2.0 and 8.8 Hz, H6’), 7.94 (1H, d,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
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58
59
60
J = 2.0 Hz, H2’), 7.21 (1H, d, J = 8.8 Hz, H5’), 6.82 (1H, d, J = 2.0 Hz,
H8), 6.53 (1H, d, J = 2.0 Hz, H6), 3.97 (6H, s, OMe), 3.95 (3H, s, OMe),
3
1
1
.76 (3H, s, OMe). 13C-NMR (acetone-d
) δ 192.5 (C4), 166.1 (C7),
6
62.9 (C5), 153.7 (C4’), 153.4 (8a), 151.6 (C3), 150.5 (C3’), 149.9 (C2),
24.2 (C6’), 123.1 (C1’), 118.6 (C4a), 112.9 (C2’), 112.8 (C5’), 100.7
(
C6), 93.6 (C8), 59.6 (C3-OMe), 56.7 (OMe), 56.4 (OMe), 56.3 (OMe).
77Se-NMR (acetone-d ) δ 768.3 (Se4). MS (ESI) m/z 425 [M( Se)+H]+
82
6
8
0
+
78
+
77
(
18), 423 [M( Se)+H] (80), 421 [M( Se)+H] (42), 420 [M( Se)+H]+
20), 419 [M( Se)+H]+ (19). HRIEMS m/z [M( Se)]
76
82
+
(
calcd. for
calcd. for
calcd. for
calcd. for
calcd. for
82Se 424.0265, found 424.0335; [M( Se)]
80
+
C
C
C
C
C
19
19
19
19
19
H
H
H
H
H
18
18
18
18
18
O
O
O
O
O
6
6
6
6
6
80Se 422.0263, found 422.0251; [M( Se)]
78
+
78Se 420.0271, found 420.0258; [M( Se)]
77
+
77Se 419.0290, found 419.0184; [M( Se)]
76
+
76Se 418.0290, found 418.0252.
Synthesis
of
5,7-hydroxy-2-phenyl-4H-chromene-4-selenone
(SeChry, 8). A solution of Chry (2) (50 mg, 197 mol) in acetonitrile (3
mL) was prepared in a microwave reaction vial, and WR (42 mg, 79

mol) was then added. The suspension was heated by microwave
irradiation at 150 ºC (MW power 175 W) for 5 minutes. The solvent was
distilled under reduced pressure and the residue obtained was purified
by silica gel flash chromatography (dichloromethane) affording SeChry
1
as a yellow solid, in 62% yield (31 mg). MP 190-192ºC. H NMR (DMSO-
d
6
) δ 13.20 (1H, exchange with D O, s, OH-5), 11.47 (1H, exchange with
2
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1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
D
2
O, bs, OH-7), 8.22 (2H, d, J = 7.5 Hz, H2’ and H6’), 7.92 (1H, s, H3),
7
=
1
.32 (1H, t, J = 7.3, H4’), 7.64-7.61 (2H, m, H3’ and H5’), 6.67 (1H, d, J
2.2, H8), 6.45 (1H, d, J = 2.2, H6). 13C NMR (DMSO-d
6
) δ 193.8 (C4),
64.8 (C7), 161.3 (C5), 153.4 (C8a), 152.0 (C2), 132.4 (C4’), 129.7
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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5
6
7
8
9
0
(C1’), 129.4 (C3’ or C5’), 126.9 (C2’ or C6’), 122.8 (C3), 116.1 (C4a),
01.2 (C6), 95.2 (C8). 77Se NMR (DMSO-d
) δ 816.2 (Se4). MS (APCI)
1
6
82
+
80
+
78
+
m/z 321 [M( Se)+H] (37), 319 [M( Se)+H] (100), 317 [M( Se)+H]
7
7
+
76
+
(
[
86), 316 [M( Se)+H] (72), 315 [M( Se)+H] (51). HRIEMS m/z
8
2
+
82
80
+
M( Se)] calcd. for C15
H
10
O
3
Se 319.9797, found 319.9808; [M( Se)]
80Se 317.9795, found 317.9803; [M(78Se)] calcd. for
+
calcd. for C15
H
10
O
3
78Se 315.9803, found 315.9804; [M( Se)]
77Se 314.9829, found 314.9796; [M( Se)]
76Se 313.9822, found 313.9812.
77
+
calcd. for
calcd. for
C
C
C
15
15
15
H
H
H
10
10
10
O
O
O
3
3
3
76
+
Thioderivatives
Synthesis
of
2-(3,4-di-hydroxyphenyl)-3,5,7-tri-hydroxy-4H-
chromene-4-thione (SQue, 5). Boron tribromide (18 eq., 414 µL, 4.4
mmol) 1 M solution in dichloromethane was added dropwise to a solution
of SPQue (4) (91 mg, 0.24 mmol), in dry dichloromethane (5 mL) at -78
ºC. The resulting mixture was stirred for 24 h at room temperature under
nitrogen atmosphere. The reaction mixture was cooled to -78 ºC and
quenched by addition of ice. The aqueous layer was extracted with ethyl
acetate (3 x 30 mL). The combined organic layers were dried over
anhydrous magnesium sulphate, affording 67 mg (86%) of SQue (5), as
1
a red solid. MP > 300ºC. H-NMR (CD
3
OD)  14.06 (1H, s, OH-5), 13.06
(
1H, s, OH), 11.07 (1H, s, OH), 9.91 (1H, s, OH), 7.76 (1H, s, H2`), 7.70
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(
(
1H, d, J = 8.5, H6`), 6.90 (1H, d, J = 8.5, H5`), 6.54 (1H, s, H8), 6.34
1H, s, H6). 13C-NMR (CD
52.6 (C8a), 149.1 (C4’), 145.4 (C3’), 142.7 (C2 or C3), 142.4 (C2 or
C3), 121.7 (C6’), 120.8 (C1’), 115.9 (C2`and C5’), 111.3 (C4a), 100.4
OD) 181.7 (C4), 163.5 (C7), 160.2 (C5),
3
1
10
11
12
13
14
15
16
17
18
19
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59
60
+
+
(C6), 93.9 (C8). MS (FI) m/z 318 [M] . HRMS-EI m/z [M] calcd. for
S 318.0198, found 318.0200.
C
15
H
10
O
6
Synthesis of 5,7-hydroxy-2-phenyl-4H-chromene-4-thione (SChry,
6
).Compound SChry (6) was prepared according to procedure reported
by Elisei et al.39 Specifically, a solution of 2 (500 mg, 2.0 mmol) and
P
2
S (444 mg, 2.0 mM) in acetonitrile (35 mL) was refluxed during 40
5
min. The solvent was distilled under reduced pressure and the residue
obtained was purified by silica gel flash chromatography (n-hexane/ethyl
acetate 1:1) affording 3 in 2.6 % yield (14 mg). MP 219-220ºC (MP lit.
2
7
7
18ºC).28 1H NMR (CDCl
3
) δ 13.7 (1H, exchange with D O, s, OH-5),
2
.93 (2H, d, J = 8.1, H2’ and H6’), 7.58-7.52 (3H, m, H3’, H4’ and H5’),
1
.41 (1H, s, H3), 6.52 (1H, d, J = 2.6, H8), 6.41 (1H, d, J = 2.6, H6). H
NMR (DMSO-d
6
) δ 13.64 (1H, exchange with D
2
O, s, OH-5), 11.3 (1H,
exchange with D
2
O, bs, OH-7), 8.16 (2H, d, J = 7.3, H2’ and H6’), 7.65-
7.58 (4H, m, H3, H3’, H4’ and H5’), 6.62 (1H, s, H8), 6.34 (1H, s, H6).
13C NMR (DMSO-d
6
) δ 195.9 (C4), 164.7 (C7), 161.9 (C5), 154.3 (C2 or
C8a), 154.2 (C2 or C8a), 132.4 (C4’), 129.7 (C1’), 129.4 (C2’ and C6’),
1
26.9 (C3’ and C5’), 117.4 (C3), 112.6 (C4a), 100.8 (C6), 94.8 (C8). MS
(
ESI+) m/z 271 [M+H]+.
Radical Scavenging activity. A solution of the tested compound (with final
concentration ranging the mM to M scale) in ethanol was added to freshly prepared
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6
ethanolic DPPH solution (final concentration100 μM). The mixtures were incubated for
1h in absence of light and the absorbance was measured at 660 nm. The blank assay
was performed with a 100 μM solution of DPPH in ethanol in the absence of the tested
compounds. All measurements were obtained in duplicate. The capability to scavenge
the DPPH radical was calculated using the following equation:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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9
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9
0
1
2
3
4
5
6
7
8
9
0
DPPH radical scavenging activity (%)= (Ablank-Aantiox)/Ablank x 100
In vitro catalytic properties as GPx mimics in a model system. DTTred (32.4 mol)
and a catalytic amount of the tested compound (3.24 mol) were dissolved in CD OD
3
1
(
300 l) and the reaction was initiated by addition of H
2
O
2
(32.4 mol). H NMR spectra
were recorded at distinct time points at 25ºC, using 3 mm tubes. Similar reaction was
performed in the absence of catalyst. The relative concentration of DTTred, and DTTox
1
were determined by the area of the corresponding H NMR signals.
Computational Methods. All calculations were performed using the Gaussian09 rev
85
A.01 suite of quantum chemical programs at the B3LYP/6-31+G(2d,p) level (using the
UB3LYP approach for radicals)86-89 The geometry of each species was fully optimized
on the gas phase, followed by a single-point frequency and energy calculation; no
imaginary frequencies were observed.
Cell cultures. Human colon (HCT-15) and pancreatic (BxPC3) carcinoma cell lines
along with melanoma (A375) cell line and non-transformed embryonic kidney (HEK293)
cells were obtained by American Type Culture Collection (ATCC, Rockville, MD). A431
and A431/Pt cisplatin-sensitive and -resistant human cervical carcinoma cells,
respectively, were kindly supplied by Prof. F. Zunino (Division of Experimental
90
Oncology, Istituto Nazionale dei Tumori, Milan, Italy). MCF-7 and its MDR phenotype,
MCF-7 ADR, human breast carcinoma cell lines, were kindly provided by Prof. N.
Colabufo (Dept. of Farmacy,Bari University, Italy).The human ovarian cancer cell line
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008 and its cisplatin resistant variant C13* were kindly provided by Prof. G. Marverti
(
Dept. of Biomedical, Metabolical and Neural Sciences of the University of Modena,
Italy).91 Cell lines were maintained in the logarithmic phase at 37 C in a 5 % carbon
dioxide atmosphere using the culture media specified below to which was added 10%
fetal calf serum (Euroclone, Milan, Italy), antibiotics (50 units mL−1 penicillin and
10
11
12
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−
1
50 mg mL streptomycin), and 2 mM l-glutamine. The RPMI-1640 medium (Euroclone)
was used for MCF-7, MCF-7 ADR, HCT-15, BxPC3, A431, A431/Pt, 2008, and C13*
cells; the F-12 HAM’S medium (Sigma Chemical Co.) was used for A549 cells; and the
DMEM medium was used for A375 and HEK293 cells.
Cytotoxicity assays. The growth inhibitory effect towards tumor cell lines was
evaluated by means of the MTT assay.92 Briefly, 3–8 × 10 cells/well, dependent upon
the growth characteristics of the cell line, were seeded in 96-well microplates in growth
medium (100 μL) and then incubated at 37 °C in a 5 % carbon dioxide atmosphere.
After 24 h, the medium was removed and replaced with a fresh one containing the
compound to be tested at the appropriate concentration. Triplicate cultures were
established for each treatment. After 48 h each well was first treated with 10 μL of a
3
−
1
5
mg mL MTT saline solution and incubated for five additional hours and then treated
with 100 μL of a sodium dodecylsulfate (SDS) solution in HCl 0.01 M. After an
overnight incubation, the inhibition of cell growth induced by the tested compound was
detected by measuring the absorbance of each well at 570 nm using a Bio-Rad 680
microplate reader. Mean absorbance for each drug dose was expressed as percentage
of the control and plotted vs drug concentration. Dose-response curves were fitted and
IC50 values were calculated with 4-PL model (P < 0.05). IC50 values represent the drug
concentrations that reduce the mean absorbance at 570 nm to 50 % of those in the
untreated control wells.
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5
Clonogenic assay. 2 × 10 human ovarian MCF-7 cancer cells were seeded in Petri
2
dishes (6 cm ) and incubated overnight. Then the cells were treated with increasing
concentrations of the tested compounds, incubated for 6 h, then washed with saline
phosphate buffer and harvested. Aliquots of 500 cells were reseeded in a growth
medium in triplicate for 10 days; the colonies were fixed and stained with crystal violet
solution (0.5 %) in acetic acid (50 %) and ethanol (50 %). The colonies were counted
discarding those containing less than 50 cells. The efficiency of clonal growth was
calculated by the ratio between the number of colonies formed and the number of cells
seeded.
0
1
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8
9
0
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
2
Cellular uptake. MCF-7 cells (7.5x10 ) were seeded in 75 cm flasks in growth
medium (20 mL). After 24 h the medium was replaced and the cells were incubated for
1
2 and 24 h with tested compounds. Subsequently, cells were washed with PBS and
harvested. Samples were subjected to three freezing/thawing cycles at -80 °C, and
then vigorously vortexed. Aliquots were removed for the determination of protein
content by the BioRad protein assay (BioRad). 1 mL of highly pure nitric acid (Se:
≤0.005 μg/kg, TraceSELECT® Ultra, Sigma Chemical Co.) was added to the samples
and those were transferred into a microwave teflon vessel. Subsequently, samples
were submitted to standard procedure using a speed wave MWS-3 Berghof instrument
(
Eningen, Germany). After cooling, each mineralized sample was analyzed for silver
amount by using a Varian AA Duo graphite furnace atomic absorption spectrometer
Varian, Palo Alto, CA; USA) at the wavelength 196 nm. The calibration curve was
(
obtained using known concentrations of standard solutions purchased from Sigma
Chemical Co.
2
Inhibition of redox enzymes in cells. MCF-7 cells were grown in 75 cm flasks at
confluence and treated with tested derivatives at increasing concentrations for 24 h. At
the end of the incubation time, cells were collected, washed with PBS and centrifuged.
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Each sample was then lysed with RIPA buffer modified as follows: 150 mM NaCl, 50
mM Tris-HCl, 1% Triton X-100, 1% SDS, 1% DOC, 1 mM NaF, 1 mM EDTA, to which it
was added, immediately before use, an antiprotease cocktail (Roche, Basel,
Switzerland) containing PMSF and supplemented with protease inhibitors. Samples
were tested for thioredoxin reductase (0.1 mg proteins) activity as described above as
well as by means of an end-point insulin assay,93 with minor modifications in order to
allow 96-well plate measurements. Glutathione reductase activity (0.1 mg proteins)
was estimated at 25 °C in 0.1 M Tris/HCl (pH 8.1) containing 0.2 mM NADPH.
10
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44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Reactions were started by the addition of
1
mM GSSG and followed
spectrophotometrically at 340 nm. Glutathione peroxidase activity (0.5 mg proteins)
was estimated at 25 °C in 50 mM Hepes/Tris (pH 7.0) and EDTA 3 mM, 0.3 mM
NADPH, 5 mM GSH and 0.25 mM tert-butyl hydroperoxide according to Rigobello et
al.94
5
Determination of total and oxidized glutathione. MCF-7 cells (2 × 10 ) were grown
in a six-well plate and incubated for 24 and 48 h with IC50 concentrations of tested
compounds. After treatment, cells were washed with PBS, treated with 6%
metaphosphoric acid, kept on ice for 15 min, and centrifuged. The pellet was dissolved
in RIPA buffer and utilized for protein determination by the BioRad protein assay
(
BioRad). Aliquots of the supernatant were neutralized with Na
3
PO and assayed for
4
oxidized glutathione following the procedure reported by Bindoli et al. 95
5
Assay of total thiol groups. MCF-7 cells (4·10 ) were seeded in a six-well plate in
growth medium (4 mL). After 24 h cells were incubated for 24 and 48 h with IC50
concentrations of tested compounds. Subsequently cells were washed with PBS buffer
and centrifuged. Supernatants were removed and cells treated with 7 M guanidine in
0.2 M Tris–HCl (pH 7.5) containing 10 mM EDTA. Samples were subjected to two
freezing/thawing cycles in liquid nitrogen and then vortexed vigorously and treated with
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