Selenium-Containing Chrysin and Quercetin Derivatives: Attractive

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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

Selenium-containing Chrysin and Quercetin Derivatives: Attractive scaffolds for cancer therapy.

Inês L. Martinsⱡ**, Catarina Charneiraⱡ**, Valentina Gandin‡*, João L. Ferreira da Silvaⱡ, Gonçalo C. Justinoⱡ, João P. Teloⱡ, 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.

ACS Paragon Plus Environment


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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,7 chemopreventive8-10 or apoptotic activities.11 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 Cisoprenylated 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 selenocarbonylcontaining 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 groups by methylation with dimethylsulfate37 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 phosphorous pentasulphide for 3 days at room temperature in THF,38 affording 4 in 26% yield. Following reaction with a large excess of boron tribromide in dichloromethane at room temperature, 4 was efficiently deprotected giving SQue (5) in 86% yield. The sulphur-containing derivative SChry (6) was obtained from Chry (1) by 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 WR40 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.

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 selenocarbonyl existence was provided by

77

Se NMR, where signals at 815.2 and

768.3 ppm were obtained for SeCry (8) and SePQue (7), respectively, which are in accordance with the

77

Se NMR shifts of selenocarbonyl groups reported in the

literature.42 Moreover, despite the use of different solvents, it is noticeable that

13

C

NMR signals observed for C-4 position of 7 and 8 present the expected downfield shift as compared with the parent oxo-derivatives.43 Furthermore, definitive proof of SePQue (7) formation was obtained by X-ray diffraction. This compound crystallizes in acetone in a monoclinic P21/c space group: a = 13.8279(19) Å; b = 15.465(2) Å ; c = 7.9179(11) Å;  = 90;  = 90.154(7); = 90; V = 1693.2(4) Å3; Z = 4. After refinement


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the residual factor, R1, 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 CSD.44,45 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-H5O5 = 153.8). The existence of this hydrogen bonding is also in accordance with the 1H NMR chemical shift obtained for this hydroxyl proton (OH-5, 13.84 ppm). This resonance was unambiguously assigned to this position due to the 1H-13C 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 1H and

13

C NMR where the absence of signals compatible

with methoxyl groups revealed effective deprotection.

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 the electronic contribution of ring substituents.50 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 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 1H NMR-based methodology57-61 that consists in monitoring the oxidation of reduced dithiothreitol (DTTred) into oxidized dithiothreitol (DTTox) mediated by H2O2 in the presence of a catalytic amount (10%) of the tested 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 H2O2 in the 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.

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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.

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* (cisplatinresistant ovarian adenocarcinoma cells). The IC50 values, calculated from the dosesurvival 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 9fold 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.

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 enhanced repair of DNA damage.62 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.

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 on mammary cancer chemoprevention9 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 selenoderivatives, 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.

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 selenoderivatives 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 selenoderivatives. 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). 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 Cterminal. 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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Page 16 of 67

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 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.

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) depletion generally precedes apoptotic cell death.75 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 production.3 On these bases, we thought of interest to measure the hydrogen peroxide basal production in MCF-7 cells treated with increasing concentrations of selenoderivatives 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 dithiothreitol (DTTox), mediated by H2O2, in the presence of a catalytic amount of the 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 H2O2. Taken together, these results show that the selenation of the 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* (cisplatinresistant 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 cisplatinresistant 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.

■ 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) 5. All other 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 published procedure,38 involving reaction with P2S2 in THF at RT for 3 days. Whenever necessary, solvents were purified by standard methods.79 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 Ltd) sprectrometer operated in the electronic impact ionization mode. 1H NMR spectra were recorded on Bruker Avance III 500 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) operating at 500 MHz.

13

C 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 D2O.

77

Se NMR

spectra were recorded on a Bruker Avance II 500 operating at 95.4 MHz, using Me2Se as external reference. Resonance and structural assignments were based on the analysis of coupling patterns, including the

C-1H coupling profiles obtained in

13

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 97.82 and refined with full-matrix least-squares refinement against F2 using SHELXL-97.83 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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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: +44-1223336033; e-mail: [email protected]; 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 8min 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-deri vati ves

Synthesis of 2-(3’, 4’-dimethoxyphenyl ) -5-hydroxy-3,7-dimethoxy- 4Hchromen- 4-selenone (SePQue, 7). A solut ion of PQue (3) ( 50 mg, 140 mol) in acetonitrile (3 mL) was prepar ed in a microwave reaction vial. WR (30 mg, 56 m ol) was then added. The suspension was heated by microwave irradiat ion at 150 º C ( MW power 175 W ) f or 5 minutes. The solvent was dist illed under reduced pressure and the residue obtained was pur if ied by silica gel f lash chromatography (dichloromet hane)


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aff ording 34 mg (83%) as a red solid. MP 168-170ºC. IR (KBr)  m a x . 3441 (OH), 1627 (C=C).

1

H-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, 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, O Me), 3.95 (3H, s, O Me), 3.76 (3H, s, O Me).

13

C-NM R (acet one -d 6 ) δ 192.5 (C4), 166.1 (C7),

162.9 (C5), 153.7 (C4’), 153.4 (8a), 151. 6 (C3), 150.5 (C3’), 149.9 (C2), 124.2 (C6’), 123.1 ( C1’), 118.6 (C4a), 112.9 (C2’), 112.8 ( C5’), 100.7 (C6), 93.6 ( C8), 59. 6 (C3 -O Me), 56.7 ( OMe), 56.4 (O Me), 56.3 (O Me). 77

Se- NMR (acet one- d 6 ) δ 768.3 (Se4). MS (ESI) m/z 425 [M( 8 2 Se)+H] +

(18), 423 [ M( 8 0 Se)+ H] + (80), 421 [ M( 7 8 Se)+H] + (42), 420 [ M( 7 7 Se)+H]+ (20),

419

[ M( 7 6 Se) +H] +

(19).

HRI EMS

[M( 8 2 Se)] +

m/z

calcd.

f or

C 1 9 H 1 8 O 6 8 2 Se

424.0265 ,

f ound

424. 0335;

[ M( 8 0 Se)] +

calcd.

f or

C 1 9 H 1 8 O 6 8 0 Se

422.0263 ,

f ound

422. 0251;

[ M( 7 8 Se)] +

calcd.

f or

C 1 9 H 1 8 O 6 7 8 Se

420. 0271 ,

calcd.

f or

C 1 9 H 1 8 O 6 7 7 Se

419.0290 ,

calcd.

f or

f ound f ound

420.0258; 419. 0184;

[ M( 7 7 Se)] + [ M( 7 6 Se)] +

C 1 9 H 1 8 O 6 7 6 Se 418.0290, f ound 418.0252.

Synthesis

of

5,7-hydroxy-2- phen yl- 4H-chromene-4-selenone

(SeChry, 8) . A solut ion of Chry ( 2) (50 mg, 197 mol) in acetonitr ile (3 mL) was prepar ed in a microwa ve reaction vial, and WR ( 42 mg, 79 mol) was then added. The suspension was heated by microwave irradiat ion at 150 ºC (MW power 175 W ) f or 5 minutes. The solvent was dist illed under reduced pressur e and the residue obtained was purif ied by silica gel f las h chromatography (dichloromethane) aff ording SeChry as a yellow solid, in 62% yield (31 mg). MP 190-192ºC. 1 H NMR (DMSO d 6 ) δ 13.20 (1H, exchange with D 2 O, s, OH-5), 11.47 (1H, exchange with



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Page 24 of 67

D 2 O, bs, OH-7), 8.22 (2H, d, J = 7. 5 Hz, H2’ and H6’), 7.92 (1H, s, H3), 7.32 (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).

13

C NMR (DMSO -d 6 ) δ 193.8 (C4),

164.8 ( C7), 161.3 ( C5), 153.4 (C8a), 152.0 (C2), 132.4 (C4’), 129.7 (C1’), 129.4 ( C3’ or C5’), 126. 9 ( C2’ or C6’), 122. 8 (C3), 116.1 (C4a), 101.2 ( C6), 95.2 ( C8).

77

Se NMR ( DMSO -d 6 ) δ 816.2 (Se4). MS ( APCI)

m/z 321 [ M( 8 2 Se)+H] + (37), 319 [ M( 8 0 Se)+H] + (100), 317 [ M( 7 8 Se)+H] + (86),

316

[ M( 7 7 Se) +H] +

(72),

315

[ M( 7 6 Se)+H] +

(51).

HRIEMS m/z

[ M( 8 2 Se)] + calcd. f or C 1 5 H 1 0 O 3 8 2 Se 319. 9797, f ound 319.9808 ; [M( 8 0 Se)] + calcd. f or C 1 5 H 1 0 O 3 8 0 Se 317.9795, f ound 317.9803; [ M( 7 8 Se)] + calcd. f or C 1 5 H 1 0 O 3 7 8 Se

315.9803,

f ound

315. 9804;

[ M( 7 7 Se)] +

calcd.

f or

C 1 5 H 1 0 O 3 7 7 Se

314.9829,

f ound

314. 9796;

[ M( 7 6 Se)] +

calcd.

f or

C 1 5 H 1 0 O 3 7 6 Se 313.9822, f ound 313.9812.

Thioderi vatives

Synthesis

of

2 -(3,4-di-hydroxyphenyl)-3, 5,7-tri-hydroxy-4H-

chromene-4-thione (SQue, 5). Boron tr ibrom ide (18 eq., 414 µL, 4.4 mmol) 1 M solution in dichloromethane was added dropwise t o a solution of SPQue (4) (91 mg, 0.24 mmol), in dr y dichloromethane ( 5 mL) at -78 ºC. The resulting mixture was stirr ed f or 24 h at room temper ature under nitrogen atmosphere. The reaction mixt ure was cooled to -78 ºC and quenched by addit ion of ice. The aqueous layer was extrac ted with et hyl acetate ( 3 x 30 m L). The combined organic layers were dried over anhydr ous m agnesium sulphate, aff ording 67 mg (86%) of SQue (5), as a red solid. MP > 300ºC. 1 H-NMR ( CD 3 O D)  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


Page 25 of 67

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(1H, d, J = 8.5, H6`) , 6.90 (1H, d, J = 8. 5, H5`), 6.5 4 (1H, s, H8), 6.34 (1H, s, H6).

13

C- NM R (CD 3 OD) 181.7 (C4), 163.5 (C7), 160.2 (C5),

152.6 (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 (C2ànd C5’), 111 .3 ( C4a), 100.4 (C6), 93.9 (C8). M S (FI) m/z 318 [ M] + . HRMS-EI m/z [ M] + calcd. f or C 1 5 H 1 0 O 6 S 318.0198, f ound 318.0200.

Synthesis

of

5,7 - hydroxy-2-phenyl-4H-chromene-4-thi one

(SChry,

6). Compound SChry (6) was prepared according to procedure reported by Elisei et al. 3 9 Specif ically, a solut ion of 2 (500 mg, 2.0 mmol) and P 2 S 5 (444 mg, 2.0 mM) in acetonitr ile (35 mL) was ref luxed dur ing 40 min. The solvent was dist illed under reduced pressure and t he residue obtained was pur if ied by silica gel f lash chromatography (n -hexane/ ethyl acetate 1:1) aff ording 3 in 2.6 % yield ( 14 mg). MP 219-220ºC ( MP lit. 218ºC). 2 8

1

H NMR (CDCl 3 ) δ 13.7 (1H, exchange with D 2 O, s, OH-5),

7.93 (2H, d, J = 8.1, H2’ and H6’), 7.58 - 7.52 (3H, m, H3’, H4’ and H5’), 7.41 (1H, s, H3), 6.52 (1H, d, J = 2.6, H8), 6.41 (1H, d, J = 2.6, H6). 1 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). 13

C 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’), 126.9 (C3’ and C5’), 117. 4 (C3), 112.6 (C4a), 100.8 (C6), 94.8 (C8). M S (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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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: 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 CD3OD (300 l) and the reaction was initiated by addition of H2O2 (32.4 mol). 1H 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 were determined by the area of the corresponding 1H NMR signals.

Computational Methods. All calculations were performed using the Gaussian09 rev A.0185 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 Oncology, Istituto Nazionale dei Tumori, Milan, Italy).90 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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2008 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 50 mg mL−1 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 × 103 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 5 mg mL−1 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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Clonogenic assay. 2 × 105 human ovarian MCF-7 cancer cells were seeded in Petri dishes (6 cm2) 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.

Cellular uptake. MCF-7 cells (7.5x105) were seeded in 75 cm2 flasks in growth medium (20 mL). After 24 h the medium was replaced and the cells were incubated for 12 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. Inhibition of redox enzymes in cells. MCF-7 cells were grown in 75 cm2 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. 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 Determination of total and oxidized glutathione. MCF-7 cells (2 × 105) 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 Na3PO4 and assayed for oxidized glutathione following the procedure reported by Bindoli et al. 95 Assay of total thiol groups. MCF-7 cells (4·105) were seeded in a six-well plate in growth medium (4 mL). After 24 h cells were incubated for 24 and 48 h with IC 50 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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3 mM DTNB to determine the total content of thiol groups. The change in absorbance was measured spectrophotometrically at 412 nm.

Oxidant species production. The production of oxidants was measured in MCF-7 cells (104/well) grown for 24 h in a 96-well plate in RPMI 1640 without phenol red. Cells were then washed in PBS/10 mM glucose and loaded with 10 μM 5-(and -6)chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2-DCFDA) (Molecular Probes-Invitrogen, Eugene, OR) for 20 min, in the dark. Afterwards, the cells were washed with the same medium and incubated with IC50 concentration of tested compounds. Fluorescence increase of DCFDA was estimated utilizing the wavelengths of 485 nm (excitation) and 527 nm (emission) in a Fluoroskan Ascent FL (Labsystem, Finland) plate reader. Antimycin (3 μM, Sigma-Aldrich), a potent inhibitor of Complex III in the electron transport chain, was used as positive control. Measurement of membrane potential. Depletion of the mitochondrial membrane potential (Δψ) was determined by measuring the fluorescence of cells stained with the dye tetramethylrhodamine methyl ester (TMRM, Molecular Probes). Cells were treated for 24 or 48 h with IC50 concentration of tested compounds and subsequently washed with PBS. They were harvested and incubated for 15 min. at 37°C in PBS with TMRM at a final concentration of 10 nM, freshly prepared from a 10 mM stock solution in DMSO. Fluorescence emissions from the 488 nm excited TMRM were collected with a flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with a 585/42-nm band-pass filter. The data were analysed by using FACSDiva software 5.3 (Becton Dickinson).

Caspase-3 activation. Caspase-3 activation was detected with the ApoAlert Caspase3 Fluorescent Assay Kit (Clontech, Mountain View, CA) according to the producer’s recommended procedures. MCF-7 cells (1 × 106) were treated for 12 or 24 h with IC50 doses of the tested compounds and lysed on ice in 50 μL of lysis buffer for 10 min and


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then treated with 50 μL of reaction buffer containing dithiothreitol (DTT) and 5 μL of caspase-3 substrate solution (Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl-coumarin (DEVD-AFC, Clontech). Fluorescence was determined on a Perkin-Elmer 550 spectrofluorimeter (excitation 440 nm, emission 505 nm). Caspase-3 activity was expressed as the increase in AFC-emitted fluorescence.

Induction of apoptosis. MCF-7 cells were seeded into 8-well tissue-culture slides (BD Falcon, Bedford, MA, USA) at 5 × 104 cells/well (0.8 cm2). After 24 h, cells were washed twice with PBS and, following 24 or 48 h treatment with IC50 doses of tested compounds, fixed in 4 % freshly prepared, ice-cold, paraformaldehyde, postfixed in ethanol, and air-dried. Slides were then stained for 5 min with 10 μg/mL of Hoechst 33258

(2′-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole

trihydrochloride hydrate, Sigma–Aldrich) in PBS before being examined by fluorescence microscopy (Olympus BX41, Cell F software, Olympus, Munster, Germany).

Statistical analysis. Data are the mean of at least four independent experiments. Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by Dunnett post hoc analysis. **P < 0.01; *P < 0.05.

■ ABBREVIATIONS ABC-transporters, ATP-binding cassette transporters; BDE, bond dissociation enthalpy; CM-DCFDA, chloromethyl derivative of dichlorodihydrofluorescein diacetate; DTTred, dithiotreitol reduced; DPPH, 2,2-diphenyl-1-picrylhydrazyl; DTTox, dithiotreitol oxidized; GF-AAF, Graphite Furnace Atomic Absorption Spectrometry; GPx, glutathione peroxidase; MDR, multidrug resistance; MMP, mitochondrial membrane potential; RF, resistance factor; ROS, reactive oxygen species; TMRM, tetramethyl rhodamine methyl ester; Trx, thioredoxin; TrxR, thioredoxin reductase;



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Page 32 of 67

■ AUTHOR INFORMATION Corresponding author AMMA: Tel: (+351) 218417627; E-mail: [email protected]. VG: Tel: (+39) 049 8275365; E-mail: [email protected].

Notes The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

This work was supported in part by Fundação para a Ciência e a Tecnologia (FCT), Portugal,

through

strategic

(UID/QUI/00100/2013),

RECI

funds projects

to

Centro

de

Química

(RECI/QEQ-QIN/0189/2012)

Estrutural and

the

University of Padova (Progetto di Ateneo CPDA131114/13). ILM also acknowledge FCT for doctoral (SFRH/BD/75426/2010) fellowship. AMMA would like to acknowledge FCT, “Programa Operacional Potencial Humano” and the European Social Fund for the IF Program (IF/01091/2013). AJSCV acknowledges COST Action CM1201. We thank Dr Ana Charas for access to her microwave reactor and Dr Conceição Oliveira for performing the HRMS measurements. Portuguese NMR and MS networks (IST-UTL Center) are also acknowledged for providing access to the facilities.

■ ASSOCIATED CONTENT Supplementary Material 1

H NMR, 13C NMR, 77Se NMR, HSQC and HMBC spectra and HPLC chromatograms of

all compounds prepared, relevant details of X-ray data analysis and selected bond


Page 33 of 67

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length and angles of 7 and the effects of low doses of flavonoids on HEK293 cells viability, Oxidant production and TrxR activity. This material is available free of charge via the Internet at http://pubs.acs.org

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Figures Caption

Figure 1. Structures of the flavonoids quercetin (1, Que) and chrysin (2, Chry).

Figure 2. ORTEP46,47 diagram, drawn with 50% probability ellipsoids, showing the atomic labelling scheme for compound SePQue (7).

Figure 3. Structures of the tested Chry- and PQue-derivatives.

Figure 4. Glutathione peroxidase-like activity of the tested compounds: plot of percentage of DTTred vs time. The oxidation of DTTred mediated by H2O2 in presence of a catalytic (10%) amount of the tested compound was monitored by 1H NMR according to Iwaoka et al. 57 methodology.

Figure 5. Cytotoxicity in non-transformed cells: HEK293 cells were treated for 72 h with increasing concentrations of tested compounds. Cytotoxicity was assessed by MTT test. IC50 values were calculated by four parameter logistic model (P < 0.05). S.I. = avearage IC50 non-trasformed cells/avearage IC50 malignant cells.

Figure 6. Clonogenic assay on MCF-7 cells treated for 6 h with increasing concentrations of 7 or 8.

Figure 7. Estimation of Se uptake in MCF-7 cells. Cells were incubated for 12 or 24 h with 12.5 or 25 μM of seleno-derivatives 7 and 8. Se cellular content was estimated by means of GF-AAS analysis. Results are expressed as μg Se/L/mg of proteins.



1 2 3 4 5 6 7 8 9 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 54 55 56 57 58 59 60

Figure 8. Effects of seleno-derivatives 7 and 8 on TrxR in human breast cancer cells. MCF-7 cells were incubated for 24 h with increasing concentrations of tested compounds. TrxR activity was assayed by means of DTNB reduction assay.

Figure 9. Effects of 7 and 8 on redox enzymes glutathione peroxidase (GPx) (A) and glutathione reductase (GR) (B) in human breast cancer cells. MCF-7 cells were incubated for 24 hours with increasing concentration of tested compounds. Subsequently, cells were washed twice with PBS and lysed. GR and GPx activities were followed at 340 nm.

Figure 10. Estimation of glutathione and total thiol groups in breast cancer cells treated with 7 and 8: (A) MCF-7 cells were incubated for 24 or 48 h with IC50 concentrations of 7 or 8. Total glutathione and oxidized glutathione (inset a) were measured spectrophotometrically at 412 nm; (B) MCF-7 cells were incubated for 24 h with IC50 concentrations of 7 or 8 The amounts of thiol groups were determined by the DTNB assay. Figure 11. Effect of 7 and 8 on caspase activation and apoptosis induction: (A) Cellular morphological changes of MCF-7 cells upon treatment with IC50 of 7 and 8 for 48 h and stained with the fluorescent dye Hoechst 33258; (B) Caspase-3 activity upon incubation of MCF-7 cells for 48 h with IC50 of 7 and 8. (C) Effects of 7 and 8 on cellular mitochondrial membrane potential. MCF-7 cells were treated for 24 oh 48 h with IC50 of 7 or 8 and stained with TMRM (10 nM). The percentage of cell with hypopolarized mitochondrial membrane potential was determined by flow cytometer analysis; Figure 12. Oxidant production in MCF-7 cells. Cells were pre-incubated in PBS/10 mM glucose medium for 20 min at 37 °C in presence of 10 mM CM-DCFDA and then


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1 2 3 4 5 6 7 8 9 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 54 55 56 57 58 59 60


treated with 25, 12.5 and 6.25 μM of 7 or 8 or antimycin (3 or 6 μM ). Fluorescence of DCF was measured.



1 2 3 4 5 6 7 8 9 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 54 55 56 57 58 59 60

Scheme Caption Scheme 1. Synthetic route for the chalcogenation of protected quercetin, PQue (3): when X=S a: P2S5 , THF, rt, 3 days;38 when X=Se a: WR, acetonitrile, MW, 175W, 150ºC, 5 min.

Scheme 2. Synthetic route for Chry direct chalcogenation: when X=S a: P2S5, acetonitrile,min.;39 when X=Se a: WR, acetonitrile, MW, 175W, 150ºC, 5 min.


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1 2 3 4 5 6 7 8 9 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 54 55 56 57 58 59 60


Table Caption

Table 1. Bond dissociation enthalpy (BDE) for each OH bond in kJ mol-1 computed with the B3LYP/6-31+G(2d,p), gas phase approach and the IC50 values obtained for the free DPPH radical scavenging activity of the tested compounds.

Table 2. Cytotoxicity studies in human cancer cells.



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Table of Contents Graphic


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1 2 3 4 5 6 7 8 9 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 54 55 56 57 58 59 60



1 2 3 4 5 6 7 8 9 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

Scheme 1 ACS Paragon Plus Environment

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1 2 3 4 5 6 7 8 9 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


Scheme 2



1 2 3 4 5 6 7 8 9 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

Figure 1


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1 2 3 4 5 6 7 8 9 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


Figure 2



1 2 3 4 5 6 7 8 9 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

Figure 3


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1 2 3 4 5 6 7 8 9 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


Figure 4



Viability (%)

1 2 3 4 5 6 7 8 9 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

Chry (2) SChry (6) SeChry (8) Que (1) PQue (3) SePQue (7)

100

80

60

40

20

Compound

HEK293 IC50 (μM)±S.D.

S.I.

Chry (2)

77.45± 4.32

1.9

SChry (6)

68.27± 3.74

1.2

SeChry (8)

8.13± 1.26

3.7

Que (1)

54.65± 3.73

2.5

PQue (3)

141.87± 8.36

1.6

SePQue (7)

17.34± 1.75

5.1

Cisplatin

14.12± 2.53

2.3

0 0

20

40

60

80

100

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120

Concentration (µM)

Figure 5


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100

Survival fraction (%)

1 2 3 4 5 6 7 8 9 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


80

60

40

20

0 0

5

10

15

20

Concentration (µM)

Figure 6


25

30


500

SeChry (8) SePQue (7)

400

µg/L/mg protein

1 2 3 4 5 6 7 8 9 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

Page 60 of 67

300

200

100

0 12.5 µM 12h

25 µM 12 h

12.5 µM 24h

Figure 7 ACS Paragon Plus Environment

25 µM 24h

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120

100

TrxR activity %

1 2 3 4 5 6 7 8 9 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


80

60

0 0.75 µM 1.25 µM 2.5 µM 5 µM 10 µM 20 µM

*

* *

*

** **

40 ** 20

0 SeChry (8)

SePQue (7)

Figure 8



120

GPx activity %

100

1.25 µM 2.5 µM 5 µM 10 µM 20 µM

A

80

60

40

20

0 SeChry (8)

SePQue (7)

120

100

1.25 µM 2.5 µM 5 µM 10 µM 20 µM

B

80

GR activity %

1 2 3 4 5 6 7 8 9 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

60

40

20

0 SeChry (8)

SePQue (7)

Figure 9


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20

GSSG (%)

24 h 48 h

A

120

**

a

15

**

10

0

7) 8) Ctr hry ( ePQue ( SeC S

**

**

**

B

**

100

**

5

mmol/mg protein

1 2 3 4 5 6 7 8 100 9 10 11 12 80 13 14 15 16 60 17 18 19 20 40 21 22 23 24 25 20 26 27 28 29 0 30 31 32 33 34 35 36 37 38 39 40 41 42


**

% sulfhydryl groups

Page 63 of 67

80

60 **

**

SeChry (8)

SePQue (7)

40

20

0

Ctr

SeChry (8)

SePQue (7)

Figure 10


Ctr

3,5 Journal of Medicinal Chemistry

A

Fluorescence (u.a.)

7

B

**

3,0 **

2,5

2,0

1,5

1,0

0,5

0,0

D D 8) 7) Ctr ry ( zVA ue ( ) + zVA h + Q C ) P (7 (8 Se Se hry Que P e SeC S

8

24 h 48 h

C

** SePQue (7)

** **

SeChry (8)

**

1 2 3 4 5 6 7 8 9 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

Ctr

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Figure 11 Ctr

0 5 ACS Paragon Plus Environment

10

15

20

% cells with depleted mitochondrial membrane potential

25

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10

8

6

Ctr (8), 25 µM (8), 12,5 µM (8), 6,25 µM (7), 25 µM (7), 12,5 µM (7), 6,25 µM antimycin 6 µM antimycin 3 µM

A

4

2

0 0 60 360 780 900 1260 1860 1980 2220 2700 2940 3300 3480 3840 4020 4200 4560 4740 5100 5280 5460 5820 6000 6180 6540 6720 7080

Fluorescence (au)

1 2 3 4 5 6 7 8 9 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


Time (sec)

Figure 12



1 2 3 4 5 6 7 8 9 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

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Table 1.

OH BDE (Kcal/mol-1)a

DPPH radical scavenging activity, IC50 (μM)

Compound

OH-5

OH-7

OH-3

OH-3’

OH-4’

Chry (2)

104.5

92.5

-

-

-

b

SChry (6)

92.0

92.6

-

-

-

4600

SeChry (8)

85.8

92.0

-

-

-

659

Que (1)

99.5

91.2

85.4

88.2

76.9

28

PQue (3)

102.2

.-

-

-

-

b

SPQue (4)

91.4

-

-

-

-

b

SePQue (7)

84.9

-

-

-

-

74

Ascorbic Acid

-

-

-

-

-

6

Table 1. Bond dissociation enthalpy (BDE) for each OH bond in kJ mol-1, computed with B3LYP/6-31+G(2d,p) gas phase approach, along with IC50 values obtained for the free DPPH radical scavenging activity of the tested compounds.a BDE OH bond dissociation enthalpy, computed as BDE(ArOH)=BDEexp(PhOH)+[Ecalc(ArO.)-Ecalc(ArOH)]-[Ecalc(PhO.)-Ecalc(PhOH)]52, with BDEexp(PhOH)=87.6 Kcal mol-1;53 b Not measurable, the IC50 exceeds the solubility range.


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Table 2.

1 2 3 4 5 IC50 (µM) ± S.D. 6 7 Compound 8 MCF-7 9 HEK293 A375 HCT-15 BxPC3 MCF-7 A431 A431/Pt 2008 C13* ADR 10 11 51.04±1.73 60.13±2.85 Chry (2) 33.23±1.23 54.43±2.03 54.27±1.65 50.32±2.09 29.39±2.86 28.62±2.51 49.66±3.01 (1.7) 12 (1.0) (2.0) 13 72.27±2.96 98.40±2.26 58.54±1.96 36.48±1.69 56.59±3.28 64.41±1.96 70.43±2.27 66.31±1.27 41.12±2.56 14 SChry (6) (1.0) (1.6) (1.4) 15 1.96±0.77 4.04±1.28 16 SeChry (8) 1.09±1.21 3.17±0.97 2.31±1.93 2.25±0.67 1.61±1.07 3.18±0.91 (2.0) 2.87 ±1.32 (0.9) (1.4) 17 22.15±2.14 34.37±3.22 37.62±3.82 Que (1) 18 25.13±2.62 16.35±2.29 24.12±1.85 20.90±3.44 23.04±1.07 21.18±1.84 (1.1) (1.5) (1.8) 19 20 SQue (5) N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 21 82.25±2.77 22 PQue (3) 69.72±3.32 68.11±2.35 >100 >100 >100 57.54±2.28 >100 >100 23 (1.4) 24 SPQue (4) N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 25 26 3.35±1.58 2.09±1.27 2.29±1.93 SePQue (7) 2.21±1.07 2.23±1.01 2.42±1.19 3.08±1.98 3.11±1.23 3.78±1.52 (1.2) 27 (1.1) (1.1) 28 8.41±1.22 29 Cisplatin 3.12±1.13 12.31±1.26 11.43±1.29 7.6±2.49 1.62±1.25 3.42±1.08 (2.1) 2.17±1.37 22.26±1.86 (10.3) 1.31±0.71 20.91±2.4 (16) Doxodubicine 30 31 32 33 34 S.D.= standard deviation, N.D.= not detected 35 4 -1 36Cells (3-8·10 ·mL ) were treated for 72 h with increasing concentrations of tested compounds. Cytotoxicity was assessed by MTT test. IC50 values were calculated by four parameter logistic model (P < 0.05). 37 38The numbers in parentheses are the values of RF (Resistance Factor) = (IC50-resistant cell)/(IC50-parent cell line) 39 ACS Paragon Plus Environment 40 41 42

Selenium-Containing Chrysin and Quercetin Derivatives: Attractive

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