Masked Phenolic-Selenium Conjugates: Potent and Selective Antiproliferative Agents Overcoming P-gp Resistance

Cancer accounts for one of the most complex diseases nowadays due to its multifactorial nature. Despite the vast number of cytotoxic agents developed so far, good therapeutic approaches are not always reached. In recent years, multitarget drugs are gaining great attention against multifactorial diseases in contraposition to polypharmacy. Herein we have accomplished the conjugation of phenolic derivatives with an ample number of organochalcogen motifs with the aim of developing novel antiproliferative agents. Their antioxidant, and antiproliferative properties (against six tumour and one non-tumour cell lines) were analysed. Moreover, in order to predict P-gp-mediated chemoresistance, the P-glycoprotein assay was also conducted in order to determine whether compounds prepared herein could behave as substrates of that glycoprotein. Selenium derivatives were found to be significantly stronger antiproliferative agents than their sulfur isosters. Moreover, the length and the nature of the tether, together with the nature of the organoselenium scaffold were also found to be crucial features in the observed bioactivities. The lead compound, bearing a methylenedioxyphenyl moiety, and a diselenide functionality, showed a good activity (GI50 = 0.88‒2.0 µM) and selectivity towards tumour cell lines (selectivity index: 14‒32); moreover, compounds considered herein were not substrates for the P-gp efflux pump, thus avoiding the development of chemoresistance coming from such mechanism, commonly found for widely-used chemotherapeutic agents.


Introduction
Organoselenium chemistry has provided modern organic synthesis with valuable intermediates and catalysts [1], but it has also emerged as a powerful tool in Medicinal Chemistry [2]. A plethora of selenium-containing derivatives have been designed and tested, eliciting quite diverse bioactivities, ranging from antimicrobial [3], anti-inflammatory [4], antidiabetic [5], anti-Alzheimer [6] to particularly anticancer [7][8][9]; concerning the latter activity, selenium derivatives not only exhibit relevant antiproliferative features, but have also been used in adjuvant therapies in mice [10], or as photosensitizers in photodynamic therapy [11], and are therefore considered as candidates for the

Chemistry
We envisioned the possibility of linking dichalcogenide and catechol moieties through an ester tether; for that purpose, we attempted coupling naturally-occurring hydroxytyrosol 1, the most remarkable phenolic derivative in olive tree [33] with 4,4'-dithiobisbutyric acid.
Several conditions were assayed, like conversion of the dicarboxylic acid into the corresponding acid dichloride with refluxing neat SOCl2 followed by coupling with 1 in the presence of tetrabutylammonium hydrogensulfate [42] at rt and using THF as solvent. We also attempted coupling of the transient acid chloride coming from the dicarboxylic acid, generated again with SOCl2, with 1 at rt and THF as solvent, in the presence of Ce(III) (0.5 equiv.); this catalyst has been previously used for the chemoselective esterification of hydroxytyrosol 1 on the aliphatic hydroxyl group with alkyl halides with different chain lengths [43]. A biocatalyzed process with different ratio of Candida antarctica B [44] involving 1 and the dicarboxylic acid 2 was also tested (THF, 50 °C). Unfortunately, all these procedures proved to be unsuccessful, as no conversion was observed by TLC.
Unexpectedly, treatment of 1 with the same disulfide in the presence of PyBOP, a well-known peptide coupling reagent, did not furnish 3, but the 14-membered ring bicyclic derivative 4 in moderate yield (Scheme 1); presumably, the mild basic conditions provided by Et3N enhanced nucleophilicity of the phenolic hydroxyl groups compared to the aliphatic counterpart. 1 H-NMR unambiguously proved that phenolic hydroxyl groups had been esterified, as protons H-2, H-5 and H-6 resonated above 7 ppm, as previously observed for acetylated olive polyphenols [45]. Scheme 1. Attempted esterification of hydroxytyrosol 1.

Chemistry
We envisioned the possibility of linking dichalcogenide and catechol moieties through an ester tether; for that purpose, we attempted coupling naturally-occurring hydroxytyrosol 1, the most remarkable phenolic derivative in olive tree [33] with 4,4'-dithiobisbutyric acid.
Several conditions were assayed, like conversion of the dicarboxylic acid into the corresponding acid dichloride with refluxing neat SOCl 2 followed by coupling with 1 in the presence of tetrabutylammonium hydrogensulfate [42] at rt and using THF as solvent. We also attempted coupling of the transient acid chloride coming from the dicarboxylic acid, generated again with SOCl 2 , with 1 at rt and THF as solvent, in the presence of Ce(III) (0.5 equiv.); this catalyst has been previously used for the chemoselective esterification of hydroxytyrosol 1 on the aliphatic hydroxyl group with alkyl halides with different chain lengths [43]. A biocatalyzed process with different ratio of Candida antarctica B [44] involving 1 and the dicarboxylic acid 2 was also tested (THF, 50 • C). Unfortunately, all these procedures proved to be unsuccessful, as no conversion was observed by TLC.
Unexpectedly, treatment of 1 with the same disulfide in the presence of PyBOP, a well-known peptide coupling reagent, did not furnish 3, but the 14-membered ring bicyclic derivative 4 in moderate yield (Scheme 1); presumably, the mild basic conditions provided by Et 3 N enhanced nucleophilicity of the phenolic hydroxyl groups compared to the aliphatic counterpart. 1 H-NMR unambiguously proved that phenolic hydroxyl groups had been esterified, as protons H-2, H-5 and H-6 resonated above 7 ppm, as previously observed for acetylated olive polyphenols [45].
Due to the difficulties encountered for linking dichalcogens with an ester-based scaffold, we decided to shift to an amide-type linker, and no other coupling reagents or different reactions were attempted. For that purpose, naturally-occurring dopamine 5 seemed to be a good building block. Combination of dopamine with commercially-available disulfides 6, 7 and 2 (n = 1, 2 and 3, respectively) in the presence of PyBOP as the peptide coupling reagent, furnished the corresponding symmetrical disulfides 8-10 in excellent yields (79-94%, Scheme 2); DMF was used as solvent, and Et 3 N as base.
Unexpectedly, treatment of 1 with the same disulfide in the presence of PyBOP, a well-known peptide coupling reagent, did not furnish 3, but the 14-membered ring bicyclic derivative 4 in moderate yield (Scheme 1); presumably, the mild basic conditions provided by Et3N enhanced nucleophilicity of the phenolic hydroxyl groups compared to the aliphatic counterpart. 1 H-NMR unambiguously proved that phenolic hydroxyl groups had been esterified, as protons H-2, H-5 and H-6 resonated above 7 ppm, as previously observed for acetylated olive polyphenols [45]. Due to the difficulties encountered for linking dichalcogens with an ester-based scaffold, we decided to shift to an amide-type linker, and no other coupling reagents or different reactions were attempted. For that purpose, naturally-occurring dopamine 5 seemed to be a good building block. Combination of dopamine with commercially-available disulfides 6, 7 and 2 (n = 1, 2 and 3, respectively) in the presence of PyBOP as the peptide coupling reagent, furnished the corresponding symmetrical disulfides 8-10 in excellent yields (79-94%, Scheme 2); DMF was used as solvent, and Et3N as base.
In order to diversify the functionality of the phenolic derivatives, phenyl selenide 18 and selenocyanate 20 were also prepared (Scheme 3); in both cases, 3-bromopropanoic acid 11 was the key synthetic intermediate. For accessing 18, compound 11 was subjected to a nucleophilic displacement with in situ generated sodium phenyl selenide (obtained by reduction of diphenyl diselenide with NaBH4), followed by peptide coupling with dopamine 5 (Scheme 3).
Similarly, nucleophilic displacement of 11 with KSeCN, followed by coupling with 5, furnished 20 in a moderate yield (34%) (Scheme 3). We have also carried out the synthesis of the hitherto unknown catechol-ebselen hybrid 26 (Scheme 4). Ebselen (2-phenyl-1,2-benzoselenazol-3-one) is a relevant selenoheterocycle which behaves as an excellent glutathione peroxidase (GPx) mimic [46], a selenoenzyme that exerts a natural defence against oxidative stress. Ebselen has also been proved to ameliorate neural degeneration after cerebral artery occlusion [47], and recently, it has also been found to act as a mimetic of lithium [48], what might be useful against bipolar disorder. In order to analyse the influence of the chalcogen atom on the biological properties, the corresponding seleno-isosters 15, 16 were accessed (Scheme 2); they key diselenides 13 and 14 (n = 2, 3, respectively, Scheme 2) were obtained from the corresponding ω-bromo carboxylic acids by nucleophilic displacement with freshly-prepared Na 2 Se 2 (reduction of elemental selenium black with NaBH 4 , for 13), or with KSeCN, followed by reduction with NaBH 4 (for 14, Scheme 2). PyBOP-mediated peptide coupling with dopamine 5 afforded diselenides 15, 16, which were isolated in good yields (62% and 77%, respectively, Scheme 2).
In order to diversify the functionality of the phenolic derivatives, phenyl selenide 18 and selenocyanate 20 were also prepared (Scheme 3); in both cases, 3-bromopropanoic acid 11 was the key synthetic intermediate. For accessing 18, compound 11 was subjected to a nucleophilic displacement with in situ generated sodium phenyl selenide (obtained by reduction of diphenyl diselenide with NaBH 4 ), followed by peptide coupling with dopamine 5 (Scheme 3). selenocyanate 20 were also prepared (Scheme 3); in both cases, 3-bromopropanoic acid 11 was the key synthetic intermediate. For accessing 18, compound 11 was subjected to a nucleophilic displacement with in situ generated sodium phenyl selenide (obtained by reduction of diphenyl diselenide with NaBH4), followed by peptide coupling with dopamine 5 (Scheme 3).
Similarly, nucleophilic displacement of 11 with KSeCN, followed by coupling with 5, furnished 20 in a moderate yield (34%) (Scheme 3). We have also carried out the synthesis of the hitherto unknown catechol-ebselen hybrid 26 (Scheme 4). Ebselen (2-phenyl-1,2-benzoselenazol-3-one) is a relevant selenoheterocycle which behaves as an excellent glutathione peroxidase (GPx) mimic [46], a selenoenzyme that exerts a natural defence against oxidative stress. Ebselen has also been proved to ameliorate neural degeneration after cerebral artery occlusion [47], and recently, it has also been found to act as a mimetic of lithium [48], what might be useful against bipolar disorder. Similarly, nucleophilic displacement of 11 with KSeCN, followed by coupling with 5, furnished 20 in a moderate yield (34%) (Scheme 3).
We have also carried out the synthesis of the hitherto unknown catechol-ebselen hybrid 26 (Scheme 4). Ebselen (2-phenyl-1,2-benzoselenazol-3-one) is a relevant selenoheterocycle which behaves as an excellent glutathione peroxidase (GPx) mimic [46], a selenoenzyme that exerts a natural defence against oxidative stress. Ebselen has also been proved to ameliorate neural degeneration after cerebral artery occlusion [47], and recently, it has also been found to act as a mimetic of lithium [48], what might be useful against bipolar disorder. Treatment of anthranilic acid with sodium nitrite and HCl afforded diazonium salt 22, which was in turn coupled with in situ generated sodium diselenide to afford diselenide 24 (Scheme 4); reaction with thionyl chloride and DMF as a catalyst furnished transient aryl selenyl choride 25 [49].  Next structural modification involved the use of O-protected phenolic derivatives, and the removal of the amide-type linker (Schemes 5 and 6). For that purpose, we chose the methylenedioxyphenyl moiety, which is present in natural antioxidants known as sesame lignans (the major components of sesame oil), and also in species, like black pepper [50]. Numerous compounds containing such acetal functionality act as inhibitors of cytochrome P450, a family of heme-containing enzymes that catalyse the oxidative metabolization of hydrophobic substrates, either endogenous or exogenous, into hydrophilic derivatives [50]. Cytochrome P450 has been considered as a target in cancer therapies [41] so incorporation of a methylenedioxyphenyl scaffold in the compounds prepared herein might improve the profile of the selenium-containing derivatives by interacting simultaneously with another key target of tumour cells.
Thus, reduction of O-protected carboxylic acids 27, 28 achieved by the combination of NaBH4 and I2 [51] afforded the corresponding alcohols (Scheme 5); Appel-type reaction (Ph3P + tetrahaloalkanes) on 29-31 was attempted for inserting a bromine atom on the ω-position of the linker (Scheme 5). Although such reaction proceeded with high yields for the preparation of 33, 34 (n = 1, 2, 82% and 72%, respectively), when applied to piperonyl alcohol 29 (n = 0), a spontaneous decomposition of the brominated derivative took place, as revealed by TLC; accordingly, in that case, bromine atom was replaced by chlorine as an attempt to reduce the intrinsic reactivity of the benzyl bromide derivative (Scheme 5); crude 32 was used without any further purification. Treatment of anthranilic acid with sodium nitrite and HCl afforded diazonium salt 22, which was in turn coupled with in situ generated sodium diselenide to afford diselenide 24 (Scheme 4); reaction with thionyl chloride and DMF as a catalyst furnished transient aryl selenyl choride 25 [49]. Final coupling with dopamine hydrochloride 5 under basic conditions (Et 3 N) furnished ebselen derivative 26 in a moderate yield (30%) (Scheme 4).
Next structural modification involved the use of O-protected phenolic derivatives, and the removal of the amide-type linker (Schemes 5 and 6). For that purpose, we chose the methylenedioxyphenyl moiety, which is present in natural antioxidants known as sesame lignans (the major components of sesame oil), and also in species, like black pepper [50]. Numerous compounds containing such acetal functionality act as inhibitors of cytochrome P450, a family of heme-containing enzymes that catalyse the oxidative metabolization of hydrophobic substrates, either endogenous or exogenous, into hydrophilic derivatives [50]. Cytochrome P450 has been considered as a target in cancer therapies [41] so incorporation of a methylenedioxyphenyl scaffold in the compounds prepared herein might improve Finally, a combination of the amide functionality contained in diselenides 15/16 and the O-methylidene acetal scaffold of derivatives 39-41 was envisioned (Scheme 6). Thus, nucleophilic displacement of NaN3 on brominated derivative 33, followed by reduction of the azido moiety, and PyBOP-promoted peptide coupling with 4-selenocyanato butyric acid 44 furnished 45 in a 44% overall yield (three steps, Scheme 6). Subsequent reduction gave symmetrical diselenide 46 in a 63% yield, which incorporated a protected catechol moiety, an amide-based tether and a diselenide functionality as the key structural motifs.

Antioxidant Properties
In vitro antioxidant properties of key unprotected phenolic derivatives were analyzed using two different methodologies: free radical scavenging capacity, using the DPPH method (2,2-diphenyl-1-picrylhydrazyl), a HAT (hydrogen atom transfer) procedure, widely-used for both natural and synthetic derivatives, and also the capacity for scavenging H2O2, one of the most common ROS in degenerative diseases, including cancer.

Free Radical Activity (DPPH Method)
DPPH is a commercially-available stable free radical commonly used for in vitro testing of radical scavenging properties of antioxidants [52]. Its methanolic solution exhibits a deep purple color (λmax = 515 nm); the strong absorption is consequently decreased upon reaction with an Finally, a combination of the amide functionality contained in diselenides 15/16 and the O-methylidene acetal scaffold of derivatives 39-41 was envisioned (Scheme 6). Thus, nucleophilic displacement of NaN3 on brominated derivative 33, followed by reduction of the azido moiety, and PyBOP-promoted peptide coupling with 4-selenocyanato butyric acid 44 furnished 45 in a 44% overall yield (three steps, Scheme 6). Subsequent reduction gave symmetrical diselenide 46 in a 63% yield, which incorporated a protected catechol moiety, an amide-based tether and a diselenide functionality as the key structural motifs.

Antioxidant Properties
In vitro antioxidant properties of key unprotected phenolic derivatives were analyzed using two different methodologies: free radical scavenging capacity, using the DPPH method (2,2-diphenyl-1-picrylhydrazyl), a HAT (hydrogen atom transfer) procedure, widely-used for both natural and synthetic derivatives, and also the capacity for scavenging H2O2, one of the most common ROS in degenerative diseases, including cancer.

Free Radical Activity (DPPH Method)
DPPH is a commercially-available stable free radical commonly used for in vitro testing of radical scavenging properties of antioxidants [52]. Its methanolic solution exhibits a deep purple color (λmax = 515 nm); the strong absorption is consequently decreased upon reaction with an Thus, reduction of O-protected carboxylic acids 27, 28 achieved by the combination of NaBH4 and I 2 [51] afforded the corresponding alcohols (Scheme 5); Appel-type reaction (Ph 3 P + tetrahaloalkanes) on 29-31 was attempted for inserting a bromine atom on the ω-position of the linker (Scheme 5). Although such reaction proceeded with high yields for the preparation of 33, 34 (n = 1, 2, 82% and 72%, respectively), when applied to piperonyl alcohol 29 (n = 0), a spontaneous decomposition of the brominated derivative took place, as revealed by TLC; accordingly, in that case, bromine atom was replaced by chlorine as an attempt to reduce the intrinsic reactivity of the benzyl bromide derivative (Scheme 5); crude 32 was used without any further purification.
Nucleophilic substitution accomplished with KSeCN in DMF (Scheme 5) furnished the expected ω-selenocyanato derivatives 35-37 in good to excellent yields (72-91%); such compounds were the key synthetic intermediates for the preparation of Se-Bn derivative 38 upon NaBH4-promoted reduction of the selenocyanato moiety (Scheme 5) and in situ alkylation of the transient sodium selenide with benzyl bromide, and also for the preparation of symmetrical diselenides 39-41 upon reduction with NaBH 4 in the presence of oxygen (Scheme 5).
Finally, a combination of the amide functionality contained in diselenides 15/16 and the O-methylidene acetal scaffold of derivatives 39-41 was envisioned (Scheme 6). Thus, nucleophilic displacement of NaN 3 on brominated derivative 33, followed by reduction of the azido moiety, and PyBOP-promoted peptide coupling with 4-selenocyanato butyric acid 44 furnished 45 in a 44% Pharmaceuticals 2020, 13, 358 7 of 22 overall yield (three steps, Scheme 6). Subsequent reduction gave symmetrical diselenide 46 in a 63% yield, which incorporated a protected catechol moiety, an amide-based tether and a diselenide functionality as the key structural motifs.

Antioxidant Properties
In vitro antioxidant properties of key unprotected phenolic derivatives were analyzed using two different methodologies: free radical scavenging capacity, using the DPPH method (2,2-diphenyl-1-picrylhydrazyl), a HAT (hydrogen atom transfer) procedure, widely-used for both natural and synthetic derivatives, and also the capacity for scavenging H 2 O 2 , one of the most common ROS in degenerative diseases, including cancer.
Free Radical Activity (DPPH Method) DPPH is a commercially-available stable free radical commonly used for in vitro testing of radical scavenging properties of antioxidants [52]. Its methanolic solution exhibits a deep purple color (λ max = 515 nm); the strong absorption is consequently decreased upon reaction with an antiradical agent, being followed spectrophotometrically. Table 1 depicts the EC 50 values for the compounds prepared herein, including hydroxytyrosol (HT 1, an olive tree antioxidant [33]) and dopamine (used herein as the building block for the synthesis of the majority of the compounds) as reference compounds. Herein, following Bahorun's methodology [53], in vitro levels of H 2 O 2 were measured in an indirect fashion, by registering the H 2 O 2 -mediated oxidation of phenol red in the presence of horseradish peroxidase, to give a chromophore with a maximum absorption at 610 nm. Accordingly, in the presence of an antioxidant agent, there is an inverse correlation between the observed absorbance, and the scavenging properties. Table 2 depicts the H 2 O 2 -scavenging properties of compounds prepared herein, and tested at a final 50 µM concentration.

Antiproliferative Activity
Organochalcogen derivatives prepared herein were evaluated in vitro as potential antiproliferative agents, using a panel of six human tumour cell lines: A549 (non-small cell lung), HBL-100 (breast), HeLa (cervix), SW1573 (non-small cell lung), as drug sensitive lines, T-47D (breast) and WiDr (colon) as drug resistant lines. In addition to precursors 2, 6, 7, 13, 14, 17, 19, ebselen and cisplatin (CDDP) were used as reference drugs. Besides tumour cells, the most active compounds were also tested against the non-tumour cell line BJ-hTert (fibroblasts). In both cases, the NCI protocol, with minor modifications [54], was followed; selected data, including the selectivity index (S.I.) can be found in Table 3 and full data in Supporting Information (Table S1).
Not soluble

P-glycoprotein Assay
This assay allows prediction of whether a certain chemotherapeutic agent could develop chemoresistance when behaving as a P-gp substrate [55], one of the most common efllux-pumps used for the extrusion of cell xenobiotics. For that purpose, a cell line-based assay was used, in particular the lung cancer cell line in its wild type (SW1573), and its variant overexpressing P-gp (Sw1573/Pgp) [56]. Moreover, compounds were tested in the presence and in the absence of verapamil, which is known to inhibit P-gp [57]. Chemotherapeutic agents paclitaxel (PTX) and vinblastine (VB) are used herein as reference compounds. In this assay, the resistance factor (Rf) for a certain compound is defined as the ratio of GI 50 in the cell line overexpressing P-gp and the wild one (Table 4), high values of Rf denoting that the compound behaves as a P-gp substrate, and thus, chemoresistance through this mechanism will probably take place.

Discussion
The role of ROS in cancer cells is highly debated nowadays; although they are known to be present at high levels during carcinogenesis and cancer progression, it is claimed that they have a dual activity [58]. Thus, on the one hand, they can facilitate cell proliferation by acting as signalling molecules, a capacity that is increased by rising ROS levels, and a concomitant improved antioxidant machinery; nevertheless, on the other hand, ROS can also provoke cell death by inducing apoptosis [59].
Free phenolic derivatives can scavenge free radicals upon reaction with free phenolic hydroxyl groups, to give quinone-type intermediates [33] (sacrificial antioxidants), converting ROS into non-radical species, like alcohols or water; in this sense, phenolic compounds act as chain-breaking or primary antioxidants, probably the most important family of natural compounds having such antioxidant mechanism. The antioxidant properties of organochalcogen, particularly organoselenium derivatives, are diverse, and therefore, they might be useful against different targets in oxidative stress, an activity elicited by their exceptional redox properties. Such activities include scavenging of free radicals [60], metal complexation [61], or mimicry of natural antioxidant enzymes for the removal of deleterious hydrogen peroxide and alkyl peroxides (e.g., GPx), the so-called bioinspired antioxidants [46]. Interestingly, diselenides and selenides can be substrates in vivo for mammalian thioredoxin reductase (TrxR), furnishing selenols/selenolates, stronger nucleophiles than their sulfur counterparts; as a result, the corresponding selenol has a stronger reducing power than isosteric thiol [62,63].
Concerning the antiradical properties, data depicted in Table 1 clearly show the influence of the length of the hydrocarbon chain of disulfides 8-10 (See structures in Scheme 2). Thus, compound 9, with a three-carbon length tether, was the best compound in the series (EC 50 = 4.2 µM). The same tendency, although to a lower extent, was found for selenoisosters 15, 16 (See structures in Scheme 2). Accordingly, it seems that the combination of a phenolic scaffold with a dichalcogenide motif provokes a synergic effect in the antiradical properties, leading to improved derivatives compared to reference compounds. Furthermore, the ebselen-dopamine hybrid 26 (See structure in Scheme 4) showed stronger antiradical activity compared with phenyl selenide 18 and selenocyanate 20 (See structures in Scheme 3). Replacement of the phenolic structure with a methylidene moiety led to a complete impairment of activity (compounds 39, 46, See structures in Schemes 5 and 6, respectively), indicating that the diselenide moiety lacks antiradical properties itself, but through a synergic effect, improves that of the phenolic motif; in fact, disulfide 9 exhibited a roughly three-fold increased activity compared to reference compounds (hydroxytyrosol and dopamine).
H 2 O 2 is considered to be one of the most common ROS in living organisms, and in particular in tumor cells [64], being produced by the mitochondria-based respiratory chain cascade through a complex enzymatic machinery [65]. For that purpose, we also envisioned the possibility of analyzing the scavenging properties of compounds prepared herein against such ROS. In a similar way to what was observed in the antiradical assay, disulfide 9 (See structure in Scheme 2) was again the most potent antioxidant within the series, capable of scavenging roughly 80% of the initial H 2 O 2 level. Although to a lower extent, diselenide 16 (See structure in Scheme 2) and ebselen analogue 26 (See structure in Scheme 4) were also remarkable examples, capable of scavenging roughly 50% of the ROS. Removal of the catechol functionality (compounds 4, 39, 46, See structures in Schemes 1, 5 and 6) led to a complete loss of ROS scavenging properties.
This capacity for scavenging ROS could suggest a potential chemoprotective action of title compounds; as it can be observed, some of the sulfur-containing derivatives exhibited a similar or even a slightly better antioxidant profile than selenium-counterparts. Accordingly, it could be claimed that some of the sulfur derivatives prepared herein could act as good chemoprotective agents, with probably reduced toxicological concerns than isosteric selenium. However, when shifted to the antiproliferative activity, as below discussed, the sulfur derivatives showed a negligible activity, unlike selenium-containing compounds. Interestingly for some other selenoderivatives we have previously detected a pro-oxidant activity in the presence of tumour cells, with a significant increase in ROS levels, and thus, potentially a pro-apoptotic effect [13].
With regards to antiproliferative activity, derivatives prepared herein can be categorized into a series of families; the first of them is comprised of disulfides 4, 8-10 (See structures in Schemes 1 and 2), their selenium isosters 15, 16 (See structures in Scheme 2), selenides 18, 20 (See structures in Scheme 3) and ebselen analogue 26 (See structure in Scheme 4). All of them have in common some structural motifs: the presence of an unprotected catechol moiety, and the connection of the phenolic and organoselenium fragments via an amide tether. According to data depicted in Table 3 and Table  S1, a series of valuable structure-activity relationships can be obtained; firstly, the length of the tether was found to be again crucial for the bioactivities, although with different behaviour for every family of compounds; thus, whereas disulfides 8 and 10, with a 2-and 4-atom carbon tether, respectively, turned out to have negligible activities at the highest tested concentration (100 µM), derivative 9 (with a 3-carbon tether) exhibited a moderate activity, with GI 50 values lower than 45 µM for 5 cell lines.
Interestingly, isosteric replacement of sulfur with selenium furnished considerable stronger derivatives (15,16), again with a remarkable influence on the tether length; thus, diselenide 16, with a longer tether, exhibited GI 50 values lower than 6.0 µM for all the cell lines, although lacking selectivity (GI 50 = 3.5 µM for fibroblasts). Moreover, the nature of the organoselenium motif was also found to clearly influence the activity, as selenides 18, 20 and ebselen analogue 26 only showed moderate antiproliferative properties.
With these data in hand, we attempted to improve both, the activity and selectivity by accomplishing structural modifications. For that purpose, we decided to install a methylidene acetal protecting group on the phenolic scaffold, and to eliminate the amide tether, giving access to derivatives 35-41 (See structures in Scheme 5). Remarkably, such structural modification afforded low micromolar-submicromolar activities for selenocyanates 35, 36 and diselenides 39, 40. Again, distance between both pharmacophores, the protected phenolic residue, and the organoselenium motif were found to be crucial structural elements, as diselenide 41, unlike its counterparts 39, 40, was found to be only a moderate antiproliferative agent. Therefore, activity increased from a 2 to 3-carbon linker, but was clearly diminished for the four-carbon structure (41). It is also important to highlight that a rather good increase in selectivity was achieved for selenocyanate 36 (4-13-fold), and particularly for diselenide 40 (up to 32-fold).
We next designed a family containing the key pharmacophoric motifs present in the previous two families of derivatives, giving access to selenocyanate 45 and diselenide 46 (See structures in Scheme 6). In both cases, a methylidene acetal, and an amide-type tether are present simultaneously in the molecule. Such compounds turned out to be the most potent compounds of the series, with activities in the submicromolar range for most of the tested cell lines (0.36-1.6 µM for 45 and 0.12-0.30 µM for 46), although reduced selectivity in comparison to 40.
Remarkably, diselenide 46 exhibited up to a 104-fold increase in activity compared to CDDP in multidrug resistant cell lines (T-47D, WiDr), showing also a selectivity index of 3-8 compared to non-tumor cell lines.
Accordingly, in view of GI 50 plot range (Figure 2), the three most potent derivatives were found to be 40, 45 and 46, now with the longest tether, the former exhibiting the best profile in terms of selectivity. The influence of the free or protected phenolic scaffold can be clearly analysed by comparing the activities of 16 (free catechol) and 46 (acetal-containing derivative), both with the same tether and the same selenium functionality (diselenide). Although toxicity towards non-tumour cells is slightly increased, the potency against tumour cell lines is improved up to 30-fold, so furnishing a better overall selectivity index for protected 46. Probably, a reduced polarity of 46 in comparison to 16 enables transportation through cell membranes, thus, improving the bioavailability of the compound. Some of the least cytotoxic compounds towards Bj-h-Tert lack the amide functionality, so it seems that removal of such moiety might contribute to the improvement of the selectivity index. were also tested against the non-tumour cell line BJ-hTert (fibroblasts). In both cases, the NCI protocol, with minor modifications [54], was followed; selected data, including the selectivity index (S.I.) can be found in Table 3 and full data in Supporting Information (Table S1).

P-glycoprotein Assay
This assay allows prediction of whether a certain chemotherapeutic agent could develop chemoresistance when behaving as a P-gp substrate [55], one of the most common efllux-pumps used for the extrusion of cell xenobiotics. For that purpose, a cell line-based assay was used, in particular the lung cancer cell line in its wild type (SW1573), and its variant overexpressing P-gp (Sw1573/Pgp) [56]. Moreover, compounds were tested in the presence and in the absence of micromolar-submicromolar concentration, particularly interesting against the two multidrug resistant cell lines (T-47D, breast) and WiDr (colon). Moreover, the selectivity index (SI) was found to be 14-32, thus showing a clear improvement of the antiproliferative profile in comparison to the reference drug (cisplatin), whose reduced selectivity is partially responsible for its numerous side-effects.
It is also interesting to mention that Desai and co-workers designed a selenocyanate and a diselenide as SAHA analogues [28], an hydroxamic acid that behaves as a strong histone deacetylase inhibitor, enzymes overexpressed in certain tumours for regulating cancer progression [66]. Such selenoderivatives, more potent histone deacetylase inhibitors than parent SAHA, were reported to be particularly active against melanoma [67], and it was hypothesized that both functionalities could be reduced under cell conditions to give a transient selenide that binds histone deacetylase [68].
In connection with this fact, it is important to mention that the most relevant antiproliferative properties found herein are coming from selenocyanates and diselenides; dialkyl diselenides have been found to exert pro-oxidant properties at low concentrations [69]. Besides targeting ROS level, it could be hypothesized that title compounds could be reduced [63] in the tumour microenvironment, giving a transient selenol/selenolate that might inhibit histone deacetylases in a similar way found for the Se-analogues of SAHA.
One major challenge when developing chemotherapeutic agents for the treatment of cancer progression is to avoid chemoresistance. Although there are many mechanisms of resistance to drugs, like changes in mitochondria, induced DNA-repair, oncogenes or tumor suppressor genes, among others, as indicated previously, a classical one is comprised of transporter pumps (e.g., P-glycoproteins), that act by extruding xenobiotics from the cell in an ATP-dependent manner; such glycoproteins are overexpressed in a number of tumors. Therefore, knowledge if chemotherapeutic agents are P-gp substrates or not becomes a crucial aspect in order to predict chemoresistance mediated by these efflux pumps [70]. Although they do not constitute the only mechanism developed by cancer cells to eliminate drugs, it is one of the most studied ones, and now considered to be one the key target to overcome chemoresistance [71], either by inhibition or by evasion; many of the most widely-used chemotherapeutic agents (e.g., doxorubicin, etoposide, paclitaxel, etc.) are susceptible to P-gp-mediated efflux. Table 4 depicts GI 50 values after a 48 h-exposure of compounds with wild SW1573 cell line, or the one overexpressing P-gp, with (w) or without (w/o) the presence of the P-gp inhibitor verapamil. Overall, the low values found for Rf denote that our compounds do not act as substrates for P-gp and thus cannot develop chemoresistance via this pump efflux mechanism, in clear contrast with the reference compounds used herein.

General Procedures
1 H (300.1 and 500.1 MHz) and 13 C (75.5 and 125.7 MHz) NMR spectra were recorded at 25 • C on a Bruker Avance 300, or on a Bruker Avance III 500 MHz spectrometer; the latter, equipped with a TCI cryoprobe). 2D NMR experiments (COSY, HSQC) were used for the assignment of 1 H and 13 C signals. Mass spectra (ESI) were recorded on a QExactive mass spectrometer. TLCs were performed on aluminium pre-coated sheets (E. Merck silica gel 60 F 254 ); spots were visualized by UV light, and by charring with 1% phosphomolybdic acid in EtOH, 1.5% vanillin in EtOH containing 1% of H 2 SO 4 , or with 0.3% ninhydrin in EtOH. Column chromatography was performed using E. Merck silica gel 60 (40-63 µm). The antioxidant assays were performed in a Hitachi U-2900 spectrophotometer with a thermostated cuvette holder, using PS cuvettes (1 cm × 1 cm × 4.5 cm). Reported methodology [52] was used with minor modifications. To a 60 µM methanolic DPPH solution (1.17 mL), the antioxidant solution (5-7 different solutions, 30 µL) or solvent (control, 30 µL) were added. After 30 min. of incubation in the dark, absorbance was measured at 515 nm, and EC 50 values were calculated.

Antiproliferative Activity
The in vitro antiproliferative activity was assayed using minor modifications of the protocol of the National Cancer Institute (NCI) of the United States against the six human solid tumor cell and the non-tumor cell lines tested [54].

Conclusions
We have successfully accomplished the conjugation of phenolic residues with organochalcogen motifs in the search for new potent and selective antiproliferative agents.
A large variety of compounds have been accessed, modifying the nature of the phenolic substituents (either free hydroxyl groups, or the methylenedioxyphenyl moiety), the kind of chalcogen atom (sulfur, selenium), the nature and length of the linker, and also the nature of the chalcogen-containing functionality (disulfide, diselenide, selenide, selenocyanato, benzoselenazolone). Such diverse structural modifications have allowed us to carry out structure-activity relationships searching for the best profile.
Unprotected derivatives, including those with moderate antiproliferative activity, might exert valuable antioxidant activities and act as chemopreventive agents.
Remarkably, selenoderivatives tested herein were not found to be substrates for the P-gp efflux pump, so presumably such compounds will not undergo chemoresistance via this transport pump for excreting xenobiotics.