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

pharmaceuticals Article Masked Phenolic-Selenium Conjugates: Potent and Selective Antiproliferative Agents Overcoming P-gp Resistance Paloma Begines 1,†, Lucía Sevilla-Horrillo 2,†, Adrián Puerta 3, Rebecca Puckett 2, Samuel Bayort 1, Irene Lagunes 3, Inés Maya 1, JoséM. Padrón3,* , Óscar López 1,* and JoséG. Fernández-Bolaños 1,* 1Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, Apartado 1203, E-41071 Seville, Spain; [email protected] (P.B.); [email protected] (S.B.); [email protected] (I.M.) 2Escuela Politécnica Superior, Universidad de Sevilla, Virgen de África 7, E-41011 Seville, Spain; [email protected] (L.S.-H.); [email protected] (R.P.) 3 BioLab, Instituto Universitario de Bio-Org á nica “Antonio Gonz á lez” (IUBO-AG), Universidad de La Laguna, c/Astrof í sico Francisco S á nchez 2, E-38206 La Laguna, Spain; [email protected] (A.P.); [email protected] (I.L.) *Correspondence: [email protected] (J.M.P.); [email protected] (Ó.L.); [email protected] (J.G.F.-B.); Tel.: +34-922-316-502 (J.M.P.) ext. 6126; +34-954-559-997 (Ó.L.); +34-954-550-996 (J.G.F.-B.) †These authors contributed equally to this work. Received: 22 September 2020; Accepted: 28 October 2020; Published: 31 October 2020   Abstract: 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 (GI 50 =0.88-2.0 µ M) 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. Keywords: phenolics; organoselenium; antioxidant; antiproliferative; P-gp 1. 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 – 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 Pharmaceuticals 2020,13, 358; doi:10.3390/ph13110358 www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2020,13, 358 2 of 22 development of new anticancer therapies. Interestingly, inorganic selenium sources usually provoke higher genotoxic stress, leading to more hazardous effects than their organic counterparts [12]. Organoselenium motifs have been combined with many other pharmacophores, like phenolic compounds [ 13 , 14 ], iminosugars [ 15 ], steroids [ 16 , 17 ], non-steroidal anti-inflammatory drugs [ 18 ], heterocycles [ 19 – 21 ], nucleosides [ 22 ], quinones [ 23 ], or metal coordination complexes [ 24 ] among others, or incorporated into the structures of natural products, like carbohydrates, giving access to the so called selenosugars [ 25 ]. The combination of chitosan and selenium nanoparticles has recently been reported as a vector for the transportation of mRNA for amplifying immune response in the potential development of cancer vaccines [26]. The mechanism involved in the anticancer properties of organoselenium derivatives still remains unclear. Some of them have proved to inhibit key enzymes which are abnormally overexpressed in tumour cells, like carbonic anhydrases (IX and XII isoforms) [ 27 ] or histone deacetylases [ 28 ]. However, what makes selenium unique, compared to its chalcogen analogue sulfur is, besides differences in bond lengths and energies, its strong redox character [ 29 ]. In fact, organoselenium compounds might behave as antioxidant agents, thus with a chemopreventive utility, or as pro-oxidant agents, provoking cell death probably through an apoptotic pathway upon exacerbation of oxidative stress [ 30 , 31 ]. In that sense, selenium-containing drugs can act as redox modulators, many of them showing preference to malignant cells [ 12 ]; pro-oxidant activity can be attributed to reactive oxygen species (ROS) induction [ 32 ], oxidation of thiol functionalities in proteins, and modification of chromatin. As a result, a series of biological downstream effects take place, affecting the activity of kinases, caspases, DNA repair machinery, or transcription factors [12]. The main purpose pursued herein has been the access to new chalcogen-based multifunctional drugs (Figure 1) with potential utility against cancer as redox chemotherapeutics. We have combined a series of organosulfur and organoselenium motifs (e.g., disulfide, diselenide, selenide, selenocyanato, benzoselenazolone) with an o-dihydroxyphenyl residue (catechol moiety), a functionality commonly found in phenolic structures produced as secondary metabolites by plants [ 33 ] and endowed with relevant biological properties, like anti-inflammatory [ 34 ], neuroprotective [ 35 ], or antiproliferative [36–38] , mainly due to their strong antioxidant properties [ 33 ]. The combination of the organoselenium and phenolic motifs, both showing either antioxidant (ROS-user) or pro-oxidant (ROS-enhancer) properties will provide compounds with two redox centres. Both pharmacophores can complement each other, and have a synergic effect in the modulation of the bioactivities of the compounds, in particular, their anticancer properties. For instance, these two redox centres might be involved in the generation of high levels of ROS in malignant cells, thus leading to cell death through an apoptotic pathway. This pattern, with two redox centres is a motif that has been reported for organoselenium/quinones [ 23 ] or ferrocene/phenols [ 39 ] as an interesting approach for the development of new chemotherapeutic agents targeting the redox dysregulation in cancer; compounds with even three and four redox centres combining chalcogen atoms and quinones have also been reported [ 40 ] to be strong antiproliferative agents. Modification of the nature of the chalcogen atom, the nature and the length of the tether and substituents on the aryl moiety can provide valuable structure-activity relationships. Besides targeting redox status, incorporation of certain selenium functionalities (e.g., selenocyanato, diselenide, Figure 1) might diversity the potential targets when tackling cancer, as such scaffolds could interfere overexpressed enzymes in tumour cells, like histone deacetylases. Moreover, we also envisioned the possibility of blocking the catechol functionality with a methylenedioxy function, a structural motif that might afford a better bioavailability when crossing cell membranes, and might also allow the inhibition of cytochrome P450 [ 41 ], a therapeutic target against cancer. Pharmaceuticals 2020,13, 358 3 of 22 Pharmaceuticals 2020, 13, x FOR PEER REVIEW 3 of 21 Figure 1. General structure of the chalcogen-polyphenol conjugates prepared herein. 2. Results 2.1. 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 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. 1H-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. Figure 1. General structure of the chalcogen-polyphenol conjugates prepared herein. 2. Results 2.1. 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 Et3N as base. Pharmaceuticals 2020,13, 358 4 of 22 Pharmaceuticals 2020, 13, x FOR PEER REVIEW 3 of 21 Figure 1. General structure of the chalcogen-polyphenol conjugates prepared herein. 2. Results 2.1. 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 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. 1H-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. Scheme 1. Attempted esterification of hydroxytyrosol 1. Pharmaceuticals 2020, 13, x FOR PEER REVIEW 4 of 21 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 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 Na2Se2 (reduction of elemental selenium black with NaBH4, for 13), or with KSeCN, followed by reduction with NaBH4 (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). Scheme 2. Synthesis of symmetrical phenolic disulfides and diselenides. 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). Scheme 3. Synthesis of phenolic aryl selenide 18 and selenocyanate 20. 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. Scheme 2. Synthesis of symmetrical phenolic disulfides and diselenides. 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 NaBH4), followed by peptide coupling with dopamine 5(Scheme 3). Pharmaceuticals 2020,13, 358 5 of 22 Pharmaceuticals 2020, 13, x FOR PEER REVIEW 4 of 21 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 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 Na2Se2 (reduction of elemental selenium black with NaBH4, for 13), or with KSeCN, followed by reduction with NaBH4 (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). Scheme 2. Synthesis of symmetrical phenolic disulfides and diselenides. 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). Scheme 3. Synthesis of phenolic aryl selenide 18 and selenocyanate 20. 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. Scheme 3. Synthesis of phenolic aryl selenide 18 and selenocyanate 20. 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. Pharmaceuticals 2020, 13, x FOR PEER REVIEW 5 of 21 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 (Et3N) furnished ebselen derivative 26 in a moderate yield (30%) (Scheme 4). Scheme 4. Synthesis of catechol-ebselen hybrid 26. 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. 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 NaBH4 in the presence of oxygen (Scheme 5). Scheme 4. Synthesis of catechol-ebselen hybrid 26. 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 5and 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 Pharmaceuticals 2020,13, 358 6 of 22 the profile of the selenium-containing derivatives by interacting simultaneously with another key target of tumour cells. Pharmaceuticals 2020, 13, x FOR PEER REVIEW 6 of 21 Scheme 5. Preparation of O-protected phenolic-based selenides, selenocyanates and diselenides. 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. Scheme 6. Preparation of O-protected selenocyanato 45 and diselenide 46. 2.2. Biological Assays 2.2.1. 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 Scheme 5. Preparation of O-protected phenolic-based selenides, selenocyanates and diselenides. Pharmaceuticals 2020, 13, x FOR PEER REVIEW 6 of 21 Scheme 5. Preparation of O-protected phenolic-based selenides, selenocyanates and diselenides. 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. Scheme 6. Preparation of O-protected selenocyanato 45 and diselenide 46. 2.2. Biological Assays 2.2.1. 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 Scheme 6. Preparation of O-protected selenocyanato 45 and diselenide 46. 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 NaBH4in 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. 2.2. Biological Assays 2.2.1. 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 ( λmax =515 nm); the strong absorption is consequently decreased upon reaction with an antiradical agent, being followed spectrophotometrically. Table 1depicts 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. Table 1. Antiradical activity for organochalcogen derivatives. Compound EC50 (µM) 4>175 812.3 ±0.5 94.2 ±0.1 10 15.3 ±0.1 15 7.8 ±0.2 16 9.3 ±1.3 18 23.2 ±0.6 20 10.8 ±0.4 26 7.8 ±0.2 39 >250 46 >250 HT 13.4 ±0.6 Dopamine 11.4 ±1.2 H2O2Scavenging Properties 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 2depicts the H 2 O 2 -scavenging properties of compounds prepared herein, and tested at a final 50 µM concentration. Pharmaceuticals 2020,13, 358 8 of 22 Table 2. H2O2scavenging properties 1. Compound % Scavenging 4—2 826.5 ±2.1 979.8 ±0.8 10 11.8 ±0.5 15 40.0 ±3.1 16 52.9 ±3.4 18 19.0 ±0.8 20 25.8 ±1.0 26 53.8 ±2.3 39 — 46 — Dopamine 46.1 ±4.6 1At 50 µM; 2No scavenging properties were observed at the tested concentration. 2.2.2. 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 3and full data in Supporting Information (Table S1). Table 3. Selected GI50 values (µM) for the antiproliferative activity of organochalcogen derivatives. Compound A549 (Lung) HBL-100 (Breast) HeLa (Cervix) T-47D (Breast) WiDr (Colon) BJ-hTert (Fibroblasts) S.I. 15 5.4 ±1.1 29 ±6 3.6 ±1.1 39 ±5 31 ±7—1— 16 3.7 ±0.6 2.4 ±0.6 2.3 ±0.4 5.8 ±0.9 3.9 ±0.6 3.5 ±0.6 0.6–1.5 18 33 ±5 34 ±1 23 ±1 71 ±5 85 ±21 — — 20 28 ±8 37 ±2 19 ±8 35 ±1 41 ±10 — — 35 3.1 ±0.7 3.5 ±1.4 3.5 ±0.8 3.4 ±0.2 2.0 ±0.1 8.7 ±3.2 2.5–4.4 36 1.8 ±0.7 2.1 ±0.2 1.2 ±0.2 1.1 ±0.3 1.3 ±0.6 14 ±2 6.7–13 37 Not soluble 38 19 ±2 14 ±3 5.2 ±1.0 4.7 ±1.2 14 ±2 — — 39 1.2 ±0.3 2.9 ±0.6 2.4 ±1.4 2.9 ±0.4 1.6 ±0.3 3.9 ±1.0 1.3–3.3 40 1.6 ±0.5 2.0 ±0.9 0.95 ±0.07 0.88 ±0.17 1.2 ±0.1 28 ±5 14–32 41 32 ±8 93 ±10 31 ±8 29 ±5 29 ±8 — 45 0.41 ±0.03 1.6 ±0.3 0.36 ±0.09 0.40 ±0.04 0.41 ±0.03 1.8 ±0.7 1.1–5.0 46 0.27 ±0.02 0.25 ±0.02 0.12 ±0.01 0.19 ±0.04 0.25 ±0.06 1.0 ±0.1 3.7–8.3 Ebselen 25 ±9 13 ±3 26 ±8 90 ±14 >100 — — CDDP 4.9 ±0.2 1.9 ±0.2 2.0 ±0.3 15 ±2 26 ±6 14 ±2 0.5–7.4 1Not tested. 2.2.3. 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 Pharmaceuticals 2020,13, 358 9 of 22 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. Table 4. Antiproliferative activity (GI 50 ) of organochalcogen derivatives in SW1573 and SW1573/P-gp cell lines 1. w/o Verapamil w Verapamil SW1573 SW1573/PGP Rf SW153 SW1573/PGP Rf 15 3.3 ±0.4 5.0 ±0.1 1.5 5.0 ±0.7 5.3 ±0.3 1.1 16 2.7 ±0.3 5.1 ±0.6 1.9 2.3 ±0.3 5.1 ±0.4 2.2 35 2.3 ±0.5 7.1 ±0.6 3.1 3.1 ±1.0 7.0 ±0.6 2.3 36 4.2 ±0.7 6.8 ±0.4 1.6 3.1 ±0.4 5.3 ±0.6 1.7 38 27 ±2 29 ±0.2 1.1 15 ±3 25 ±2 1.6 39 3.0 ±0.5 18 ±0.1 6.0 2.5 ±0.1 5.1 ±0.4 2.0 40 1.9 ±0.1 3.9 ±0.2 2.0 1.8 ±0.2 4.5 ±1.0 2.5 45 1.1 ±0.2 3.3 ±0.6 3.1 1.1 ±0.1 2.5 ±0.1 2.3 PTX 1.5 ±0.5 196 ±53 128 1.6 ±0.2 4.2 ±0.9 2.6 VB 0.9 ±0.3 2051 ±682 2388 0.8 ±0.2 1.0 ±0.5 1.3 1 Paclitaxel (PTX) and vinblastine (VB) were used as reference drugs. GI 50 values are given in µ M for organochalcogen derivatives and in nM for PTX and VB. Standard deviation was calculated from at least two independent experiments. Rf represents the ratio between GI50 values in SW1573/PGP and SW1573 cells. 3. 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 1clearly 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 µ M). 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 Pharmaceuticals 2020,13, 358 16 of 22 stirred under Ar and in the dark, at rt, 13h for 35 and at 75 ◦ C, 5 h for 36 , 37 . After that, the crude was concentrated to dryness and the residue was partitioned between CH 2 Cl 2 (20 mL) and H 2 O (20 mL); the aqueous layer was further extracted with CH 2 Cl 2 (3 × 10 mL). The combined organic fractions were dried over Na 2 SO 4 , filtered, and the filtrate was concentrated to dryness; the residue was purified by column chromatography using the eluant indicated in each case. (30,40-Methylenedioxyphenyl)methyl selenocyanate (35) (3 0 ,4 0 -methylenedioxyphenyl)methyl chloride 32 (141.6 mg, 0.83 mmol) was used. Column chromatography (10:1 cyclohexane-EtOAc) gave 35 as a white solid. Yield: 172.4 mg (87%); R F 0.29 (9:1 cyclohexane-EtOAc); 1 H-NMR (300 MHz, CDCl 3 ) δ 6.85-6.81 (m, 2H, H-2, H-6), 6.77 (d, 1H, J 5,6 =8.4 Hz, H-5), 5.98 (s, 2H, OCH 2 O), 4.26 (s, 2H, CH 2 Se) ppm; 13 C-NMR (125.7 MHz, CDCl 3 ) δ 148.3, 148.2 (C-3, C-4), 129.0 (C-1), 123.0 (C-6), 109.3 (C-2), 108.8 (C-5), 102.0 (SeCN), 101.6 (OCH 2 O), 33.5 (CH2Se) ppm; HRESI-MS calcd for C9H7NNaO280Se ([M+Na]+): 263.9534, found: 263.9527. 2-(30,40-Methylendioxyphenyl)ethyl selenocyanate (36) 2-(3 0 ,4 0 -Methylenedioxyphenyl)ethyl bromide 33 (190 mg, 0.83 mmol) was used. Column chromatography (5:1 cyclohexane-EtOAc) afforded 36 . Yield: 192 mg, 91%; R F 0.66 (6:1 cyclohexane-EtOAc); 1 H-NMR (300 MHz, CDCl 3 ) δ 6.76 (d, 1H, J 50,60 =7.7 Hz, H-5 0 ), 6.68-6.65 (m 2H, H-2 0 , H-6 0 ), 5.94 (s, 2H, OCH 2 O), 3.23, 3.10 (2brt, 2H each, J H,H =7.0 Hz, CH 2 -CH 2 ) ppm; 13 C-NMR (75.5 MHz, CDCl 3 ) δ (ppm): 148.0, 146.8 (C-3 0 , C-4 0 ), 132.3 (C-1 0 ), 121.7 (C-6 0 ), 108.9, 108.6 (C-2 0 , C-5 0 ), 101.5 (CN), 101.1 (OCH 2 O), 36.6 (CH 2 Ar), 30.8 (CH 2 Se); HRESI-MS calcd for C 10 H 9 NNaO 280 Se ([M+Na]+): 277.9691, found 277.9687. 3-(3’,4’-Methylenedioxyphenyl)propyl selenocyanate (37) 3-(3’,4’-Methylenedioxyphenyl)propyl bromide 34 (201.5 mg 0.83 mmol) was used. Column chromatography (6:1 cyclohexane-EtOAc) gave 37 . Yield: 160.1 mg, 72%; R F 0.34 (6:1 cyclohexane-EtOAc). 1 H-NMR (300 MHz, CDCl 3 ) δ 6.74 (d, 1H, J 50,60 =7.9 Hz, H-5 0 ), 6.67 (brd, 1H, J 20,60 =1.7 Hz, H-2 0 ), 6.62 (brdd, 1H, H-6 0 ), 5.93 (s, 2H, OCH 2 O), 3.01 (t, 2H, J H,H =7.2 Hz, CH 2 Se ), 2.70 (t, 2H, J H,H =7.1 Hz, CH 2 Ar), 2.19 (quint, 2H, CH 2 -CH 2 Se) ppm; 13 C-NMR (125.7 MHz, CDCl 3 ) δ 148.0, 146.3 (C-3 0 , C-4 0 ), 133.7 (C-1 0 ), 121.5 (C-6 0 ), 109.0, 108.5 (C-2 0 , C-5 0 ), 101.4 (SeCN), 101.1 (OCH 2 O), 34.7 (CH 2 Ar), 32.4 (CH 2 -CH 2 Se), 28.7 (CH 2 Se) ppm; 77 Se-NMR (95 MHz, CDCl 3 ) δ 207.57 ppm; HRESI-MS calcd for C11H11NNaO280Se ([M+Na]+): 291.9847, found 291.9839. 4.2.8. 1-Benzylseleno-2-(3’, 4’-methylenedioxyphenyl)ethane (38) To a solution of 2-(3 0 ,4 0 -methylenedioxyphenyl)ethyl selenocyanate 36 (129.1 mg, 0.51 mmol) and benzyl bromide (0.24 mL, 2.02 mmol, 4.0 equiv.) in MeOH (5 mL) at 0 ◦ C, was slowly added NaBH 4 (21.1 mg, 0.56 mmol, 1.1 equiv.). The resulting mixture was stirred at 0 ◦ C, under inert atmosphere (N 2 ) and in the dark for 2 h. Then, the crude reaction was concentrated to dryness and the residue was partitioned between EtOAc (8 mL) and sat. aq. NH 4 Cl (8 mL). The water layer was further washed with EtOAc (3x5 mL); the combined organic fractions were dried over MgSO 4 , filtered and concentrated to dryness. The residue was purified by column chromatography (cyclohexane) to give 38 . Yield: 72.2 mg, 44%; R F 0.76 (6:1 ciclohexane-EtOAc); 1 H-NMR (300 MHz, (CD 3 ) 2 CO) δ 7.36–7.20 (m, 5H, Ar-H), 6.74 (d, 1H, J 50,60 =7.9 Hz, H-5 0 ), 6.71 (d, 1H, J 20,60 =1.5 Hz, H-2 0 ) 6.65 (d, 1H, H-6 0 ), 5.93 (s, 2H, OCH 2 O), 3.82 (s, 2H, CH 2 Ph), 2.86-2.65 (m, 4H, CH 2 -CH 2 ) ppm; 13 C-NMR (75.5 MHz, (CD 3 ) 2 CO) δ 148.6, 146.9 (C-3 0 , C-4 0 ), 140.8 (Ar-C ipso ), 136.3 (C-1 0 ), 129.8, 129.2 (Ar-C o , Ar-C m ) 127.3 (Ar-Cp), 122.1 (C-6 0 ), 109.6, 108.8 (C-2 0 , C-5 0 ), 101.7 (OCH 2 O), 37.4 (CH 2 Ar), 27.1 (CH 2 Se), 25.7 (CH 2 Ph) ppm; 77 Se-NMR (95 MHz, CDCl 3 ) δ 262.17 ppm; HRESI-MS calcd for C 16 H 16 NaO 280 Se ([M+Na] + ): 343.0208, found 343.0200. Pharmaceuticals 2020,13, 358 17 of 22 4.2.9. General Procedure for the Preparation of Diselenides 39-41 To a solution of ω -(3’, 4’-methylenedioxyphenyl)alkyl selenocyanates 35 - 37 (0.69 mmol) in absolute EtOH (7 mL) at 0 ◦ C was slowly added NaBH 4 (13.5 mg, 0.36 mmol). The corresponding mixture was stirred for 15 h at rt. The crude reaction was concentrated to dryness and the residue was purified by column chromatography using the eluant indicated in each case. Bis[(3’, 4’-methylenedioxyphenyl)methyl]diselenide (39) (3 0 ,4 0 -Methylenedioxyphenyl)methyl selenocyanate 35 (167 mg, 0.69 mmol) was used. Column chromatography (cyclohexane → 4:1 cyclohexane-EtOAc) gave 39. Yield: 87 mg, 59%; R F 0.64 (4:1 cyclohexane-EtOAc); 1 H-NMR (300 MHz, CDCl 3 ) δ 6.74 (d, 1H, J 50,60 =7.7 Hz, H-5 0 ), 6.74 (brd, 1H, J 20,60 =1.6 Hz, H-2 0 ), 6.69 (brdd, 1H, H-6 0 ), 5.94 (s, 2H, OCH 2 O), 3.84 (s, 2H, CH 2 Se) ppm; 13 C-NMR (125.7 MHz, CDCl 3 ) δ 147.8, 146.9 (C-3 0 , C-4 0 ), 132.9 (C-1 0 ), 122.4 (C-6 0 ), 109.5, 108.3 (C-2 0 , C-5 0 ), 101.2 (OCH 2 O), 33.0 (CH 2 Se) ppm; HRESI-MS calcd for C 16 H 14 NaO 480 Se 2 ([M+Na] + ): 452.9115, found: 452.9109. Bis[2-(30,40-methylenedioxyphenyl)ethyl]diselenide (40) 2-(3 0 ,4 0 -Methylenedioxyphenyl)ethyl selenocyanate 36 (177.7 mg, 0.69 mmol) was used. Column chromatography (40:1 → 30:1 cyclohexane-EtOAc) gave 40 as a yellow oil. Yield: 61 mg, 39%; R F 0.41 (6:1 cyclohexane-EtOAc); 1 H-NMR (300 MHz, CDCl 3 ) δ 6.74 (d, 1H, J 50,60 =7.9 Hz, H-5 0 ), 6.69 (brd, 1H, J 20,60 =1.4 Hz, H-2 0 ), 6.65 (brdd, 1H, H-6 0 ), 5.93 (s, 2H, OCH 2 O), 3.10 (brt, 2H, JH,H =7.5 Hz , CH 2 ), 2.95 (brt, 1H, CH 2 ) ppm; 13 C-NMR (125.7 MHz, CDCl 3 ) δ 147.8, 146.2 (C-3 0 , C-4 0 ), 134.7 (C-1 0 ), 121.5 (C-6 0 ), 109.0, 108.4 (C-2 0 , C-5 0 ), 101.0 (OCH 2 O), 37.4 (CH 2 Ar), 31.2 (CH 2 Se) ppm; HREI-MS calcd for C18H18O480Se2([M]+): 457.9530, found: 457.9527. Bis[3-(3’, 4’-methylenedioxyphenyl)propyl]diselenide (41) 3-(3’, 4’-Methylenedioxyphenyl)propyl selenocyanate 37 (184 mg, 0.69 mmol) was used. Column chromatography (cyclohexane → 4:1 cyclohexane-EtOAc) gave 41 . Yield: 121.3 mg, 73%; R F 0.71 (4:1 cyclohexane-EtOAc); 1 H-NMR (300 MHz, CDCl 3 ) δ 6.72 (d, 1H, J 50,60 =7.9 Hz, H-5 0 ), 6.67 (brd, 1H, J 20,60 =1.4 Hz, H-2 0 ), 6.62 (brdd, 1H, H-6 0 ), 5.92 (s, 2H, OCH 2 O), 2.88 (t, 2H, JH,H =7.2 Hz , CH 2 Se), 2.63 (t, 2H, J H,H =7.2 Hz, CH 2 Ar), 2.01 (quint, 2H, CH 2 -CH 2 Se) ppm; 13 C-NMR (125.7 MHz, CDCl 3 ) δ 147.8, 145.9 (C-3 0 , C-4 0 ), 135.2 (C-1 0 ), 121.4 (C-6 0 ), 109.0, 108.3 (C-2 0 , C-5 0 ), 100.9 (OCH 2 O), 35.2 (CH 2 Ar), 32.7 (CH 2 -CH 2 Se), 29.1 (CH 2 Se) ppm; 77 Se-NMR (95 MHz, CDCl 3 ) δ 303.09 ppm; HRESI-MS calcd for C20H22O480Se2([M]+): 485.9849, found: 485.9837. 4.2.10. 1-Azido-2-(30,40-methylenedioxyphenyl)ethane (42) A solution of 2-(3 0 ,4 0 -methylenedioxyphenyl)ethyl bromide 33 (1.52 g, 6.64 mmol) and NaN 3 (1.49 g, 22.9 mmol, 3.4 equiv.) in DMF (25 mL) was heated at 70 ◦ C overnight. After that, the crude was partitioned between H 2 O (15 mL) and CH 2 Cl 2 (3 × 20 mL); combined organic fractions were dried over MgSO 4 , filtered, and the filtrate was concentrated to dryness. The residue was purified by column chromatography (cyclohexane → 1:4 cyclohexane-Et 2 O) to give 42 as a colourless oil. Yield: 1.02 g, 80%. R F 0.76 (1:4 cyclohexane-Et 2 O); 1 H-NMR (300 MHz, CDCl 3 ) δ 6.66 (d, 1H, J 50,60 =8.0 Hz, H-5 0 ), 6.61 (m, 1H, H-2 0 ), 6.57 (m, 1H, H-6 0 ), 5.84 (s, 2H, OCH 2 O), 3.36 (t, 2H, J H,H =7.2 Hz, CH 2 ), 2.71 (t, 2H, CH 2 ) ppm; 13 C-NMR (75.5 MHz, CDCl 3 ) δ 147.9, 146.5 (C-3 0 , C-4 0 ), 131.9 (C-1 0 ), 121.9 (C-6 0 ), 109.2, 108.5 (C-2 0 , C-5 0 ), 101.1 (OCH 2 O), 52.8 (CH 2 N 3 ), 35.2 (CH 2 Ar) ppm; HRESI-MS calcd for C 9 H 9 N 3 O 2 [M]+: 191.0695, found: 191.0683. 4.2.11. N-[20-(30,40-Methylenedioxyphenyl)ethyl]-4-selenocyanatobutanamide (45) To a solution of 2-(3,4-methylendioxyphenyl)ethylamine 43 (194 mg, 1.17 mmol) in anhydrous DMF (3 mL), were added PyBOP (630 mg, 1.21 mmol), 4-selenocyanatobutanoic acid 44 (231 mg, Pharmaceuticals 2020,13, 358 18 of 22 1.20 mmol) and anhydrous Et 3 N (0.25 mL, 1.80 mmol, 1.5 equiv.). The corresponding mixture was stirred overnight at rt, under Ar and in the dark. Then, the crude reaction was concentrated to dryness, and the residue was purified by column chromatography (CH 2 Cl 2→ 80:1 CH 2 Cl 2 -MeOH) to give 45 as an orange oil. Yield: 279 mg, 70%; R F 0.60 (EtOAc); 1 H-NMR (300 MHz, CDCl 3 ) δ 6.74 (d, 1H, J50,60=7.8 Hz , H-5 0 ), 6.66 (brd, 1H, J 20,60 =1.6 Hz, H-2 0 ), 6.61 (dd, 1H, H-6 0 ), 5.92 (s, 2H, OCH 2 O), 5.62 (brs, 1H, NH), 3.46 (q, 2H, J H,H =6.9 Hz, CH 2 N), 3.09 (t, 2H, J H,H =6.8 Hz, CH 2 Ar), 2.72 (t, 2H, J H,H =6.9 Hz, CH 2 CO), 2.31 (brt, 2H, J =6.7 Hz, CH 2 ), 2.20 (quint, 2H, CH 2 CH 2 CH 2 ) ppm; 13 C-NMR (75.5 MHz, CDCl 3 ) δ 171.2 (CO), 148.0, 146.4 (C-3 0 , C-4 0 ), 132.5 (C-1 0 ), 121.7 (C-6 0 ), 109.1, 108.5 (C-5 0 , C-2 0 ), 101.9 (SeCN), 101.1 (OCH 2 O), 40.8 (CH 2 N), 35.4 (ArCH 2 ), 34.8 (CH 2 CO), 29.1 (CH 2 Se), 26.5 (CH 2 CH 2 CH 2 ) ppm; HRESI-MS m/z calcd for C 14 H 16 N 2 NaO 3 Se [M+Na] + : 363.0218, found: 363.0218. 4.2.12. 4,4’-Diselenobis{N-[20’-(30”,40”-Methylenedioxyphenyl)ethyl]butanamide} (46) To a solution of selenocyanate 45 (128.8 mg, 0.38 mmol) in EtOH (2 mL) was added NaBH 4 (14.4 mg, 0.38 mmol). The corresponding mixture was stirred at rt overnight; then it was concentrated to dryness and the residue was partitioned between EtOAc (25 mL) and sat. aq. NH 4 Cl (20 mL). The aqueous layer was further washed with EtOAc (3x20 mL); the combined organic fractions were dried over MgSO 4 , filtrated and the filtrate was concentrated to dryness. The residue was purified by column chromatography (1:1→1:3 cyclohexane-EtOAc) to give 46 as a white solid. Yield: 75.4 mg, 63%; R F 0.38 (EtOAc); 1 H-NMR (300 MHz, CDCl 3 ) δ 6.73 (d, 1H, J 50”,60” =7.9 Hz, H-5 0 ”), 6.66 (brd, 1H, J 20”,60” =1.5 Hz, H-2 0 ”), 6.61 (dd, 1H, H-6 0 ”), 5.92 (s, 2H, OCH 2 O), 5.86 (brs, 1H, NH), 3.44 (q, 2H, J H,H =6.9 Hz, CH 2 N), 2.87 (t, 2H, J H,H =7.1 Hz, ArCH 2 ), 2.72 (t, 2H, J H,H =6.9 Hz, CH 2 CO), 2.24 (t, 2H, J H,H =6.9 Hz, CH 2 Se), 2.03 (quint, 2H, CH 2 CH 2 CH 2 ) ppm; 13 C-NMR (75.5 MHz, CDCl 3 ) δ 172.2 (CO), 147.9, 146.3 (C-3 0 ”, C-4 0 ”), 132.7 (C-1 0 ”), 121.6 (C-6 0 ”), 109.1, 108.5 (C-2 0 ”, C-5 0 ”), 101.0 (OCH 2 O), 40.9 (CH 2 N), 35.9 (ArCH 2 ), 35.5 (CH 2 CO), 29.0 (CH 2 CH 2 CH 2 ), 26.7 (CH 2 Se) ppm; HRESI-MS m/z calcd for C26H33N2O6Se2[M+H]+: 629.0664, found: 629.0662. 5. 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. Regarding antiproliferative properties, diselenide 40 , bearing a methylenedioxyphenyl residue, and an ethylene tether between both pharmacophoric units, was found to be the lead compound in terms of potency (GI 50 =0.88-2.0 µ M) and selectivity (selectivity index: 14-32). Diselenide 46 , incorporating an amide-type linker was found to be the most potent derivative within the series (GI 50 =0.12-0.27 µM), but with diminished selectivity (selectivity index: 4-8). 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. Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8247/13/11/358/s1. Table S1: GI 50 values ( µ M) for the antiproliferative activity of organoselenium derivatives. 1 H- and 13 C-NMR spectra of compounds 4,8,9,10,15,16,18-20,26,35-42,45,46. Pharmaceuticals 2020,13, 358 19 of 22 Author Contributions: Compound synthesis and characterization was carried out by P.B., L.S.-H., R.P., S.B. and supervised by Ó .L., J.G.F.-B. and I.M. Antioxidant assays were conducted by P.B. Antiproliferative and P-gp assays were accomplished by A.P. and I.L., and supervised by J.M.P. The manuscript was written by Ó .L. and revised by Ó .L., J.M.P. and J.G.F.-B. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Spanish MCIU, grant numbers CTQ2016-78703-P (MCIU/FEDER) and PGC2018-094503-B-C22 (MCIU/AEI/FEDER, UE), FEDER (501100008530) and Junta de Andalucía (FQM134). Acknowledgments: P.B. thanks University of Seville for the award of a fellowship. A.P. thanks the EU Social Fund (FSE) and the Canary Islands ACIISI for a predoctoral grant TESIS2020010055. I.L. thanks CONACYT for a postdoctoral fellowship. We thank CITIUS NMR Facility (University of Seville) for the NMR experiments. Conflicts of Interest: There are no conflict of interest to declare. References 1. Mukherjee, A.J.; Zade, S.S.; Singh, H.B.; Sunoj, R.B. Organoselenium chemistry: Role of intramolecular interactions. Chem. 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