Selenocoumarins as new multitarget antiproliferative agents: Synthesis, biological evaluation and in silico calculations

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published by Elsevier in European Journal of Medicinal Chemistry on 1 October 2019, available at: https://doi.org/10.1016/j.ejmech.2019.06.073 .” Elsevier Editorial System(tm) for European Journal of Medicinal Chemistry Manuscript Draft Manuscript Number: EJMECH-D-18-03428R2 Title: Selenocoumarins as new multitarget antiproliferative agents: synthesis, biological evaluation and in silico calculations Article Type: VSI: Multi-Target Drugs Keywords: Coumarin, organoselenium, antiproliferative, ROS, multitarget, in silico calculations, histone deacetylase Corresponding Author: Dr. José M Padrón, PhD Corresponding Author's Institution: Universidad de La Laguna First Author: Irene Lagunes Order of Authors: Irene Lagunes; Paloma Begines; Adrián Silva; Alexis R Galán; Adrián Puerta; Miguel X Fernandes; Inés Maya; José G Fernández- Bolaños; Óscar López; José M Padrón, PhD Abstract: Herein we report a straightforward preparation of new antiproliferative agents based on the hybridization of a coumarin skeleton and an organoselenium motif. Three families were obtained: isoselenocyanate, selenocarbamates and selenoureas. The main purpose of these hybrid structures is the development of new antiproliferative agents with a multitarget mode of action. A strong correlation between the nature of the organosenium scaffold and the antiproliferative activity was observed. Thus, whereas selenocarbamates proved to be inactive, or moderate antiproliferative agents, isoselenocyanate and most of the selenoureas behaved as strong antiproliferative agents, with GI50 values within the low micromolar range. Interestingly, a good selectivity toward tumor cell lines was found for some of the compounds. Moreover, an increase in the ROS level was observed for tumor cells, and accordingly, these pro-oxidant species might be involved in their mode of action. Overall, title compounds were found not to be substrates for P-glycoprotein, which is overexpressed in many cancer cells as a way of detoxification, and thus, to develop drug resistance. In silico calculations revealed that the selenoderivatives prepared herein might undergo a strong interaction with the active site of HDAC8, and therefore, be potential inhibitors of histone deacetylase 8. In vitro assessment against HDAC8 revealed a strong inhibition of such enzyme exerted by selenoureas, particularly by symmetrical coumarin-containing selenourea. Two compounds showed good antiproliferative data and appear as plausible leads for further testings. The symmetrical coumarin 6 displays the best in vitro inhibition of HDAC8, but is affected by P-gp. In contrast, the N-butyl selenourea coumarin derivative 5a escapes P-gp resistance but has lower HDAC8 inhibition activity. *Graphical Abstract (for review) Click here to download high resolution image Selenocoumarins as new multitarget antiproliferative agents: synthesis, biological evaluation and in silico calculations Irene Lagunes,1# Paloma Begines,2# Adrián Silva,2 Alexis R. Galán,1 Adrián Puerta,1 Miguel X. Fernandes,1 Inés Maya,2 José G. Fernández-Bolaños,2,* Óscar López2,*, José M. Padrón1,* 1 BioLab, Instituto Universitario de Bio-Orgánica “Antonio González” (IUBO-AG), Centro de Investigaciones Biomédicas de Canarias (CIBICAN), Universidad de La Laguna, Apartado 456, E-38071 La Laguna, Spain. e-mail: [email protected], Tel.: +34 922 316 502 ext. 6126 2 Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, Apartado 1203, E-41071 Sevilla, Spain. Email: [email protected], [email protected] #These authors contributed equally Highlights  A straightforward preparation of coumarin-based selenoderivatives was achieved  Isoselenocyanate and selenoureas were strong and selective antiproliferative agents  An increased level of ROS was detected in their mode of action  Non-substrates for P-glycoprotein  Coumarin-selenoderivatives proved inhibitors of HDAC8 *Highlights (for review) 1 Selenocoumarins as new multitarget antiproliferative agents: synthesis, biological evaluation and in silico calculations Irene Lagunes,1# Paloma Begines,2# Adrián Silva,2 Alexis R. Galán,1 Adrián Puerta,1 Miguel X. Fernandes,1 Inés Maya,2 José G. Fernández-Bolaños,2,* Óscar López2,*, José M. Padrón1,* 1 BioLab, Instituto Universitario de Bio-Orgánica “Antonio González” (IUBO-AG), Centro de Investigaciones Biomédicas de Canarias (CIBICAN), Universidad de La Laguna, Apartado 456, E-38071 La Laguna, Spain. e-mail: [email protected], Tel.: +34 922 316 502 ext. 6126 2 Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, Apartado 1203, E-41071 Sevilla, Spain. Email: [email protected], [email protected] #These authors contributed equally Graphical Abstract Highlights  A straightforward preparation of coumarin-based selenoderivatives was achieved  Isoselenocyanate and selenoureas were strong and selective antiproliferative agents  An increased level of ROS was detected in their mode of action  Non-substrates for P-glycoprotein  Coumarin-selenoderivatives proved inhibitors of HDAC8 *Revised Manuscript Click here to view linked References 2 Abstract Herein we report a straightforward preparation of new antiproliferative agents based on the hybridization of a coumarin skeleton and an organoselenium motif. Three families were obtained: isoselenocyanate, selenocarbamates and selenoureas. The main purpose of these hybrid structures is the development of new antiproliferative agents with a multitarget mode of action. A strong correlation between the nature of the organosenium scaffold and the antiproliferative activity was observed. Thus, whereas selenocarbamates proved to be inactive, or moderate antiproliferative agents, isoselenocyanate and most of the selenoureas behaved as strong antiproliferative agents, with GI50 values within the low micromolar range. Interestingly, a good selectivity toward tumor cell lines was found for some of the compounds. Moreover, an increase in the ROS level was observed for tumor cells, and accordingly, these pro-oxidant species might be involved in their mode of action. Overall, title compounds were found not to be substrates for P-glycoprotein, which is overexpressed in many cancer cells as a way of detoxification, and thus, to develop drug resistance. In silico calculations revealed that the selenoderivatives prepared herein might undergo a strong interaction with the active site of HDAC8, and therefore, be potential inhibitors of histone deacetylase 8. In vitro assessment against HDAC8 revealed a strong inhibition of such enzyme exerted by selenoureas, particularly by symmetrical coumarin-containing selenourea. Two compounds showed good antiproliferative data and appear as plausible leads for further testings. The symmetrical coumarin 6 displays the best in vitro inhibition of HDAC8, but is affected by P-gp. In contrast, the N-butyl selenourea coumarin derivative 5a escapes P-gp resistance but has lower HDAC8 inhibition activity. Keywords Coumarin, organoselenium, antiproliferative, ROS, multitarget, in silico calculations, histone deacetylase 3 1. Introduction Coumarin (2H-chromen-2-one), whose name comes from coumarou, the vernacular name for tonka beans (Dipteryx odorata Wild.), from where it was isolated in 1820 [1], is an ubiquitous phytochemical [2] mainly found in plants, but also in bacteria and fungi. So far, over 1300 coumarins have been isolated from such sources [3]. Naturally-occurring or synthetic [4] coumarins are currently considered as privileged scaffolds [5], endowed with a plethora of biological activities [6]. Although one of the most known properties is their strong antioxidant character [7], a wide spectrum of other bioactivities has been reported for numerous coumarin-containing derivatives: anti-HIV and anti-HCV [8], antituberculosis [9], anti-leishmanial [10], anticancer [11], anti-inflammatory [12], and anti-Alzheimer’s [13], among others. The planar structure and the presence of a lactone scaffold in chromenones allows the establishment of non-covalent interactions, promoting the inhibition of key enzymes currently considered as therapeutic targets, such as cholinesterases (Alzheimer’s) [14], glycosidases (type-2 diabetes, lysosomal storage disorders) [15] MAO (Alzheimer’s, Parkinson) [16, 17], or carbonic anhydrase (cancer) [18]. The coumarin core skeleton has inspired the development of new therapeutic agents. In fact, there are some coumarin-based marketed drugs, among which, the anticoagulant agents warfarin and dicoumarol are relevant examples [19]. These vitamin K antagonists are effective in thrombosis prevention, but are also frequently implicated in adverse drug reactions, among which bleeding threatens the life of treated patients [20]. It has been demonstrated [21] that alkylation on C-3 and/or C-4 positions of coumarin strongly reduces the hepatoxicity of such template, probably by the diminished formation of the transient 3,4- epoxide in the metabolization of coumarin. The preparation of hybrid structures derived from coumarins is currently an emerging area of interest within Medicinal Chemistry [22, 23]. Such hybrid structures have proved relevant properties against Alzheimer’s disease [24] and cancer [25]. The high complexity associated to multifactorial diseases, among which cancer is a relevant example, has obliged to change the design of new drugs, shifting from the one-target one-drug approach to the multitarget paradigm. Classical drug cocktails required for tackling the diverse targets usually lead to severe side-effects or undesirable drug-drug interactions. In this scenario, multitarget drug approach emerged as a rational way of combatting several targets simultaneously upon combination of a series of pharmacophores within the drug structure [26].Accordingly, the 4- methyl coumarin scaffold seemed to be a good starting point in the synthetic strategy to reduce side-effects in the final compounds (Fig. 1). Figure 1. General bioactivity pattern for the selenium-containing coumarins. 4 Herein we report the preparation of novel multitarget coumarin-based derivatives by combination of the chromenone scaffold with an organoselenium motif. We have successfully accomplished the preparation of selenium-containing hybrid derivatives of iminosugars [27], steroids [28, 29], and polyphenols [30] as novel antiproliferative agents. The organoselenium motif can be useful for altering the tumor cell redox status [31] and probably induce cell death. Moreover, coumarin-containing derivatives have been found to exhibit inhibition of histone deacetylases (HDACs) [32, 33]. HDACs catalyze the removal of the acetyl group of ε-N-acetyl lysine on histone, achieving a more compact chromatin structure upon ionic interactions between histones and DNA, and thus controlling genetic transcription. HDAC inhibitors have been reported to act as anticancer agents [34]. To the best of our knowledge, the combination of selenium and coumarin scaffolds has rarely been described only in literature. A selenocyanato-containing umbelliferone has recently been reported to exert myeloprotection in therapies based on the administration of carboplatin, by diminishing the level of pro-oxidant species, and restoration of the glutathione redox pool [35], and at the same time, it potentiated the anti-tumor properties of the cytotoxic agent. 2. Results and discussion 2.1. Chemistry Herein we present the design of three different families of coumarin-based selenoderivatives as potential novel antiproliferative agents: isoselenocyanate, selenocarbamates and selenoureas. Modification of the selenium-containing functional group, paired with different stereoelectronic effects of the substituents might modulate the biological properties of the target compounds, allowing the establishment of valuable structure-activity relationships for designing the lead compound. The synthesis of the selenocoumarins was started from commercially-available 7- amino-4-methylcoumarin (AMC, 1). Derivative 1 was transformed into formamide 2 (Scheme 1) upon treatment with freshly-prepared acetic formic anhydride (AFA) [36]. Attempts to accomplish the synthesis of 2 using a biphasic medium (CH2Cl2sat. aq. NaHCO3), similar to what was reported for sugar-derived formamides [37] produced moderate yields (36%). Replacement of biphasic medium with pyridine produced 2 [38] with a 93% yield. Next, formamide was transformed into the hitherto unknown coumarin-based isoselenocyanate 3 (Scheme 1) in a one-pot two-step procedure, which involves triphosgenebased dehydration of 2 into a transient non-isolated isocyanide, followed by addition of elemental selenium black (73% overall yield) [37, 39]. 13C-NMR signals around 130 ppm confirmed the presence of the isoselenocyanate group. Isoselenocyanates are quite versatile synthetic intermediates, due to the pronounced electrophilicity of the heterocumulene moiety [31]. This feature allowed us the straightforward transformation of 3 into selenocarbamates 4 (5389%), N-alkyl(aryl) selenoureas 5, and dimeric 6 (53quant.) upon reaction with alcohols, amines, and AMC (1) (Scheme 1), respectively. 13C-NMR resonances (190, 180 ppm) confirmed the presence of the selenocarbamate or selenoureido motif, respectively. Attempts to purify selenoureas 5 and 6 5 using column chromatography (silica gel or alumina as the stationary phase) led to extensive decomposition. To our delight, precipitation of title compounds from the crude reaction mixture led to pure selenoureas 5 and 6 as stable crystalline solids. This observation strongly contrasts with selenocarbamates 4, which could be purified by silica gel column chromatography without decomposition. Presumably, the higher +R effect of the NH group compared to oxygen leads to increased electronic density on selenium atom, and a longer CSe bond, making selenoureas more prone to chemical transformations. N-Phenyl selenourea 5b was reported recently as a chromo-fluorogenic chemosensor for anion recognition. It was prepared in a modest yield (37%) by coupling phenyl isoselenocyanate with AMC (1) [40]. Scheme 1. a) HCO2COCH3, Py, rt, 24 h, 93%; b) i) Triphosgene, Et3N, CH2Cl2, N2, reflux, 2.5 h; ii) black Se, N2, dark, 24 h, 73%; c) ROH, N2, 65 ºC, dark, 5389%; d) RNH2, N2, rt (40 ºC for 5e), dark, 53%-quant.; e) 1, THF, N2, dark, 3 h, 60%. 2.2. Antiproliferative activity The antiproliferative activity of selenocompounds reported in Scheme 1 was studied in five human solid tumor cell lines. In addition to precursor AMC (1), the standard anticancer drug cisplatin (CDDP) was used as reference compound. Besides tumor cells, active compounds in all cell lines were tested against the non-tumor cell line BJ-hTERT for selectivity. We followed the NCI protocol [41] and the GI50 vales are shown in Table 1. Overall, the data on antiproliferative activity shows that most of the tested selenocompounds exhibit growth inhibition in all tumor cell lines, whilst AMC (1) is inactive (GI50 > 100 µM) against all cells. Based on the GI50 range (Fig. 2), the most active compounds of the series are selenoureas 5a and 5d, and dimer 6. These three products show 12 Figure 5. Inhibition of HDAC8 activity by selenocoumarins. TSA = trichostatin A. 3. Conclusions AMC (1) was efficiently transformed into the corresponding 7-isoselenocyanato derivative 3, which in turn reacted with alcohols and alkyl/aryl amines to produce coumarin-derived selenocarbamates 4 and selenoureas 5-6. Strong activities, within the low micromolar range, were found for isoselenocyanate 3 and some selenoureas 5-6, which were accompained with good selectivity towards cancer cells. High levels of ROS were detected after treatment of HeLa cells with selenocoumarins 5-6, which might be involved in the antiproliferative mechanism. Furthermore, selenoderivatives did not act as P-glycoprotein substrates, thus avoiding the rise of drug resistance. Moreover, in silico calculations predicted that all selenoderivatives prepared herein had a more favorable interaction with HDAC8 (a key therapeutic target against cancer) than parent AMC (1). This interaction was confirmed in vitro using a HDAC8 inhibition assay. Overall, two compounds could be selected as lead for further testing, namely selenoureas 5a and 6. Although both compounds show good antiproliferative profile, dimer 6 displays the best in vitro inhibition of HDAC8, but it can be extruded by P-gp. In contrast, the advantage of compound 5a relies in avoiding P-gp resistance, but has lower HDAC8 inhibition activity. 4. Materials and methods 4.1. Materials Melting points (uncorrected) were recorded on an Electrothermal apparatus. 1H (300.1 and 500.1 MHz) and 13C (75.5 and 125.7 MHz) NMR spectra were recorded at 25 °C on a Bruker 13 Avance 300//Avance III 500 MHz spectrometers (CDCl3 and DMSO-d6). The assignments of 1H and 13C signals were confirmed by COSY and HSQC bidimensional experiments. Mass spectra (ESI) were recorded on a QExactive mass spectrometer. TLCs were performed on aluminium pre-coated sheets (E. Merck Silica gel 60 F254); spots were visualized by UV light, and by charring with 10% vanillin in EtOH containing 1% of H2SO4. Column chromatography was performed using E. Merck Silica Gel 60 (40–63 μm), using the eluant indicated in each case. 4.2. Experimental procedures 4.2.1. 7-Isoselenocyanato-4-methyl-2H-chromen-2-one (3) To a solution of N-(4-methyl-2-oxo-2H-chromen-7-yl)formamide 2 (325.6 mg, 1.60 mmol), Et3N (960 μL, 6.88 mmol, 4.3 equiv) and CH2Cl2 (4 mL) a solution of triphosgene (253.4 mg, 0.85 mmol, 0.53 mol. equiv.) in CH2Cl2 (4 mL). was slowly added. The resulting mixture was refluxed for 2.5 h in the dark and under inert atmosphere. Then, black selenium (254.3 mg, 3.22 mmol, 2.0 equiv) was added, and the mixture was refluxed for further 24 h. After that, the crude reaction was filtered through a Celite® pad and washed with cold CH2Cl2. The filtrate was concentrated to dryness and the residue was purified by column chromatography (CH2Cl2) to give 3 as a solid. Yield: 310.6 mg (74%). Rf 0.92 (20:1 CH2Cl2MeOH); mp: 172 ºC (dec.); 1H-NMR (300 MHz, CDCl3) δ 7.59 (d, 1H, J5,6 = 9.0 Hz, H-5), 7.23 (d, 1H, J6,8= 2.0 Hz, H-8), 7.21 (dd, 1H, J5,6 = 9.0 Hz, J6,8= 2.0 Hz, H-6), 6.31 (q, 1H, J3,CH3 = 1.2 Hz, H-3), 2.43 (d, 3H, J3,CH3 = 1.2 Hz, Ar-CH3) ppm; 13C-NMR (125.7 MHz, CDCl3) δ 159.9 (CO), 154.1 (C-4), 151.5 (C-8a), 133.7, 132.6 (C-7, NCSe), 126.0 (C-5), 122.4 (C-6), 119.7 (C-4a), 115.7 (C-3), 114.5 (C-8), 18.8 (Ar-CH3) ppm; HREI-MS m/z calculated for C11H7NO280Se ([M]+): 264.9637, found: 264.9644. 4.2.2. General procedure for the preparation of selenocarbamates 4a  d A solution of isoselenocyanate 3 (142.6 mg, 0.54 mmol) in the corresponding alcohol (8 mL) was heated (refluxing conditions for 4a, 4b; 65 ºC for 4c, 4d) in the dark and under inert atmosphere during the time indicated below. After that, the crude reaction was concentrated in vacuo and the residue was purified as indicated in each case. 4.2.2.1. O-Methyl-N-(4-methyl-2-oxo-2H-chromen-7-yl)selenocarbamate (4a) Reaction took place for 2 h, and the residue was purified by column chromatography (cyclohexane→5:1 cyclohexaneEtOAc) to give 4a as a white solid. Yield: 92.5 mg (58%). Rf 0.79 (20:1 CH2Cl2MeOH); mp: 167 ºC (dec.). 1H-NMR (300 MHz, DMSO-d6) δ: 12.0 (s, 1H, NH), 7.73 (d, 1H, J5,6 = 8.6 Hz, H-5), 7.45 (m, 2H, H-6, H-8), 6.33 (brs, 1H, H-3), 4.18 (s, 3H, OCH3), 2.40 (brd, 3H, J3,CH3 = 0.7 Hz, Ar-CH3) ppm; 13C-NMR (125.7 MHz, DMSO-d6) δ: 191.6 (CSe), 159.9 (CO), 153.3, 153.0 (C-4, C-8a), 140.3 (C-7), 126.0 (C-5), 117.4 (C-6), 116.4 (C-4a), 113.3 (C-3), 108.5 (C-8), 61.8 (OCH3), 18.0 (Ar-CH3) ppm; HRESI-MS m/z calculated for C12H12NO380Se [M+H]+: 297.9977, found: 297.9977. 4.2.2.2. O-Ethyl-N-(4-methyl-2-oxo-2H-chromen-7-yl)selenocarbamate (4b) 14 Reaction took place for 2 h, and the residue was purified by column chromatography (CH2Cl2) to give 4b as a yellow solid. Yield: 89.1 mg (53%). Rf 0.66 20:1 (CH2Cl2MeOH); mp: 193 ºC (dec). 1H-NMR (300 MHz, DMSO-d6) δ: 11.94 (s, 1H, NH), 7.74 (d, 1H, J5,6 = 8.7 Hz, H-5), 7.45 (m, 2H, H-6, H-8), 6.32 (brq, 1H, J3,CH3 = 1.0 Hz, H-3), 4.68 (q, 2H, JH,H = 6.8 Hz, OCH2), 2.40 (d, 3H, J3,CH3 = 1.0 Hz, Ar-CH3), 1.40 (t, 3H, JH,H = 6.8 Hz, OCH2CH3) ppm; 13C-NMR (125.7 MHz, DMSO-d6) δ: 190.1 (CSe), 159.8 (CO), 153.3, 152.9 (C-4, C- 8a), 140.3 (C-7), 126.1 (C-5), 117.3 (C-6), 116.3 (C-4a), 113.2 (C-3), 108.4 (C-8), 71.7 (OCH2CH3), 18.0 (Ar-CH3), 14.0 (OCH2CH3) ppm; HRESI-MS m/z calculated for C13H13NNaO380Se [M+Na]+: 333.9953, found: 333.9951. 4.2.2.3. N-(4-Methyl-2-oxo-2H-chromen-7-yl)-O-propylselenocarbamate (4c) Reaction took place for 3.5 h, and the residue was purified by column chromatography (10:1 cyclohexaneEtOAc) to give 4c as a white solid. Yield: 140.0 mg (80%). Rf 0.54 (60:1 CH2Cl2MeOH); mp: 167-169 ºC; 1H-NMR (300 MHz, DMSO-d6) δ: 11.96 (s, 1H, NH), 7.74 (d, 1H, J5,6 = 8.7 Hz, H-5), 7.45 (m, 2H, H-6, H-8), 6.32 (d, 1H, J3,CH3 = 1.1 Hz, H-3), 4.59 (t, 2H, JH,H = 6.3 Hz, OCH2), 2.40 (d, 3H, J3,CH3 = 1.1 Hz, Ar-CH3), 1.81 (sext, 2H, JH,H = 7.1 Hz, OCH2CH2), 0.97 (t, 3H, JH,H = 7.1 Hz, O(CH2)2CH3) ppm; 13C-NMR (125.7 MHz, DMSO-d6) δ: 190.3 (CSe), 159.8 (CO), 153.3 152.9 (C-4, C-8a), 140.4 (C-7), 126.0 (C-5), 117.3 (C-6), 116.4 (C-4a), 113.2 (C-3), 108.4 (C-8), 77.2 (OCH2), 21.4 (OCH2CH2), 18.0 (Ar- CH3), 10.3 (O(CH2)2CH3) ppm; HRESI-MS m/z calculated for C14H15NNaO380Se [M+Na]+: 348.0109, found: 348.0106. 4.2.2.4. O-Butyl-N-(4-methyl-2-oxo-2H-chromen-7-yl)selenocarbamate (4d) Reaction took place for 3.5 h, and the residue was purified by crystallization (CH2Cl2cyclohexane) to give 4d as a yellow solid. Yield: 163.0 (89%). Rf 0.46 (60:1 CH2Cl2MeOH); mp: 129-131ºC; 1H-NMR (300 MHz, 90ºC, DMSO-d6) δ: 11.96 (brs, 1H, NH), 7.75 (d, 1H, J5,6 = 8.6 Hz, H-5), 7.42 (m, 2H, H-6, H-8), 6.33 (d, 1H, J3,CH3 = 1.1 Hz, H- 3), 4.64 (m, 2H, OCH2), 2.41 (brd, 3H, J3,CH3 = 1.1 Hz, Ar-CH3), 1.76 (m, 2H, OCH2CH2), 1.43 (sext, 2H, JH,H = 7.4 Hz, CH2CH3), 0.94 (t, 3H, JH,H = 7.4 Hz, O(CH2)3CH3) ppm; 13C- NMR (125.7 MHz, DMSO-d6) δ: 190.3 (CSe), 159.9 (CO), 153.5, 153.0 (C-4, C-8a), 140.5 (C-7), 126.2 (C-5), 118.6 (C-6), 116.4 (C-4a), 113.1 (C-3), 108.4 (C-8), 75.4 (OCH2), 30.0 (OCH2CH2), 18.7, 18.0 (CH2CH3, Ar-CH3), 13.6 (O(CH2)3CH3) ppm; HRESI-MS m/z calculated for C15H17NNaO380Se [M+Na]+: 362.0266, found: 362.0263. 4.2.3. General procedure for the preparation of selenoureas 5a-e, 6 A solution of 3 (100.4 mg, 0.38 mmol) and the corresponding amine (1.0 equiv.) in anhydrous THF (4 mL) was stirred under N2 and in the dark (temperature and time indicated in each case). After that, the crude reaction was concentrated to dryness and purified by precipitation. 4.2.3.1. N-Butyl-N’-(4-methyl-2-oxo-2H-chromen-7-yl)selenourea (5a) 15 Butylamine (38 μL, 0.38 mmol) was used, and the reaction was kept at rt for 30 min. After removal of the solvent under reduced pressure, the residue was purified by precipitation (CH2Cl2-cyclohexane) to give 5a as a yellow-green solid. Yield: 68.5 mg (53%). Rf 0.69 (20:1 CH2Cl2MeOH), mp: 165 ºC (dec). 1H-NMR (300 MHz, DMSO-d6) δ: 10.19 (s, 1H, NH’), 8.58 (t, 1H, JNH,CH2 = 5.9 Hz, NH), 7.71 (d, 1H, J5,6 = 8.6 Hz, H-5), 7.58 (d, 1H, J6,8 = 2.1 Hz, H-8), 7.34 (brdd, 1H, J5,6 = 8.6 Hz, J6,8 = 2.1 Hz, H-6), 6.30 (brs, 1H, H-3), 3.57 (q, 2H, JH,H = 6.2 Hz, NHCH2), 2.41 (d, 3H, J3,CH3 = 0.8 Hz, Ar-CH3), 1.57 (quint., 2H, JH,H = 7.3 Hz, NHCH2CH2), 1.33 (sext, 2H, JH,H = 7.4 Hz, CH2CH3), 0.91 (t, 3H, JH,H = 7.4 Hz, (CH2)3CH3) ppm; 13C-NMR (125.7 MHz, DMSO-d6) δ: 179.1 (CSe), 160.0 (CO), 153.4 (C-4), 153.1 (C- 8a), 142.6 (C-7), 125.8 (C-5), 118.5 (C-6), 115.6 (C-4a), 112.5 (C-3), 109.0 (C-8), 46.6 (NHCH2), 30.3 (NHCH2CH2), 19.6 (CH2CH3), 18.0 (Ar-CH3), 13.7 (CH2-CH3) ppm; HRESIMS m/z calculated for C15H19N2O280Se [M+H]+: 339.0606, found: 339.0602. 4.2.3.2. N-(4-Methyl-2-oxo-2H-chromen-7-yl)-N’-phenylselenourea (5b) Aniline (35 μL, 0.38 mmol) was used, and the reaction was kept at rt for 2.5 h. After removal of the solvent under reduced pressure, the residue was purified by precipitation (CH2Cl2cyclohexane) to give 5b as a yellow-green solid. Yield: 89.0 mg (66%). Rf 0.75 (20:1 CH2Cl2MeOH); mp: 164 ºC (dec.); 1H-NMR (300 MHz, DMSO-d6) δ: 10.49 (s, 2H, NH, NH’), 7.72 (d, 1H, J5,6 = 8.7 Hz, H-5), 7.59 (d, 1H, J6,8 = 2.1 Hz, H-8), 7.46 (dd, J5,6 = 8.7 Hz, J6,8 = 2.1 Hz, H-6), 7.44-7.33 (m, 4H, Ar-H), 7.23-7.18 (m, 1H, Ar-H), 6.32 (d, 1H, J3,CH3 = 1.2 Hz, H-3), 2.42 (d, 3H, J3,CH3 = 1.2 Hz, Ar-CH3) ppm; 13C-NMR (125.7 MHz, DMSO-d6) δ: 179.0 (CSe), 159.9 (CO), 153.2 (C-4, C-8a), 143.2 (C-7), 139.5 (Ar-C-ipso), 128.7 (Ar-C-m), 125.5, 125.4 (Ar-C-p, C-5), 124.6 (Ar-C-o), 119.8 (C-6), 116.1 (C-4a), 112.9 (C-3), 110.4 (C-8), 18.1 (Ar-CH3) ppm; HRESI-MS m/z calculated for C17H15N2O280Se [M+H]+: 359.0293, found: 359.0290. 4.2.3.3. N-(4-Methoxyphenyl)- N’-(4-Methyl-2-oxo-2H-chromen-7-yl)selenourea (5c) p-Anisidine (46.8 mg, 0.38 mmol) was used, and the reaction was kept at rt for 30 min and at 40 ºC for 1.5 h. After removal of the solvent under reduced pressure, the residue was purified by precipitation (CH2Cl2cyclohexane) to give 5c as a light green solid. Yield: 101.5 mg (69%). Rf 0.54 (CH2Cl2-MeOH 20:1), mp: 163 ºC (dec). 1H-NMR (300 MHz, DMSO-d6) δ: 10.36, 10.29 (2s, 1H each, NH, NH’), 7.73 (d, 1H, J5,6 = 8.7 Hz, H-5), 7.59 (d, 1H, J6,8 = 2.0 Hz, H-8), 7.45 (dd, 1H, J5,6 = 8.7 Hz, J6,8 = 2.0 Hz, H-6), 7.30 (m, 2H, Ar-H-o), 6.93 (m, 2H, Ar-H-m), 6.31 (d, 1H, J3,CH3= 1.1 Hz, H-3), 3.75 (s, 3H, OCH3), 2.41 (d, 3H, J3,CH3= 1.1 Hz, Ar-CH3) ppm; 13C-NMR (125.7 MHz, DMSO-d6) δ: 179.5 (CSe), 160.8 (CO), 157.8 (Ar-C- p), 154.0 (C-4), 153.4 (C-8a), 143.6 (C-7), 132.5 (Ar-C-ipso), 127.4 (Ar-C-o), 125.9 (C-5), 120.6 (C-6), 116.7 (C-4a), 114.5 (Ar-C-m), 113.3 (C-3), 111.2 (C-8), 55.8 (OCH3), 18.5 (Ar- CH3) ppm; HRESI-MS m/z calculated for C18H17N2O380Se [M+H]+: 389.0399, found: 389.0394. 4.2.3.4. N-(4-Bromophenyl)-N’-(4-methyl-2-oxo-2H-chromen-7-yl)selenourea (5d) p-Bromoaniline (65.5 mg, 0.38 mmol) was used, and the reaction was kept at rt for 4 h. After removal of the solvent under reduced pressure, the residue was purified by precipitation 16 (cyclohexane) to give 5d as a yellow solid. Yield: 165.8 mg (quant.). Rf 0.43 (20:1 CH2Cl2MeOH), mp: 178 ºC. 1H-NMR (300 MHz, DMSO-d6) δ: 10.54 (brs, 2H, NH, NH’), 7.73 (d, 1H, J5,6 = 8.7 Hz, H-5), 7.58 (d, 1H, J6,8 = 2.1 Hz, H-8), 7.55 (m, 2H, Ar-H-m), 7.45 (dd, 1H, J5,6 = 8.7 Hz, J6,8 = 2.1 Hz, H-6), 7.40 (m, 2H, Ar-H-o), 6.33 (m, 1H, H-3), 2.42 (d, 3H, J3,CH3 = 1.1 Hz, CH3) ppm; 13C-NMR (125.7 MHz, DMSO-d6) δ: 179.2 (CSe), 159.9 (CO), 153.3, 153.1 (C-4, C-8a), 143.0 (C-7), 139.0 (Ar-C-ipso), 131.5 (Ar-C-m), 126.7 (Ar- C-o), 125.5 (C-5), 120.5, 119.8 (Ar-C-p, C-6), 116.2 (C-4a), 112.9 (C-3), 110.5 (C-8), 18.5 (Ar-CH3) ppm; HRESI-MS m/z calculated for C17H14BrN2O2Se [M+H]+: 436.9398, found: 436.9396. 4.2.3.5. N-(4-Methyl-2-oxo-2H-chromen-7-yl)-N’-(4-trifluoromethyl)selenourea (5e) p-Trifluoromethylaniline (48 µL, 0.38 mmol) was used, and the reaction was kept at 40 ºC for 2 h. After removal of the solvent under reduced pressure, the residue was purified by precipitation (cyclohexane) to give 5e as a yellow-green solid. Yield: 162.5 mg (quant.). Rf 0.44 (20:1 CH2Cl2MeOH); mp: 186 ºC. 1H NMR (500 MHz, DMSO-d6) δ: 10.74 (brs, 2H, 2 NH), 7.74 (d, J5,6 = 8.6 Hz, 1H, H-5), 7.71 (m, 2H, Ar-H-m), 7.68 (m, 2H, Ar-H-o), 7.59 (d, 1H, J8,6 = 2.1 Hz, H-8), 7.47 (dd, 1H, J5,6 = 8.6 Hz, J6,8 = 2.1 Hz, H-6), 6.34 (d, 1H, J3,CH3 = 1.1 Hz, H-3), 2.42 (d, 3H, J3,CH3 = 1.1 Hz, Ar-CH3) ppm; 13C NMR (125.7 MHz, DMSO-d6) δ: 179.6 (CSe), 159.8 (CO), 153.1, 153.0 (C-8a, C-4), 143.3, 142.8 (Ar-C-ipso, C-7), 125.0 (q, 2JC,F = 32.7 Hz, Ar-C-p), 125.5 (C-5), 124.3 (q, 1JC,F = 270.3 Hz, CF3), 125.7 (c, 3JC,F = 3.5 Hz Ar-C-m), 124.3 (Ar-Co), 119.7 (C-6), 116.3 (C-4a), 113.2 (C-3), 110.5 (C-8), 18.0 (Ar-CH3) ppm; HRESI-MS m/z calculated for C18H14F3N2O280Se [M+H]+: 427.0167, found: 427.0167. 4.2.3.6. N,N’-Bis(4-methyl-2-oxo-2H-chromen-7-yl)selenourea (6) AMC 1 (66.5 mg, 0.38 mmol) was used, and the reaction was kept at 45 ºC for 3 h, and at rt for further 18 h. After removal of the solvent under reduced pressure, the residue was washed with Et2O, EtOAc and a 1:1 cyclohexaneEtOAc mixture to give 6 as a yellow solid. Yield: 126.3 mg (76%). Rf 0.26 (20:1 CH2Cl2MeOH); mp: 199 ºC (dec). 1H-NMR (300 MHz, DMSO-d6) δ: 10.78 (brs, 2H, 2NH), 7.75 (d, 2H, J5,6 = 8.7 Hz, H-5), 7.61 (d, 2H, J6,8 = 2.1 Hz, H-8), 7.48 (dd, 2H, J5,6 = 8.7 Hz, J6,8 = 2.1 Hz, H-6), 6.33 (m, 2H, H-3), 2.42 (brd, 6H, J3,CH3= 1.0 Hz, Ar-CH3) ppm; 13C NMR (125.7 MHz, DMSO-d6) δ: 179.2 (CSe), 159.8 (CO), 153.2, 153.1 (C-4, C-8a), 143.0 (C-7), 125.5 (C-5), 119.8 (C-6), 116.4 (C-4a), 113.0 (C-3), 110.5 (C-8), 18.1 (Ar-CH3) ppm; HRESI-MS m/z calculated for C21H17N2O480Se [M+H]+: 441.0348, found: 441.0342. 4.3. Cell lines and growth conditions Cell lines used in this study were kindly provided by Dr. Godefridus J. Peters (Cancer Center Amsterdam, Vrije Universiteit, Amsterdam, The Netherlands). All cells were grown in RPMI 1640 supplemented with 1 mM glutamine, 5% FBS and antibiotics. Cells were grown at 37 ºC in a humidified atmosphere of 5% CO2 and maintained at low passage. 17 4.4. Antiproliferative activity The antiproliferative activity was tested in vitro against human cancer cells using the protocol of the National Cancer Institute (NCI) of the USA [49] with minor modifications [41]. Samples for testing were dissolved initially in DMSO at 40 mM, i.e. 400 times the maximum test concentration. For each compound, the cells were exposed to serial decimal dilutions in the range of 0.001–100 μM for a period of 48 h. 4.5. ROS assay To measure the level of ROS we used the commercially available kit ROS-GloTM H2O2 Assay (Promega Corporation, WF, USA). Cells were exposed to compounds at the indicated doses for 48 h, after which time the non-lytic assay was performed following manufacturer’s indications. Luminescence was measured on a Synergy HTX multimode microplate reader (BioTek, VT, USA). 4.6. Molecular modeling Peptidyl-prolyl cis-trans isomerase A (PDB ID: 1VBS), mitochondrial U Peptidyl-prolyl cistrans isomerase F (PDB ID: 3R49) and U Histone deacetylase 8 (PDB ID: 5FCW) were downloaded from Protein Data Bank. We used HyperChem software (Hypercube Inc., FL, USA) to draw and optimize the molecular structures and OpenBabel application to convert the files into adequate format before exporting them to docking software Glide v8.2 (Schrödinger, MA, USA). For docking calculations, we removed ligands and non-essential water molecules present in the crystal structure and added polar hydrogen atoms to the protein chain before running any docking calculations. We used “Standard Precision” protocol in Glide with OPLS 2005 forcefield with post-docking minimization. To assess docking reliability we performed redocking of co-crystallized ligands for all used structures. The RMSDs for the co-crystallized ligand of PPIase was 0.15Å, ligand of PPIF was 0.18Å and for the ligand of histone deacetylase 8 was 0.49Å which ensures that the docking protocol is valid. 4.7. HDAC8 inhibition assay To study the inhibitory activity of selenocompounds against HDAC8 we used the commercially available kit Histone Deacetylase 8 (HDAC8) Inhibitor Screening Kit (Sigma- Aldrich, MO, USA). Compounds were tested at 10 µM in 96-well plates according to manufacturer’s indications. Fluorescence was measured on a FLUOstar Omega microplate reader (BMG Labtech, Germany). Acknowledgements P.B. thanks University of Seville for a predoctoral fellowship. M.X.F. thanks Cabildo de Tenerife for an “Agustín de Betancourt” contract. Financial support from the Spanish Government CTQ2016-78703-P (MICINN/FEDER, UE) and PGC2018-094503-B-C22 (MCIU/AEI/FEDER, UE), the Junta de Andalucía (FQM134/FEDER 501100008530, UE). We thank Servicio de Resonancia Magnética Nuclear (CITIUS, University of Seville) for the NMR experiments. 18 Appendix A. Supplementary data Supplementary data related to this article can be found at _______ Declarations of interest: none References [1] A. Vogel, Darstellung von benzoesäure aus der Tonka‐bohne und aus den meliloten‐oder steinklee‐blumen, Ann. Phys. 64 (1820) 161166. https://doi.org/10.1002/andp.18200640205. [2] F.G. Medina, J.G. Marrero, M. Macías-Alonso, M.C. González, I. Córdova-Guerrero, A.G. Teissier García, S. Osegueda-Robles, Coumarin heterocyclic derivatives: chemical synthesis and biological activity, Nat. Prod. Rep. 32 (2015) 14721507. https://doi.org/10.1039/C4NP00162A [3] A. Stefanachi, F. Leonetti, L. Pisani, M. Catto, A. 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