Biotinylated selenocyanates: Potent and selective cytostatic agents

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published by Elsevier in Bioorganic Chemistry on April 2023, available at: https://doi.org/10.1016/j.bioorg.2023.106410 .” 1 Biotinylated selenocyanates: potent and selective cytostatic agents Jesús M. Roldán-Peña,a Adrián Puerta,b Jelena Dinić,c Sofija Jovanović Stojanov,c Aday González-Bakker,b Francisco J. Hicke,a Atreyee Mishra,d Akkharadet Piyasaengthong,d,e Inés Maya,a James W. Walton,d Milica Pešić,c,* José M. Padrón,b,* José G. Fernández-Bolaños,a,* Óscar Lópeza,* aOrganic Chemistry Department, Faculty of Chemistry, University of Seville, PO box 1203, E-41071 Seville, Spain. e-mail: [email protected], o[email protected] bBioLab, Instituto Universitario de Bio-Orgánica “Antonio González” (IUBO-AG), Universidad de La Laguna, Astrofísico Francisco Sánchez 2, E-38206 La Laguna, Spain, e-mail: jmp[email protected] cInstitute for Biological Research “Siniša Stanković”, National Institute of the Republic of Serbia, University of Belgrade, Despota Stefana 142, 11060 Belgrade, Serbia, e-mail: [email protected] dDepartment of Chemistry, Durham University, Lower Mountjoy, South Road, Durham DH1 3LE, UK eBioscience Program, Faculty of Science, Kasetsart University, Bangkok 10900, Chatuchak, Thailand Graphical abstract Highlights  Biotinylation of the pharmacophore selenocyanate led to a very strong antiproliferative agent  Nanomolar activities were obtained in most of the tested cell lines for the lead compound  Outstanding selectivities were achieved (S.I. up to > 7700)  Depolarization of tumour mitochondrial membrane, leading to apoptosis was observed  MDR cells were efficiently targeted by co-administration with tariquidar  Cytostatic action was observed for the lead compound against SW1573 cells using continuous cell-living imaging Revised Manuscript Click here to view linked References 2 Abstract Most of the currently available cytotoxic agents for tackling cancer are devoid of selectivity, thus causing severe side-effects. This situation stimulated us to develop new antiproliferative agents with enhanced affinity towards tumour cells. We focused our attention on novel chalcogen-containing compounds (thiosemicarbazones, disulfides, selenoureas, thio- and selenocyanates), and particularly on selenium derivatives, as it has been documented that this kind of compounds might act as prodrugs releasing selenium-based reactive species on tumour cells. Particularly interesting in terms of potency and selectivity was a pharmacophore comprised by a selenocyanato-alkyl fragment connected to a p-phenylenediamine residue, where the nature of the second amino moiety (free, Bocprotected, enamine-protected) provided a wide variety of antiproliferative activities, ranging from the low micromolar to the nanomolar values. The optimized structure was in turn conjugated through a peptide linkage with biotin (vitamin B7), a cellular growth promoter, whose receptor is overexpressed in numerous cancer cells; the purpose was to develop a selective vector towards malignant cells. Such biotinylated derivative behaved as a very strong antiproliferative agent, achieving GI50 values in the low nM range for most of the tested cancer cells; moreover, it was featured with an outstanding selectivity, with GI50 > 100 µM against human fibroblasts. Mechanistic studies on the mode of inhibition of the biotinylated selenocyanate revealed (Annexin-V assay) a remarkable increase in the number of apoptotic cells compared to the control experiment; moreover, depolarization of the mitochondrial membrane was detected by flow cytometry analysis, and with fluorescent microscopy, what supports the apoptotic cell death. Prior to the apoptotic events, cytostatic effects were observed against SW1573 cells using label-free cell-living imaging; therefore, tumour cell division was prevented. Multidrug resistant cell lines exhibited a reduced sensitivity towards the biotinylated selenocyanate, probably due to its P-gp-mediated efflux. Remarkably, antiproliferative levels could be restored by co-administration with tariquidar, a P-gp inhibitor; this approach can, therefore, overcome multidrug resistance mediated by the P-gp efflux system. Keywords: biotin, organochalcogen, selenocyanate, antiproliferative, apoptosis; cytostatic agent 3 1. Introduction Cancer, a conglomerate of over 100 different diseases [ 1 ], depending on the tissue or organ affected, is nowadays a leading cause of morbidity and mortality worldwide [ 2 ]. In fact, cancer became in 2019 the first or second cause of death in more than 100 countries for people below 70 years old [ 3 ]. This rise is partially due to the decrease in mortality provoked by cardiovascular diseases. In 2020, 19.3 million new cancer cases and roughly 10 million deaths were estimated [3], and cancer screening was strongly hampered by COVID19 pandemic [ 4 ]. In low-income countries, roughly 30% of cancer incidence has its origin in cancer-causing infections, like hepatitis and human papillomavirus (HPV) [ 5 ]. The enormous complexity of this multifactorial disease, with etiologies not even fully understood [ 6 ], together with the severe side-effects caused by most of the chemotherapeutic agents due to lack of selectivity [ 7 ], the development of multidrug resistance (MDR) [ 8 ] or the high economic burden underwent by the patient and the society (in this context, the term financial toxicity was coined) [ 9 ] are some of the key aspects to be faced by the new anticancer therapies. Such therapies seek the more efficient elimination of tumour cells, the increase of rate survival and the improvement of the patient’s life quality. Several recent outstanding advances in cancer treatment can be highlighted, in the attempt of boosting the immune system against tumour cells [ 10 ]; among such strategies, monoclonal antibodies for the development of cancer vaccines [ 11 ], personalized anticancer therapies by reprogramming T-cells (chimeric antigen receptor T, CAR-T, cells therapy) [ 12 ], or immune checkpoint blockades [ 13 ] are remarkable. However, primary tumour treatment still requires the use of cytotoxic agents in most of the cases; in this context, the US FDA approved 332 anticancer drugs in the period 2009–2020 [ 14 ]. Herein, we pursued the preparation of potential drug candidates to increase the arsenal of cytotoxic agents available so far. For that purpose, we envisioned the possibility of using organochalcogen derivatives; sulfur- and selenium-containing compounds exhibit a plethora of beneficial biological activities, which are frequently enhanced in the selenium isosters. Selenium is present in the active site of 25 selenoproteins, most of them belonging to the antioxidant protective machinery, and is 4 currently considered to be an essential micronutrient, whose deficit leads to several diseases and disorders [ 15 ]. Among the numerous biological properties found in organoselenium derivatives [ 16 , 17 ], the following ones can be highlighted: strong redox properties [ 18 ], antiviral (including anti-SARS-COV-2 effects) [ 19 ], antidepressant-like properties [ 20 ], anti-inflammatory [ 21 ], antinociceptive [ 22 ], antiprotozoal [ 23 , 24 ], therapeutic enzymes inhibitors like those against monoamino oxidase (MAO) and/or carbonic anhydrases [ 25 , 26 ], O-GlcNAcase (OGA) and/or cholinesterases [ 27 , 28 ], histone deacetylases (HDACs) [ 29 , 30 ], or glycosidases [ 31 ], reduction of Aβ aggregation [ 32 ], or anticancer activities [ 33 ‒ 40 ], among others. In order to enhance the selectivity towards tumour cells, we envisioned the possibility of conjugating the organoselenium motif to a residue of biotin (vitamin B7), a cell growth promoter; biotin receptor is overexpressed in numerous cancer cell lines [ 41 ], and is therefore, considered as an attractive therapeutic target for the selective vectorization of drugs towards cancer cells. Biotinylation of cytotoxic agents, like taxoids [ 42 ], doxorubicin [ 43 ], or gemcitabine [ 44 ], among others, has successfully been reported. 2. Results and discussion 2.1. Chemistry In the search for more selective antiproliferative agents, we decided to carry out the conjugation of biotin to organochalcogen scaffolds, pharmacophores widely employed in our group [23, 27‒29, 31, 39, 40]. Thus, connection with phenolic thio(seleno)semicarbazones, selenoureas, thio(selenocyanates), and disulfides/diselenides was envisioned (Figure 1). 5 Figure 1. General structure of the biotinylated organochalcogen derivatives attempted herein Biotin and the active pharmacophores were connected through an amidecontaining linker; although the use of thionyl chloride has been reported [ 45 ] to generate the corresponding acyl chloride and the subsequent transformation into amides, the intrinsic high instability of this biotin-derived intermediate encouraged us to use a more versatile synthetic pathway. We then shifted to the activation of the carbonyl group with isobutyl chloroformate, previously used by Zheng et al. for linking biotin and γ-aminobutiric acid [ 46 ], followed by coupling of the transient mixed anhydride with p-phenylenediamine (Scheme 1). In this context, Scheme 1 depicts conjugation of biotin with phenolic thiosemicarbazones (derivatives 9‒11) in five steps: activation of the carbonyl group, coupling with the aromatic diamine 3, transformation of the free amino group of 4 into isothiocyanate 5 (using thiophosgene as the thionating agent in a triphasic medium), treatment with hydrazine and condensation of the transient thiosemicarbazide with commercially-available phenolic aldehydes 6‒8 with different numbers of hydroxyl groups. Such phenolic scaffold is endowed with strong redox properties, and has previously been used in our group to develop antiproliferative agents [40]. 6 Scheme 1. Preparation of biotinylated thiosemicarbazones 9‒11 With the aim of analysing the influence of the biotin residue on the antiproliferative activity, thiosemicarbazone 15 was also accessed (Scheme 2); in this case, the biotinyl residue was replaced by a Boc fragment. For that purpose, monoprotection of p-phenylenediamine with (Boc)2O was achieved using the methodology reported by Togashi et al. [ 47 ]. In a similar way as depicted in Scheme 1, subsequent treatment with thiophosgene, hydrazine and 3,4-dihydroxybenzaldehyde (protocatechuic aldehyde) afforded 15 (16% overall yield, four steps). 7 Scheme 2. Preparation of N-Boc-protected thiosemicarbazone 15 The influence of the free or protected phenolic hydroxyl groups on the bioactivity was also evaluated; for that purpose, 3,4-dihydroxybenzaldehyde was acetylated prior to conjugation with biotin (compound 17, Scheme 3). This can provide information about the potential effect of increasing the lipophilicity of the molecule. Scheme 3. Preparation of O-protected biotinylated thiosemicarbazone 17 8 Other functionalities connected to the biotin residue were the thiocyanate and disulfide scaffolds (Scheme 4). Initially, the use of Boc-protecting group was attempted. Thus, N-alkylation of 12 with 1,6-dibromohexane, and subsequent nucleophilic reaction with KSCN furnished thiocyanate 19 in good yield; unfortunately, attempts to carry out the acidic deprotection of the N-Boc moiety were unsuccessful, as extensive decomposition was observed. We then shifted to the use of enamine as the N-protecting group by coupling pphenylenediamine 3 with diethyl ethoxymethylenemalonate [ 48 ] (Scheme 4). Following this approach, and starting from 20, targeted thiocyanate 23, with the free p-amino group could be successfully accessed in a three-step procedure: N-alkylation, nucleophilic displacement with KSCN and efficient N-deprotection with ethylenediamine. The presence of the thiocyanato motif was evidenced in compounds 19, 22 and 23 in the 13C-NMR spectra (resonances at 112.4‒112.5 ppm). Scheme 4. Preparation of of N-protected and N-unprotected thiocyanates 19, 22, 23 15 2.2.2. Antiproliferative effects on sensitive/multidrug resistant cell lines According to data depicted in Table 1, biotinylated selenocyanate 31 can clearly be considered as the lead compound, and therefore, a good candidate to further assays for the elucidation of its mode of action. The same assays were initially planned also for the non-biotinylated counterpart 30; nevertheless, its reduced solubility under the assays conditions, precluded its evaluation. An important issue to be considered when developing new anticancer drugs is their behaviour towards resistant cell lines. Multidrug resistance (MDR) is indeed one of the major problems and challenges to be solved in chemotherapies, as it accounts for 90% of deaths [ 52 ]. Although numerous mechanisms responsible for the development of MDR are currently known, special attention should be given to ATP-binding cassette (ABC) superfamily of transporters, among which P-glycoprotein efflux pump (P-gp) is overexpressed in numerous tumours [8]; as a result, cytotoxic chemotherapeutic agents are extruded from the cells, leading to failure of the anticancer therapy. Moreover, the faster the cancer cells can adapt to unfavourable situations (e.g. through metabolic pathways, redox changes, autophagy, increased capacity of repairing DNA, etc.) provoked by the chemotherapeutic agents, the higher resistance will be observed [ 53 ]. In this context, the effects of 31 on cell growth were studied in NCI-H460 (lung), NCI-H460/R (lung, resistant cell line), and MRC-5 cells (fibroblasts, nontumour). Table 2 summarizes the GI50 values obtained by the sulforhodamine B (SRB) assay. Although the potency of 31 against the lung cell lines used in Table 2 is sensibly lower that against the six cell lines depicted in Table 1, the possibility of analyzing the effects on a sensitive/resistant couple deserves attention. Biotinylated selenocyanate 31 inhibited the growth of drug-sensitive cancer cells, with remarkable selectivity towards cancer cells. Furthermore, considerable difference in sensitivity between sensitive and MDR cells was also noticed. Specifically, as indicated in Table 2, NCI-H460/R cells (which overexpress P-glycoprotein) were approximately 10-fold more resistant to 31 compared to NCI-H460. Multidrug resistant NCI-H460/R cell line, which is a lung carcinoma cell line resistant to DOX and cross-resistant to etoposide, paclitaxel, vinblastine and 16 epirubicin, was selected from parental NCI-H460 cells after continuous exposure to increasing concentrations of DOX [ 54 ]. We next addressed the interaction of 31 with P-glycoprotein. The potential of 31 (10 and 100 µM) to modulate the P-glycoprotein function was evaluated by rhodamine 123 accumulation assay and was compared to tariquidar (TQ), a third-generation P- glycoprotein inhibitor (Fig. 2). Treatment with 50 nM TQ successfully inhibited activity of this efflux pump in NCI-H460/R cells, resulting in the accumulation of rhodamine 123 similar to NCI-H460 cells. However, 31 displayed P-glycoprotein substrate properties which was evident by dose-dependent competitive reduction in cellular efflux of fluorescent rhodamine 123, a substrate of P-glycoprotein. Namely, the addition of 10 μM 31 did not modulate the P-glycoprotein function, while 100 μM treatment increased rhodamine accumulation by approximately 50%. Table 2. GI 50 values (µM) for the antiproliferative activity of 31 in NCI-H460, NCI-H460/R, and MRC-5 cells Compound NCI-H460 (Lung) NCI-H460/R (Lung) MRC-5 (Fibroblasts) (non-tumour) S.I. Resistance factor (NCI-H460/R / NCI-H460) 31 11.1±1.29 116.6±1.46 136.1±1.26 12 10.5 17 Figure 2. Effect of 31 on P-gp activity. (A) Flow-cytometry assay for the accumulation of rhodamine 123 in in NCI-H460/R cells treated with 31. (B) Rhodamine 123 fluorescence in NCI-H460/R cells treated with 31. Statistical significant difference (ANOVA test) compared to NCI-H460/R control cells: p < 0.05 (*), p < 0.001 (***). To evaluate if the activity of P-glycoprotein is responsible for the resistance of NCI-H460/R cells to 31, the compound’s effects on cell growth inhibition in combination with TQ was tested by the sulforhodamine B assay (Figure 3). TQ, without effects on the growth of cells, improved 31 efficacy in resistant cells. Specifically, the GI50 value of 31 in NCI-H460/R was lowered from 116.6 µM to 33.5 µM upon the 18 addition of TQ. The cell growth inhibition profiles of 31 in NCI-H460/R cells with or without the addition of TQ are shown in Figure 3, and compared to the cell growth inhibition profile of 31 in sensitive parental cell line. Therefore, the observed resistance of NCI-H460/R cells to 31 is likely a result of its lower cellular accumulation due to P-glycoprotein activity. Figure 3. Effect of administration of TQ + 31 against NCI-H460/R cells (nonlinear regression) 2.2.3. Cell death analysis (Annexin-V assay) The effect of the compound on cell death induction was then analysed. Flowcytometric analysis of cell death showed that 31 considerably increased a number of apoptotic NCI-H460 cells. The amount of NCI-H460 cells in early apoptosis (AV+ PI−) increased from 0.3% in control (untreated cells) to 12% for cells treated with 31 (10 µM, Fig. 4A and B). Viable (AV− PI−) and necrotic (AV− PI+) cells are also depicted in Figure 4A and B. The number of cells in late apoptosis (AV+ PI+) increased from 0.3% in control to 24% for cells treated with compound 31 (Fig. 4). Conventional chemotherapeutic DOX (doxorubicin) was used as a positive control for inducing cell death. 19 Figure 4. Annexin-V/PI staining assay on NCI-H460 cells treated with 31 (10 µM); [DOX] = 400 nM 2.2.4. Analysis of mitochondrial transmembrane potential During apoptosis, outer and inner mitochondrial membranes increase their permeability [ 55 ]; for that reason, mitochondrial membrane potential can be considered as an early marker for apoptosis. Tetramethylrhodamine (TMRE) staining was used to evaluate the effect of 31 on the mitochondrial membrane potential in NCI-H460 cells. Depolarization of mitochondrial membrane was detected by flow cytometry in cells treated with 10 µM 31 after 48 h (Fig. 5A), which corresponded with apoptosis observed in this cell line under the same conditions (Fig. 4). Carbonyl cyanide m- 20 chlorophenyl hydrazone (CCCP), a mitochondrial uncoupling agent was used as a positive control for dissipation of mitochondrial membrane potential. The inability of depolarized mitochondria to sequester TMRE in 31-treated NCI- H460 cells was also visualized by fluorescent microscopy (Fig. 5B). Figure 5. Mitochondrial transmembrane potential changes provoked by 31. TMRE staining was used in the treatment of NCI-H460 cells with 31 (10 µM, 48 h). (A) Flow-cytometric profiles of TMRE-stained cells after treatments with 31 and 10 µM CCCP (left panel). Fluorescence intensity of TMRE (arbitrary units) of NCI-H460-treated cells (right panel). Statistical significant difference (ANOVA test) compared to control cells: p < 0.001 (***). (B) Fluorescence microscopy on NCI-H460-treated cells 2.2.5. Continuous Live-Cell Imaging To study in more detail the phenotypic effects of compound 31, we used labelfree continuous live-cell imaging microscopy. With this technique, we monitored (every 5 minutes) at single cell level the effects of the exposure to 31 (100 nM) for 20 h on the lung cancer cells SW1573. As a result, cytostatic effects were induced by 31 on treated cells (Supplementary material, videos S1 and S2). Untreated cells moved freely and 21 divided, almost doubling the population at the end of the experiment. In contrast, the number of treated cells remained constant and no cell division was observed. Cell morphology experienced changes as time progressed, distinguishing several apoptotic hallmarks. This observation is consistent with the results obtained in the cell death analysis. 2.2.6. Measurement of the inhibition of histones deacetylases (HDACs) Histones are small metalloproteins that bind DNA strands to furnish chromatin, whose post-translational modifications (methylation, phosphorylation, acetylation, deacetylation) regulates gene expression. In particular, acetylation and deacetylation of histones depend on the enzymes histone acetyltransferases (HATs) and deacetylases (HDACs) [ 56 ]. The latter enzymes are overexpressed in numerous tumours, yielding aberrant deacetylation in the terminal lysine units of histones. As a result, a more compact chromatine is obtained, causing the repression of certain genes involved in the control of cell growth and death, and thus, allowing the development of tumour cells [ 57 ]. Therefore, inhibition of HDACs is currently considered as an attractive therapeutic target in the development of new anticancer agents. The most common family of such inhibitors is comprised of hydroxamates, which chelate the Zn2+ ion of the enzyme active site. In this context, SAHA (suberoylanilide hydroxamic acid, Vorinostat®) was approved by FDA against cutaneous T cell lymphoma [ 58 ]. SelSA, a selenocyanate structurally related to 30 was reported to be a good inhibitor of HDAC-6 [30]. For that purpose, derivatives 30 and 31 were also tested as potential inhibitors of HDACs to determine if such activity also contributes to their antiproliferative mechanism. Unfortunately, derivatives 30 and 31 were found to be considerably weaker inhibitors than SAHA (IC50= 0.27 ± 0.08 μM) and SelSA (Figure 6), and although inhibition of HDACs can not be ruled out, it would be a minor contribution to their antiproliferative mode of action. The lower activity compared to SAHA is likely due to the monodentate coordination to Zn, which will be weaker compared to the bidentate coordination of SAHA. Furthermore, SAHA benefits from a hydrogen bond interaction between its PhNHCO- group and the enzyme active site cavity entrance, whereas this type of interaction is not present in 30, 31, due to the absence of such amide moiety. 22 Moreover, the behaviour of both compounds in a dose-dependent inhibition experiment (Figure 6) could suggest a weak interaction that does not involve binding in the enzyme active site. Figure 6. HDAC activity at various inhibitor concentrations (30, 31). SAHA was used as a positive control 3. Conclusions In conclusion, conjugation of biotin with organochalcogen scaffolds for the development of antiproliferative agents was found to be highly dependent on the nature of the organochalcogen motif. This approach turned out to be particularly effective in the case of selenocyanates, providing very strong and selective antiproliferative agents, with activities reaching the nM range in six out of the seven human tumour cell lines tested. Apoptosis and depolarization of the mitochondria membrane were found to be involved in the mode of action of biotinylated selenocyanates Prior to the apoptotic events, cytostatic effects were observed against SW1573 cells using label-free cellliving imaging; therefore, tumour cell division was prevented. Moreover, as a way of avoiding P-gp-mediated chemoresistance, coadministration with tariquidar turned out to be a valid alternative. 0 10 20 30 40 50 60 70 80 90 100 1.6 8 40 82.2 HDAC Activity (%) Inhibitor Concentration / μM SAHA 30 31 23 Therefore, biotinylated selenocyanate 31 could be considered as an attractive lead compound in the development of new families of chemotherapeutic agents against cancer. 4. Experimental section 4.1. Materials and methods 4.1.1. General procedures The same general procedures concerning chromatography, NMR spectroscopy and MS spectrometry as reported previously [ 59 ] were used. 4.1.2. Antiproliferative activities The in vitro antiproliferative activity against the panel of 6 tumour cell lines, and the non-human line (BJ-hTert) was assayed using minor modifications of the protocol of the US National Cancer Institute (NCI) [50]. The tumour cell lines were a kind gift from Professor G.J. Peters (UV Medical Center, Amsterdam, The Netherlands), whereas the non-tumour cell line was obtained from Dr. R. Freire (HUC, Tenerife, The Canary Islands). For the assays against the lines NCI-H460 and NCI-H460/R, the conditions reported previously were used [59]. DOX, CCCP, and TQ) were obtained from Sigma- Aldrich (Germany). RPMI 1640 and Minimum Essential Medium (MEM) were purchased from Capricorn Scientific GmbH (Germany). Penicillin-streptomycin solution, amphotericin B solution, L-glutamine, and trypsin were purchased from Biowest (France). Fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), sulforhodamine B (SRB), rhodamine 123 (Rho 123) and tetramethylrhodamine (TMRE) were obtained from Sigma-Aldrich. Bovine serum albumin (BSA) was from Serva (Heidelberg, Germany). Annexin-V- FITC (AV) and propidium iodide (PI) Apoptosis Detection Kit was purchased from Abcam (Cambridge, UK). Human non-small cell lung carcinoma cell line NCI-H460 was obtained from American Type Culture Collection (Rockville, MD, United States). MRC-5 cell line 24 (normal human embryonic lung fibroblasts) was purchased from American Type Culture Collection. 4.1.3. Sulforhodamine B assay The same conditions as reported previously [59] were used; compound 31 was tested in a 1 nM ‒ 100 μM concentration range. 4.1.4. Rhodamine 123 accumulation assay The same conditions as reported previously [59] were used; NCI-H460/R cells were treated with 10 µM and 100 µM 31. 4.1.5. Cell death analysis by flow cytometry The same conditions as reported previously [59] were used; NCI-H460 cells were incubated at a density of 200,000 cells/well. The cells were treated with 10 µM 31 for 48 h. As a positive control, DOX was used at a 400 nM concentration. 4.1.6. Mitochondrial transmembrane potential analysis The effect of 31 on mitochondrial membrane potential was analysed using the cell-permeant dye TMRE [ 60 ]. NCI-H460 cells were seeded in 6-well tissue culture plate (200,000 cells/well), incubated overnight, and treated with 10 µM 31 for 48 h. As a positive control, cells were also treated with 10 µM CCCP for 30 min prior to TMRE staining. Next, the cells were stained with 500 nM TMRE for 30 min at 37 °C in the dark. To visualize the effect of 31 on mitochondrial membrane potential, the cells were imaged live on ZOE Fluorescent Cell Imager (Bio-Rad Laboratories, Hercules, CA, USA), 20× objective. TMRE fluorescence was also quantified on a CyFlow Space flow cytometer (Partec, Münster, Germany) by measuring fluorescence emission in FL2 channel. At least 20,000 events were recorded per each sample. 31 a solution of thiocyanate 23 (68.1 mg, 0.27 mmol) in DMF (1 mL), cooled down at 0 ºC; reaction was kept stirring at that temperature for further 5 min, and then, 2.5 h at rt. After that, the mixture was concentrated to dryness and the residue was purified by column chromatography (CH2Cl2 → 20:1 CH2Cl2-MeOH) to give 24 as a syrup. Yield: 68.4 mg (53%); [α]D 25 = +31 (c 1.15, (CD3)2SO); 1H-NMR (500 MHz, (CD3)2SO) δ 9.41 (s, 1H, Ar-NH-CO), 7.25 (m, 2H, Ar-H), 6.47 (m, 2H, Ar-H), 6.42 (s, 1H, s, NH biotin), 6.34 (s, NH biotin), 5.29 (t, 1H, JH,H = 5.6 Hz, CH2-NH), 4.30 (m, 1H, CH-N), 4.14 (m, 1H, CH-N), 3.11 (m, 1H, CH-S), 2.95 (q, 2H, JH,H = 6.7 Hz, CH2), 2.82 (dd, 1H, JH,H = 5.2 Hz, JH,H = 12.5 Hz, H-CH-S), 2.58, (d, 1H, JH,H = 12.3 Hz, H-CH-S), 2.22 (t, 2H, JH,H = 7.3 Hz, CH2-CO), 1.54 (m, 14H, 7CH2) ppm; 13C-NMR (125.7 MHz, (CD3)2SO) δ 170.1 (C=O), 162.7 (C=O), 145.3, 128.3, 120.9 (Ar-C), 113.2 (SCN), 111.7 (Ar-C), 61.0 (NH-CH), 59.2 (NH-CH), 55.4 (S-CH), 43.1, 36.0, 33.2, 29.5, 28.5, 28.3, 28.1, 27.1, 26.0, 25.3 (CH2) ppm; HRESI-MS calcd. for C23H34N5O2S2 ([M+H]+): 476.2148, found: 476.2148. 6,6’-Dithiobis[N-(4’’-biotinylamino)phenyl]aminohexyl (25). To a solution of thiocyanate 24 (58.1 mg, 0.12 mmol) in DMF (1.5 mL) was added NaBH4 (10.0 mg, 0.26 mmol, 2.2 equiv.); reaction was kept stirring at rt for 20 h. Then, it was concentrated to dryness and the residue was purified by column chromatography (CH2Cl2 → 5:1 CH2Cl2-MeOH) to give 25 as a syrup. Yield: 34.4 mg (64%). [α]D 25 = +29 (c 0.64, DMSO); 1H-NMR (300 MHz, (CD3)2SO) δ 9.47 (s, 2H, NH), 7.25 (m, 4H, Ar-H), 6.46 (m, 4H, Ar-H), 6.44 (s, 2H, NH biotin), 6.36 (s, 2H, NH biotin), 5.28 (t, 2H, JH,H = 5.2 Hz, NH-CH2), 4.30 (m, 2H, CH-NH), 4.13 (m, 2H, CH-NH), 3.11 (m, 2H, CH-S), 2.92 (q, 4H, JH,H = 5.9 Hz, 2CH2), 2.82 (dd, 2H, JH,H = 5.1 Hz, JH,H = 12.5 Hz, H-CH-S), 2.69 (t, 4H, JH,H = 7.2 Hz, CH2-S), 2.57 (d, 2H, JH,H = 12.3 Hz, H-CH-S), 2.22 (t, 4H, J = 7.2 Hz, 2CH2-CO), 1.48 (m, 28H 14CH2) ppm; 13C-NMR (75.5 MHz, (CD3)2SO) δ 170.1 (C=O), 162.7 (C=O), 145.3, 128.3, 120.9, 111.7 (Ar-C), 61.1(NHCH), 59.2 (NH-CH), 55.4 (S-CH), 43.1, 37.8, 36.0, 28.6, 28.6, 28.3, 28.1, 27.6, 26.3, 25.3 (CH2) ppm; HRESI-MS calcd. for C44H67N8NaO4S4 ([M+H]+): 899.4163, found: 899.4144. N-[4-(Biotinylamino)phenyl]formamide (26). A solution of amino derivative 4 (120.0 mg, 0.36 mmol) and AcOH (21 µL, 0.36 mmol, 1.0 equiv.) in ethyl formate (5 mL) was 32 refluxed for 3.5 h. The resulting precipitate was filtered, washed with ethyl formate (5 mL) and centrifuged at 3,000 rpm for 2 min twice to give 26 as a white solid. Yield: 118.1 mg (98%). Mp: 188-196 ºC; [α]D 24 = +41 (c 0.81, DMSO); 1H-NMR (300 MHz, (CD3)2SO) δ 9.45 (s, 1H, CHO), 7.21 (m, 2H, Ar-H), 6.50 (m, 2H, Ar-H), 6.44 (s, 1H, NH), 6.36 (s, 1H, NH), 5.04 (s, 2H, 2NH), 4.30 (m, 1H, CH-NH), 4.13 (m, 1H, CHNH), 3.11 (m, 1H, CH-S), 2.82 (dd, 1H, JH,H = 5.2 Hz, JH,H = 12.5 Hz, H-CH-S), 2.57 (d, 1H, JH,H = 12.2 Hz, H-CH-S), 2.22 (t, 2H, JH,H = 7.3 Hz, CH2), 1.48 (m, 6H, 3CH2) ppm; 13C-NMR (75.5 MHz, (CD3)2SO) δ 170.1 (C=O), 162.7 (C=O), 144.0, 128.9, 120.8, 114.0 (Ar-C), 61.0 (NH-CH), 59.2 (NH-CH), 55.4 (S-CH), 39.8 (S-CH2), 36.0, 28.2, 28.1, 25.3 (CH2) ppm; HRESI-MS calcd. for C17H23N4O3S ([M+H]+): 363.1485, found: 363.1500. N-[4-(Biotinylamino)phenyl]-N’-(4-iodophenyl)selenourea (27). To a solution of amino derivative 4 (46.0 mg, 0.14 mmol) in DMF (2 mL) was added 4-iodophenyl isoselenocyanate (50.3 mg, 0.16 mmol, 1.1 equiv.). The resulting mixture was heated at 45 ºC in the darkness under Ar for 2 h. Then, it was concentrated to dryness, and the residue was washed with EtOH (15 mL) and centrifuged at 3,000 rpm during 2 min twice, to give 27 as a grey solid. Yield: 51.6 mg (57%). Mp: 187‒195 ºC; [α]D 23 = +24 (c 0.80, DMSO); 1H-NMR (300 MHz, (CD3)2SO) δ 10.16 (s, 1H, NH), 10.00 (s, 1H, NH), 9.91 (s, 1H, NH), 7.65 (m, 2H, Ar-H), 7.55 (m, 2H, Ar-H), 7.24 (m, 4H, Ar-H), 6.43 (s, 1H, biotin), 6.36 (s, 1H, NH biotin), 4.30 (m, 1H, CH-N), 4.14 (m, 1H, CH-N), 3.12 (m, 1H, CH-S), 2.82 (dd, 1H, JH,H = 12.4 Hz, JH,H = 5.0 Hz, H-CH-S), 2.57 (d, 1H, JH,H = 12.6 Hz, H-CH-S), 2.30 (t, 2H, JH,H = 7.3 Hz, CH2-CO), 1.51 (m, 6H, 3CH2) ppm; 13C- NMR (75.5 MHz, (CD3)2SO) δ 179.0 (C=Se), 171.6 (C=O), 163.2 (C=O), 140.2, 137.6, 137.4, 134.7, 127.5, 125.9, 119.7 (Ar-C), 90.2 (Ar-C-I), 61.5 (CH-NH), 59.7 (CH-NH), 55.9 (CH-S), 36.7, 28.7, 28.6, 25.6 (CH2) ppm; HRESI-MS calcd. for C23H26IN5NaO2SSe ([M+Na]+): 665.9909, found: 665.9892. tert-Butyl {4-[(6’-selenocyanatohexyl)amino]phenyl}carbamate (28). A solution of bromo-derivative 18 (224.5 mg, 0.60 mmol) and KSeCN (258.8 mg, 1.80 mmol, 3.0 equiv.) in DMF (6 mL) was heated at 70 ºC in the darkness under Ar for 2.5 h. Then it was concentrated to dryness and the residue was purified by column chromatography (cyclohexane → 1:4 EtOAc‒cyclohexane) to give 28 as a red syrup. Yield: 124.7 mg 33 (52%). 1H-NMR (300 MHz, CDCl3) δ 6.93 (m, 2H, Ar-H), 6.69 (m, 2H, Ar-H), 3.77 (brs, 2H, 2NH), 3.54 (t, 2H, JH,H = 7.2 Hz, CH2), 3.01 (t, 2H, JH,H = 7.3 Hz, CH2), 1.86 (m, 2H, CH2), 1.42 (m, 6H, 3CH2), 1.40 (s, 9H, C(CH3)3) ppm; 13C-NMR (300 MHz, CDCl3) δ 155.4 (C=O), 144.5, 134.3, 128.4, 116.0 (Ar-C), 101.7 (SeCN), 79.8 (C(CH3)3), 49.9, 30.9, 29.6, 28.9 (CH2), 28.5 (C(CH3)3), 27.7, 26.0 (CH2) ppm; HRESIMS calcd. for C18H28N3O280Se ([M+H]+): 398.1341, found: 398.1335. Diethyl 2-{[(4’-((6’’-selenocyanatohexyl)amino)phenyl)amino]methylene}malonate (29). A solution of bromo-derivative 21 (100.6 mg, 0.23 mmol) and KSeCN (97.8 mg, 0.68 mmol, 3.0 equiv.) in DMF (2 mL) was heated at 70 ºC in the darkness under Ar for 3.5 h. Then, it was concentrated to dryness and the residue was purified by column chromatography (cyclohexane → 1:2 EtOAc‒cyclohexane) to give 29 as a syrup. Yield: 95.4 mg (90%); 1H-NMR (300MHz, (CD3)2CO) δ 10.85 (d, 1H, JH,H = 13.9 Hz, Ar-NH- C=), 8.38 (d, 1H, JH,H = 14.0 Hz, CH=), 7.10 (m, 2H, Ar-H), 6.68 (m, 2H, Ar-H), 5.01 (t, 1H, JH,H = 5.0 Hz, NH), 4.21 (q, 2H, JH,H = 7.1 Hz, CH2-CH3), 4.14 (q, 2H, JH,H = 7.1 Hz, CH2-CH3), 3.19 (t, 2H, JH,H = 7.1 Hz, CH2), 3.13 (t, 2H, JH,H = 7.1 Hz, CH2), 1.93 (m, 2H, CH2), 1.67 (m, 2H, CH2), 1.52 (m, 4H, 2CH2), 1.27 (m, 6H, 2CH2-CH3) ppm; 13C-NMR (75.5 MHz, (CD3)2CO) δ 169.6 (C=O), 166.0 (C=O), 153.1 (=CH-N-), 148.1, 130.1, 119.9, 113.8 (Ar-C), 102.7 (SeCN), 92.2 (C=CH), 60.2 (CH2-CH3), 59.9 (CH2- CH3), 44.3, 31.9, 30.1, 29.5, 27.1 (CH2), 14.8, 14.7 (2CH3) ppm; HRESI-MS calcd. for C21H30N3O480Se ([M+H]+): 468.1396, found: 468.1396. N-(6’-Selenocyanatohexyl)benzene-1,4-diamine (30). To a solution of enamine 29 (241.3 mg, 0.52 mmol) in EtOH (6 mL) was added ethylenediamine (175 µL, 2.62 mmol, 5.0 equiv.), and the corresponding mixture was stirred at rt in the darkness under Ar for 16 h. Then, it was concentrated to dryness and the residue was purified by column chromatography (cyclohexane → 2:1 EtOAc‒cyclohexane) to give 30 as a syrup. Yield: 105.5 mg (68%); 1H-NMR (300 MHz, (CD3)2SO) δ 6.38 (m, 4H, Ar-H), 4.39 (brs, 3H, NH, NH2), 3.08 (t, 2H, JH,H = 7.2 Hz, CH2), 2.88 (m, 2H, CH2), 1.81 (m, 2H, CH2), 1.45 (m, 6H, 3CH2) ppm; 13C-NMR (75.5 MHz, (CD3)2SO) δ 140.6, 138.6, 115.6, 113.6, (Ar-C), 104.6 (SeCN), 44.1, 30.8, 29.8, 28.3, 26.0 (CH2) ppm; HRESI-MS calcd. for C13H20N3Se ([M+H]+): 298.0817, found: 298.0815. 34 N-Biotinyl-N’-(6-selenocyanatohexyl)benzene-1,4-diamine (31). To a solution of D- biotin (73.9 mg, 0.30 mmol) and Et3N (96 µL, 0.69 mmol, 2.3 equiv.) in DMF (4.5 mL) was slowly added isobutyl chloroformate (57 µL, 0.44 mmol, 1.5 equiv.); after the addition, the reaction was kept stirring at rt under Ar for 10 min. Then, this mixture was added on a solution of selenocyanate 30 (88.8 mg, 0.30 mmol, 1.0 equiv.) in DMF (1 mL), cooled down at 0 ºC; reaction was kept stirring at that temperature for further 5 min, and then, 2 h at rt. After that, the mixture was concentrated to dryness and the residue was purified by column chromatography (cyclohexane → 5:1 CH2Cl2-MeOH) to give 31 as a grey solid. Yield: 79.2 mg (51%). Mp: 141‒150 ºC; [α]D 25 = +30 (c 0.92, DMSO); 1H-NMR (300 MHz, (CD3)2SO) δ 9.43 (s, 1H, NH), 7.25 (m, 2H, Ar-H), 6.47 (m, 2H, Ar-H), 6.43 (s, 1H, NH biotin), 6.35 (s, 1H, NH biotin), 5.30 (s, 1H, NH), 4.30 (m, 1H, CH-N), 4.14 (m, 1H, CH-N), 3.12 (m, 1H, CH-S), 3.08 (t, 2H, JH,H = 7.3 Hz, CH2), 2.95 (m, 2H, CH2), 2.82 (dd, 1H, JH,H = 5.1 Hz, JH,H = 12.4 Hz, H-CH-S), 2.57 (d, 1H, JH,H = 12.5 Hz, H-CH-S), 2.22 (t, 2H, JH,H = 7.2 Hz, CH2-CO), 1.82 (m, 2H, CH2), 1.49 (m, 12 H, 6CH2) ppm; 13C-NMR (75.5 MHz, (CD3)2SO) δ 170.6 (C=O), 163.2 (C=O), 145.7, 128.8, 121.4, 112.2 (Ar-C), 105.1 (SeCN), 61.5 (NH-CH), 59.7 (NH-CH), 55.9 (S-CH), 43.6, 36.5, 31.2, 30.3, 29.1, 28.7, 28.6, 26.4, 25.8 (CH2) ppm; HRESI-MS calcd. for C23H34N5O2S80Se ([M+H]+): 524.1593, found: 524.1583. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We thank Grant PID2020-116460RB-I00 funded by MCIN/AEI/10.13039/501100011033, and Junta de Andalucía (FQM134) for financial support. J.R.-P. thanks the Ministerio de Educación y Ciencia for the award of a predoctoral grant. A.P. and J.M.P. thank the Spanish Government (Project PID2021- 123059OB-I00 funded by MCIN/AEI /10.13039/501100011033 / FEDER, UE) for financial support. A.P. thanks the EU Social Fund (FSE) and the Canary Islands ACIISI for a predoctoral grant TESIS2020010055. J.D., S.J.S. and M.P thank the Ministry of Education, Science and Technological Development of the Republic of Serbia 35 for financial support (451-03-68/2022-14/200007). A.P.d,e acknowledges the Thailand Development and Promotion of Science and Technology (DPST) Talents Project for funding. This work was performed within the framework of COST Action CA17104 STRATAGEM ”New diagnostic and therapeutic tools against multidrug resistant tumors”. 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