Aromatic selenocyanates as a new class of non-mutagenic 2 antimicrobial selenium compounds with pronounced activity against 3 multidrug resistant ESKAPE bacteria 4

Selenocyanates form an interesting class of organic selenium compounds as they serve as 21 multifunctional agents (being the precursors of seleninic acids and diselenides in synthetic 22 chemistry and as antimicrobial and cytotoxic agent in biology) and, due to their similarity with 23 better known thiocyanates promise high biological activity. Yet whilst selenocyanates are common 24 in synthetic chemistry, they are rarely considered in pharmaceutical design. Arylmethyl 25 selenocyanates (1-13) have been synthesized and an insight into their structural properties using 26 X-ray crystallography has been obtained. The compounds subsequently have been evaluated for 27 their potential antimicrobial, nematicidal and cytotoxic activity. ADMET properties in vitro, using 28 mutagenicity (AMES) and permeability (PAMPA) tests, have been determined. The compounds 29 exhibit pronounced activity against various strains of bacteria (both Gram-positive and 30 Gram-negative) and yeasts. Among them, benzylselenocyanate (1) represents the most active 31 anti-ESKAPE agent, with potent antibacterial activity, especially against multidrug resistant MRSA 32 strains (HEMSA 5). Our results demonstrate that the arylmethyl selenocyantes are not only 33 non-mutagenic but also possess moderate cytotoxic activity against cancer cells. 34


Introduction
Since the discovery of the first modern antibiotics almost one hundred years ago, countless lives have been saved in the skirmish against pathogenic bacteria.Antibiotics such as the penicillin have served as important and effective weapons -and often as double-edged swords -to prevent the onset of contagious ailments as prophylactic as well as post-infection curative agents.Since then, the quality of life (QoL) has been improving remarkably until the deadly enemy started to learn from our mistakes and instigated to develop resistance against antibiotics.Among various other reasons, overuse, misuse or extensive utilisation of antibiotics have led to the emergence of resistance in pathogenic bacteria, which has now become one of the biggest threats facing humanity [1].The phenomenon of "resistance" to currently available antibiotics has attracted the attention of scientist for well over two decades now, and there is a considerable demand for development of antibiotics against these resistant strains of bacteria.
Nature itself is an affluent source of antibiotics [2][3][4].Natural substances, acquired from medicinal as well as culinary plants like garlic and mustard, have been extensively employed as antibiotics [5][6][7].These substances are usually enriched with potent phytochemicals like alkaloids, flavonoids, terpenes and polyphenols and oragnosulfur compounds [8][9][10][11].Organosulfur compounds are widely dispersed in nature e.g.allicin in garlic, ergothioneine in mushrooms, thiocyanates and isothiocyanates in cruciferous vegetables [12][13][14].Isothiocyanates and thiocyanates are organosulfur compounds and are well known for their potential antimicrobial and cytotoxic potential [14][15][16][17].A remarkable upsurge in activity as well as reactivity has been observed for the selenium analogues of organosulfur compounds [18].Although organotellurium compounds are generally more potent than sufur and selenium analogues, yet these compounds are rather toxic and unstable, therefore, not suitable for drug design [19].Organoselenium compounds have, therefore, attracted the attention of scientists quite recently and certain compounds like ebselen have already entered the clinical trials [20].
Selenium analogues of naturally occurring thiocyanates (selenocyanates) represent an interesting class of organoselenium compounds [21].These compounds are multifunctional in nature as they are employed as a precursor for the synthesis of various other organoselenium compounds like seleninic acids or diselenides in synthetic chemistry and as an effective cytotoxic agent in biology [22][23][24].Although some organoseleno compounds have been reported as active compounds against microbes [25][26][27], little is known about antimicrobial activity of aromatic selenocyantes.Moreover, the literature also reveals that this class of selenocompounds has mostly been studied when attached to some "bioactive" scaffolds which may also influence their biological activity [24].Since hardly any evidence of -chemically quite stable -benyzlselenocyanates can be found in the biological literature, we have turned our attention to this class of compounds and have focussed on the synthesis of some of the most basic arylmethyl selenocyanates (1-13, Figure 1), which indeed exhibit a rather encouraging effectiveness against a broad spectrum of biologically highly significant since antibiotic-resistant targets.

Crystallographic studies
The structures of compounds 1-13 were confirmed by 1 H and 13 C-NMR and the molecular masses as well purity were confirmed by LC-MS.Additionally, a deeper insight into the structures of two selected compounds (1 and 12) was stipulated by employing X-ray crystallographic analysis (Table 1, Figure 2 and Figure 3).
The molecular structures and atomic-numbering schemes of 1 and 12 are presented in Figure 2.
In  The packing of the molecules in the crystal structures can be characterized by intermolecular interactions listed in Table 1.In both structures, only weak C-H•••N contacts with another arrangement of molecules are observed (Figure 3).
Symmetry codes

Antimicrobial activity
Compounds 1-13 were initially assessed for their potential antimicrobial activity against selected bacteria (both Gram-positive and Gram-negative), fungi, and multicellular nematodes.
Minimum inhibitory concentration (MIC) values have been determined against bacteria and fungi and have been compared to the reference antimicrobial agents (Table 2) [26,[32][33][34][35][36].Most of the compounds displayed significant antimicrobial activity against both Gram-positive and Gram-negative members of dangerous ESKAPE family [27].In the case of Gram-positive bacteria, the compounds have been examined against the reference strain (ATCC 25923) and the multidrug resistant (MDR) clinical isolate of S. aureus (HEMSA 5).It is noticeable that all compounds, except 11, displayed MIC values much lower than that of reference oxacillin against the MDR strain [26,32].In the case of the most active compound (1), an excellent antimicrobial action was observed against HEMSA 5 which falls into the range of oxacillin against the susceptible strain ATCC 25923 (Table 2).
Moreover, seven compounds (2, 3, 6, 8, 9, 12 and 13) demonstrated anti-staphylococcal activity with MIC values lower than 10 µg/ml.Interestingly, the series of compounds did not discriminate between referent and MDR S. aureus strains and displayed similar antibacterial potency against both or even slightly stronger against the MDR strain (12).This indicates the ability of the selenocyanates to overcome bacterial MDR, most likely by by-passing the components responsible for resistance, which is of great significance when searching for new therapeutic solutions in the face of a significant drop in antibiotic effectiveness today.As for Gram-negative bacteria, compound 1 demonstrated excellent antimicrobial activity, especially against pathogenic A. baumannii and P. aeruginosa.In these cases, compound 1 was more potent than the reference drugs, i.e. oxacillin and piperacillin [33,34].Interestingly, a significant decrease of antibacterial activity of 1 was observed in the case of non-pathogenic S. carnosus which highlights the selective targeting of this compound towards pathogenic bacteria.Compounds 2, 3, 6, 9, 12 and 13 also demonstrated a pronounced activity against A. baumannii (MIC< 20 µg/ml).
albicans and inhibited the growth at concentrations even below 20 µg/ml.The highest activity, almost in the range of clinically relevant antifungal drugs [35,36], has been observed for compounds 5, 7 and 11 with MIC < 10 µg/ml (Table 2).The apparent activity of these compounds against C. albicans suggests their possible application as potent antifungal agents.The aromatic selenocyanates, however, have not exhibited a significant activity against non-pathogenic S. cerevisiae.Here, just compound 3 inhibited the growth at the concentration below 20 µg/ml (i.e. at 6.59 µg/ml), yet its activity against this pathogenic fungus was distinctly lower than that of the benchmark fungicide itraconazole [35,36].

Nematicidal activity
In order to extend the scope of preliminary studies focused on unicellular organisms, such as bacteria and yeasts, another assay involving multicellular organisms of parasites has been employed.Thus, the whole series (1-13) has been evaluated for nematicidal potential against the agricultural nematode S. feltiae, which represents a simple and reliable multicellular model system (Figure 4).Compounds have been evaluated at the concentrations ranging from 3.75 µM to 30 µM and all of them caused a remarkable, concentration-dependent decrease in the viability, which was pronounced even at the lowest concentrations tested.As for the Gram-negative bacteria compound 1 stands out as it exhibited either similar or even higher nematicidal activity as compared to other compounds, with the exception of compounds 9 and 11.Although agricultural applications are more speculative at this time, these results indicate that the selenocyanates may also serve as excellent nematicidal agents, possibly also against pathogenic nematodes affecting animals and humans.

Influence of arylmethyl selenocyantes on mammalian cells
In order to investigate any selectivity for microorganisms and to rule out any major cytotoxicity against mammalian cells, compounds 1-12were investigated for their possible cytotoxicity against the normal NIH/3T3 mouse embryonic fibroblast cell line and two cancer cell lines of mouse T-lymphoma, i.e. the sensitive (PAR) and the multidrug resistant cell line (MDR) transfected with the human MDR1 (ABCB1) gene that codes for the ABC transporter.Doxorubicin was used as positive control (Table 3).Furthermore, the efflux pump inhibitory activity of selected compounds (1, 2, 4, 5, 7 and 11) was investigated in both cancer cell lines (PAR and MDR) using the rhodamine 123 accumulation assay, that allowed to evaluate rhodamine 123 (R123) retention by flow cytometry.

Cytotoxicity in cancerous and non-cancerous cell lines
According to the results (Table 3), none of the compounds exhibited any significant inhibitory activity against the non-cancerous NIH/3T3 mouse fibroblast cells.In both T-lymphoma cell lines, compounds 4, 5, 7 and 10 were considerably less cytotoxic when compared to the reference doxorubicin.Intriguingly, compounds 1-3, 6, 8, 9, 11 and 12 displayed significant cytotoxic effects on the parental and multidrug-resistant sublines of mouse T-lymphoma cells.Although the compounds investigated (1-12) did not exhibit any distinct selectivity, the substantial activity of compounds 1, 3 and 12 against MDR cells is worth noticing.Thus, aromatic selenocyanates 1-3, 6, 8, 9, 11 and 12 may not only be of interest in the context of antimicrobial action -as anticipated initially -but also as a starting point in further search for anticancer agents with MDR-reversing properties.

Cancer MDR efflux pump inhibitory properties
As some of the selenocyanates demonstrated cytotoxic activity against MDR subline of mouse T-lymphoma cells, the compounds were evaluated for their potential as the inhibitors of efflux pump.None of the compounds exhibited significant ABCB1 inhibitory activity as compared to the positive control (verapamil, 20 µM) on the cell line under investigation ( lower than that of the 11.None of the compounds, however, demonstrated any significant inhibitory activity (ranging between 5.52-11.75%)at lower concentration (2 µM) when compared to positive control (verapamil) and previously evaluated selenoesters and selenoanhydride [38].

ADMET studies in vitro
Since the series of selenocyanates under investigation demonstrated pronounced antimicrobial and nematicidal activities, it was important to investigate their "drug-likeness" profile.Four compounds (1, 2, 4 and 13) were selected for the in vitro assays on safety and absorption properties.

Mutagenicity
The Ames fluctuation experiments were performed in triplicate to provide a mutagenic index (MI), computed according to the method described previously (Table 5) [27].Binomial B-values were calculated according to the manufacturer's instructions (Xenometrix AG, Allschwil, Switzerland).
Results of the experiments indicate very clearly that neither the selenocyanates (1, 2, 4, 13) nor the reference ebselen displayed any mutagenic potential at a concentration of 1 µM.Compounds 1, 2 and 13 were also non-mutagenic at the higher concentration of 10 µM.Solely compound 4, at the higher concentration (10 µM), exhibited an increased Binomial B -value (B = 1.0), which may point towards a probable mutagenic potential.The mutagenicity of this derivative is rather ambiguous, as the value of the second parameter indicative of mutagenicity (MI = 1.75) was still below the threshold of 2.0, and substantially lower than the MI value for the mutagenic reference NQNO with a MI = 6.91 calculated at a concentration of 0.5 µM (Figure 5, Table 5).

In vitro PAMPA permeability
The PAMPA permeability screening test imitates the structural and biological conditions of the cell membrane and allows for a quick and simple determination of a given compound's passive transport through biological membranes, characterized by a permeability coefficient (Pe).A pre-coated PAMPA Plate System Gentest™ (Corning, Tewksbury, MA, USA) was employed, which guarantees good predictability and correlation of data obtained for human absorption in the Caco-2 cell line.The concentrations of the compounds tested in the donor and acceptor compartments were estimated by the capillary electrophoresis method (CE) as described previously [40][41][42].The permeability results obtained were compared with the data for the reference drugs, i.e. high permeable caffeine and low permeable norfloxacin (Table 5).All compounds (1, 2, 4 and 13) exhibited good permeability with Pe values above the threshold for highly permeable compounds (>1.5 × 10 −6 cm/s) [42].The results calculated for compound 13 may, however, be ambiguous due to the instability of the compound in phosphate buffered saline (pH 7.4), as around 50 % decomposition was determined by controlled LC/MS analysis (data not shown).Ebselen -reference compound in Ames test.Reference compounds in PAMPA: high-permeable drug, caffeine; low-permeable drug, norfloxacin.MI -mutagenic index (the quotient of the number of revertant colonies induced in a test sample and the number of revertants in a negative control).B -Binomial B value, nd -not determined; Pe -permeability coefficient.

SAR-discussion
Although a variety of the biological activities seemingly associated with compounds 1-13 is, undeniably, associated with the reactive selenocyanate motif, the size and substituents of the aromatic fragments seem to present significant factors that affect both, the direction as well as strength of the identified activities, and for this reason also selectivity.
The SAR analysis indicates that the unsubstituted benzyl was evidently the most favourable aromatic fragment (compound 1), responsible for the highest antibacterial properties against all ESKAPE pathogens involved in this study, with special accent on an excellent potency against the MDR S. aureus (HEMSA 5), placing compound 1 in the range of the best antimicrobial drugs.In comparison to the rest of compounds investigated (2-13), the benzylselenocyanate (1) also demonstrated remarkable antifungal as well as nematicidal properties.Compound 1 also exhibited the strongest cytotoxic action on both, parental and multidrug resistant T-lymphoma cells.
Untypically, the β-naphthyl derivative (12) was more active against the MDR strain (HEMSA 5) at the lower concentration as compared to the non-resistant reference strain (ATCC 25923).The comparable MICs of benzyl and naphthyl derivatives against both S. aureus strains, or even lower for the MDR one, indicate not only antibacterial but also distinct reversal-MDR mechanisms of action for these Reactive Selenium Species.Although the MDR mechanism of HEMSA 5 is rather complex and has not been thoroughly explored until now, a predominant contribution of protein efflux pumps is highly probable.The potent and most effective bacterial efflux pump inhibitors (EPIs), e.g.
peptidomimmetics or naphthylmethylpiperazine (NMP) [43] contain benzyl and/or naphthyl moieties in their chemical structures.This strongly supports the notion that the interesting antibacterial activities, observed for compounds 1, 12 and 13, were conditioned by the reactive SeCN-fragment on one hand, and by the unsubstituted benzyl or naphthyl moieties which are likely to inhibit the MDR efflux mechanisms, on the other.Interestingly, the 1-naphthyl derivative (13) also exhibited some toxicity against non-pathogenic S. carnosus, whilst the 3-methylbenzyl (3), was active against all bacteria and yeast at the concentration below 30 µM.
In the case of antifungal activity against C. albicans, the role of the substituent position at the benzene ring seems to play a crucial role.Thus, the three most active agents were para-substituted with fluorine (5), chlorine (7) and NO2 (11), respectively.Interestingly, the p-nitrobenzylselenocyanate (11) exhibited the most selective activity against C. albicans compared to the rest of the microbes tested, yet was not active against agricultural pest nematodes.Although the strongest nematicidal effect was observed for 3,4-dichlorobenzyl compound ( 9), the second most active compound was the 4-nitrobenzyl derivative (11), which was one of the three most active cytotoxic agents effective against both, parental and multidrug resistant mouse T-lymphoma cells.
Generally, elemental selenium as well as simple organic selenium compounds interact with the cellular thiolstat and modulate the redox state of cancer cells [44].Several simple organoselenium compounds, such as aromatic selenoesters and anhydrides exhibit excellent cytotoxicity [45] and Pgp-inhibitory activities in the corresponding assays [38,46].The combination of the selenocyanate functional group with arylmethyl moiety, however, does not seem to be effective for cytotoxic or MDR-reversing activity in cancer cells.For cytotoxicity SAR, the methyl (2 and 3) and the unsubstituted benzyl (1) and β-naphthyl (12) fragments exhibited the highest cytotoxic activity against T-lymphoma cell lines, with a slight discrimination between parental and MDR cells.The cancer MDR inhibitory activities were almost negligible for the selenocyanates tested, with an exception of the 4-nitrobenzyl selenocyante (11), which demonstrated moderate activity i.e. 2-fold lower than that of verapamil.
Similar selenocompounds (incuding benzoselenophene-diones and various selenoesters) have also been evaluated in murine lymphoma and human adenocarcinoma cell lines overexpressing the ABCB1 transporter [38].The cyclic selenoanhydride and the various selenoesters were several times more potent than verapamil, but the nature of their substituents at the aromatic ring seemed to influence the SAR properties to a lesser extent.In the case of the selenocyanates, the modification of the substituents in the para-position significantly influenced the efflux pump inhibitory properties.
The strong electron-withdrawing NO2 group has been observed to play a promoting role.The methylselenocyante substituent at an aromatic ring, presumably due to some bio-isosteric analogy with the carboxylic moiety, seems to be a main factor responsible for a considerable decrease in the ABCB1 modulatory properties of all aromatic selenocyanates (1-13) as compared to previously investigated selenoesters [38,46].

Conclusions
The comprehensive studies presented in the previous sections, have provided new insight into the chemistry and biological activity of small aromatic selenocyanates, which may be useful in the hunt for new antimicrobial and anticancer agents.An overview on the full spectrum of biological screening results allows to perceive a general trend for the arylmethyl selenocyanates (1-13) that comprises of the pronounced antimicrobial and antiparasitic properties with moderate cytotoxic and weak efflux pump inhibitory properties in cancer cells as well as a lower toxic activity against non-pathogenic organisms and cell lines.SAR studies indicate that benzylselenocyanate (1) can be selected as the best lead structure, distinctly the most active one during these studies.Indeed, compound 1 not only showed an excellent activity against multidrug resistant S. aureus, but also a significant action against Gram-negative pathogens, pathogenic yeasts, parasites and cancer T-lymphoma growth.Our initial ADMET screening in vitro has also confirmed the lack of mutagenic effects and a good permeability for compound 1.Noteworthy, antibacterial properties were also observed for naphthyl derivatives (12,13), and antifungal for 4-fluorobenzylselenocyanate (5).
Additionally, methylbenzyl (2 and 3) and β-naphthyl derivatives (12) have been identified as more attractive lead structures in the ongoing search for anticancer agents.
Since the arylmethyl selenocyanates have demonstrated considerable activity against both, pathogenic bacteria and a yeast, and were also rather potent against the problematic MDR microorganisms, future studies in the field of antimicrobial agents should consider these simple structures as part of innovative drug design.

Chemical synthesis
1 H NMR and 13 C NMR spectra were recorded on a Varian Mercury-VX 300 MHz PFG instrument in DMSO-d6 at ambient temperature using the solvent signal as an internal standard.
The values of the chemical shifts are expressed in δ values and the coupling constants (J) in Hz.Mass spectra were recorded on a UPLC-MS/MS system consisted of a Waters ACQUITY® UPLC® (Waters Corporation, Milford, MA, USA) coupled to a Waters TQD mass spectrometer (electrospray ionization mode ESI-tandem quadrupole).The UPLC/MS purity of all the final compounds was confirmed to be higher than 95%.Retention times (tR) are given in min.Thin-layer chromatography was performed on pre-coated Merck silica gel 60 F254 aluminium sheets.The reactions at a fixed temperature were carried out using a magnetic stirrer with a contact thermometer Heidolph MR 2001.

X-ray crystallography
Single crystals suitable for X-ray analysis were obtained from ethanol for 1 and butan-2-ol for 12 by slow evaporation of the solvent at room temperature.Intensity data of 1 was collected on the Bruker-Nonius Kappa CCD four circle diffractometer, whereas of 12 was collected on Oxford Diffraction SuperNova Diffractometer equipped with a Mo (0.71069 Å) Kα radiation source.Position of non-hydrogen atoms were determined by direct method using SIR-2014 program [50].Hydrogen atoms bonded to carbons atoms were included at idealized positions and were refined using a riding model.The aryl hydrogen atoms were constrained with C-H 0.93 Å, the methylene groups with C-H 0.97 Å and Uiso(H) = 1.2Ueq.The final refinements were performed by SHELXL program [51], ORTEP [52] and MERCURY [53] programs were employed for molecular graphics.Clinical and Laboratory Standard Institute (CLSI) recommendations [54].The compounds (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13) were evaluated for their antimicrobial activity against a broad spectrum of microorganisms, including Gram-positive bacteria (S. carnosus and S. aureus), Gram-negative bacteria (A.baumanni and P. aeruginosa) and yeasts (C.albicans and S. cerevisiae).The values of MIC were recorded after incubation of compounds for 20 h and 24 h with bacteria and yeast, respectively.The experiments were performed in triplicate and on three different occasions (i.e. a total of nine times).

Nematicidal activity
The compounds were also evaluated for their nematicidal activity against model nematode (Steinernema feltiae).S. feltiae was obtained from Sautter and Stepper GmbH (Ammerbuch, Germany).
The assay was performed according to the protocol mentioned in the literature [55,56].Results are represented as means ± SD.GraphPad Prism 5 was used to perform the statistical analysis.Statistical significances were calculated by employing GraphPad Prism 5 and calculations were performed using one-way ANOVA, with p < 0.05 considered to be statistically significant.The cell lines were incubated at 37˚C, in a 5% CO2, 95% air atmosphere.
The cell line was incubated at 37˚C, in a 5% CO2, 95% air atmosphere.

Assay for cytotoxic effect
Cytotoxicity assays were performed following the procedure described in the literature [58,59].
Parental and multidrug resistant mouse T-lymphoma cells and NIH/3T3 non-cancerous mouse embryonic fibroblast cell lines were used to determine the effect of the selenocyanates on the growth of cells.The effects of increasing concentrations of selenocyanates on cell growth were tested in 96-well flat-bottomed microtiter plates.The compounds were diluted in a volume of 100 µL of medium.
The adherent mouse embryonic fibroblast cells were cultured in 96-well flat-bottomed microtiter plates, using DMEM supplemented with 10% heat-inactivated fetal bovine serum.The culture plates were incubated at 37°C, in a 5% CO2, 95% air atmosphere.In a separate plate, the respective dilutions of the selenocyanates were prepared.The density of the cells was adjusted to 1x10 4 cells per well, the cells were seeded for 4 h at 37°C, 5% CO2, then the medium was removed from the plates containing the cells, and the dilutions previously made were added to the cells.
In case of the mouse T-lymphoma cells, the two-fold serial dilutions were prepared in 100 µL of Results have been expressed in terms of IC50, defined as the inhibitory dose that reduces the growth of the cells exposed to the tested compounds by 50%, each representing the mean of a minimum of three independent experiments.

Evaluation of rhodamine 123 (R123) retention by flow cytometry
The potency of the tested compounds as inhibitors of the ABCB1 efflux pump was determined using a fluorescence-based detection system, described previously in the literature [38,59].
Verapamil was applied as a reference inhibitor of the ABCB1 transporter.The parental (PAR) and

Figure 2 .
Figure 2. The molecular structure of (a) 1 and (b) 12, with the appropriate atomic-numbering scheme.Displacement ellipsoids are drawn at 30% probability level.

Figure 3 .
Figure 3. Partial packing views, indicating the intermolecular interactions in a layer of (a) selenocyanate 1 and (b) selenoyanate 12.The intermolecular interactions are depicted as dashed lines.
McCoy's 5A, horizontally.The parental (PAR) and multi-drug resistant (MDR) mouse T-lymphoma cells were adjusted to a density of 1×10 4 cells in 100 µL of McCoy's 5A medium and were added to each well, with the exception of the medium control wells.The culture plates were incubated at 37˚C for 24 h; at the end of the incubation period, 20 µL of MTT (thiazolyl blue tetrazolium bromide, Sigma) solution (from a stock solution of 5 mg/ml) were added to each well.After incubation at 37˚C for 4 h, 100 µL of sodium dodecyl sulfate (SDS) (Sigma) solution (10% in 0.01 M HCI) were added to each well and the plates were further incubated at 37˚C overnight.Cell growth was determined by measuring the optical density (OD) at 540/630 nm with Multiscan EX ELISA reader (Thermo Labsystems, Cheshire, WA, USA).Inhibition of the cell growth was determined according to the formula below: multidrug resistant (MDR) mouse T-lymphoma cells were adjusted to a density of 2×10 6 /ml, re-suspended in serum-free McCoy's 5A medium and distributed in 0.5 ml aliquots into Eppendorf centrifuge tubes.The tested compounds (1 and 10 µl, from a stock solution of 1mM) were added at different concentrations (2 µM and 20 µM final concentrations, respectively), and the samples were incubated for 10 min at room temperature.Subsequently, 10 µl (with a final concentration of 5.2 µM) of rhodamine 123 was added to the samples and the cells were incubated for 20 minutes at 37˚C, washed twice and re-suspended in 0.5 ml phosphate buffered saline (PBS) for analysis.The fluorescence intensity of the cell population was measured with a Partec CyFlow flow cytometer (Partec, Munster, Germany).Verapamil was used as a positive control at 20 µM final concentration in the rhodamine 123 exclusion experiments.The mean fluorescence intensity (%) was calculated for the treated MDR and PAR mouse T-lymphoma cells as compared to the untreated cells.The fluorescence activity ratio (FAR) was calculated based on the following equation which relates the measured fluorescence values: Geben Sie hier eine Formel ein.4.6.Mutagenicity assay 4.6.1.Reagents for microplate fluctuation Ames test 4-nitroquinoline-N-oxide (NQNO), DMSO, ebselen, bromocresol purple, NADP + , glucose-6-phosphate sodium salt and glucose-6-phosphate dehydrogenase were purchased from Preprints (www.preprints.org)| NOT PEER-REVIEWED | Posted: 22 February 2018 doi:10.20944/preprints201802.0142.v1

Table 1 .
Parameters of intermolecular C-H•••N interactions for 1 and 12

Table 3 .
Cytotoxic effects of arylmethyl selenocyanates on both, non-cancerous and cancer cells.

Table 4 .
Effects of selenocyanates on rhodamine 123 accumulation by multidrug resistant (MDR)mouse T-lymphoma cells.
VER, verapamil; FAR, fluorescence activity ratio; FAR quotient: FAR of a compound related to FAR of VER tested at 20 µM.