Highly bioactive novel aryl-, benzyl-, and piperazine-selenoureas: synthesis, structural characterization and in vitro biological evaluation

Selenoureas are widespread as useful elements for constructing important species and biologically active molecules. Finding an efficient and straightforward method to prepare this motif and biologically screen derivatives thereof is crucial. Herein, we demonstrate the effectiveness of using ethanol as a solvent in the preparation of various substituted aryl-, benzyl-, and piperazine-selenoureas from isoselenocyanates and amines. The synthetic method includes mild reaction conditions, large substrate scope, and good isolated yields. Biological evaluation of the prepared products on MDA-MB-231 and MCF-7 cancer cell lines revealed several remarkably active compounds (IC50 < 10 μΜ) with the best one exhibiting IC50 values of 1.8 μΜ and 1.2 μΜ observed against the challenging former triple-negative breast cancer cell line and the latter one, respectively. The chemical structures of all new compounds were fully characterized by multinuclear nuclear magnetic resonance (NMR) spectroscopy and high accuracy mass measurements.


Introduction
Selenoureas are organoselenium compounds which contain two amine (-NH) groups flanking the sides of a selenocarbonyl (C¼Se) function. In recent years, the synthesis, and applications of selenourea derivatives have received growing attention and have been the subject of several reviews [1,2]. Such species are close structural analogs of thiourea and urea compounds, although the presence of larger and more polarizable selenium atom introduces significant changes to their chemical and biological properties [2,3]. As such, they have been widely used as key precursors to make biologically important heterocycles [4,5,6,7] and have been utilized in many applications in the fields of organic and analytical chemistry, agriculture, and medicine.
In analytical chemistry, selenoureas have proven useful receptors for the binding, recognition, and transport of various anions [13] and can generate mono-and bi-coordinated adducts [14]. Recently, molecular logic gate founded on selenoureas/anions host-guest interaction using substrate 13 was reported to perform a ternary (conditional) logic operation employing proton NMR to measure response output [15]. Another innovative application exploiting the binding properties of selenoureas involves utilizing them as suitable phasing agents for macromolecular X-ray crystal structures in parallel to the traditional practice of heavy-atom derivatization. In this strategy, selenourea (SeC(NH 2 ) 2 ) (5) is introduced into protein crystals and binds to reactive moieties at the surface of macromolecules via H-bonding, where the amide groups may act as donors and selenium atom as acceptor [16]. Bis-selenoureas in the form of bis(imidazoline selone) (9) have been employed as ligands for iridium photocatalyst complexes to promote oxidative coupling reaction of benzylamines to imines with visible light [17]. Such bidentate organochalcogens have also found success as ligands for iridium(III) and rhodium(III) complexes for application as catalysts for norbornene polymerization [18]. Other cyclic selenoureas like N 0 -dimethylimidazole selone have been used as starting materials to zinc selenides [19] and copper complexes of biological importance [20,21,22]. The related NHC-derived cyclic selenourea 7 and its related derivatives, beneficial for the quantification of NHC electronic properties, have also been explored in the coordination to gold(I) [23]. N,N, N 0 -trisubstituted selenourea 10 and other derivatives serve as precursors to lead selenide (PbSe) nanocrystals, useful for optoelectronics and photovoltaic devices [24].
Biologically, selenourea derivatives containing diselenide linkage 14 were found cytotoxic against Leishmania infantum axenic intracellular amastigotes and demonstrated improved antileishmanial activity in infected macrophages over the reference drug miltefosine (EC50 < 2.84 μM). N-Aliphatic substituents such as hexyl in selenourea was necessary for Figure 1. Examples of prevalence of the selenourea motif in several products and its usefulness in chemical and bioactive agents. optimum leishmanicidal activity [25]. In biological systems, hydroxyl radical produced from the reaction of H 2 O 2 with Cu þ damages DNA oxidatively, causing cancer, ageing, and other diseases. Selenium-containing compounds like N, N 0 -dimethylimidazole selone can act as antioxidants by coordinating to copper as one justification for their observed antioxidant activity [19]. The related pyrrolidine-1carboselenoamide (11) exhibits superoxide scavenging activity (SOSA) and scavenges superoxide radical species from 4-β-phorbol-12-myristate-13-actate (PMA) (71.1% inhibition) [26]. Generally, selenoureas were several folds more active than their corresponding thioureas. The existence of selenoureas as zwitterions in biological systems is a crucial property that reverses their interactions with biomolecules, restoring their original redox state. This distinctive advantage enhances their redox efficiency in comparison to sulfur analogs. The sulfur analogues of cyclic selenourea 12 are in use as drugs for the therapy of hyperthyroidism, rendering the latter as potential antithyroid agent as it significantly reduces the tyrosine nitration of both cytochrome c and BSA [27]. Presently, various forms of selenium species are being utilized as a preventative agent for cancer. Recently, some of the ferrocene incorporated selenoureas 8 [28,29] were reported to exhibit electrostatic DNA binding capabilities through the positively charged Fe(II) with the negatively charged phosphate backbone of DNA, confirming their efficiency against cancer initiation, propagation, and termination. In some cases, derivatives of 8 interacted with DNA through partial intercalation, indicating that 8 can be useful as antitumor agent. Studies exploring the antibacterial potential of selenoureas have been scarce. In a recent study, selenoureadihydropyrrol-2-one derivatives showed promising antibacterial activity, notably exceeding the activity of the sulfur series of analogs, highlighting the importance of selenoureas as antibacterial agents [30].
Cancer is one of the principal reasons of human death [31] where seventy percent of cancer-related mortality happens in under-developed and developing nations [31,32,33]. There is an increasing trend of deaths resulting from various types of cancers worldwide, with a projected twelve million deaths in the year 2030 according to WHO [34]. Despite the progress in cancer therapy, the success of cancer treatment is a significant challenge due to the varied responses of tumor cells and toxicity associated with existing chemotherapeutic drugs. Henceforth, developing new chemotherapeutic agents for cancer therapy continues to represent an inspiring endeavor for chemists.
As of the end of 2020, the number of women who were diagnosed with breast cancer (BC) in the previous five years had reached 7.8 million, rendering it the world's most frequently diagnosed type of cancer [35].
Despite showing similar pathological characteristics, patients react differently to treatments and show considerably different outcomes, highlighting the variety of BC subtypes. Thus, BC is classified into molecular subtypes according to their gene expression profile. Indeed, proteomic, genomic, transcriptional, and epigenetic results have revealed that BC cells may differ in progression, pathway activity [36], proliferative potential, apoptosis, vascularization, and ability to metastasize and invade tissues [37,38]. Consequently, this provided us the impetus to contribute to cancer research to address some of the above shortcomings. Thus, the intention of this investigation was to assess the cytotoxic effect of selenoureas 21-37 against MDA-MB-231 ER(À)/PR(À)/HER2(À) and MCF-7 ER(þ)/PR(þ)/HER2(À) breast cancer cell lines as models. These were selected because they show key differences regarding the presence of receptors for estrogen (ER), progesterone (PR), and epidermal growth factor (HER2) [39]. Additionally, these cancer cell lines vary in the level of malignancy [40]. Tumors that are negative for hormone receptors are more aggressive, causing limited treatment options [41] than positive ones [40].

Chemical synthesis of 21-35
Herein, we report the efficient synthesis and characterization of a new set of novel aryl, benzyl, and piperazine selenoureas 21-35, and demonstrate their in vitro biological activities. Although several methods in the literature describe the synthesis of selenoureas [42,43,44], the best-known protocol remains the reaction of isoselenocyanates with primary and secondary amines. The critical step in the synthetic process is the preparation of isoselenocyanates, which, unlike their isothiocyanates equivalents, are commercially unavailable. There is several synthetic strategies that have been reported in the literature towards their preparation [45,46,47]. Among these, using elemental black selenium to oxidize isocyanides appears the most reliable synthetic method [48,49,50,51]. In the current work, the first coupling partner for the preparation of the desired selenoureas, 4-methylphenyl isoselenocyanate 17a and its 4-chloro analogue 17b [25], were prepared in 55 and 85 % yields, respectively, from toluidine and 4-chloroaniline (Scheme 1). These isoselenocyanates were prepared in one pot according to the published procedure by Zakrzewski [40] which, incidentally, is one of the most useful and good yielding protocols. The preparation of 17a,b initially involves in situ preparation of isocyanides 16a,b by a Hofmann isonitrile synthesis utilizing anilines 15a,b as reactants together in CHCl 3 and 50% aqueous NaOH solution and the ammonium salt Aliquat 336 as phase transfer reagent. Both isoselenocyanates 17a and 17b were fully characterized by mp, Infrared, nuclear magnetic resonance, and high accuracy mass measurement techniques and the acquired data is consistent with what has been reported in the literature [25].
At the outset of our current work, we were aiming to prepare selenoureas in high purity without the need of resorting to purification by column chromatography since they could not be separated and purified by this method due to their instability and similar R f values to the corresponding requisite amines. Pleasingly, the choice of chemical solvent and duration of reaction was key to obtaining pure products. Thus, using the less electrophilic isoselenocyanate 17a as a model substrate and 2nitroaniline as the least nucleophilic reactant, we explored several reaction conditions to determine the optimal conditions for best yields and purity of the target products. Initially, the reaction between 17a and the anilines was stirred overnight at ambient temperature using a series of solvents including DMF, DMSO, and acetonitrile. Equimolar amount of triethylamine was added to foster mild basic conditions which are more suitable for the formation of the acid-sensitive selenourea. Unfortunately, the desired product was not observed as the ortho nitro group was detrimental to the nucleophilicity and reactivity of the aniline due to its very strong negative mesomeric effect. As an alternative aniline derivative, the more electron-rich p-toluidine was used instead, and the reaction was re-run under the same conditions outlined earlier. Progress of chemical reaction was followed carefully by 1H NMR spectroscopy. While extensive decomposition of the selenourea product was observed in DMSO and DMF solvents overnight as indicated by the observed copious quantities of black selenium deposited in the reaction vessel, acetonitrile proved a more suitable solvent, although affording only 36% yield of selenourea 21. However, with ethanol as solvent and shorter reaction time to circumvent decomposition of the selenourea product, 1,3-di-p-tolylselenourea (21) crashed out of the reaction and was successfully isolated by filtration using Whatman filter paper in 76% yield.
Having established the optimized reaction conditions (1:1:1 equimolar amounts of the aniline, isoselenocyanate, and TEA, rt, 2h), the applicability of this synthetic route was then verified using 4-methylphenyl isoselenocyanate (17a) and 4-chlorophenyl isoselenocyanate (17b) on a variety of substituted aniline, benzyl and piperazine derivatives. Aniline and derivatives 18 carrying substituents able of exhibiting positive and negative mesomeric (þM, ÀM) and inductive (þI and -I) effects furnished the desired selenoureas 21-37 in 59-94% yield. While 2-nitroaniline was completely unreactive because of the strong negative mesomeric (ÀM) effect of the nitro group, the corresponding aniline analogs with negative inductive effect substituents (ÀI) like 4-Cl, produced the desired selenoureas 22 and 30, although in low yields, in 63% and 59% yields, respectively. Interestingly, 22 could be prepared in 82% yield from selenourea 17b, highlighting the crucial impact of the aromatic substituents on the reactivity of the aniline derivative and formation of selenourea product. Thus, aniline analogs with positive inductive effect (þI), positive mesomeric (þM) effect, and no substituents are expected to be more suitable reactants. Indeed, the yields of the resulting selenoureas were generally higher and reactions were faster with p-toluidine (Table 1, compound 21, 76%), aniline ( Table 1, compound 23, 81%; compound 32, 77%), and p-anisidine (Table 1, compound 31, 80%). As expected, the reactivity of the benzylamine substrates was not impacted by the nature of aromatic substituent and these species were more reactive nucleophiles, as anticipated, than the aniline analogs. For instance, having strongly electron withdrawing substituents such as 4-F (ÀI) afforded the product 27 in 97% yield, meanwhile using an amine with a very strong electron donating group like 4-OMe (þM) afforded the selenourea 35 in a relatively lower 78% yield (Table 1). In many cases, higher yields of products ranging from 63 to 97% were obtained with benzylamines. Finally, the substituted piperazine, which presents a more sterically challenging scenario, afforded the selenourea products in high yields (Table 1, compound 36, 94%; compound 37, 88%). The above synthetic approach could be executed on gram scale as proven by the synthesis of selenoureas 22 and 24 on a larger quantities from p-touidine and benzylamine (10 mmol scale) to afford the products in 79% and 75% yields, respectively.
The resulting light peach/off-white/brown/grey products could be separated cleanly as solids without the need for purification by chromatography and are reasonably stable. All products were amenable to storage at ambient temperature for months. High-resolution mass spectrometry (HRMS) and NMR measurements validated the proposed chemical structures of selenoureas 21-37 shown in Table 1.
The chemical structures of all new selenoureas (21-36) ( Table 1) and formation of the key R-N-C¼Se(NH)-Ar bonds were confirmed and initially verified by routine spectroscopic and analytical techniques including melting point, IR, 1-Dimension NMR, and high resolution mass spectrometry. In addition, 2-dimension homonuclear and heteronuclear NMR spectroscopic measurements ( 1 H-1 H-gDQFCOSY, 1 H-13 C-gHSQC, 1 H-13 C-gHMBC, and 1 H-1 H-ROESYAD) were performed on one member of each group of derivatives (aryl, benzyl and piperazine) as representative examples to trace the 1 H-1 H and 1 H-13 C connectivity and identify the chemical shifts of each carbon and proton (see supporting information section S9-S84). The physical and IR/NMR spectral data of known selenoureas agreed with those stated in the literature (see experimental and supporting information sections). The most distinguishing and informative IR signals of the selenoureas are those of the N-H and C¼Se groups which appear around 3100-3400 cm À1 and 1500-1600 in the IR region, respectively.
Next, using N-(4-chlorophenyl)-4-(pyrimidin-2-yl)piperazine-1-carboselenoamide (37) as a representative model example for NMR analysis and studies, the relevant 1-D and 2-D NMR spectra that were employed for structural elucidation and chemical shift assignments are shown in Figure 2. The 13 C-CRAPT NMR spectrum ( Figure 2, spectrum b) clearly confirms the occurrence of the expected 10 signals (4 aromatic CH's, 3 aromatic quaternary carbons, 1 C¼Se carbon, and 2 piperazine methylenes which is agreeable with 5 carbon atoms being magnetically equivalent. The most prominent aspect of the 13  Having totally matched the chemical shift values to the protons of the three independent spin systems of the pyrimidine, piperazine, and chlorophenyl, the respective 13 C chemical shifts were traced and paired with the proton chemical shifts through the 1 H-13 C-gHSQC spectrum ( Figure 2d and Table 2). Finally, the quaternary carbons were identified through the 1 H-13 C-gHMBC spectrum (Figure 2e). While the most deshielded C¼Se carbon (δ 181.2 ppm) was identified through longrange HMBC correlation with the C 2 0 -H protons (δ 4.10/181.2 ppm), the pyrimidine C 4 0 chemical shift was established by a strong cross peak with the C 3 0 -H protons (δ 3.87/161.2 ppm). Interestingly, the latter correlation proves the creation of the new selenourea N-C¼Se quaternary center and is an indication of a successful coupling between the piperazine and isoselenocyanatestarting material. The remaining chlorophenyl quaternary carbons C 1 and C 4 were identified through strong correlation contours with C 3 H ( 3 J, δ 7.36/141 ppm) and C 2 H ( 3 J, δ 7.33/ 129.5 ppm), respectively. The chemical shifts of the assigned 1 H and 13 C NMR of selenourea 37 have been summarized in Table 2 along with the structure showing the most relevant NOE and HMBC correlations.

Biological evaluation
The MTT assay was employed to assess the cytotoxic potentials of 21-37 on MDA-MB-23v1 ER(À)/PR(À)/HER2(À) and MCF-7 ER(þ)/PR(þ)/HER2(À) cells. The selenoureas were tested at concentrations ranging from 0.006 to 123.4 μg/mL to examine the impact of different concentrations on cancer cell survival. The correlation between surviving fraction and concentration of each compound was plotted to determine the survival curve of each cancer cell line against each selenourea. Generally, both cell lines were very responsive to increase in drug dose between 10 and 40 μg/mL as shown by the subsequent reduction in cell surviving fraction, while higher dose-response was less evident between 40 and 125 μg/mL (Figures 3a-f). Notably, the cell lines showed variation in cytotoxic responses, as would be anticipated. The observed variation in cytotoxic responses suggest different cytotoxicity on tumor cell lines, which could be due to differential expression of receptors in these cells, involving numerous intracellular signaling paths [52]. Because of the overlapping nature of the dose-response curves in Figures 3a-f and the high number of data points collected, we elected to utilize the half-maximal inhibitory concentration (IC 50 ) values extracted from the plotted dose-response curves and converted to μM since this comprises a more convenient method to compare the cytotoxic activities of 21-37 as antitumor agents (see Table 3). Table 3, all synthesized selenoureas showed mild to strong cytotoxic activity with IC 50 Table 3) tested in this study. Triple-negative breast cancer constitutes about 15% of all BC incidents and exemplifies a therapeutic challenge because of its poor prognosis and unavailability of standard treatment [53]. Likewise, the efficacy of these species in the MDA-MB-231 cell line emphasizes their capacity to conquer shortcomings in treating triple-negative breast cancer.

As indicated in
The sharp variation in cytotoxic activity among selenoureas in the same series is a tribute to the impact wielded by manipulating the N- (1) H and N-(3)-H is not a requirement for high activity, it seems that both may be needed for optimum dual potency. Selenium-based molecules have shown efficacy as chemotherapeutic compounds [54]. Noteworthy, different Se compounds have been reported for their anti-cancer effects based on different mechanisms of action. Selenium compounds may trigger cell death through apoptosis, and non-apoptotic events such as cell cycle arrest [55,56], oxidative stress [57], necrosis [58], autophagy [56], ferroptosis [59], necroptosis [60], entosis [61] and anoikis [62].
The antitumor, antiviral, antibacterial and antifungal properties of imidazolidineiminothiones and their derivatives have been systematically established over the past recent years [64,65,66,67,68,69,70]. While the 1-position contains a carbonyl (C¼O) group flanked by two nitrogen atoms (N 1 and N 3 ), the heterocyclic ring contains adjoining imino and thione functional groups in the 4 and 5 locations, respectively. This type of arrangement serves as a reactive template for subsequent cyclizations in which one or both exocyclic nitrogen and oxygen can be integrated into further fused heterocyclic rings. Imidazolidineiminothiones and the many heterocycles based on them have been demonstrated to exhibit broad scale of pharmacological impacts against tumor cells, and viral, microbial, and fungal strains. In this context, we were further interested in transforming the selenoureas, as part of planned future extension to this work, to N1-, N3-disubstituted selenoxo-imidazolidine-4,5-diones. There are no known or reported general literature methods to prepare such species [71], which will not only serve as potentially bioactive Se analogs to imidazolidineiminothiones and their derivatives but will be crucial in finding the impact of masking the N-(1)-H and N-(3)-H of selenoureas and determining their role in obtaining optimum potency. Thus, after solvent, temperature, base and reagent optimization, we found that treatment of selenourea 22 with oxalyl chloride (2 equivalents) in DCM with triethylamine as base (2 equivalents) were the best conditions to furnish the desired selenoxo-imidazolidine-4,5-dione 38 in 77% yield (Scheme 2). In continuation to the work reported herein, the newly developed methodology will be employed to prepare a series of selenoxo-imidazolidine-4, 5-dione derivatives for biological testing and comparative analysis.

Reagents
All chemical transformations were performed with magnetic stirring in oven-dried glassware. All chemical reagents and reaction solvents were used as received from MilliporeSigma. Analytical thin-layer chromatography (TLC) used to track the progress of reactions or test purity of isolated products was performed on precoated silica gel plates (HSGF 254) and visualized under UV irradiation at λ ¼ 254 nm.

1
H and 13 C NMR spectra were recorded in DMSO-d 6 on a Varian 400 MHz NMR spectrometer. The NMR chemical shifts (δ) are reported in parts per million (ppm) relative to the residual solvent peak ( 1 H-NMR δ 2.50 for DMSO-d 6 ; δ 39.52 for DMSO-d 6 ). The following abbreviations were used to explain NMR peak multiplicities: br s ¼ broad signal, s ¼ singlet, d ¼ doublet, t ¼ triplet, and m ¼ multiplet. IR spectra were recorded using a Thermo Nicolet Nexus 470 FT-IR. High-resolution mass analyses (HRMS) were obtained using a Waters Q-TOF Premier mass spectrometer [electrospray ionization (ESI)]. Melting points were 3.2.1.14-Methylphenyl isoselenocyanate (17a). A mixture of the amine (0.015 mol), chloroform (2.4 g, 1.62 mL, 0.02 mol), dichloromethane (10 mL), Aliquat 336 (0.2 g, 0.0005 mol), and NaOH (6.84 g, 4.5 mL, 0.086 mol) was vigorously stirred and heated at 45 C for 12 h. The reaction was cooled to room temperature, chloroform (10 mL) and water (mL) were added, and the mixture was further heated at 45 C for another 6 h. Progress of the reaction and consumption of the amine was monitored by 1 HNMR. The mixture was cooled to room temperature, and finely divided black elemental selenium powder (1.5 g, 0.019 mol) was added. The heterogeneous mixture was vigorously stirred at 45 C for 1 h. The isoselenocyanate formation was monitored by 1 HNMR. The reaction mixture was cooled to room temperature, then water (15 mL) and dichloromethane (15 mL) were added, and the remaining unreacted selenium was filtered off under reduced pressure using a sintered glass Büchner funnel (250 mL capacity). The organic layer was separated and dried using anhydrous MgSO 4 . The drying agent was then removed by filtration and the solvent was evaporated under reduced pressure in a well-ventilated fume hood due to a strong stench. The residue was purified by column chromatography on silica gel using a gradient of petroleum ether and ethyl acetate as the mobile phase (gradient elution starting with 100% pet. Ether, followed by 1-3% EtOAc-pet.ether) to afford 15 g (50%) of the isoselenocyanate 4 as a light pink solid; R f ¼ 0. 33
The reaction mixture was stirred in dark under nitrogen conditions at room temperature for maximum 2 h. The formed precipitate was filtered using Whatman filter paper, washed with minimum amount of ethanol and hexane, and air-dried to afford the desired selenourea product.
3.2.7.2. 7Cytotoxicity assay. The cells viability was investigated by MTT assay [63]. Cells were trypsinized (0.25% trypsin) and aliquots of 120 μL of the suspended cells (5 Â 10 4 cells/mL) were added to 96-well plates to yield 6000 cells/well. After 24h, 60 μL of serially diluted compounds in DMSO were added to the prepared cells. After 48 h, 10 μL of a 5% MTT solution in PBS was added for 2 h. The medium was aspirated, and the formazan crystals were dissolved in 200 μL of DMSO for 10 min on a shaker. The optical density was read in a microplate reader (ChroMate, USA) at 570 nm.