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European Journal of Medicinal Chemistry 122 (2016) 55e71

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European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Research paper

Combinatorial synthesis, in silico, molecular and biochemical studiesof tetrazole-derived organic selenides with increased selectivityagainst hepatocellular carcinoma

Saad Shaaban a, e, *, Amr Negm b, Abeer M. Ashmawy c, Dalia M. Ahmed d,Ludger A. Wessjohann e, **

a Organic Chemistry Division, Department of Chemistry, Faculty of Science, Mansoura University, El-Gomhorya Street, 35516 Mansoura, Egyptb Biochemistry Division, Department of Chemistry, Faculty of Science, Mansoura University, El-Gomhorya Street, 35516 Mansoura, Egyptc Cancer Biology Department, National Cancer Institute, Cairo University, Egyptd Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Egypte Leibniz Institute of Plant Biochemistry, Department of Bioorganic Chemistry, Weinberg 3, D-06120 Halle (Saale), Germany

a r t i c l e i n f o

Article history:Received 2 March 2016Received in revised form8 May 2016Accepted 4 June 2016Available online 6 June 2016

Keywords:OrganoseleniumDiselenidesQuinoneTetrazoleAzido-Ugi reactionRedox modulatorsCaspase-8ApoptosisKi-67Bcl-2

Abbreviations: DOS, diversity-oriented synthesishepatocellular carcinoma; MCF-7, breast adenocarcintution; GPx, glutathione peroxidase; ROS, reactive onitrogen species; IMCRs, isocyanide-based multicomdiphenyl-1-picrylhydrazyl.* Corresponding author. Organic Chemistry Divisio

Faculty of Science, Mansoura University, El-GomhoEgypt.** Corresponding author.

E-mail addresses: dr_saad_chem@mans.edu.eg (S.halle.de (L.A. Wessjohann).

http://dx.doi.org/10.1016/j.ejmech.2016.06.0050223-5234/© 2016 Elsevier Masson SAS. All rights re

a b s t r a c t

Novel tetrazole-based diselenides and selenoquinones were synthesized via azido-Ugi and sequentialnucleophilic substitution (SN) strategy. Molecular docking study into mammalian TrxR1 was used topredict the anticancer potential of the newly synthesized compounds. The cytotoxic activity of thecompounds was evaluated using hepatocellular carcinoma (HepG2) and breast adenocarcinoma (MCF-7)cancer cells and compared with their cytotoxicity in normal fibroblast (WI-38) cells. The correspondingredox properties of the synthesized compounds were assessed employing 2,2-diphenyl-1-picrylhydrazyl(DPPH), glutathione peroxidase (GPx)-like activity and bleomycin dependent DNA damage. In general,diselenides showed preferential cytotoxicity to HepG2 compared to MCF-7 cells. These compoundsexhibited also good GPx catalytic activity compared to ebselen (up to 5 fold). Selenoquinones 18, 21, 22and 23 were selected to monitor the expression levels of caspase-8, Bcl-2 and Ki-67 molecular bio-markers. Interestingly, these compounds downregulated the Bcl-2 and Ki-67 expression levels andactivated the expression of caspase-8 in HepG2 cells compared to untreated cells. These results indicatethat some of the newly synthesized compounds possess anti-HepG2 activity.

© 2016 Elsevier Masson SAS. All rights reserved.

1. Introduction

Diversity-oriented synthesis (DOS) has emerged as a powerfultool in pharmaceutical industry and now it is routinely used, for

; OS, oxidative stress; HCC,oma; SN, nucleophilic substi-xygen species; RNS, reactiveponent reactions; DPPH, 2,2-

n, Department of Chemistry,rya Street, 35516 Mansoura,

Shaaban), wessjohann@ipb-

served.

drug discovery, but mostly for improvement of desired propertiesusing focused libraries [1]. Lead candidates in this context can beidentified and optimized via screening of skeletally diverse smallmolecules for specific biological target(s) [2]. Recent years havewitnessed the developments of new DOS pathways; however,multicomponent reactions (MCRs) are among the extensivelyinvestigated synthetic strategy for the efficient generation of smallchemical entities with increased molecular diversity andcomplexity. Fortunately, isocyanide-based multicomponent re-actions (IMCRs), the most common subclass of MCR, offer astraightforward way to introduce structural diversity and molecu-lar complexity in a single step [3]. Amongst all known IMCRs,Passerini and Ugi reactions have emerged as a robust mean in theconstruction of peptide-like compounds by giving access to dep-sipeptides [4,5], N-methyl peptides [6], and peptoids [7e9],including dimeric ones.

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Despite the potential of the classical, basic form of Passerini andUgi products, they are limited by their nature; that is, ester and/amide based structures. These compounds are therefore; prone tometabolism, i.e. in vivo they might be cleaved or modified topotentially more or less active or desirable metabolites. The po-tential of Passerini and Ugi reaction was accordingly extended toattain more constrained scaffolds i.e. heterocyclic moieties. This iswhere Groebke and azido-Ugi reactions come to play [10]. Thesereactions give access to bioactive imidazole- and tetrazole-based,constrained motifs [11,12].

Another alternative approach to attain further diversity andcomplexity is to tag the Ugi reaction with subsequent post-condensation modifications, often via attaching orthogonal func-tional group(s) at the backbone of the initial Ugi product [12e14].The incorporation of bifunctional building block allows subsequentsecondary transformations (e.g., nucleophilic substitution (SN) re-actions, cyclocondensation and cycladdition) and attains the sec-ond level of diversity [15]. In view of the former, sequentialcoupling of Ugi reaction to other reactions have been reported withdifferent post-modification reactions such as Heck, Diels-Alder,PicteteSpengler, Petasis, Mannich, Wittig and Click reactions forthe efficient synthesis of a number of therapeutically importantheterocyclic scaffolds with dense structural features and func-tionalities [12,15e20]. This double-layered approach has gainedvast importance, and now it is routinely used for drug discovery.

Recently, we employed a multicomponent strategy (e.g., Pass-erini, Ugi and Ugi/SN, Michael reactions) for the synthesis of hybridstructures containing diverse organoselenium libraries coupled tobioactive pharmacophores (e.g., quinones, naphthalene, cyclic im-ides) or pharmacologically relevant heterocycles (e.g., thiazolidi-none, pyrazole and thiazolopyrimidine) [21e31]. Some of thesecompounds exhibit cytotoxicity at sub-micromolar concentrationsagainst various types of cancer cells, such as hepatocellular carci-noma (HepG2), breast adenocarcinoma (MCF-7), A-498 (humankidney carcinoma) and A-431 (human epidermoid carcinoma) celllines. It is worthwhile to mention that the toxicity was more pro-nounced in case of HepG2 and MCF-7 cells. Furthermore, some ofthese compounds show lower cytotoxicity when tested againstnormal cells such as HUVEC (human umbilical vein endothelial),WI-38 (human lung fibroblast) and HF (primary human fibroblast)cell lines. The underlying cytotoxicity and selectivity mechanismsare quite interesting since these compounds can either function asantioxidant or pro-oxidants relying on their redox properties andthe intracellular redox environment in which they are placed[21,25,27].

In normal cells, organoselenium compounds act as antioxidantsand thus protect cells form oxidative damage. On the other hand,these compounds become pro-oxidants in oxidatively stressed cells(e.g., MCF-7 and HepG2) [30,32,33]. This bimodal function proposesorganoselenium compounds not only as chemopreventive agents,but also as selective chemotherapeutics [34e36].

Although one can only speculate about the organoseleniumpossible mode(s) of action, their cytotoxicity has been attributedprimarily to caspase 3/7 activation and subsequent induction ofapoptosis. Additionally, different phenotypical changes wereobserved and these included endoplasmic reticulum, actin cyto-skeleton and cellular morphology alterations as well as cell cyclearrest and various biochemical changes (e.g., ROS and GSH levels)[21,22,26,27,29]. Interestingly, we found that selenium based qui-nones were among the most active compounds exhibitingincreased anticancer activity [22,29,31].

In continuation of our program directed towards the develop-ment of therapeutically promising organoselenium agents, weherein report the development of a facile route towards symmet-rical diselenide and selenoquinone based-tetrazoles via azido-Ugi

and azido-Ugi/SN methodology. The respective mode-of-action(s)of the newly synthesized compounds are assessed in twofold: a)addressing their corresponding cytotoxicity in cell assays usingHepG2, MCF-7 and normal cells (WI-38) as well as estimating theircorresponding effect on the expression levels of caspase-8, Bcl-2and Ki-67 molecular biomarkers; b) exploration of the redoxmodulation activities of the synthesized compounds employingDPPH, GPx-like activity and bleomycin dependent DNA damageassays. Furthermore, in silicomolecular modeling studies, includingfield alignment and docking studies, will be applied as a pre-liminary prediction tool to estimate the antioxidant and cytotoxicproperties of the compounds.

2. Results and discussions

2.1. Design and synthesis

Until recently, the synthesis of organic selenides was not an easytask and included the use of expensive/toxic starting materials.Recent years have witnessed significant progress in the synthesis ofdifferent classes of organoselenium compounds such as selenahe-terocyclic, selenocyanates, selenides and diselenides [25,37e42].

As a part of our project aimed towards the development oforganoselenium-based chemotherapeutic agents, we herein reportthe synthesis of tetrazole-based symmetrical diselenide and sele-noquinone compounds (6e22) synthesized via azido-Ugi andsequential SN strategy (Fig. 1).

Based on recent findings reported by our group, we envisionedthat tetrazole-based organoselenium scaffolds thus might be moreefficient anticancer agents. Tetrazoles have received considerableattention from the pharmaceutical market as they constitute thecore scaffold of several bioactive compounds and many marketeddrugs (e.g., pentylenetetrazol, cilostazol, ceftezole, irbesartan, los-artan) [43]. Furthermore, these compounds are of particular inter-est because tetrazole is a bioisostere for carboxylic acid group(COOH) but yet with better potency, favorable physicochemicalproperties, improved pharmacokinetic profiles and metabolic sta-bility. This is mainly due to the tetrazole larger size and superiorlipophilicity (z10 times more) which in turn leads to an increase ofthe substrate receptor interaction [44,45].

There are many methods for the construction of tetrazole ringsystem; however, the azido-Ugi reaction is superior to classicalmethods in terms of automation, reaction time and overall yields[46]. In order to guarantee a second level of diversity, 4,40-dis-elanediyldianiline (4) was used as a bifunctional key synthon as itpossesses an amino group convenient for the azido-Ugi reactionand a readily liberated selenolate nucleophile (formed in situ viareduction of the diselenide using NaBH4) suitable for subsequent SNreactions.

With regard to library construction, six structurally diverseisonitriles (e.g., tert-butylisocyanide (2a), cyclohexyl isocyanide(2b), 4-isocyanopermethylbutane-1,1,3-triol (IPB, 2c) [47], benzy-lisocyanide (2d), 4-methoxybenzylisocyanide (2e) and 2,4-dimethoxybenzylisocyanide (2f) were included for library valida-tion purposes, and partly their potential for additional post-Ugireactions. As oxo component, four different aldehydes were usedincluding both aliphatic (paraformaldehyde (1a), isobutyraldehyde(1b)) and aromatic (4-methylbenzaldehyde (1d) and furfur-aldehyde (1c))) ones, whereas trimethylsilyl azide (TMSN3) 3 wasused as the acid component (see Fig. 2).

Tetrazole-based symmetric diselenides (6e19) were synthe-sized by the addition of two equivalents of aldehyde 1 to a meth-anolic solution of 4 (one equivalent), followed by the subsequentaddition of 2.5 equivalents of TMSN3 and isocyanide 2. Aftercompletion of the reactions, reduction of diselenides (6e19) with

Fig. 1. Synthesis of functionalized diseleno- and selenium-based quinone compounds via sequential Ugi/SN and herein emphasizes azido-Ugi/SN reactions.

Fig. 2. Aldehydes, isonitriles, azide (“acid”), amine, and quinone used in the azido-Ugi and subsequent reduction/SN reactions.

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NaBH4 afforded the corresponding sodium selenolate which in turnreacts with 2-bromo-3-methyl-1,4-naphthoquinone (5) via a SNmechanism (or addition/elimination sequence) to give tetrazole-based selenoquinones (20e22).

It is noteworthy that the reaction sequence can be also per-formed in a one pot (five components) set up without the isolationof the initial azido-Ugi products. Overall, the syntheses of 6e22proceeded smoothly and compounds were obtained in excellentyields (up to 96%, Fig. 3).

Fig. 3. Structures of tetrazole/napthoquinone-base

2.2. In silico prediction of biological activity

Molecular modeling techniques have been shown to be of a highvalue in antineoplastic drug development. Therefore, they wereused in order to virtually assess the redox modulation and thepotential anticancer properties of the newly synthesized com-pound prior to the biological screening. Field alignment wasapplied for the 3D similarity study between the symmetrical dis-elenides of this work and two related reference compounds with

d organoselenium redox modulators (6e23).

S. Shaaban et al. / European Journal of Medicinal Chemistry 122 (2016) 55e71 59

known GPx-like activity. Furthermore, a docking study was alsoperformed at the Thioredoxin reductase (TrxR1) [48e50] dimer-ization interface domain to explore the ability of these compoundsto act as TrxR1 inhibitors.

2.2.1. 3D similarity study3D similarity study is performed for a focused library of sym-

metrical diselenides to figure out if these compounds possesssimilar electrostatic pattern, conformation and/or shape to refer-ence compounds with known GPx mimetic activity. Compounds(Z)-N-(4-methylbenzylidene)-1-(2-((2-(1-((E)-4-methylbenzylideneamino)ethyl)phenyl)diselanyl)phenyl)ethan-amine (24) [51] and 2,20-((diselanediylbis(4,1-phenylene))bis(ace-tylazanediyl))bis(N-(tert-butyl)-3-methylbutanamide) (25) [21],with recognized GPx mimetic activities, were used as templatesand for comparison purposes. Field aligning technique is used toalignmolecules based on their molecular fields. Themolecules withsimilar field patterns to 24 and 25 are expected to possess similarpatterns of biological activity, especially in redox reactions [52]. Asmolecular similarity approaches one, the alignment and accord-ingly the electrostatic structural similarities are better.

Due to the lack of the availability of bioactive conformations of24 and 25, a field template was primarily generated using the FieldTemplater Module using Forge 10.4. This was followed by fieldalignment of selenol forms of our diselenides to that templateemploying Field Align Module in Forge 10.4 [53].

The selenol forms of compounds 24 and 25 were loaded assingle 2D structures and then passed to the conformation hunter togenerate a set of diverse conformations. Field Templater searchesfor common field patterns across the explored conformationalspace of a set of molecules. The top-ranked field template wasselected (scores: molecular similarities: 0.713, field similarity:0.656, raw field score: �67.014, shape similarity: 0.769 and rawshape score: 154.4). The generated field template shows thenegative field point (blue) localized around the selenol groupswhile multiple positive field points (red) surround the para sub-stitution region. Conformers of compound 25 showed some addi-tional negative field points (Fig. 4).

After applying Field Alignment, the most probable conformer ofeach test molecules was selected on the basis of the pair-wisematching. The average molecular similarity is about 0.619e0.713.

Fig. 4. Field alignment of diselenide intermediates of template molecules, yellow and greeandnegative (blue), Van der Waals (yellow), and hydrophobic (orange) field points are reppoints. (For interpretation of the references to colour in this figure legend, the reader is re

The score values for the compounds after alignment over thetemplate generated are illustrated in Table 1. The essential fieldpoints are also present in our test compounds (Fig. 5). Accordingly,the field alignment technique demonstrated that our novel dis-elenides share the same electrostatic pattern and e at least in themore stable conformations e shape with reference compounds 24and 25, suggesting that they would have similar GPx mimeticactivity.

2.2.2. Docking studyMany organoselenium compounds were reported to inhibit

Thioredoxin Reductase (TrxR) in some cancer cells and therefore,activate an apoptosis pathway [54]. To predict the anticancer ac-tivity and apoptotic ability of our compounds, docking intomammalian TrxR enzyme was performed. TrxR1 shows a homo-dimeric quaternary arrangement of its four monomers. Eachmonomer has two main regions: FAD and NAD binding domains atthe N-terminal and the dimerization interface domain at the flex-ible C-terminal side [50,55e57].

Wang et al [54] reported the in silico simulation model of theproposed interaction mechanism of the organoselenium com-pound ethaselen 26 with mammalian TrxR1. They provided anevidence-based explanation that ethaselen 26 targets the C-ter-minal active site of the mammalian TrxR1 at the dimerizationinterface domain forming two intermolecular selenenyl sulfide(SeeS) and selenenyl selenide (SeeSe) covalent bonds with theCys497 and Sec498 amino acids of the protein, respectively; andthus it is acting as an irreversible inhibitor for this enzyme [54].

Due to the lack of human TrxR1 3D structure complexes co-crytallized with an inhibitor, flexible docking was applied usingFlexible Docking protocol. This protocol allows some receptorflexibility during the docking of the flexible ligands [58]. The side-chains of the specified amino acids are allowed to move duringdocking. This allows the receptor to adapt to the different ligands inan induced-fit model.

Ethaselen 26 was successfully docked and the ideal pose wasselected i.e. the posewhich orients the selenium atoms towards theinteracting cystein residues 497 and 498 while pointing the phenylgroup of the other selenenylbenzene group towards His 472 (Fig. 6).Such pose is similar to that reported and proposed by themoleculardynamics simulation study [59].

n 24 and purple and pink 25 in lowest energy conformations; with the positive (red)resented as balls or cubes or polygons, while black arrows indicate the essential fieldferred to the web version of this article.)

Table 1Scores values for diselenides (7e19) after alignment over field template reference compounds 24 and 25.

Compound Conformer no. Molecular similarity Field similarity Shape similarity

7 3 0.709 0.660 0.7588 28 0.648 0.630 0.6659 546 0.634 0.577 0.69110 9 0.694 0.644 0.74411 829 0.619 0.562 0.67712 45 0.671 0.645 0.69613 615 0.633 0.597 0.66914 132 0.682 0.674 0.68915 1 0.713 0.666 0.76116 1 0.629 0.581 0.67617 48 0.700 0.642 0.75918 149 0.678 0.621 0.73419 414 0.641 0.610 0.673

Fig. 5. Field alignment of diselenide forms of the test molecules (silver) on the right over reference templates 24, 25 on the left in a separate display showing the presence of thesame essential field points.

Fig. 6. Docked pose of ethaselen 26 at the dimerization interface domain as predictedby Flexible Docking by Discovery studio 2.5 showing the orientation of selenium atomapproaching Cys 497 and 498 and the other selenylphenyl moiety towards His 472.

S. Shaaban et al. / European Journal of Medicinal Chemistry 122 (2016) 55e7160

The test compounds were docked using Flexible Docking pro-tocol, and the best pose for each structure was selected. Compound23 showed the best -CDOCKER energy (32.17 kcal/mol). Thecarbonyl oxygen of the tertiary amide interacts with Cys 497 viaintermolecular hydrogen bonding (1.5 Å). Also, there is an addi-tional hydrogen bond between Glu 477 and NH of the secondaryamide (1.6 Å). Such interactions orient the selenium atom close tothe second interacting Cys 498 (corresponding to human Sec 498 inhuman TrxR) within a distance of 5.9 Å (Fig. 7a and b).

Furthermore, compounds 21 and 22 exhibited also higherbinding energies, 7.07 and 13.92, respectively, compared to etha-selen 26 with 0.75 kcal/mol. On the other hand, compound 18manifested the poorest binding energy (see Supplementarymaterial).

The binding modes of structures 21 and 22 show p-p in-teractions between the aromatic ring of His 472 or Phe 406 and thetetrazole ring (Fig. 7d and f). Morever, the 22 docked pose showsadditional interactions: s-p interaction between Gln 494 and thequinone ring, two hydrogen bonds between Ile 478 and the tetra-zole ring nitrogens (N3 and N4) of 2.3 Å each and a hydrogen bondbetween Ser 404 and the carbonyl oxygen of quinone (2.5 Å). Theseinteractions anchor the compounds 21 and 22 in the binding site ina such way that a selenium atom gets closer to the cystein residue498 with a distance of 6.8 Å and 5.8 Å, respectively.

2.3. Pharmacology, toxicity and antioxidant profiles

2.3.1. Cytotoxic activity of redox active compounds against humancell lines

In order to check if the chemistry developed here has led topotentially interesting candidates in cancer therapy, our initial aimis directed towards checking if the newly synthesized compoundspossess any anticancer activities as predicted by the in silico study.

Fig. 7. Docked pose and 3D binding mode of structures 23 (a, b), 21 (c, d) and 22 (e, f) showing H bonds in green dotted lines and s-p and p-p interactions as orange lines. (Forinterpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Cytotoxicity screen was therefore performed against twomammalian cancer cell lines, HepG2 and MCF-7 cells. These celllines were chosen based on our previous broad-spectrum screeningresults, which showed that the toxicity of organoselenium com-pounds are generally more pronounced in case of MCF-7 andHepG2 cells. Furthermore, these cell lines are rather suitablemodels for drug targeting. On the other hand, WI-38 normal lungfibroblast cells were also used to gain an initial insight into theselective cytotoxicity of these compounds and to narrow down the

number of most interesting compounds for further in-depthstudies.

Cells were incubated with different concentrations of the testcompounds for 48 h and the viability was estimated by quantifyingthe number of metabolically active cells using the standard color-imetric3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide (MTT) assay. 5-FU was used as positive control because it iscommonly used in adjuvant and palliative cancer chemotherapy.Cytotoxicity was estimated from the respective dose response

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curves and is expressed as the compounds’ concentration that re-duces the absorbance of the treated cells by 50% (IC50) i.e. 50%growth inhibition. The therapeutic index (TI) is defined as the ratioof the concentration of compound that inhibits 50% of the normalWI-38 cells viability to the concentration that inhibits 50% ofHepG2 cell viability (Table 2).

Initial screening indicates that some of the compounds wereable to reduce the viability of MCF-7 and HepG2 with moderateIC50s. Furthermore, a distinct correlation was observed betweenthe chemical structures of test compounds and their correspond-ing cytotoxicity indicating that it is not selenium cytotoxicity thatis observed. Contrary to our expectations, tetrazol-based sym-metrical diselenides were generally more cytotoxic than theanalogous tetrazol-based selenoquinones. Moreover, cytotoxicitywas more pronounced in HepG2 compared to MCF-7 cells.Notably, phenotypic changes, including cell morphology and cell-cell adhesion were also observed, in culture, after the treatmentwith organoselenium compounds. Additionally, cell blebs forma-tion, shrinkage and detachment were also noticed in the cultureplate, and these can be considered as an early morphological signof apoptosis.

Interestingly, a clear difference in the toxicity zones was observedbetween the normal (WI-38) and cancer HepG2 cells. Therefore, TIwas calculated in correspondence to HepG2 cells (IC50WI-38/IC50HepG2). Compounds 9 and 18were found to be the most selectiveones (selectivity index ¼ 32 and 11, respectively). Moreover, com-pounds 6,11,14 and 20 have TI values up to 3which in turnwere alsohigher than the standard benchmark compound 5-FU.

Of course, such initial studies are not directly transferable andtherefore, require more investigations using a wider arsenal ofnormal and tumor cells, and eventually organismic experiments.

2.3.2. Evaluation of caspase-8, Bcl-2 and Ki-67 molecularbiomarkers in HepG2 cells

In order to further study the possibly addressed signalingpathways and obtain hints on the mode(s) of action of the mostpromising compounds 18, 21, 22 and 23 at the molecular level, theexpression levels of selected tumor proliferation, anti-apoptotic

Table 2GIC50 values* in mM of the compounds (6e23) from MTT viability assays of HepG2,MCF-7 (both cancer) and WI-38 (fibroblast) cell lines. TI ¼ therapeutic index*.

Compd. No. MCF-7 HepG2 WI-38 TI

5-FU 3 ± 0.13 8 ± 1.07 4 ± 0.63 0.56 a 37 ± 2.31 a >37 28 ± 2.07 44 ± 4.58 21 ± 1.34 e

8 62 ± 2.34 46 ± 3.15 41 ± 3.47 e

9 70 ± 4.51 2 ± 0.34 64 ± 2.33 3210 a a a e

11 a 47 ± 1.98 a >212 14 ± 1.35 51 ± 1.03 11 ± 0.97 e

13 a a 88 ± 2.36 e

14 a 36 ± 2.32 83 ± 2.21 215 a a 68 ± 2.58 e

16 78 ± 1.52 a 56 ± 3.67 e

17 52 ± 1.27 a 43 ± 2.82 e

18 a 10 ± 0.86 a >1019 a a a e

20 42 ± 3.24 33 ± 2.08 48 ± 3.25 1.521 a 47 ± 3.14 36 ± 2.98 0.822 a 9 ± 0.64 5 ± 0.38 0.623 34 ± 2.11 53 ± 3.67 29 ± 1.54 >0.5

*The metabolic activity of the cells was measured after 48 h of incubation withdifferent concentrations of the investigated compounds by means of MTT assay. TheIC50 (mM) was determined from the dose-response curves as the mean of two par-allel experiments; 5-Fluorouracil was used as a positive control; aGrowthinhibition � 100 mM (inactive). TI is the therapeutic index and calculated as follow:IC50WI-38/IC50HepG2.

and apoptotic protein markers were assessed in HepG2 cells. Inview of the former, the Ki-67 proliferative marker was chosen dueto its strict association with cell proliferation, whereas pro-apoptotic caspase-8 and anti-apoptotic Bcl-2 proteins wereselected to monitor apoptosis induction.

As shown in Fig. 8, compounds 18, 21, 22 and 23 were able toupregulate the expression of caspase-8 and downregulate theexpression of Ki-67 and Bcl-2 compared with untreated cells. It isworthwhile to mention that a distinct correlation was observedbetween the chemical structures of test compounds and theircorresponding expression modulation activity showing that it isnot a general selenium cytotoxic effect. Not surprisingly, and inagreement with TrxR1 docking study, the selenoquinones 21, 22and 23 were more active compared to the symmetric diselenides.Interestingly, 23 exhibited a superior activity within the quinoidanalogues and this worth further study. These results were in linewith our previous studies, in which selenium based quinonesinduced a chain of biochemical alterations among which is the cellcaspases activation and apoptosis induction [27,29]. These alter-ations seem to take place primarily in specific cells (e.g., HepG2)with a disturbed intracellular redox balance.

While it is premature to explain why compound 23 was amongthe most active agents in these assays, one may speculate that thiscompound may hit more than one specific cellular target(s) andcause widespread modification of proteins and enzymes for thebenefit of activation. Furthermore, it’s likely that 23 might also betaken up by cells and modified in vivo into active metabolic in-termediates. As these are unknown, it is too early to speculate overdetails on its exact metabolism, pharmacokinetics in animals andenrichment in specific tissues or degradation, although these issuesare clearly important and will form part of our future studies. Ul-timately, as the structure of these compounds provides consider-able scope for modifications, and the synthesis of derivatives is nowstraightforward, this will become a promising starting point forfuture studies of structural variants.

2.3.3. Assessment of antioxidant activityRecently, Organoselenium compounds have acquired much

attention, particularly from the public health and pharmaceuticalindustry, due to their chemotherapeutic potential as antitumoragents. Accordingly, different modes of action have been proposedfor such compounds. However, ROS-modulation has been acceptedas a key hallmark mechanism for organoselenium compounds[32,59].

Owing to the fact that organoselenium compounds are onoccasion considered as redox-modulators [59e62], the redox ac-tivities of the synthesized compound are further estimatedemploying different biochemical assays such as DPPH, GPx-likeactivity and bleomycin-dependent DNA damage assays. The redoxstatus can be crucial in fighting and differentiating tumor cells fromnormal ones [63,64].

2.3.3.1. DPPH free radical scavenging assay. There are manymethods used for the evaluation of the antioxidant properties oforganic compounds; however, the in vitro DPPH chemical assay isfrequently used to estimate the radical scavenging activities oforganoselenium compounds and nutritional products due to itssimplicity and rapidity, but with limited in vivo relevance. Theantioxidant activity of a compound is estimated by its ability toreduce DPPH. radical (purple color in methanol) to DPPHH (color-less) and the corresponding radical-scavenging activity is evaluatedby the decrease in the absorbance at 517 nm [65,66]. The standardwater soluble antioxidant, vitamin C was used as the positivecontrol in this assay.

As depicted in Table 3, tetrazole-selenoquinones 20 and 21were

Fig. 8. Expression levels of Ki-67, Bcl-2, and caspase-8 in HepG2 cells after 48 h incubation with compounds 18, 21, 22 and 23 at their respective IC50s compared to untreated cells.

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the most active compounds in this assay showing a free-radicalscavenging activity compared to ascorbic acid. This was in excel-lent agreement with our previous report that quinones are goodantioxidants at lower concentrations [67].

2.3.3.2. Bleomycin DNA damage assay. The bleomycin-iron DNAdamage assay was used to evaluate the pro-oxidant activity of thesynthesized compound. This assay has been established as an ac-curate and distinct method to test potential of drugs and food an-tioxidants [68,69]. Bleomycin antibiotics exert their anticanceractivity via ferrous (Fe2þ) ions assisted damage of DNA. Bleomycinforms a complex with Fe2þ, which in turn catalyzes DNA degra-dation. In brief, pro-oxidant compounds reduce the bleomycin-Feþ3

to bleomycin-Feþ2, under aerobic conditions, and thus induce DNAdegradation, which can be followed spectrophotometrically by theincrease in absorbance at 532 nm (Table 3). In this assay, vitamin C

Table 3The redox modulation activity of the compounds.

Redox modulation activity

Compd. No. DPPH assay

Antioxidant activity %

Vitamin Ca 96.5 ± 1.26 38.6 ± 1.67 44.3 ± 0.98 30.4 ± 1.59 31.8 ± 1.810 30.5 ± 1.111 26.3 ± 1.312 24.0 ± 1.113 18.8 ± 1.514 49.5 ± 1.915 19.4 ± 1.916 22.9 ± 1.217 22.9 ± 2.118 20.5 ± 1.119 22.1 ± 1.920 69 ± 1.721 93 ± 1.422 95.3 ± 1.6

a Ascorbic acid is used as standard for antioxidant in case of DPPH assay while it is used3 replicates.

is used as a reducing agent and reduces bleomycin-Feþ3 complex toinduce DNA degradation.

Using a modified assay [70,71], as absorbance increased, morebleomycin-Fe3þis converted into bleomycin-Fe2þ, thereby inducingthe DNA degradation supporting the idea of the pro-oxidant activityof these compounds. Compounds 6, 9, 10, 13 and 16 suppressed thereduction of bleomycin-Feþ3, thus diminishing the bleomycin-Feþ2

chromogen formation and protecting the DNA. On the other hand,compounds 14, 19 and 22 induced DNA degradation significantlymore than the other investigated compounds (Table 3).

In accordance to the ROS-modulation theory, that explains thecytotoxic activity of the organoselenium compounds. Compound22 primarily is exerting its cytotoxic effect via its pronounced pro-oxidant properties. On the other hand, there are likely othermechanisms for the other compounds involved in their cytotoxicactivity which need further investigation.

Bleomycin-dependent DNA damage assay

Fold Absorbance

1 310 ± 3.80.4 67 ± 0.430.5 93 ± 0.890.3 98 ± 0.930.3 81 ± 0.810.3 77 ± 0.560.3 94 ± 0.720.3 108 ± 1.50.2 81 ± 0.690.5 222 ± 3.90.2 96 ± 1.90.3 79 ± 0.980.2 99 ± 1.30.2 92 ± 1.70.2 130 ± 2.10.8 103 ± 1.61.0 90 ± 1.81.0 138 ± 2.7

as a reducing agent in bleomycin-dependent DNA damage assay; Values aremeans of

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2.3.3.3. Glutathione peroxidase-like activity assay. Selenium traceelement is a pivotal component of many enzymes, including GPx[72] and thioredoxin reductases [73]. Most of these enzymescontain selenium cofactor as selenocysteine amino acid, which isresponsible for their corresponding antioxidant properties. Sincethe discovery of organoselenium compounds as good GPx mimics,and many of these agents have been developed for the control ofdifferent illness related to oxidative stress, such as cancer, Alz-heimer and inflammatory diseases [74,75].

The GPx-like activities of the novel compounds were evaluatedusing NADPH-reductase coupled assay [76e81]. This assay isfrequently used for the assessment of the GPx-like activity oforganoselenium compounds. Reduction of peroxides by GPx isfollowed by subsequent conversion of the oxidized glutathione(GSSG) to its reduced form and oxidation of NADPH to NADPþ.Therefore, the activity of GPx is monitored spectrophotometricallyby the decrease in absorbance (340 nm) due to oxidation of NADPHto NADPþ. The standard GPx-like seleno-organic ebselen was usedas the positive control.

As shown in Fig. 9, most of the compounds exhibited moderateto good GPx-like activity. Symmetrical diselenides exhibited higheractivity in this assay compared to their selenoquinone analogues, ascan be expected from theory. Furthermore, compounds 9,12, 14,19,and 21 were even more active, up to 5 fold compared to the GPxmimetic ebselen.

3. Conclusion

New symmetrical organodiselenides and selenium-based qui-nones containing tetrazole moiety were synthesized, employingsequential azido-Ugi/SN reactions in one step and in good yields. Insilico prediction of the compounds’ antioxidant activity using 3Dsimilarity studies revealed a comparable electrostatic pattern andshape with reference compounds suggesting a similar GPx mimeticactivity which was further validated by biological screening.Furthermore, docking study performed into mammalian TrxR1enzyme, showed that compounds 21, 22 and 23 have promisingbinding energies and binding mode that orients the selenium atomtowards Cys 498 for interaction; and thus they may act as TrxRinhibitors.

Fig. 9. Glutathione peroxidase-like activity assay in mM. min�1 of the investigated compthroughout the entire time course.

Moreover, compoundswere evaluated for their cytotoxic activityagainst HepG2 and MCF-7 cell lines and compared with their cyto-toxicity tonormalWI-38employing standardMTTassays. In general,diselenides were found to be more cytotoxic than selenoquinones,and this was more pronounced in HepG2 compared to MCF-7 cells.Furthermore, a distinct selectivity was noticed by comparing thecytotoxicity in HepG2with that of normalWI-38 cells. Interestingly,compounds 9 and 18 exhibited a selectivity index of ca. 32 and 11,respectively. Moreover, compounds 6, 11, 14 and 20 showed a ther-apeutic index up to 3 for the model cell lines, which in turnwere allhigher than that for the benchmark drug 5-FU. To this point, some ofthe compounds exhibited a preferential cytotoxicity, and this wasobvious in HepG2 compared to MCF-7 cells. Furthermore, distinctselectivity patterns were observed by comparing the cytotoxicity inHepG2 with that of normal WI-38 cells.

Although it might appear that, compared to some standarddrugs, these compounds do not provide nanomolar activity, there isenough evidence of selectivity and the perspective of an unknown(and thus potentially new) mode of action to warrant furtherstudies. Within this context, we are fully aware that a clear QSARwill require large and diverse sets of compounds including sulfur-and for completion maybe even tellurium-analogues, to ascertain aspecific role of organoselenium and to screen for compounds withimproved activity and selectivity.

As expected, diselenides showed superior GPx catalytic activityand among the compounds, 9, 12, 14, 19 and 21 were more active(up to 5 fold) than ebselen. Additionally, tetrazole-selenoquinones20 and 21 showed the highest radical scavenging activity in theDPPH assay which was also in line with our previous reports,ascertaining that quinones are efficient antioxidants at lower con-centrations. Further the pro-oxidant activity of the synthesizedcompounds evaluated with the bleomycin DNA damage assayrevealed that compounds 14, 19 and 22 were able to induced sig-nificant DNA degradation more than the other investigated com-pounds (e.g., 6, 9, 10, 13 and 16).

Overall, the selenium-based quinones were the most promisingcandidates and therefore, were selected for further in-depthstudies of the underlying molecular mechanism. Treatment ofHepG2 cells with compounds 18, 21, 22 and 23 revealed a signifi-cant downregulation in the expression levels of Bcl-2 and Ki-67 and

ounds. The reaction was monitored to completion and the reaction rate was linear

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activation of caspase-8 compared to untreated cells. Importantly,23 showed the highest modulation activity the three biomarkers.These results support themolecular docking study intomammalianTrxR1 that predicted the best binding energies for compounds 23,21 and 22 than 18. TrxR1 inhibition increases caspases level andthus induces apoptosis.

At this point, it should be noted that the results presented hereare preliminary in nature and need more in-depth studies in thefuture. In consequence, a more in-depth studies and additionalexperiments to investigate the exact mode(s) of action and toidentify possible intracellular targets (such as specific organelles, ormembranes) are warranted. Furthermore, optimization of diaryldiselenides scaffold is required to improve their pharmcodynamicand pharmacokinetic properties. This opens plenty of room forfurther, multi-disciplinary studies involving synthetic, bioorganicand medicinal chemistry, cell biology, and pharmacology in orderto develop a strategy to treat cancer applying organoseleniumcompounds.

4. Experimental protocols

4.1. Material and methods

All chemical reagents for the synthesis of the compounds werepurchased from Sigma-Aldrich-Fluka or Merck (AMD) and usedwithout further purification unless stated otherwise. Reactions ininert atmospherewere carried out under argon (4.6) using standardSchlenck techniques. Silica gel 60 (Macherey-Nagel, 50e200 mm)was used for column chromatography. Unless noted otherwise, thedimensions of columns used were 2.5 cm (diameter) and 25e30 cm(height of silica gel). TLC plates (silica gel 60 F254, 0.20 mm) werepurchased from Merck. NMR spectroscopy: 1H NMR spectra wererecorded at 500 MHz, 13C NMR spectra at 100 MHz on a VarianINOVA spectrometer. Chemical shifts are reported in d (ppm),expressed relative to the solvent signal at 7.26 ppm (CDCl3, 1HNMR) and at 77.16 ppm (CDCl3, 13C NMR), as well as 3.31 ppm (1HNMR, CD3OD) and 49.00 ppm (13C NMR, CD3OD).

Coupling constants (J) are given in Hz. MS analysis: analyseswere performed using a TSQ quantummass spectrometer equippedwith an ESI source and a triple quadrupole mass detector (ThermoFinnigan). High-resolution mass spectrometry (HRMS) was per-formed on an Accela UPLC-system (Thermo-Fisher) coupled to alinear trap-FT-Orbitrap combination (LTQ-Orbitrap), operating inpositive ionization mode. These spectra indicated �99% MS-purityof the prepared compounds. DNA (Calf Thymus type1), bleomycinsulfate, thiobarbituric acid (TBA), 1,1-diphenyl-1-picrylhydrazyl,ethylenediaminetetraacetic acid (EDTA) and ascorbic acid wereobtained from Sigma. All other chemicals were of analytical grade.Compounds were prepared and purified according to the experi-mental and purification procedures in the Supplementary Mate-rials. Further details on the spectral data and copies of 1H and 13CNMR spectra for all synthesized compounds can also be found inSupplementary Materials. 4-(2-(4-aminophenyl)diselanyl)benzen-amine (4) and 23 were synthesized according to a literature re-ported method [82,83].

4.2. Molecular modeling

4.2.1. Preparation of TrxR1The mammalian TrxR1 protein (PDB id: 1H6V) was retrieved

from Protein Data Bank (PDB) for docking. Only one monomer wasusedwhile others were deleted. All watermolecules were removed.Protonation of the protein, completing missed residues, atomordering corrections if necessary and checking bonds and bondordering and their correction if necessary were performed by the

use of the clean protein tool under protein report and utilities ofDiscovery Studio 2.5. CHARMm force field was applied followed byenergy minimization using Adopted Basis NR algorithm withmaximum steps of 2000 while other parameters maintained asdefault. The binding site of TrxR1 was defined by: finding sites fromreceptor cavities, selecting the one present at the interface domainand definition of the docking sphere with dimensions X: 9, Z: 10.36and Y: 149.54 and a radius of 13 Å.

4.2.2. Ligands preparation and dockingLigand test molecules were prepared using the prepare ligands

protocol under general purposes, whereby generation of allpossible isomers have been considered to be generated. Whilegeneration of tautomers, application of Lipiniski’s rule filtration andchanging ionization parameters were set to false. For the dockingprocess, flexible docking protocol was applied. The selected resi-dues for generation of flexible side chain conformations were thatof dimerization interface domain (368e499). Generation of 10protein conformations of the selected residues was determinedwith a maximum number of 8 residues. Generation of ligand con-formations was enabled as fast method by generating 25 differentconformers of each test compound with energy threshold of20 kcal.

All other parameters of docking remained as default. Afterdocking run finished, the highest binding energy pose was selectedto visualize the ligand protein interactions as reported above (Seesupplementary material).

4.3. Biological assays

4.3.1. Cytotoxicity assayThe HepG2 human liver carcinoma, breast adenocarcinoma

(MCF-7) and lung fibroblast (WI-38) cell lines were purchased fromAmerican Type Culture Collection (HTB-37; Rockville, MD, USA).The cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM) supplemented with 10% (v/v) calf serum (Hyclone Labo-ratories, Ogden, UT), 60 mg/mL penicillin G and 100 mg/mLstreptomycin sulfate maintained at 37

�C in a humidified atmo-

sphere containing about 15% (v/v) CO2 in air.MTT [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium

bromide] (Sigma) was used to measure the metabolic activity ofcells which are capable of reducing it by dehydrogenases to a violetcolored formazan product. Briefly, 120 mL aliquots of a cell sus-pension (50,000 cells mL�1) in 96-well microplates were incubatedat 37 �C and 10% CO2 and allowed to grow for two days. Then 60 mLof serial dilutions of the test compounds were added. After 48 h ofincubation at 37 �C and 10% CO2, 75 mL MTT in phosphate bufferedsaline (PBS) were added to a final concentration of 0.5 mg mL�1.After 2 h the precipitate of formazan crystals was centrifuged andthe supernatant discarded. The precipitate was washed three timeswith 100 mL PBS and dissolved in 100 mL DMSO. The resulting colorwas measured at 590 nm using an ELISA plate reader. 5-FU wasused as a positive control because it is commonly used in adjuvantand palliative cancer chemotherapy. All investigations were carriedout in two parallel experiments. The IC50 values were determinedas the concentrations of tested materials, which showed 50% of theabsorbance of untreated control cells as estimated from the dose-response curves. The therapeutic index (TI) is defined as the ratioof the concentration of compound that inhibits 50% of the growthof the normal WI-38 cells to the concentration that inhibits 50% ofHepG2 cell viability.

4.3.2. In vitro studies4.3.2.1. DPPH free radical scavenging activity. The hydrogen atom orelectron donation ability of the corresponding compounds was

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measured by the bleaching of the purple color of a methanolicsolution of DPPH. This spectrophotometric assay uses stable radicaldiphenylpicrylhydrazyl as a reagent. The test compounds weredissolved in methanol to obtain a final concentration of 1 mM200 mL of each sample were added to 0.4 mL of 0.1 mM DPPH inmethanol. After 30 min of incubation in the dark, the absorbancewas read against a blank at 517 nm. Ascorbic acid (vitamin C) wasused as standard antioxidant (positive control). Blank sample wasrun without DPPH and using methanol instead of sample. Negativecontrol samplewas runwithmethanol instead of tested compound.The radical scavenging activity was calculated from the followingequation:

I% ¼ (Ablank�Asample)/(Ablank) � 100

4.3.2.2. Glutathione peroxidase like activity. GPx kit (Biodiagnostic,Egypt) was used for the determination of GPx according to Pagliaet al. [84]The reaction mixture contained 1 mL assay buffer (50 mMphosphate buffer containing 0.1% triton X-100) and 0.1 mL NADPHreagent (24 mmol Glutathione, 12 unit Glutathione reductase and4.8 mmol NADPH) and 0.01 mL (41 mM) tested compounds and thereactionwas started by the addition of H2O2 (0.8 mM). The contentswere mixed well and the absorbances were recorded at 340 nmover a period of 3 min against deionized water. The change ofabsorbance per minute (A340nm/min) was estimated using ebselen(41 mM) as the positive control. The values represented in Fig. 9 areexpressed after background correction for the reaction with H2O2and GSH. In case of colored compounds, their activities were esti-mated after subtracting their own absorbance at the used wavelength.

4.3.2.3. Bleomycin-dependent DNA damage. The reaction mixturecontained calf thymus DNA (0.5 mg/mL), bleomycin sulfate(0.05 mg/mL), MgCl2 (5 mM), FeCl3 (50 mM), samples to be tested(2 mM) and L-ascorbic acid was used as positive control. Themixture was incubated at 37 �C for 1 h. The activity of test com-pounds was evaluated as malondialdehyde (MDA) equivalents.Thiobarbituric reactive substances which arose from deoxyribosedegradation of DNAwere assessed. The reaction was terminated byaddition of 0.05 mL EDTA (0.1 M). The color was developed byadding 0.5 mL thiobarbituric acid (1% w/v) and 0.5 mL HCl (25% v/v). The tube was capped with a screw cap and heated at 80 �C for30 min. After cooling in ice water, the mixture was then shaken andcentrifuged and the extent of DNA damage was measured by in-crease in absorbance at 532 nm.

4.3.2.4. Detection of caspase-8 protein expression levels.HepG2 cells were treated with the corresponding IC50 of each drugand incubated for 48 h. The cells were detached by trypsin andlysed by freezing at liquid nitrogen and then thawing with gentlemixing. Cell lysates incubated with Horseradish Peroxidase conju-gated anti-CASP8 antibody for 30min at 37 �C. The reaction productwas detected at 450 nm using enzyme-linked immunosorbentassay (Platinum ELISA; Biospess).

4.3.2.5. Detection of Bcl-2 protein expression levels. Bcl-2 levelswere evaluated in HepG2 cells treated with the corresponding IC50of each compound and incubated for 48 h and compared with theirlevels in control untreated HepG2 cell line. The cells were harvestedby applying trypsin and lysed by freezing with liquid nitrogen andthen thawing with gentle mixing. According to the instructions ofthe manufacturer of (Platinum ELISA; eBioscience©), cell lysateswere incubated with biotin-conjugate for 2 h 25 �C and then with

streptavidin-HRP for 1 h at 25 �C. The reaction product wasdetected at 450 nm.

4.3.2.6. Detection of Ki-67 protein expression levels. HepG2 cellswere treated with the IC50 dose of each compound for 48 h, thenwere detached by trypsin and lysed by ultrasonication for 4 times.The reaction product was detected at 450 nm using enzyme-linkedimmunosorbent assay (Platinum ELISA; Biomatic) according to theinstructions of the manufacturer.

4.3.2.7. Statistical analysis. Data analysis was performed using thestatistical package for the social sciences software, release 15.0 forWindows (SPSS version 15.0, Chicago: SPSS Inc). All results wereexpressed as mean ± SD. A P-value <0.05 was considered statisti-cally significant. Statistical analysis was performed by analysis ofvariance (ANOVA) with LSD and Dunnett’s test for PostHoc.

4.4. Synthesis and characterization

4.4.1. General procedure I for the preparation of symmetricaldiselenide based-tetrazoles derivatives (6e19) by azido-Ugi reaction

Under argon, a mixture of 4 (1 mmol), aldehyde (2.2 mmol),TMSN3 (2.5 mmol) and isonitrile (2.5 mmol) in 1 mL methanol wasstirred overnight at room temperature. Upon completion (moni-tored by TLC), 10 mL dichloromethane were added to dissolve thesticky product, washed with water (3 � 50 mL). The organic layerwas separated, dried over anhydrous Na2SO4 and evaporated underreduced pressure. The residue was purified by chromatography onsilica gel employing petroleum ether: ethyl acetate (4:3) to giveyellow solid pure compound.

4.4.2. General procedure II for the preparation of selenoquinonebased-tetrazoles (20e22)4.4.2.1. General procedure IIA: two-step synthesis of selenoquinonebased-tetrazoles (20e22) via sequential azido-Ugi/SN methodology.To a solution of the isolated azido-Ugi adduct (1 mmol), 2-methyl-3-bromo-l, 4-naphthoquinone (5) (552.2 mg, 2.2 mmol) in ethylacetate (20 mL) and water (20 mL) heterogeneous solvent system,was added tricaprylmethylammonium chloride (45 mg, 5% mol).NaBH4 (189.15 mg, 5 mmol) was then added in small portions withcaution and the mixture was stirred at room temperature forfurther 2 h. Upon completion (monitored by TLC), the organic layerwas separated and solvent was dried over anhydrous Na2SO4 andremoved under reduced pressure and the residue was purified bychromatography on silica gel employing petroleum ether and ethylacetate as eluent to give reddish brown solid.

4.4.2.2. General procedure IIB: one-potsequential azido-Ugi/reduc-tion/SN synthesis of selenoquinone based-tetrazoles (20e22).Upon completion of the azido-Ugi reaction (monitored by TLC), 2-methyl-3-bromo-l, 4-naphthoquinone (5) (552.2 mg, 2.2 mmol)and tricaprylmethylammonium chloride (45 mg, 5% mol) wasadded to a heterogeneous solvent system of ethyl acetate (20 mL)and water (20 mL). NaBH4 (189.15 mg, 5 mmol) was then added insmall portions with caution and the mixture was stirred at roomtemperature for further 2 h. Upon completion (monitored by TLC),the organic layer was separated, dried over Na2SO4 and solvent wasremoved under reduced pressure and the residue was purified bychromatography on silica gel employing petroleum ether and ethylacetate as eluent to give reddish brown solid.

4.4.3. 4,40-Diselanediylbis(N-(1-(1-(tert-butyl)-1H-tetrazol-5-yl)-2-methylpropyl)aniline) (6)

Compound 6 was synthesized according to the general proce-dure I from 4,40-diselanediyldianiline (4) (344 mg, 1 mmol),

S. Shaaban et al. / European Journal of Medicinal Chemistry 122 (2016) 55e71 67

isobutyraldehyde (1b) (201 mL, 2.2 mmol), trimethylsilyl azide (3)(332 mg, 2.5 mmol) and tert-butyl isocyanide (2a) (283 mL,2.5 mmol). Its formation was monitored by TLC petrol ether: ethylacetate ¼ 4:1, Rf ¼ 0.36, purified by column chromatography onsilica gel with petrol ether: ethyl acetate¼ 3:1. Yield: 620mg (88%).1H NMR (400 MHz, CDCl3) d 7.34 (d, J ¼ 8.6 Hz, 4H, AreH),6.56e6.46 (m, 4H, AreH), 4.91 (s, 2H, 2NH), 4.38 (d, J ¼ 7.4 Hz, 2H,2CH), 2.43e2.30 (m, 2H, 2CH), 1.76 (s, 18H, 6CH3), 1.13 (J ¼ 6.6 Hz,6H, 2CH3), 1.02 (d, J ¼ 6.7 Hz, 6H, 2CH3).13C NMR (101 MHz, CDCl3)d 155.74 (C-tetrazol), 147.30 (CeAr), 136.05 (CHeAr), 135.98(CHeAr), 119.36 (CeAr), 114.41 (CHeAr), 70.79 (C-t-butyl), 61.53(CH), 55.53 (CH), 34.53 (CH-isopropyl), 30.23 (CH3-t-butyl), 19.99(CH3), 18.04 (CH3). MS (ESI): m/z ¼ found 727.6 [MþþNa]; calcd.704.21[MþþNa]; HRMS calcd. for C30H44N10Se2 [MþþNa]: 727.1998,found 727.1973 [MþþNa].

4.4.4. 4,40-Diselanediylbis(N-((1-(tert-butyl)-1H-tetrazol-5-yl)methyl)aniline) (7)

Compound 7 was synthesized according to the general proce-dure I from 4,40-diselanediyldianiline (4) (344 mg, 1 mmol), para-formaldehyde (1a) (66 mg, 2.2 mmol), trimethylsilyl azide (3)(332 mg, 2.5 mmol) and tert-butyl isocyanide (2a) (283 mL,2.5 mmol). Its formation was monitored by TLC petrol ether: ethylacetate ¼ 4:1, Rf ¼ 0.31, purified by column chromatography onsilica gel with petrol ether: ethyl acetate¼ 3:1. Yield: 533mg (86%).1H NMR (400 MHz, CDCl3) d 7.50e7.38 (m, 4H, AreH), 6.67e6.55(m, 4H, AreH), 4.86 (s, 2H, 2NH), 4.66 (s, 4H, 2CH2), 1.80 (s, 18H,6CH3).13C NMR (101 MHz, CDCl3) d 151.67 (C-tetrazol), 147.12(CeAr), 136.05 (CHeAr), 119.78 (CeAr), 113.80 (CHeAr), 61.45 (C-t-butyl), 39.98 (CH2), 29.59 (CH3-t-butyl). MS (ESI): m/z ¼ found643.4 [MþþNa]; calcd. 643.1 [MþþNa]; HRMS calcd. forC24H32N10Se2 [MþþNa]: 643.0998, found 643.10396 [MþþNa].

4.4.5. 4,40-Diselanediylbis(N-((1-(tert-butyl)-1H-tetrazol-5-yl)(p-tolyl)methyl)aniline) (8)

Compound 8 was synthesized according to the general proce-dure I from 4,40-diselanediyldianiline (4) (344 mg, 1 mmol), 4-methylbenzaldehyde (1d) (264 mg, 2.2 mmol), trimethylsilylazide (3) (332mg, 2.5 mmol) and tert-butyl isocyanide (2a) (283 mL,2.5 mmol). Its formation was monitored by TLC petrol ether: ethylacetate ¼ 4:1, Rf ¼ 0.34, purified by column chromatography onsilica gel with petrol ether: ethyl acetate¼ 3:1. Yield: 680mg (85%).1H NMR (400 MHz, CDCl3) d 7.39e7.28 (m, 4H, AreH), 7.21 (d,J ¼ 8.2 Hz, 4H, AreH), 7.16 (d, J ¼ 8.1 Hz, 4H, AreH), 6.52 (dd,J ¼ 12.2, 6.0 Hz, 4H, AreH), 6.09 (s, 2H, 2CH), 5.01 (s, 2H, 2NH), 2.32(s, 6H, 2CH3), 1.68 (s, 18H, 6CH3).13C NMR (101MHz, CDCl3) d 155.04(C-tetrazol), 146.22 (CeAr), 138.73 (CeAr), 135.89 (CHeAr), 134.73(CeAr), 129.85 (CHeAr), 127.64 (CHeAr), 119.66 (AreC), 114.40 (Ar-CH), 61.78 (C-t-butyl), 53.99 (CHe), 30.05 (CH3-t-butyl), 21.09(CH3). MS (ESI): m/z ¼ found 825.21 [Mþþ2 þ Na]; calcd. 823.21[MþþNa]; HRMS calcd. for C38H44N10Se2 [MþþNa]: 823.1998, found823.19729 [MþþNa].

4.4.6. 4,40-Diselanediylbis(N-(1-(1-(2,4-dimethoxybenzyl)-1H-tetrazol-5-yl)-2-methylpropyl)aniline) (9)

Compound 9 was synthesized according to the general proce-dure I from 4,40-diselanediyldianiline (4) (344 mg, 1 mmol), iso-butyraldehyde (1b) (201 mL, 2.2 mmol), trimethylsilyl azide (3)(332 mg, 2.5 mmol) and 1-(isocyanomethyl)-2,4-dimethoxybenzene(2f) (443 mg, 2.5 mmol). Its formation was monitored by TLCpetrol ether: ethyl acetate ¼ 4:1, Rf ¼ 0.31, purified by columnchromatography on silica gel with petrol ether: ethyl acetate ¼ 3:1.Yield: 732 mg (82%). 1H NMR (400 MHz, CDCl3) d 7.19 (dd, J ¼ 23.9,7.3 Hz, 4H, AreH), 7.05 (dd, J¼ 8.4, 4.1 Hz, 2H, AreH), 6.49e6.42 (m,4H, AreH), 6.30 (d, J ¼ 7.7 Hz, 4H, AreH), 5.58e5.44 (s, 4H, 2CH2),

4.61 (dd, J ¼ 9.2, 7.2 Hz, 2H, 2CH), 4.35 (d, J ¼ 9.4 Hz, 2H, 2CH), 3.79(s, 12H, 4CH3), 2.09 (dd, J ¼ 13.5, 6.7 Hz, 2H, 2CH), 1.65 (s, 2H, 2NH),1.03 (d, J ¼ 6.8 Hz, 6H, 2CH3), 0.85 (d, J ¼ 6.0 Hz, 6H, 2CH3).13C NMR(101 MHz, CDCl3) d 161.72 (OeCeAr), 157.67 (OeCeAr), 155.47 (C-tetrazol), 147.02 (CeAr), 135.87 (CHeAr), 131.01 (CHeAr), 119.24(CeAr), 113.99 (CHeAr), 105.00 (CHeAr), 98.80 (CHeAr), 55.49(H3CeO), 54.12 (H3CeO), 45.70 (CH2), 33.08 (CH-isopropyl), 19.14(CH3), 18.61 (CH3). MS (ESI): m/z ¼ found 915.5 [MþþNa]; calcd.915.22[MþþNa]; HRMS calcd. for C40H48N10O4Se2 [MþþNa]:915.2098, found 915.20992[MþþNa].

4.4.7. 4,40-Diselanediylbis(N-(1-(1-(4-methoxyphenyl)-1H-tetrazol-5-yl)-2-methylpropyl)aniline) (10)

Compound 10 was synthesized according to the general proce-dure I from 4,40-diselanediyldianiline (4) (344 mg, 1 mmol), iso-butyraldehyde (1b) (201 mL, 2.2 mmol), trimethylsilyl azide (3)(332 mg, 2.5 mmol) and 1-(isocyanomethyl)-4-methoxybenzene (2e)(368 mg, 2.5 mmol). Its formation was monitored by TLC petrolether: ethyl acetate ¼ 4:1, Rf ¼ 0.34, purified by column chroma-tography on silica gel with petrol ether: ethyl acetate ¼ 3:1. Yield:675mg (84%). 1H NMR (400MHz, CDCl3) d 7.32e7.25 (m, 4H, AreH),7.22e7.12 (m, 4H, AreH), 7.04 (ddd, J ¼ 17.1, 7.8, 2.1 Hz, 4H, AreH),6.37 (dd, J ¼ 18.2, 5.9 Hz, 4H, AreH), 4.49 (dd, J ¼ 9.6, 8.4 Hz, 2H,2CH), 4.20e4.07 (m, 2H, 2CH), 3.91 (s, J ¼ 8.1 Hz, 6H,2CH3),2.26e2.13 (m, 2H, 2CH), 1.65 (s, 2H, 2NH), 1.04 (d, J ¼ 9.3 Hz,6H, 2CH3), 0.92 (d, J ¼ 7.8 Hz, 6H, 2CH3).13C NMR (101 MHz, CDCl3)d 161.29 (OeCeAr), 156.37 (C-tetrazol), 146.58, 135.85, 127.32,125.97, 119.64 (CeAr), 114.89 (CHeAr), 114.34 (CHeAr), 55.75(H3CeO), 54.57 (H3CeO), 33.35 (CH-isopropyl), 19.17 (CH3-isopro-pyl). MS (ESI): m/z ¼ found 827.5 [MþþNa]; calcd. 827.17[MþþNa];HRMS calcd. for C36H40N10O2Se2 [MþþNa]: 827.1598, found827.15482[MþþNa].

4.4.8. 4,40-Diselanediylbis(N-(2-methyl-1-(1-(2,4,4-trimethoxybutyl)-1H-tetrazol-5-yl)propyl)aniline) (11)

Compound 11 was synthesized according to the general proce-dure I from 4,40-diselanediyldianiline (4) (344 mg, 1 mmol), iso-butyraldehyde (1b) (201 mL, 2.2 mmol), trimethylsilyl azide 3(332 mg, 2.5 mmol) and IPB (2c) (433 mg, 2.5 mmol). Its formationwas monitored by TLC petrol ether: ethyl acetate ¼ 4:1, Rf ¼ 0.32,purified by column chromatography on silica gel with petrol ether:ethyl acetate ¼ 3:1. Yield: 796 mg (90%). 1H NMR (400 MHz, CDCl3)d 7.39e7.25 (m, 4H, AreH), 6.61e6.49 (m, 4H, AreH), 4.73 (dd,J ¼ 16.4, 8.7 Hz, 2H, 2CH), 4.62e4.52 (m, 2H, 2CH), 4.48e4.29 (m,4H, 2CH2), 3.85e3.78 (m, 2H, 2CH), 3.37 (s, 6H, 2CH3), 3.35 (s, 3H,CH3), 3.34 (s, 3H, CH3), 3.13e3.11 (s, 3H, CH3), 3.01 (s, 3H, CH3),2.42e2.25 (m, 2H, 2CH), 1.95e1.84 (m, 4H, 2CH2), 1.09 (d, J¼ 6.7 Hz,6H, 2CH3), 1.02e0.94 (d, J ¼ 6.7 Hz, 6H, 2CH3).13C NMR (101 MHz,CDCl3) d 156.35 (C-tetrazol), 147.08 (CeAr), 135.89 (CHeAr), 119.18(CeAr),113.99 (CHeAr),101.51 (CHeAr), 54.45 (CH), 53.98 (CH3eO),53.44 (CH3eO), 51.33 (CH2), 34.47 (CH2), 33.18 (CH-isopropyl),32.73 (CH), 19.20 (CH3), 18.63 (CH3). MS (ESI): m/z ¼ found 907.6[MþþNa]; calcd. 907.2[MþþNa]; HRMS calcd. for C36H56N10O6Se2[MþþNa]: 907.2598, found 907.26106 [MþþNa].

4.4.9. 4,40-Diselanediylbis(N-((1-(tert-butyl)-1H-tetrazol-5-yl)(furan-2-yl)methyl)aniline) (12)

Compound 12 was synthesized according to the general proce-dure I from 4,40-diselanediyldianiline (4) (344 mg, 1 mmol), 4-furan-2-carbaldehyde (1c) (182 mg, 2.2 mmol), trimethylsilyl azide(3) (332 mg, 2.5 mmol) and tert-butyl isocyanide (2a) (283 mL,2.5 mmol). Its formation was monitored by TLC petrol ether: ethylacetate ¼ 4:1, Rf ¼ 0.30, purified by column chromatography onsilica gel with petrol ether: ethyl acetate¼ 3:1. Yield: 646mg (86%).1H NMR (400 MHz, CDCl3) d 7.44e7.33 (m, 6H, AreH), 6.61 (d,

S. Shaaban et al. / European Journal of Medicinal Chemistry 122 (2016) 55e7168

J ¼ 8.4 Hz, 4H, AreH), 6.38e6.31 (s, 2H, 2NH), 6.23 (dd, J ¼ 9.8,4.7 Hz, 4H, AreH), 5.11 (s, 2H, 2CH), 1.73 (s, 18H, 6CH3).13C NMR(101 MHz, CDCl3) d 153.32 (C-tetrazol), 150.60 (C-furan), 145.80(CeAr), 142.94 (CHeAr), 135.76 (CH-furan), 135.57 (CHeAr), 133.56(CHeAr), 121.69 (CHeAr), 120.30 (CeAr), 114.66 (CHeAr), 110.92(CH-furan), 109.05 (CHeAr), 62.03 (C-t-butyl), 48.51 (CH), 29.96(CH3-t-butyl). MS (ESI): m/z ¼ found 777.4 [Mþþ2 þ Na]; calcd.775.14 [MþþNa]; HRMS calcd. for C32H36N10O2Se2 [MþþNa]:775.1298, found 775.12466[MþþNa].

4.4.10. 4,40-Diselanediylbis(N-((1-(2,4-dimethoxybenzyl)-1H-tetrazol-5-yl)(furan-2-yl)methyl)aniline) (13)

Compound 13 was synthesized according to the general proce-dure I from 4,40-diselanediyldianiline (4) (344 mg, 1 mmol), 4-furan-2-carbaldehyde (1c) (182 mg, 2.2 mmol), trimethylsilyl azide(3) (332 mg, 2.5 mmol) and 1-(isocyanomethyl)-2,4-dimethoxybenzene) (2f) (443 mL, 2.5 mmol). Its formation wasmonitored by TLC petrol ether: ethyl acetate ¼ 4:1, Rf ¼ 0.36, pu-rified by column chromatography on silica gel with petrol ether:ethyl acetate ¼ 3:1. Yield: 809 mg (86%). 1H NMR (400 MHz, CDCl3)d 7.36 (t, J ¼ 3.7 Hz, 2H, AreH), 7.30 (t, J ¼ 7.5 Hz, 2H, AreH), 7.09(dd, J ¼ 8.1, 3.7 Hz, 2H, AreH), 6.48e6.38 (m, 8H, AreH), 6.32 (dd,J ¼ 3.3, 1.9 Hz, 2H, AreH), 6.22 (d, J ¼ 2.4 Hz, 2H, AreH), 6.09 (dd,J ¼ 10.1, 6.2 Hz, 2H, AreH),5.55 (s, 4H, 2CH2), 5.01 (s, 2H, 2CH), 3.80(s, 6H, 2CH3), 3.73 (s, 6H, 2CH3).13C NMR (101 MHz, CDCl3) d 161.81(OeCeAr), 157.91 (OeCeAr), 153.25 (C-tetrazol), 149.53 (C-furan),145.80 (CeAr), 143.16 (CHeAr), 135.70 (CHeAr), 135.68 (CHeAr),131.24 (CHeAr), 119.95 (CeAr), 114.18 (CHeAr), 113.40 (CeAr),110.79 (CHeAr), 108.68 (CHeAr), 104.85 (CHeAr), 98.74 (CHeAr),55.47 (CH), 47.07 (H3CO), 46.32 (CH2). MS (ESI): m/z ¼ found 963.4[MþþNa]; calcd. 963.1[MþþNa]; HRMS calcd. for C42H40N10O6Se2[MþþNa]: 963.1398, found 963.13525[MþþNa].

4.4.11. 4,40-Diselanediylbis(N-((1-cyclohexyl-1H-tetrazol-5-yl)(furan-2-yl)methyl)aniline) (14)

Compound 14 was synthesized according to the general proce-dure I from 4,40-diselanediyldianiline (4) (344 mg, 1 mmol), 4-furan-2-carbaldehyde (1c) (182 mg, 2.2 mmol), trimethylsilyl azide(3) (332 mg, 2.5 mmol) and isocyanocyclohexane (2b) (310 mL,2.5 mmol). Its formation was monitored by TLC petrol ether: ethylacetate ¼ 4:1, Rf ¼ 0.31, purified by column chromatography onsilica gel with petrol ether: ethyl acetate¼ 3:1. Yield: 715 mg (89%).1H NMR (400MHz, CDCl3) d 7.65e7.46 (m, 2H, AreH), 7.43e7.37 (m,2H, AreH), 7.37e7.28 (m, 2H, AreH), 7.20e7.10 (m, 1H, AreH),7.06e6.88 (s, 1H, NH), 6.72e6.48 (m, 3H, AreH), 6.43e6.29 (m, 3H,AreH), 6.19e5.98 (m, 2H, AreH), 5.13 (s, 1H, NH), 4.47e4.45 (s, 2H,2CH), 2.11e1.85 (m, 8H, 4CH2), 1.59e1.55 (m, 6H, 3CH2), 1.46e1.25(m, 6H, 3CH2).13C NMR (101 MHz, CDCl3) d 152.38 (C-tetrazol),149.40 (C-furan), 145.75 (CeAr), 143.18 (CHeAr), 136.12 (CHeAr),133.60 (CHeAr), 121.70 (CHeAr), 120.39 (CeAr), 115.49 (CHeAr),114.29 (CHeAr), 114.20 (CHeAr), 111.16 (CHeAr), 108.99 (CHeAr),58.74 (CH), 48.01 (CH-cyclohexyl), 32.79 (CH2-cyclohexyl), 25.37(CH2-cyclohexyl), 24.76 (CH2-cyclohexyl). MS (ESI): m/z ¼ found827.5 [MþþNa]; calcd. 827.17[MþþNa]; HRMS calcd. forC36H40N10O2Se2 [MþþNa]: 827.1598, found 827.15663[MþþNa].

4.4.12. 4,40-Diselanediylbis(N-((1-cyclohexyl-1H-tetrazol-5-yl)methyl)aniline) (15)

Compound 15 was synthesized according to the general proce-dure I from 4,40-diselanediyldianiline (4) (344 mg, 1 mmol), para-formaldehyde (1a) (66 mg, 2.2 mmol), trimethylsilyl azide (3)(332 mg, 2.5 mmol) and isocyanocyclohexane (2b) (310 mL,2.5 mmol). Its formation was monitored by TLC petrol ether: ethylacetate ¼ 4:1, Rf ¼ 0.34, purified by column chromatography onsilica gel with petrol ether: ethyl acetate¼ 3:1. Yield: 598mg (89%).

1H NMR (400 MHz, DMSO) d 7.31 (dd, J ¼ 36.9, 11.3 Hz, 4H, AreH),6.61 (d, J ¼ 8.3 Hz, 4H, AreH), 4.66 (s, 4H, 2CH2), 4.54 (s, 2H, 2NH),2.11e210 (m, 4H), 1.98e1.74 (m, 10H), 1.37 (dt, J ¼ 25.2, 11.7 Hz,6H).13C NMR (101 MHz, DMSO) d 151.90 (C-tetrazol), 147.83 (CeAr),135.35 (CHeAr), 116.80 (CeAr), 112.71 (CHeAr), 56.74 (CH), 35.88(CH2-cyclohexyl), 32.32 (CH2-cyclohexyl), 30.40 (CH-cyclohexyl),24.54 (CH2-cyclohexyl), 24.40 (CH2-cyclohexyl). MS (ESI): m/z ¼ found 695.5 [MþþNa]; calcd. 695.15[MþþNa]; HRMS calcd. forC28H36N10Se2 [MþþNa]: 695.1398, found 695.13601[MþþNa].

4.4.13. 4,40-Diselanediylbis(N-((1-cyclohexyl-1H-tetrazol-5-yl)(p-tolyl)methyl)aniline) (16)

Compound 16 was synthesized according to the general proce-dure I from 4,40-diselanediyldianiline (4) (344 mg, 1 mmol), 4-methylbenzaldehyde (1d) (264 mg, 2.2 mmol), trimethylsilylazide (3) (332 mg, 2.5 mmol) and isocyanocyclohexane (2b) (311 mL,2.5 mmol). Its formation was monitored by TLC petrol ether: ethylacetate ¼ 4:1, Rf ¼ 0.33, purified by column chromatography onsilica gel with petrol ether: ethyl acetate¼ 3:1. Yield: 724mg (85%).1H NMR (400 MHz, CDCl3) d 7.34e7.22 (m, 8H, AreH), 7.20e7.13 (m,4H, AreH), 6.54 (ddd, J ¼ 14.4, 8.6, 6.6 Hz, 4H, AreH), 5.88 (s, 2H,2CH), 5.20 (dd, J ¼ 14.6, 6.3 Hz, 2H, CH2), 4.33 (s, 2H, 2NH), 2.33 (s,6H, 2CH3), 2.11e1.89 (m, 6H, 3CH2), 1.82e1.68 (m, 6H, 3CH2),1.42e1.16 (m, 8H, 4CH2).13C NMR (101 MHz, CDCl3) d 154.23 (C-tetrazol), 146.09 (CeAr), 139.00 (CeAr), 136.17 (CHeAr), 135.97(CHeAr), 134.40 (CeAr), 129.99 (CHeAr), 127.19 (CHeAr), 119.65(CeAr), 114.09 (CHeAr), 58.38 (CH), 53.33 (CH), 32.64 (CH2-cyclo-hexyl), 25.33 (CH2-cyclohexyl), 25.21 (CH2-cyclohexyl), 21.12 (CH3-Ar). MS (ESI): m/z ¼ found 875.5 [MþþNa]; calcd. 875.24[MþþNa];HRMS calcd. for C42H48N10Se2 [MþþNa]: 875.2298, found875.22903[MþþNa].

4.4.14. 4,40-Diselanediylbis(N-(1-(1-cyclohexyl-1H-tetrazol-5-yl)-2-methylpropyl)aniline) (17)

Compound 17 was synthesized according to the general proce-dure I from 4,40-diselanediyldianiline (4) (344 mg, 1 mmol), iso-butyraldehyde (1b) (201 mL, 2.2 mmol), trimethylsilyl azide (3)(332 mg, 2.5 mmol) and isocyanocyclohexane (2b) (310 mL,2.5 mmol). Its formation was monitored by TLC petrol ether: ethylacetate ¼ 4:1, Rf ¼ 0.32, purified by column chromatography onsilica gel with petrol ether: ethyl acetate¼ 3:1. Yield: 605mg (80%).1H NMR (400 MHz, CDCl3) d 7.24 (ddd, J ¼ 29.9, 9.9, 4.1 Hz, 4H,AreH), 6.61e6.36 (m, 4H, AreH), 4.73e4.72 (m, 2H), 4.62e4.52 (m,2H), 4.43e4.35 (m, 2H), 2.29 (ddd, J ¼ 27.6, 14.0, 7.2 Hz, 2H, CH2),2.08e1.89 (m, 8H, 4CH2), 1.79 (dd, J ¼ 21.4, 10.6 Hz, 4H, 2CH2), 1.40(d, J ¼ 6.7 Hz, 6H, 2CH3), 1.15 (d, J ¼ 6.7 Hz, 6H, 2CH3), 0.94 (d,J ¼ 6.7 Hz, 4H, 2CH2), 0.87 (d, J ¼ 6.8 Hz, 2H, CH2).13C NMR(101 MHz, CDCl3) d 154.49 (C-tetrazol), 147.00 (CeAr), 136.15(CHeAr), 119.72 (CeAr), 114.08 (CHeAr), 70.64 (CH), 58.35 (CH),55.26 (CH), 33.39 (CH2-cyclohexyl), 33.07 (CH2-cyclohexyl), 25.33(CH2-cyclohexyl), 24.72 (CH2-cyclohexyl), 19.34 (CH3), 18.77 (CH3).MS (ESI): m/z ¼ found 755.0 [Mþ � 1]; calcd. 756.24[Mþ]; HRMScalcd. for C34H48N10Se2 [MþþNa]: 779.2298, found 779.22847[MþþNa].

4.4.15. 4,40-Diselanediylbis(N-(1-(1-benzyl-1H-tetrazol-5-yl)-2-methylpropyl)aniline) (18)

Compound 18 was synthesized according to the general proce-dure I from 4,40-diselanediyldianiline (4) (344 mg, 1 mmol), iso-butyraldehyde (1b) (201 mL, 2.2 mmol), trimethylsilyl azide (3)(332 mg, 2.5 mmol) and (isocyanomethyl)benzene (2d) (304 mL,2.5 mmol). Its formation was monitored by TLC petrol ether: ethylacetate ¼ 4:1, Rf ¼ 0.31, purified by column chromatography onsilica gel with petrol ether: ethyl acetate¼ 3:1. Yield: 633mg (82%).1H NMR (400MHz, CDCl3) d 7.40e7.29 (m, 6H, AreH), 7.28e7.09 (m,

S. Shaaban et al. / European Journal of Medicinal Chemistry 122 (2016) 55e71 69

8H, AreH), 6.27 (dd, J ¼ 8.5, 3.2 Hz, 4H, AreH), 5.74e5.47 (dd,J¼ 8.5, 3.2 Hz, 4H, 2CH2),4.60e4.46 (m, 2H, 2CH), 4.30 (d, J¼ 8.4 Hz,2H, 2CH), 2.11 (s, 2H, 2NH), 1.12 (d, J ¼ 6.7 Hz, 6H, 2CH3), 0.93 (d,J ¼ 6.7 Hz, 6H, 2CH3).13C NMR (101 MHz, CDCl3) d 155.64 (C-tet-razol), 146.70 (CeAr), 135.89 (CHeAr), 133.29 (CeAr), 129.21(CHeAr), 129.00 (CHeAr), 127.49 (CHeAr), 119.55 (CeAr), 113.99(CHeAr), 70.67 (CH), 54.87 (CH), 51.38 (CH2), 32.93 (CH-isopropyl),19.25 (CH3), 18.72 (CH3). MS (ESI): m/z ¼ found 795.3 [MþþNa];calcd. 795.18[MþþNa].

4.4.16. 4,40-Diselanediylbis(N-((1-(2,4-dimethoxybenzyl)-1H-tetrazol-5-yl)(p-tolyl)methyl)aniline) (19)

Compound 19 was synthesized according to the general proce-dure I from 4,40-diselanediyldianiline (4) (344 mg, 1 mmol), 4-methylbenzaldehyde (1d) (264 mg, 2.2 mmol), trimethylsilylazide (3) (332 mg, 2.5 mmol) and 1-(isocyanomethyl)-2,4-dimethoxybenzene) (2f) (443 mg, 2.5 mmol). Its formation wasmonitored by TLC petrol ether: ethyl acetate ¼ 4:1, Rf ¼ 0.36, pu-rified by column chromatography on silica gel with petrol ether:ethyl acetate ¼ 3:1. Yield: 870 mg (88%). 1H NMR (400 MHz, CDCl3)d 7.27e7.21 (m, 4H, AreH), 7.14 (dq, J ¼ 16.8, 8.4 Hz, 8H, AreH),7.06e7.00 (m, 2H, AreH), 6.47e6.40 (m, 4H, AreH), 6.38e6.29 (m,4H, AreH), 5.87 (s, 2H, 2CH), 5.42 (s, 4H, 2CH2), 4.99 (s, 2H, 2NH),3.80 (s, 6H, 2CH3), 3.75 (s, 6H, 2CH3), 2.31 (s, 6H, 2CH3).13C NMR(101 MHz, CDCl3) d 161.78 (OeCeAr), 157.77 (OeCeAr), 155.04 (C-tetrazol), 146.22 (CeAr), 138.73 (CeAr), 135.79 (CHeAr), 133.70(CeAr), 131.09 (CHeAr), 129.82 (CHeAr), 127.25 (CHeAr), 121.54(CHeAr), 119.32 (CeAr), 113.91 (CHeAr), 113.45 (CeAr), 104.89(CHeAr), 98.72 (CHeAr), 55.46 (CH), 55.46 (H3CeO), 52.34(H3CeO), 45.93 (CH2), 21.08 (CH3-Ar). MS (ESI): m/z ¼ found 989.8[Mþþ1]; calcd. 888.22[Mþ]; HRMS calcd. for C48H48N10O4Se2[MþþNa]: 1011.2098, found 1011.20776[MþþNa].

4.4.17. 2-((4-(((1-(tert-Butyl)-1H-tetrazol-5-yl)methyl)amino)phenyl)selanyl)-3-methylnaphthalene-1,4-dione (20)

Compound 20 was synthesized according to the general pro-cedure II from2-methyl-3-bromo-l,4-naphthoquinone (5)(552.2 mg, 2.2 mmol), compound 7 (620 mg, 1 mmol), NaBH4(189.15 mg, 5 mmol) and tricaprylmethylammonium chloride(45 mg, 5% mol). Its formation was monitored by TLC petrol ether:ethyl acetate ¼ 4:1, Rf ¼ 0.37, purified by column chromatographyon silica gel with petrol ether: ethyl acetate ¼ 3:1. Yield: 447 mg(93%). 1H NMR (400 MHz, CDCl3) d 8.09e8.01 (m, 2H, AreH),7.73e7.63 (m, 2H, AreH), 7.49e7.43 (m, 2H, AreH), 6.68e6.59 (m,2H, AreH), 4.93 (s, 1H, NH), 4.62 (s, 2H, CH2), 2.16 (s, 3H, CH3), 1.72(s, 9H, 3CH3).13C NMR (101 MHz, CDCl3) d 182.46 (C]O, quinone),181.96 (C]O, quinone), 151.55 (C-tetrazol), 148.45 (CeAr), 147.74(C-quinone), 146.86 (C-quinone), 136.29 (CHeAr), 133.35 (CHeAr),132.25 (C-quinone), 132.02 (C-quinone), 126.92 (CHeAr), 126.63(CHeAr), 116.86 (CeAr), 114.04 (CHeAr), 61.43 (C-t-butyl), 39.95(CH2), 29.74 (CH3-t-butyl), 17.23 (CH3-quinone). MS (ESI): m/z ¼ found 504.3 [MþþNa]; calcd. 504.10[MþþNa]; HRMS calcd. forC23H23N5O2Se [MþþNa]: 504.0898, found 504.0909[MþþNa].

4.4.18. 2-((4-(((1-(tert-Butyl)-1H-tetrazol-5-yl)(p-tolyl)methyl)amino)phenyl)selanyl)-3-methylnaphthalene-1,4-dione (21)

Compound 21 was synthesized according to the general proce-dure II from2-methyl-3-bromo-l,4-naphthoquinone (5) (552.2 mg,2.2 mmol), compound 8 (800 mg, 1 mmol), NaBH4 (189.15 mg,5 mmol) and tricaprylmethylammonium chloride (45 mg, 5% mol).Its formation was monitored by TLC petrol ether: ethylacetate ¼ 4:1, Rf ¼ 0.38, purified by column chromatography onsilica gel with petrol ether: ethyl acetate¼ 3:1. Yield: 548mg (96%).1H NMR (400 MHz, CDCl3) d 8.07e8.01 (m, 2H, AreH), 7.67 (ddd,J¼ 12.2, 5.7, 3.7 Hz, 2H, AreH), 7.38 (d, J¼ 8.6 Hz, 2H, AreH), 7.17 (q,

J¼ 8.2 Hz, 4H, AreH), 6.54 (d, J¼ 8.6 Hz, 2H, AreH), 6.05 (s, 1H, CH),2.32 (s, 3H, CH3), 2.12 (s, 3H, CH3), 1.68 (s, 9H).13C NMR (101 MHz,CDCl3) d 182.51 (C]O), 181.95 (C]O), 154.79 (C-tetrazol), 148.58(CeAr), 148.16 (C-quinone), 147.69 (CeAr), 146.02 (C-quinone),136.24 (CeAr), 133.53 (CeAr), 133.56 (CHeAr), 133.36 (CHeAr),132.25 (C-quinone), 132.05 (C-quinone), 129.88 (CHeAr), 129.22(CHeAr), 127.63 (CHeAr), 127.09 (CHeAr), 114.72 (CHeAr), 65.29(C-t-butyl), 54.05 (CH), 39.90 (CH3-t-butyl), 21.12 (CH3-Ar), 17.26(CH3-quinone).

4.4.19. 2-((4-(((1-(tert-Butyl)-1H-tetrazol-5-yl)(furan-2-yl)methyl)amino)phenyl)selanyl)-3-methylnaphthalene-1,4-dione (22)

Compound 22 was synthesized according to the general pro-cedure II from2-methyl-3-bromo-l,4-naphthoquinone (5)(552.2 mg, 2.2 mmol), compound 12 (752 mg, 1 mmol), NaBH4(189.15 mg, 5 mmol) and tricaprylmethylammonium chloride(45 mg, 5% mol). Its formation was monitored by TLC petrol ether:ethyl acetate ¼ 4:1, Rf ¼ 0.36, purified by column chromatographyon silica gel with petrol ether: ethyl acetate ¼ 3:1. Yield: 514 mg(94%). 1H NMR (400 MHz, CDCl3) d 8.11e7.99 (m, 2H, AreH),7.75e7.63 (m, 2H, AreH), 7.47e7.30 (m, 4H, AreH), 6.63 (dd,J ¼ 11.9, 8.7 Hz, 2H, AreH), 6.33 (s, 1H, CH), 6.22 (dd, J ¼ 7.5, 4.2 Hz,2H, AreH), 2.17 (s, 3H, CH3), 1.76 (s, 9H, 3CH3).13C NMR (101 MHz,CDCl3) d 182.51 (C]O, quinone), 181.93 (C]O, quinone), 153.26 (C-tetrazol), 150.58 (C-furan), 147.53 (CeAr), 146.11 (C-quinone),145.61 (CeAr), 142.94 (C-quinone), 136.21 (CHeAr), 133.60(CHeAr), 133.39 (C-quinone), 132.04 (C-quinone), 127.36 (CHeAr),126.96 (CHeAr), 126.60 (CHeAr), 114.94 (CHeAr), 110.95 (CHeAr),109.05 (CHeAr), 62.35 (C-t-butyl), 48.52 (CH), 30.92 (CH3-t-butyl),17.29 (CH3-quinone). MS (ESI): m/z ¼ found 570.2 [MþþNa]; calcd.570.11[MþþNa]; HRMS calcd. for C27H25N5O3Se [MþþNa]:570.0998, found 570.1015[MþþNa].

Acknowledgements

The authors thank the Egyptian Ministry of Higher Education,Deutscher Akademischer Austauschdienst (DAAD), Leibniz Instituteof Plant Biochemistry and Mansoura University for financialsupport.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.2016.06.005.

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