Synthesis, Structure and Antioxidant Activity of Cyclohexene-Fused Selenuranes and Related Derivatives

Synthesis, structure and antioxidant activity of new cyclohexene-fused spiroselenuranes and a spirotellurane is reported. Oxidation reactions of bis(o-formylcyclohex-1-ene)selenide/bis(2-hydroxymethylcyclohex-1-ene)selenide provide the corresponding spiroselenuranes. The glutathione peroxidase-like activity of the newly synthesized compounds has been evaluated.


Introduction
The biochemistry of selenium is of considerable current interest due to the discovery of selenocysteine in a number of enzymes. The enzymes include glutathione peroxidase [1,2], iodothyronine deiodinase [3][4][5], and thioredoxin reductase etc. [6,7]. Glutathione peroxidase (GPx) functions as an antioxidant and is responsible for the destruction of harmful peroxides in various living organisms. The enzyme's catalytic cycle involves selenol (Enz-SeH) as the active form that reduces hydroperoxides and becomes oxidized to the selenenic acid (ESeOH), which reacts with reduced glutathione (GSH) to form selenenyl sulfide adduct (ESeSG). A second molecule of glutathione then regenerates the active sites of the enzyme by OPEN ACCESS attacking the selenosulfide (ESeSG) to form the oxidized glutathione (GSSG). Thus, in the overall catalytic cycle, two equivalents of glutathione are oxidized to the disulfide and water, while the hydroperoxide is reduced to the corresponding alcohol (Scheme 1) [8,9]. Scheme 1. Catalytic mechanism of GPx enzyme.
Back and coworkers reported the synthesis of a series of substituted aromatic spirocycles. The methoxy-substituted selenurane (9f) proved the most effective catalyst for the reduction of hydrogen peroxide with benzyl thiol. Detailed GPx-like activity of related spirodiazaselenurane/ellurane (10)(11)(12)(13) have been evaluated by Back and coworkers [28] and Mugesh and coworkers [29,30]. Very recently, our group reported the synthesis and structure of bicyclic diacycloxy-and diazaselenuranes 14-15 and related compounds ( Figure 1) [31]. Although there are many reports on the synthesis, structural studies and GPx-like activities of aromatic spirodioxychalcogenuranes and spirodiazaselenurane/-tellurane, studies on the aliphatic analogues especially alicyclic are rare. In this paper, we report the synthesis, structural characterization of some new alicyclic spirodioxyselenuranes and a spirodioxytellurane.

Spectroscopy Studies
The compounds were characterized by common spectroscopic tools such as IR, 1 H-, 77 Se-NMR spectroscopy, mass spectrometry and single crystal X-ray diffraction studies (Supplementary Materials). The FT-IR spectrum of 19 exhibits the OH stretching frequency (C-OH) at 3298 cm −1 , indicating the presence of the alcohol group. The carbonyl stretching frequency of spirodioxyselenurane 20 and -tellurane 21 were observed at (νC=O) 1683 cm −1 1663 cm −1 respectively which are in good agreement with those reported for spirodioxyselenurane 6 (1695 cm −1 ) and 1,1′-spirobis(3H-2,1-benzoxatellurole)-3,3′-dione [34]. For compounds 19-21 the absence of the characteristic peak for the formyl group in 1 H-NMR spectra confirmed the reduction or oxidation of 17. This was further corroborated by single crystal X-ray analysis (vide infra). In the 1 H-NMR spectrum of compound 22, the methylene protons (-CH2-OH) are split. This is in contrast to the precursor 19, where these appeared as a singlet. It is due to the diastereotopic nature of the methylene protons. A similar pattern has been reported by Back and coworkers for 9a [35]. The 77 Se-NMR spectrum of compound 22 exhibits a signal at 908 ppm. This is significantly downfield as compared to 19 (325 ppm).

Molecular Structure of 19
The molecular structure of 19 is shown in Figure 2. The coordination geometry around the selenium atom is V-shaped with C1BA-Se-C1A bond angle being 99.10(1)°. A distinct feature of the structure is that both the hydroxyl groups are directed away from the selenium atom and there is asymmetry in the Se···OH bond distances. The Se···OH bond distances are 3.778 Å (Se-O1BA) and 4.486 Å (Se-O1A), respectively. A similar observation has been made in the case of the bis(o-formylphenyl) selenide having two formyl groups trans to the selenium [36]. Selvakumar et al. also found a similar situation in the case of a (5-(tert-butyl)-2-(phenylselanyl)-1,3-phenylene)dimethanol having two hydroxyl groups where none were coordinated to the selenium [31]. However, in di(2-hydroxybenzyl)selenide [22], out of the two OH groups, one OH group was coordinated to selenium with a bond distance of 3.008 Å. The lack of coordinating ability of the CH2OH group in 19 as well as in its aromatic analogue may be due to the poor electron donating property of the CH2OH groups and the availability of rotational degrees of freedom which could prevent the O···Se···O repulsive interaction. The compound shows hydrogen bonding interactions between the hydroxyl groups of the adjacent molecules ( Figure 3).  Interestingly, in case of 19, along with Se···OH intermolecular interaction, short C-H···Se intramolecular interactions were also observed. The short interatomic distances between selenium and one of the benzylic hydrogens (C-H···Se) are 2.710 and 2.704 Å which are shorter than the reported for diselenocin [37] (2.92 Å) and sum of the van der Walls radii (2.99 Å) [38]. The C-H···Se bond angles for 19 are 110.8° and 112.4° which are good agreement with diselenocin (101.7° and 107.0°). The solid state IR (KBr) spectrum indicates Se···H-C interaction. The C-H symmetric stretching frequency (2831 cm −1 ) is shifted towards the lower wave number as compared with normal methylene adjacent to the electronegative atom (υ = 2855 cm −1 ).

Glutathione Peroxidase-Like Activity
The glutathione peroxidase activity of the compounds was determined by the coupled reductase assay [46]. In this assay, the GPx-like activity was measured by a coupled enzyme containing nicotinamide adenine dinucleotide phosphate (NADPH) (0.01 M), glutathione (GSH) (0.05 M), catalysts (0.002 M), H2O2 (0.026 M), glutathione reductase (GR) (1.3 unit). The reduction of H2O2 by GSH was also recorded using ebselen for comparison. The decrease in NADPH concentration was monitored spectrophotometrically at 340 nm and results are summarized in Table 1. It was found that di-(o-formylcyclohex-1-ene)diselenide (16) is more efficient catalyst in comparison with ebselen, (2-phenyl-1,2-benzisoselenazol-3(2H)-one) [47], and bis(o-formylphenyl)diselenide [24]. The higher activity of 16 is probably due to the presence of stronger Se···O intramolecular interaction in comparison with bis(o-formylphenyl)diselenide. This stabilizes the selenenyl sulfide intermediate which in turn reacts with thiol to produce the disulfide. However, 17 and 19 do not show significant activity. The spirocyclic derivatives 20 and 22 where Se is in +4 oxidation state also did not show any significant activity. A similar result has been reported by Singh and co-workers [22] and Back and co-workers [23] in the case of aromatic spirocyclic derivatives. The reaction rates were similar to the control reaction rate. The GPx-like activity of compound 16 was calculated to be V0 = 49.8 ± 1.61 μM min −1 which is better than that of bis(o-formylphenyl)diselenide and standard ebselen (Table 1).

Mechanistic Studies on Di-(2-formylcyclohexenyl)diselenide 16
To understand the mechanism and identify the intermediates involved in the catalytic reaction, 77 Se-NMR spectroscopy has been used since its chemical shift is very sensitive to the environment. The three major intermediates involved in the peroxidase reduction, i.e., RSeH, RSeOH and RSeSPh, are expected to show large differences in 77 Se-NMR chemical shift values.

Reaction of 16 with PhSH Followed by TBHP
The reaction of di-(2-formylcyclohexenyl)diselenide 16 with 3 equivalents of PhSH (thiophenol) does not show any new peak corresponding to the formation of the expected selenenyl sulfide 24 even after 30 min. It indicates that the reactivity of PhSH towards 16 is very slow. The addition of tert-butylhydroperoxide (TBHP) to the above mixture results in the instantaneous formation of the selenenyl sulfide 24 (indicated by 77 Se-NMR signal at 705 ppm). The signal is shifted downfield in comparison to the reported value of diselenide 3 (545 ppm) [22]. This significant downfield shift is due to the existence of strong Se···O intramolecular interaction in selenenyl sulfide 24 which suggests that 24 is very stable in solution and is not disproportionated to the corresponding 16.
When the reaction mixture containing TBHP was treated with seven equivalents of PhSH, peaks corresponding to the intermediates selenenic acid 25, seleninic acid 26 and selenonic acid 27 disappeared. Two new peaks at 705 and 866 ppm along with peak for 16 were observed. The peaks at 705 and 866 ppm probably correspond to the selenenyl sulfide 24 and selenoxysulfide 28, respectively. Selenoxysulfide 28 is formed by the reaction of selenenic acid 25 with PhSH. The reaction of selenenic acid with PhSH is analogous to the observation by Mugesh et al. [17] where bis[2-(4,4-dimethyl-2oxazolinyl)phenyl]diselenide reacts rapidly with thiol to give corresponding selenoxysulfide intermediates. After the addition of 14 equivalents of PhSH, peaks corresponding to the 16 and other intermediates had disappeared except for the peak of selenenyl sulfide 24. Based on these observations and mechanisms reported for related diselenides, the following mechanism for the catalytic action of 16 (Scheme 3) is proposed.

General Information
All the organochalcogen derivatives were synthesized under nitrogen or argon atmosphere using standard Schlenk line techniques. Solvent were purified and dried by standard procedures and were freshly distilled prior to use [48]. All the chemicals used were reagent grade and were used as received. Melting points were recorded in capillary tubes. The NMR spectra were recorded in CDCl3 solvent.
The 1 H (400 MHz), 13 C (100 MHz), 77 Se (57.26/76.4 MHz) and 125 Te (126.3 MHz) spectra were recorded on a Varian Mercury plus or Bruker 400 MHz spectrometer. Chemical shifts cited were referenced with respect to TMS for ( 1 H and 13 C) as internal standard and Me2Se (for 77 Se), Me2Te (for 125 Te) as external standards. Elemental analysis was performed on Carlo-Erba model 1106 and Eager 300 EA112 elemental analyzers. The IR spectra were recorded in the range 400-4000 cm −1 by using KBr pellets for solid samples on a Thermo Nicolet Avatar 320 FT-IR spectrometer. Mass spectral (MS) studies were completed by using a QTOF Micro mass spectrometer with electrospray ionization mode analysis. In the case of isotopic patterns, the value is given for the most intense peak. The UV-VIS spectra in solution for GPx activity were recorded with a JASCO, V-570 spectrometer.
The single crystal X-ray diffraction measurements for compounds were performed on Oxford Diffraction Gemini diffraction measurement device with graphite monochromated Mo Kα radiation (λ = 0.7107 Å). The structures were determined by routine heavy-atom method using SHELXS 97 [49] and refined by full-matrix least-squares with the non-hydrogen atom anisotropic and hydrogen atoms with fixed isotropic thermal parameters of 0.07 Å by means of SHELXS 97 program [50]. The structure refinement parameters for compounds 19-21 are given in Table 2. CCDC-1041184 (19), CCDC-1041181 (20), CCDC-1041182 (21), contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

Coupled Reductase Assay
The GPx-like activities of 16-20 were measured by JASCO spectrophotometer according to the literature method using ebselen as the standard. The catalytic reaction was carried out at room temperature (25 °C) in 1 mL of the solution containing 100 mM potassium phosphate buffer, pH 7.5, 1 mM EDTA, 0.1 mM GSH, 0.25 mM of NADPH, 0.020 mM of catalyst and 0.26 mM of H2O2. The activity was followed by the decrease of NADPH on addition of H2O2 and absorption was measured at 340 nm (εmax = 6.22 × 10 3 M −1 cm −1 ) (chemical reactions (1)- (3)). Each of the experiments was carried out in triplicate.