Nitro-, Azo-, and Amino Derivatives of Ebselen: Synthesis, Structure, and Cytoprotective Effects

Article abstract of DOI:10.1021/acs.joc.6b02418

Novel azo-bis-ebselen compounds 7 were prepared by reduction of 7-nitro-2-aryl-1,2-benzisoselenazol-3(2H)-ones 3 and 6 with sodium benzenetellurolate, NaTeC₆H₅, and by reaction of 2-bromo-3-nitrobenzamides with Na₂Se₂

Full text of DOI:10.1021/acs.joc.6b02418

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Article
Nitro-, Azo- and Amino Derivatives of Ebselen:
Synthesis, Structure and Cytoprotective Effects
Vijay Pal Singh, Jia-fei Poon, Jiajie Yan, Xi Lu, Marjam
Karlsson Ott, Ray J. Butcher, Paul J. Gates, and Lars Engman
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b02418 • Publication Date (Web): 02 Dec 2016
Downloaded from http://pubs.acs.org on December 2, 2016
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Nitro-, Azo- and Amino Derivatives of Ebselen: Synthesis, Structure and
Cytoprotective Effects
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Vijay P. Singh,*,† Jiaꢀfei Poon,† Jiajie Yan,† Xi Lu,‡ Marjam Karlsson Ott,‡ Ray J. Butcher,§ Paul J.
Gates¶ and Lars Engman*,†
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†Department of Chemistry – BMC, Uppsala University, Box 576, SEꢀ751 23 Uppsala, Sweden
‡Division of Applied Materials Science, Department of Engineering Sciences, Uppsala University,
Sweden
§Department of Chemistry, Howard University, Washington D.C. 20059, United States
¶University of Bristol, School of Chemistry, Bristol, BS8 1TS, United Kingdom
Abstract: Novel azoꢀbisꢀebselen compounds 7 were prepared by reduction of 7ꢀnitroꢀ2ꢀarylꢀ1,2ꢀ
benzisoselenazolꢀ3(2H)ꢀones 3 and 6 with sodium benzenetellurolate, NaTeC6H5, and by reaction of
2ꢀbromoꢀ3ꢀnitrobenzamides with Na2Se2. The Xꢀray structure of 7b showed that the molecule, due to
strong intramolecular secondary Se···N interactions, is completely planar. Azoꢀcompounds 7 upon
further reaction with NaTeC6H5 were reductively cleaved to provide two equivalents of the
corresponding aromatic amine. The weak Se─N bond was not stable enough to survive the reaction
conditions and diselenides 8 were isolated after workꢀup. Whereas azoꢀbisꢀebselens 7 were poor
mimics of the glutathione peroxidase (GPx)ꢀenzymes, nitroebselens 3, 6 and 11b and diselenides 8
were 3ꢀ6 fold more active than ebselen. Based on 77Se NMR spectroscopy, a catalytic cycle for
diselenide 8b, involving aminoebselen 14, was proposed. As assessed by chemiluminescence
measurements, the good GPxꢀmimics could reduce production of reactive oxygen species (ROS) in
stimulated human mononuclear cells more efficiently than Trolox. No toxic effects of the compounds
were seen in MC3T3ꢀcells at 25 ꢀM.
Introduction
Selenocysteine (Sec, 1) is recognized as the 21st proteinogenic amino acid. It is an essential part of
the active site of selenoproteins in humans and animals. So far, 23 selenoprotein families have been
annoted.1 The human selenoproteome consists of 25 selenoproteins.2 Whereas some of these have a
known structure and function (GPx, iodothyronine deiodinases, thioredoxin reductases)3,4 others are
as yet insufficiently characterized. The substitution of selenium for sulfur in cysteine (Cys) has many
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implications.5 At physiological pH, the more acidic selenol (pKa = 5.43)6 is essentially deprotonated.
Sec is therefore a more reactive nucleophile than Cys. Also, redoxꢀcycling (oxidation/reduction)
occurs more readily in Sec than in Cys. In addition to catalase, the GPxꢀenzymes are the most
important hydroperoxideꢀdecomposing enzymes in humans.7 Gluthathione (GSH) is used as the
stoichiometric reducing agent (eq 1). In the proposed catalytic cycle for the action of the GPxꢀ
enzymes selenium is present in the form of selenol, selenenic acid and selenosulfide.8
The reports on the existence of GPx triggered a search for smallꢀmolecule compounds that could
mimic the action of the large enzyme. The benzisoselenazolone ebselen (2) was the first compound
of this kind.9 Since the mid 1980s, the suitability of ebselen as a pharmaceutical agent has been
extensively probed. Due to its GPxꢀactivity, it has been found to reduce oxidative stress.10 It is/has
been used in clinical trials for the prevention or treatment of cardiovascular diseases, arthritis, stroke
and cancer.11 Considered as a safe drugꢀlike compound with a history of use in human clinical trials,
ebselen has also been included in the National Institutes of Health Clinical Collection.12 Ebselen has
been subjected to numerous structural modifications in order to improve its GPxꢀactivity.13 An early
study by Parnham and coꢀworkers showed that the nitroꢀderivative 3 of ebselen was 9ꢀfold better
than the parent as a catalyst for the glutathioneꢀinduced reduction of tꢀBuOOH.14 However, this
finding has not been much explored and the reason for the rate enhancement has been attributed both
to electronic15 and steric16 effects.
We thought it would be interesting to prepare benzisoselenazolones carrying a variety of Nꢀ
substituents (nitroꢀ, azoꢀ and amino groups) in position 7. In the following we describe their
synthesis and structure as well as their GPxꢀlike activities and cytoprotective effects.
Results and Discussion
Synthesis: We envisaged obtaining azoꢀ and amino derivatives of ebselen by reduction of the
corresponding nitro compounds. Nitroꢀsubstituted ebselens were prepared using a slightly modified17
version of the procedure developed by Christians and coꢀworkers.18 The required benzamides 4 a-c
(eq 2) for this reaction were obtained in high yields by addition of aniline, paraꢀtoluidine and paraꢀ
anisidine,
respectively, to 2ꢀbromoꢀ3ꢀnitrobensoyl chloride. Their conversion to
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benzisoselenazolones 3/6b-c involved aromatic nucleophilic substitution with nBuSeNa, generated in
situ from Bu2Se2 and NaBH4 in ethanol, followed by bromine induced cyclization.
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Reduction of the nitro group in the presence of the weak SeꢀN bond turned out to be difficult.
Sodium benzenetellurolate, NaTePh,19 generated in ethanol by sodium borohydride reduction of
diphenyl ditelluride (Ph2Te2), was found to cleanly reduce compound 3 to azoꢀbisꢀebselen derivative
7a in 51% yield. However, in the case of 6b, the corresponding azoꢀderivative 7b was isolated as the
minor product (9%) along with diselenide 8b (16%). Obviously, in 8b, the nitro group has been
reduced all the way to an amine and the SeꢀN bond cleaved reductively. Oxidation of the resulting
selenol then provided the diselenide product. Reduction of azoꢀcompound 7b with NaTePh produced
diselenide 8b as the only product (87%).
An alternative method for the preparation of azoꢀderivatives of ebselen was also tried. This is based
on the finding that 2ꢀbromoꢀ3ꢀnitrobenzylic alcohols, when reacted with disodium diselenide
(Na2Se2) produced azoꢀderivatives 9 of 2ꢀoxaselenaindane.20 Reaction of in situꢀprepared Na2Se2
with compounds 4a-d at ambient temperature produced the corresponding azoꢀbisꢀebselens 7a
(29%), 7b (39%), 7c (21% when heated at reflux) and 7d (68%) (eq 3). Due to poor solubility, we
were unable to characterize the methoxy derivative 7c.
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Further reaction of compound 7d with NaTePh afforded diselenide 8d in 52% yield. Under the
similar reaction conditions, benzamides 5a-c, carrying a butylseleno group in position 2, were
cleanly reduced to the corresponding amines 10a-c in high yields. We recently reported on the
cytoprotective effects of the radicalꢀtrapping and hydroperoxideꢀdecomposing ebselenol 11a.21
Curious about the effects of an orthoꢀcoordinating nitro group, compound 6c was Oꢀdemethylated
with 3 equivalents of BBr3 in CH2Cl2 to afford the nitroebselenol 11b in 88% yield.
Structure: The structures of azoꢀbisꢀebselen derivative 7b and diselenide 8b were determined by Xꢀ
ray crystallography. Dark black crystals of 7b suitable for Xꢀray crystallographic analysis were
o
obtained by slow evaporation of a CHCl3ꢀsolution at ꢀ20 C. The structure (Figure 1) shows strong
intramolecular secondary Se···N interactions [Se1···N1 2.442(5) Å] notably shorter than the sum of
the van der Waals radii of Se and N (3.45 Å).22 Normally, the bond length of a Se─N covalent bond
is 1.87 Å. The strong secondary Se···N interactions causes a slight elongation of this bond (Se1─N2
= 1.940(3) Å). The coordination geometry is strongly distorted from a linear arrangement with the
bond angle N2─Se1─N1 = 156.56(13)o, thus, indicating a Tꢀshaped geometry around the Se atom.
Because of these strong interactions, compound 7b adopts a completely planar conformation. The
crystal structure of 7b showed πꢀπ stacking interactions between the benzene ring with a centroidꢀ
centroid distance of 3.711 Å (Figure S3 in the Supporting Information).
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Figure 1. ORTEP diagram (up) top view and (down) side view of 7b. Hydrogen atoms and solvatedꢀ
CHCl3 molecules are omitted for clarity. Thermal ellipsoids are set at 50% probability. Significant
bond lengths [Å] Se1···N1 2.442(5); Se1─C1 1.851(4); Se1─N2 1.940(3), and angles [o]
N2─Se1─C1 84.04(16); N2─Se1···N1 156.56(13); C1─Se1─N1 72.55(15).
Suitable orange crystals of diselenide 8b were obtained for Xꢀray crystallographic analysis by slow
evaporation of an ethyl acetate/pentane solution at room temperature. The structure (Figure 2)
indicates a Vꢀshaped geometry around the selenium atom with bond angles C1a─Se1─Se2 and
C1b─Se2─Se1 of 104.26(5)o and 103.65(5)o, respectively. The C1a─Se1─Se2─C1b dihedral angle
of 98.47(7)o indicates a “transoid” conformation. As indicated in Figure 2, selenium is hydrogen
bonded to the –NH2 group in the ortho position. The Se⋅⋅⋅H distance of 2.734 Å is significantly
shorter than the sum of the van der Waals radii of selenium and hydrogen (3.10 Å).22
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Figure 2. ORTEP diagram of 8b. Hydrogen atoms are omitted for clarity. Thermal ellipsoids are set
at 50% probability. Significant bond lengths [Å] Se1─C1a 1.9171(15); Se1─Se2 2.3602(2);
Se2─C1b 1.9156(15), and angles [o] C1a─Se1─Se2 104.26(5); C1b─Se2─Se1 103.65(5);
C1a─Se1─Se2─C1b 98.47(7).
Bonding: To find out more about the effect of intramolecular secondary Se···N/O interactions on
bonding and 77Se NMR chemical shifts, density functional theory (DFT) calculations were carried
out. The geometries of 6b, 6c, 7a, 7b, 7d, and 11b were fully optimized in the gas phase at the
B3LYP/6ꢀ311+G(d) level of theory (for optimized geometries and coordinates see the Supporting
Information). The optimized geometry of 7b showed good agreement with the Xꢀray crystal
structure. The structures of 7a and 7d were also found to be completely planar with strong Se···N
interactions. The second perturbation energies ESe···N and ESe···O shown in Table 1 were obtained by
NBO analysis. The values reflect the effectiveness of orbital interactions between the lowꢀlaying
σ*Se–N and nN/nO. The magnitude of the nN→σ*Se–N and nO→σ*Se–N orbital interactions suggests a
significant covalent interaction between the nitrogen lone pair and the lowꢀlying antibonding orbital
of the Se atom. The NBO results indicate that the Se···N interactions were much stronger than the
Se···O interactions. The calculated 77Se NMR chemical shifts were in good agreement with the
experimental values.
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Table 1. Data for 3, 6b-c, 7a-b, 7d, and 11b obtained by DFT calculations at the B3LYP/6ꢀ
311+G(d,p) level in the gas phase. The NBO analysis was calculated at the B3LYP/6ꢀ311+G(d,p)
level using the B3LYP/6ꢀ311+G(d)ꢀlevelꢀoptimized geometries.
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NPA Charges
77Se NMR
Secondꢀorder perturbation energy
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Compounds rSe···O/N [Å]
ESe···O/N
(kcal/mol)
2.593 (2.573) 12.63
r
Se─N [Å]
qSe
(in ppm)
317,23
6b
1.924(1.896) +0.753
910 (924)
905 (929)
910 (932)
904 (931)
903 (927)
925 (946)
912 (923)
2.598
2.604
2.532
12.35
12.00
17.32
1.922
1.921
1.946
+0.753
+0.750
+0.734
6c
7a
2.537 (2.442) 16.89
1.944 (1.941) +0.733
7b
2.539
2.601
16.57
12.22
1.943
1.922
+0.732
+0.751
7d
11b
[a] The 77Se NMR values are referenced to Me2Se (δ = 0). The experimental values are given in parentheses.
The topology of the electron density at the Se···N/O bond critical point (BCP) was evaluated
according to Bader’s theory of atoms in molecules (AIM) using the AIM2000 software package (for
molecular graphs, see Figure S6 in the Supporting Information). Parameters were calculated in order
to support the NBO calculations in the form of electron density (ρSe···N/O), Laplacian (∇2ρSe⋅⋅⋅N/O) and
the total electron energy density (HSe⋅⋅⋅N/O) for the Se···N/O interactions (Tables S19 and S20 in the
Supporting Information). Whereas compounds 7a-b and 7d showed negative values for HSe⋅⋅⋅N, HSe⋅⋅⋅O
for compounds 6b-c and 11b were positive, suggesting weaker electrostatic interactions between Se
and O atoms.
Glutathione Peroxidaseꢀlike Activity: The GPxꢀlike activity of antioxidants 2ꢀ3, 6b-c, 7a-b, 7d, 8b,
8d and 11b was assessed by the coupled reductase assay, using H2O2 as a substrate and GSH as a
thiol cofactor in the presence of glutathione reductase (GR). GR serves to reduce oxidized
glutathione (GSSG) formed by the action of the GPxꢀcatalyst on H2O2/GSH, using βꢀnicotinamide
adenine dinucleotide phosphate (NADPH) as a cofactor. GPxꢀactivities were assessed as the initial
rates (ν0) for the consumption of NADPH by UVꢀspectroscopy at 340 nm during the initial 10
seconds of reaction. The values shown in Table 2 were corrected for the spontaneous oxidation of
GSH by H2O2 (24.3 ± 1.2 µM⋅minꢀ1). Ebselen (46.5 ± 0.8 µM⋅minꢀ1) and 3 (111.4 ± 4.6 µM⋅minꢀ1)
were included as benchꢀmarks and references in the study. Nitroebselens 6b, 6c and 11b were found
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to be nearly three times more active than ebselen (2), and slightly better than 3. This enhanced
activity of the nitro compounds can probably be ascribed to intramolecular secondary Se···O
interactions. Such or similar effects are well described in the literature.14,17,20b,24 Azoebselens 7a, 7b
and 7d turned out to be considerably poorer GPxꢀmimics than 2 and 3. Interestingly, diselenide 8b
showed an activity (271.6 ± 5.7 µM⋅minꢀ1) almost sixꢀfold larger than recorded for ebselen. The other
diselenide 8d, carrying a bulky naphthyl group, also showed good GPxꢀactivity (133.8 ± 2.7
µM⋅minꢀ1).
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Table 2. GPxꢀlike activities of ebselen, 3, 6b-c, 7a-b, 7d, 8b, 8d and 11b as determined by the initial
rate of NADPH consumption (ν0) in the presence of H2O2, GSH and glutathione reductase
GPxꢀlike activity, ν0 (µM⋅minꢀ1)
Activity relative to
Antioxidants
ebselen
1
2, ebselen
46.5 ± 0.8
111.4 ± 4.6
135.2 ± 4.1
137.4 ± 2.3
29.5 ± 0.6
2.3 ± 0.1
2.4
3
2.9
6b
6c
7a
7b
7d
8b
8d
11b
3.0
0.6
0.05
0.2
9.7 ± 0.2
5.8
271.6 ± 5.7
133.8 ± 2.7
123.9 ± 0.7
2.9
2.7
[a] Assay conditions: Phosphate buffer (100 mM), pH 7.5 with ethylenediaminetetraacetic acid
(EDTA; 1 mM), GSH (1 mM), NADPH (0.2 mM), GR (1.3 unit⋅mLꢀ1), H2O2 (0.80 mM) and
catalysts (20 ꢀM). Stock solutions (2 mM) of catalysts 2, 3 and 6a-b were prepared in MeOH and
stock solutions of catalysts 7, 8 and 11b were prepared in DMSO. Initial rates (vo) were corrected for
the spontaneous oxidation of GSH (24.3 ± 1.2 µM⋅minꢀ1). Errors correspond to ± SD for triplicates.
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Consumption of Hydrogen Peroxide: The performance of some of the best GPxꢀmimics 8b, 8d and
11b (10 mol%) together with GSH, NADPH, GR and H2O2 in phosphate buffer was also followed
for much longer time (250 min). As shown in Figure 3, a significant decrease in the absorbance of
NADPH was seen during ca. 80 min. Thereafter, the consumption of NADPH corresponds more or
less to the background reaction. Ebselen (2), included as a reference in Figure 3, could not match the
activity of 8b, 8d and 11b.
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Figure 3. NADPHꢀconsumption with time in the absence (control) and presence of catalysts 2
(ebselen), 8b, 8d and 11b. A stock solution of catalyst 2 was prepared in MeOH and stock solutions
of catalysts 8b, 8d and 11b were prepared in DMSO. Assay conditions: reactions were carried out
with phosphate buffer (100 mM), pH 7.5, with EDTA (1 mM), GSH (0.10 mM), NADPH (0.20
mM), GR (1.3 unit⋅mLꢀ1), H2O2 (20 ꢀM) and catalyst (2 ꢀM).
Mechanistic studies: In order to obtain some mechanistic insight, 77Se NMR experiments in DMSOꢀ
d6 were carried out with diselenide 8b (δ = 369 ppm). Addition of 1 equivalent of H2O2 (Scheme 1)
did not cause much of a change in the spectrum (see the 77Se NMR spectrum in the Supporting
Information). Upon addition of another equivalent of H2O2 to the above mixture, one new signal
appeared at δ = 1156 ppm, corresponding to seleninic acid 12 (or a dehydrated product thereof ꢀ an
Seꢀoxide of aminoebselen). Now, 1 equivalent of GSH was added to the mixture produced
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containing unreacted 8b and 12. This caused the disappearance of the peak at δ = 1156 and the
appearance of two new peaks at δ = 935 ppm and δ = 344 ppm, corresponding to amino derivative 14
of ebselen and selenol 16, respectively. Presumably, compound 14 is formed by cyclization of an
intermediate selenenic acid 13. 17,24,25 Further addition of 2 equivalents of GSH to the mixture caused
the disappearance of the peaks corresponding to 12, 14 and 16 and only the peak at 369 ppm,
corresponding to diselenide 8b, remained in the 77Se NMR spectrum. When GSH (1 equivalent) was
added to a solution of 8b in DMSOꢀd6, the peak for 16 at δ = 346 ppm appeared.
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Scheme 1. Proposed mechanism for the reduction of H2O2 in the presence of GSH and diselenide 8b
It is not surprising that the peak from selenol 16 is shifted only 25 ppm upfield from that of
diselenide 8b. This is due to strong intramolecular secondary Se···O interactions with the carbonyl
group and additional substituent effects from the aromatic rings. A similar downfield chemical shift
of 232 ppm for the corresponding selenol derived from ebselen was also accounted for in terms of
strong Se···O interactions.24b In none of the above experiments did we see a peak corresponding to
selenosulfide 15. It is probably a highly reactive species. We speculate that hydrogen bonding of Se
to the orthoꢀamino group facilitates attack by thiol.
Cytoprotective effects: An overproduction of ROS/reactive nitrogen species (RNS) in biological
systems has many deleterious effects. For example, oxidative damage of DNA is directly linked to
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cardiovascular and neurodegenerative diseases.26,27 Since many of the novel antioxidants showed
excellent hydroperoxideꢀdecomposing activities, we decided to test their protective effects in cellular
systems. Freshly isolated human mononuclear cells (MNC) were stimulated with phorbol myristate
acetate (PMA) to produce ROS/RNS in the presence of antioxidants 3, 6b-c, 7a-b, 7d, 8b, 8d, 11b,
ebselen (2) and Trolox at 25 ꢀM. The total chemiluminescence (CL; extraꢀ and intracellular) was
then recorded in a luminol amplified assay. As shown in Figure 4, CL was significantly reduced for
all compounds tested. Good GPxꢀmimics such as nitro compounds 6b, 6c and 11b and diselenide 8b
all afforded better cytprotection than Trolox. Nitroebselenol 11b was by far the most potent
protective agent.
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Figure 4. Chemiluminescence (normalized to positive control) monitored at λ = 425 nm in PMAꢀ
stimulated MNCꢀcells exposed to 25 µM of antioxidants Ebselen (2), 3, 6b-c, 7a-b, 7d, 8b, 8d, 11b
and Trolox. N = 3 to 6 for each group from 4 independent experiments.
Cell Viability: In order to reveal any toxicity of antioxidants 3, 6b-c, 7a-b, 7d, 8b, 8d and 11b,
MC3T3ꢀcells (a preosteoblast cell line) were exposed to 25 µM of compound and cell viability
checked after 1 and 3 days by using the Alamar Blue assay (Figure 5). No toxic effects of the
compounds were seen after 3 days.
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Figure 5. Relative cell viability of MC3T3 cells in the presence of 25 µM of antioxidants 3, 6b-c,
7a-b, 7d, 8b, 8d and 11b as determined by Alamar Blue measurements after 1 and 3 days at λem
=
590 nm.
Conclusion
In search for novel ebselen derivatives with improved antioxidative properties we have
described straightforward syntheses of azoꢀbisꢀebselens from nitroebselens and 2ꢀbromoꢀ3ꢀ
nitrobenzamides. Reduction of the azoꢀgroup to the corresponding amine was effected by NaTePh,
but this transformation was accompanied by cleavage of the SeꢀN bond, resulting in formation of
diaryl diselenides carrying both aminoꢀ and benzamide groups in the ortho positions. As revealed by
Xꢀray crystallography and DFTꢀcalculations, the intramolecular Se···Nꢀcoordination in azoꢀbisꢀ
ebselens was much stronger than the corresponding Se···Oꢀinteraction in nitroebselens. This may be
the reason for the poor GPxꢀactivity of the azoꢀbisꢀebselens. Both nitroebselens 6 and diselenides 8
outperformed ebselen when it comes to catalysis of GSHꢀinduced reduction of hydrogen peroxide.
Considering their low toxicity and ability to inhibit ROS/RNS in stimulated human MNCꢀcells, we
feel that compounds of this sort would be useful as models and tools in the development of novel
treatments for diseases with a component of oxidative stress (cardiovascular diseases, stroke,
Alzheimer’s and Parkinson’s diseases).
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Experimental Section
2ꢀBromoꢀ3ꢀnitrobenzoic acid (98% purity) was purchased and used as such. 1H and 13C NMR spectra
for all compounds prepared were recorded on 300 MHz (1H: 300 MHz; 13C: 75 MHz) and 400 MHz
(1H: 399.97 MHz; 13C: 100 MHz) spectrometers, using the residual solvent peaks of CDCl3 (1H: δ
7.26; 13C: δ 77.2), CD3OD (1H: δ 3.31; 13C: δ 49.0) and DMSOꢀd6 (1H: δ 2.50; 13C: δ 39.5), as
indirect references to TMS (δ = 0 ppm). 77Se NMR spectra were recorded on 300 MHz (77Se: 57
MHz) and 400 MHz (77Se: 76 MHz) spectrometers with Ph2Se2 (δ = 460 ppm) as an indirect
reference to Me2Se (δ = 0 ppm). Flash column chromatography was performed using silica gel (0.04ꢀ
0.06 mm). Melting points are uncorrected. The high resolution mass spectra (HRMS) were obtained
using a time of flight (TOF) instrument equipped with electrospray ionization (ESI) and electron
impact (EI+) operating in the positive ion mode. Tetrahydrofuran (THF) was dried in a solvent
purification system by passing it through an activated alumina column before use.
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Typical procedure for benzamide formation: 2-Bromo-3-nitro-N-phenylbenzamide (4a). A
mixture of 2ꢀbromoꢀ3ꢀnitrobenzoic acid (1.0 g, 4.06 mmol) and thionyl chloride (5 mL) was refluxed
for 3 h in the presence of a catalytic amount of N,Nꢀdimethyl formamide (DMF). Excess thionyl
chloride was then removed under reduced pressure. The residue was dissolved in dichloromethane
(10 mL). Aniline (0.74 g, 8.13 mmol) in CH2Cl2 (15 mL) was added dropwise at room temperature.
The resulting solution was then stirred for overnight. The reaction mixture was poured in water (10
mL) and extracted with dichloromethane. The separated organic layers were combined, dried over
anhydrous Na2SO4 and the solvent was evaporated under reduced pressure. The crude residue was
purified by column chromatography on silica gel using ethyl acetate/nꢀpentane (1:1) as an eluent to
give the title product (1.13 g, 87%) as a solid. Physical and spectroscopic data were in good
1
agreement with the literature.17 The H and 13C NMR spectra of 4a have been included in the
Supporting Information.
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2-Bromo-3-nitro-N-p-tolylbenzamide (4b). White solid. Yield: 1.30 g (96%); Mp 166ꢀ168 C; H
NMR (CDCl3): 2.36 (s, 3H), 7.18 (d, J = 8.2 Hz, 2H), 7.48 (d, J = 8.5 Hz, 2H), 7.53 (t, J = 7.8 Hz,
1H), 7.62 (br s, 1H), 7.71 (dd, J = 1.7, 7.7 Hz, 1H), 7.76 (dd, J = 1.6, 7.9 Hz, 1H); 13C NMR
(CDCl3): 21.1, 111.6, 120.5, 126.2, 128.9, 129.9, 131.9, 134.6, 135.4, 141.5, 151.3, 164.2; HRMS
δ
δ
(TOF MS ES+) m/z calcd for C14H11N2O3Br [M+H]+: 335.0031; found: 335.0035.
2-Bromo-N-(4-methoxyphenyl)-3-nitrobenzamide (4c). The crude residue was purified by
chromatography on silica gel with 60% ethyl acetate/pentane as an eluent to afford the title
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compound as a white solid. Yield: 1.34 g (94%); Mp 180ꢀ182 oC; 1H NMR (CD3OD):
δ 3.80 (s, 3H),
6.93 (dd, J = 2.2, 6.9 Hz, 2H), 7.57 (dd, J = 2.2, 6.9 Hz, 2H), 7.66 (t, J = 7.8 Hz, 1H), 7.74 (dd, J =
1.8, 7.8 Hz, 1H), 7.89 (dd, J = 1.7, 7.9 Hz, 1H); 13C NMR (CD3OD):
55.9, 112.0, 115.1, 123.2,
126.6, 130.2, 132.3, 143.2, 152.8, 158.5, 166.9; HRMS (TOF MS ES+) m/z calcd for C14H11N2O4Br
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8
δ
9
[M+H]+: 350.9980; found: 350.9975.
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2-Bromo-N-(1-naphthyl)-3-nitrobenzamide (4d). The crude residue was washed with pentane to
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obtain the title compound of sufficient purity. Yield: 0.89 g (59%); Mp 243ꢀ246 C; H NMR
(DMSOꢀd6): 7.56ꢀ7.59 (several peaks, 3H), 7.80 (m, 3H), 7.89 (d, J = 8.0 Hz, 1H), 7.97ꢀ8.00
(several peaks, 2H), 8.10 (dd, J = 1.2, 6.8 Hz, 1H), 8.20 (t, J = 7.2 Hz, 1H), 10.75 (s, 1H); 13C NMR
(DMSOꢀd6): 110.5, 122.6, 122.9, 125.3, 125.6, 126.2, 126.3, 126.4, 128.2 (2C), 129.5, 131.8,
δ
δ
132.7, 133.8, 141.9, 150.9, 165.6; HRMS (TOF MS EI+) m/z calcd for C17H11N2O3Br [M]+:
369.9953; found: 369.9959.
Typical procedure for the introduction of a butylseleno group by nucleophilic aromatic
substitution: 2-Butylseleno-3-nitro-N-phenylbenzamide (5a). Benzamide 4a (0.75 g, 2.33 mmol)
was added to an in situꢀprepared solution of BuSeNa (4 equivalents) in EtOH (30 mL) at 0 oC under
inert atmosphere. The mixture was stirred at room temperature for 2 h and then warmed at 50 oC for
20 h. The solvent was removed under reduced pressure and the residue dissolved in CHCl3 and
washed with water. After drying over anhydrous Na2SO4, evaporation of the solvent and column
chromatography using 20% ethyl acetate/pentane as eluent, the pure title compound (0.51 g, 55%)
1
was isolated. Physical and spectroscopic data were in good agreement with the literature.17 The H,
13C and 77Se NMR spectra of 5a have been included in the Supporting Information.
2-Butylseleno-3-nitro-N-p-tolylbenzamide (5b). Orange liquid (semiꢀsolid). Solidified in the
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freezer. Yield: 0.59 g (67%); Mp 69ꢀ73 C; 1H NMR (CDCl3):
δ 0.78 (t, J = 7.3 Hz, 3H), 1.22ꢀ1.29
(several peaks, 2H), 1.45ꢀ1.55 (several peaks, 2H), 2.36 (s, 3H), 2.87 (t, J = 7.5 Hz, 2H), 7.20 (d, J =
8.2 Hz, 2H), 7.49ꢀ7.55 (several peaks, 3H), 7.83 (dd, J = 1.3, 7.9 Hz, 1H), 7.91 (dd, J = 1.4, 7.6 Hz,
1H), 8.24 (br s, 1H); 13C NMR (CDCl3):
δ 13.5, 21.1, 22.7, 31.1, 31.9, 120.0, 122.4, 125.5, 128.9,
129.9, 133.4, 135.0, 135.1, 142.9, 155.1, 165.2; 77Se NMR (CDCl3):
δ
283; HRMS (TOF MS ES+)
m/z calcd for C18H20N2O3Se [M+H]+: 393.0717; found: 393.0721.
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2-Butylseleno-N-(4-methoxyphenyl)-3-nitrobenzamide (5c). Yellow solid. Yield: 0.50 g (56%);
Mp 94ꢀ97 oC; 1H NMR (CDCl3):
δ 0.78 (t, J = 7.3 Hz, 3H), 1.26 (m, 2H), 1.51 (m, 2H), 2.88 (t, J =
6
7
8
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7.5 Hz, 2H), 3.82 (s, 3H), 6.93 (dd, J = 2.2, 7.7 Hz, 2H), 7.51 (t, J = 7.8 Hz, 1H), 7.57 (dd, J = 2.2,
5.4 Hz, 2H), 7.82 (dd, J = 1.5, 8.0 Hz, 1H), 7.90 (dd, J = 1.5, 7.6 Hz, 1H), 8.22 (br s, 1H); 13C NMR
(CDCl3):
δ 13.5, 22.7, 31.0, 31.9, 55.6, 114.5, 121.7, 122.3, 125.3, 128.8, 130.7, 133.1, 143.0, 155.0,
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157.0, 165.2; 77Se NMR (CDCl3):
[M+H]+: 409.0667; found: 409.0663.
δ
283; HRMS (TOF MS ES+) m/z calcd for C18H20N2O4Se
Typical procedure for the synthesis of nitroebselen analogues: 7-Nitro-2-phenyl-1,2-
benzisoselenazol-3(2H)-one (3). To a stirred solution of selenide 5a (0.72 g, 1.84 mmol,) in dry
CHCl3 (10 mL) was added Br2 (0.10 ml, 1.84 mmol) in dry CHCl3 (5 mL) containing Et3N (0.257
o
ml, 1.84 mmol) at 0 C under an inert atmosphere. The reaction mixture was then stirred for 3 h at
room temperature and water (10 mL) was added. The separated organic layer was dried over
anhydrous Na2SO4. Removal of the solvent and purification of the residue by silica gel column
chromatography (elution with 25% ethyl acetate/nꢀpentane) afforded the title compound (0.58 g,
95%) as a red solid. Physical and spectroscopic data were in good agreement with the literature.17
The 1H, 13C and 77Se NMR spectra of 3 have been included in the Supporting Information.
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7-Nitro-2-p-tolyl-1,2-benzisoselenazol-3(2H)-one (6b). Yield: 0.50 g (82%); Mp 184 C; H NMR
(CDCl3): 2.39 (s, 3H), 7.27 (d, J = 7.9 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H), 7.72 (t, J = 7.8 Hz, 1H),
8.46 (dd, J = 0.9, 7.5 Hz, 1H), 8.57 (dd, J = 0.9, 8.1 Hz, 1H); 13C NMR (CDCl3):
21.3, 125.2,
929;
δ
δ
127.6, 127.9, 130.3, 131.6, 135.3, 135.8, 136.6, 137.4, 142.2, 164.0; 77Se NMR (CDCl3):
δ
HRMS (TOF MS ES+) m/z calcd for C14H10N2O3Se [M+H]+: 334.9935; found: 334.9943.
2-(p-Methoxyphenyl)-7-nitro-1,2-benzisoselenazol-3(2H)-one (6c). Purification with 40% ethyl
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acetate/nꢀpentane afforded the pure title compound as a red solid. Yield: 67%; Mp 206ꢀ209 C; H
NMR (CDCl3): 3.86 (s, 3H), 6.99 (d, J = 9.0 Hz, 2H), 7.51 (d, J = 8.8 Hz, 2H), 7.73 (t, J = 7.6 Hz,
1H), 8.47 (d, J = 7.6 Hz, 1H), 8.58 (d, J = 8.1 Hz, 1H); 13C NMR (CDCl3):
55.8, 114.9, 127.1,
932; HRMS
δ
δ
127.6, 127.9, 131.0, 131.4, 135.3, 136.6, 142.2, 158.9, 164.2; 77Se NMR (CDCl3):
δ
(TOF MS ES+) m/z calcd for C14H10N2O4Se [M+H]+: 350.9884; found: 350.9876.
Typical procedure A for the preparation of azo-bis-ebselen compounds: Bis-[2-phenyl-1,2-
benzisoselenzol-3-(2H)-one-7-yl]diazene (7a). Nitroebselen 3 (275 mg, 0.86 mmol) was added to
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an in situ prepared colourless solution of PhTeNa (1.72 mmol) from the reaction of Ph2Te2 (352 mg,
0.86 mmol) and NaBH4 (65 mg, 1.73 mmol) in EtOH (10 mL) at room temperature under an inert
atmosphere. The mixture was then heated at reflux for 20 h, allowed to cool, poured into water and
extracted with CHCl3. The separated CHCl3ꢀextract was dried over anhydrous Na2SO4, the solvent
was removed under reduced pressure and the residue was purified by column chromatography on
silica gel. The column was first eluted with pentane/ethyl acetate (50:50) then with CHCl3 and finally
with CHCl3/MeOH (98:2) to give the title compound as a dark brown solid. Yield: 0.125 g (51%);
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Mp ˃320 oC; 1H NMR (CDCl3):
Hz, 4H), 7.80 (t, J = 7.6 Hz, 2H), 8.40 (d, J = 7.2 Hz, 2H), 8.61 (d, J = 7.6 Hz, 2H); 13C NMR
δ 7.32 (t, J = 7.2 Hz, 2H), 7.49 (t, J = 7.2 Hz, 4H), 7.66 (dd, J = 8.0
(CDCl3):
(CDCl3):
δ
δ
125.3, 126.7, 128.3, 129.6, 130.2, 131.7, 131.8, 132.1, 139.4, 144.5, 164.0; 77Se NMR
931; HRMS (MALDI) m/z calcd for C26H16N4O2Se2 [M+H]+: 576.9682; found: 576.9689.
Bis-[2-p-tolyl-1,2-benzisoselenzol-3-(2H)-one-7-yl]diazene (7b). Heating at reflux for 40 h.
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Purification by column chromatography using 4% MeOH/CHCl3. Yield: 30 mg (9%); Mp ˃320 C;
1H NMR (CDCl3):
Hz, 2H), 8.38 (d, J = 7.6 Hz, 2H), 8.57 (d, J = 7.6 Hz, 2H); 13C NMR (CDCl3):
δ
2.41 (s, 6H), 7.29 (d, J = 7.6 Hz, 4H), 7.52 (d, J = 8.0 Hz, 4H), 7.78 (t, J = 7.2
21.3, 125.4, 128.2,
927; HRMS (TOF MS
δ
130.2, 131.2, 131.8, 132.1, 136.7 (2C), 144.6, 164.1; 77Se NMR (CDCl3):
δ
ES+) m/z calcd for C28H20N4O2Se2 [M+H]+: 604.9995; found: 604.9970.
Typical procedure B for the preparation of azo-bis-ebselen compounds: Bis-[2-phenyl-1,2-
benzisoselenzol-3-(2H)-one-7-yl]diazene (7a). To a brown suspension of in situ prepared Na2Se2
(1.24 mmol) in dry THF (20 mL) under an inert atmosphere was slowly added a solution of
benzamide 4a (0.20 g, 0.62 mmol) in THF (10 mL) at room temperature. After heating at reflux for
5h and cooling to room temperature, water (20 mL) was added and stirring was continued for 10
min. Following extraction with CHCl3, the combined organic layers were dried over anhydrous
Na2SO4. Removal of the solvent and purification of the residue by silica gel column chromatography,
eluting first with pentane/ethyl acetate (50:50) then with CHCl3 and finally with CHCl3/MeOH
(98:2), afforded compound 7a (62 mg, 35%). Similarly, compound 7b (340 mg, 36%) was prepared
from 4b (1.00 g, 2.98 mmol).
Bis-[1-naphthyl-1,2-benzisoselenzol-3-(2H)-one-7-yl]diazene (7d). The solution of 4d and Na2Se2
was stirred for 8 h at room temperature before workup. Purification by column chromatography
using 5% MeOH/CHCl3 as an eluent afforded the title compound (68%) as a dark brown solid. Mp
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˃320 oC; 1H NMR (CDCl3):
Hz, 2H), 7.95 (t, J = 6.0 Hz, 4H), 8.42 (d, J = 7.6 Hz, 2H), 8.53 (d, J = 8.0 Hz, 2H); 13C NMR
δ
7.49ꢀ7.61 (several peaks, 8H), 7.77 (t, J = 8.0 Hz, 2H), 7.85 (d, J = 8.0
6
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(CDCl3): 123.5, 125.8, 126.8 (2C), 127.1, 128.3, 128.7, 129.1, 130.8, 130.9, 131.4, 131.7, 132.4,
δ
134.8, 135.0, 144.5, 165.3; 77Se NMR (CDCl3):
C34H20N4O2Se2 [M]+: 675.9917; found: 675.9913.
δ
946; HRMS (TOF MS EI+) m/z calcd for
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Typical procedure for the synthesis of diselenides: Bis[3-amino-N-(1-naphthyl)benzamide-2-yl]
diselenide (8d). The azoꢀbisꢀebselen 7d (50 mg, 0.074 mmol) was added to an in situꢀprepared
colorless solution of NaTePh (0.592 mmol) prepared from Ph2Te2 (61 mg, 0.148 mmol) and NaBH4
(11 mg, 0.296 mmol) in EtOH (10 mL) at room temperature under an inert atmosphere. The mixture
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was then heated at 80 C for 1 h. and allowed to cool to room temperature. After addition of water,
the mixture was extracted with ethyl acetate and dried over anhydrous MgSO4. Evaporation of the
solvent and purification by column chromatography, using ethyl acetate as an eluent afforded the
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title compound (25 mg, 52%) as a yellow solid. Mp 236ꢀ240 C; H NMR (DMSOꢀd6):
δ 5.63 (s,
4H), 6.45 (d, J = 7.1 Hz, 2H), 6.80 (d, J = 7.8 Hz, 2H), 6.90 (t, J = 7.5 Hz, 2H), 7.25 (d, J = 7.0 Hz,
2H), 7.25ꢀ7.52 (several peaks, 6H), 7.87 (d, J = 8.2 Hz, 2H), 7.91 (d, J = 8.2 Hz, 2H), 7.97 (d, J =
7.9 Hz, 2H), 10.27 (s, 2H); 13C NMR (DMSOꢀd6):
δ 110.1, 114.3, 114.8, 122.6, 123.0, 125.3, 125.5,
125.7, 125.8, 127.8, 128.4, 130.3, 133.1, 133.5, 144.5, 150.9, 169.2; 77Se NMR (DMSOꢀd6):
δ 364;
HRMS (TOF MS ES+) m/z calcd for C34H26N4O2Se2 [M+Na]+: 705.0286; found: 705.0304.
Bis[3-Amino-N-(p-tolyl)benzamide-2-yl] diselenide (8b). Orange solid. Yield: 44 mg (87%); Mp
219ꢀ221 oC; 1H NMR (DMSOꢀd6):
2.25 (s, 6H), 5.46 (s, 4H), 6.60 (d, J = 7.0 Hz, 2H), 6.79 (d, J =
8.2 Hz, 2H), 7.05 (d, J = 8.2 Hz, 4H), 7.10 (t, J = 7.8 Hz, 2H), 7.33 (d, J = 8.2 Hz, 4H), 10.08 (s,
2H); 13C NMR (DMSOꢀd6):
20.5, 110.2, 114.6, 115.0, 120.0, 128.8, 130.4, 132.4, 136.3, 144.7,
δ
δ
150.8, 167.3; 77Se NMR (DMSOꢀd6):
δ
368; HRMS (TOF MS ES+) m/z calcd for C28H26N4O2Se2
[M+H]+: 611.0464; found: 611.0475.
Diselenide 8b was also isolated in 16% yield when 6b was allowed to react with NaTePh.
Typical procedure for reduction of 2-butylseleno-3-nitrobenzamides: 3-Amino-2-butylseleno-
N-phenyl benzamide (10a). To in situꢀprepared NaTePh (0.96 mmol) from the reaction of Ph2Te2
(192 mg, 0.48 mmol) and NaBH4 (35 mg, 0.94 mmol) in EtOH (10 mL) was added 5a (96 mg 0.24
mmol) at room temperature under nitrogen. The mixture was then heated at reflux for overnight.
After cooling to room temperature the solvent was evaporated and the residue dissolved in
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dichloromethane. After treatment with water the organic layer was dried over anhydrous MgSO4 and
the solvent removed under reduced pressure. Chromatographic purification, eluting with 40% ethyl
o
acetate/pentane, afforded the title compound (70 mg, 96%) as a white solid; Mp 76ꢀ78 C; 1H NMR
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(CDCl3):
2H), 6.83 (d, J = 7.8 Hz, 1H), 6.96 (d, J = 6.9 Hz, 1H), 7.12ꢀ7.22 (several peaks, 2H), 7.37 (t, J = 7.8
Hz, 2H), 7.63 (d, J = 7.8 Hz, 2H), 7.79 (s, 1H); 13C NMR (CDCl3):
13.6, 23.0, 29.3, 32.6, 110.4,
δ 0.83 (t, J = 7.5 Hz, 3H), 1.33 (m, 2H), 1.58 (m, 2H), 2.81 (t, J = 7.5 Hz, 2H), 4.62 (s,
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δ
115.8, 117.6, 120.1, 124.5, 129.2, 130.1, 138.2, 144.4, 149.6, 168.0; 77Se NMR (CDCl3):
δ 145;
HRMS (TOF MS EI+) m/z calcd for C17H20N2OSe [M]+: 348.0741; found: 348.0742.
3-Amino-2-butylseleno-N-(p-tolyl)benzamide (10b). Yield: 82%; Mp 88ꢀ90 oC; 1H NMR
(CD3OD): 0.84 (t, J = 7.0 Hz, 3H), 1.35 (m 2H), 1.58 (m 2H), 2.32 (s, 3H), 2.77 (t, J = 7.4 Hz,
2H), 6.74 (dd, J = 1.2, 7.5 Hz, 1H), 6.86 (dd, J = 1.2, 8.2 Hz, 1H), 7.32ꢀ7.18 (several peaks, 3H),
7.52 (d, J = 8.3 Hz, 2H); 13C NMR (CD3OD):
13.9, 21.0, 23.9, 28.7, 33.6, 111.6, 116.2, 116.7,
δ
δ
121.7, 130.2, 130.9, 135.1, 137.3, 146.7, 151.6, 171.5; 77Se NMR (CD3OD):
δ 137; HRMS (TOF MS
EI+) m/z calcd for C18H22N2OSe [M]+: 362.0897; found: 362.0901.
o
1
3-Amino-2-(butylseleno)-N-(4-methoxyphenyl)benzamide (10c). Yield: 79%; Mp 100ꢀ104 C; H
NMR (DMSOꢀd6): 0.79 (t, J = 7.4 Hz, 3H), 1.25ꢀ1.31 (several peaks, 2H), 1.45ꢀ1.51 (several
δ
peaks, 2H), 2.71 (t, J = 7.4 Hz, 2H), 3.73 (s, 3H), 5.52 (br s, 2H), 6.58 (d, J = 7.0 Hz, 1H), 6.81 (d, J
= 8.3 Hz, 1H), 6.89 (d, J = 8.7 Hz, 2H), 7.12 (t, J = 7.8 Hz, 1H), 7.61 (d, J = 8.6 Hz, 2H), 9.99 (s,
1H); 13C NMR (DMSOꢀd6):
δ 13.4, 22.3, 26.9, 31.9, 55.2, 108.6, 113.7, 114.1, 114.4, 120.9, 129.4,
132.7, 145.9, 150.3, 155.2, 167.7; 77Se NMR (DMSOꢀd6):
C18H22N2O2Se [M]+: 378.0846; found: 378.0844.
δ
144; HRMS (TOF MS EI+) m/z calcd for
2-(4-Hydroxyphenyl)-7-nitrobenzisoselenazol-3(2H)-one (11b). Solution of BBr3 (0.86 mmol,
0.86 mL) was added into a stirred solution of 6c (0.27 mmol, 0.10 g) in dry CH2Cl2 (10 mL) at ꢀ78
oC under an inert atmosphere. Stirring was then continued for overnight at room temperature. The
reaction mixture was diluted with dichloromethane and poured into a solution of brine. The separated
organic layer was dried over anhydrous MgSO4. The solvent was removed under reduced pressure to
afford a red solid which was washed with diethyl ether and dried under vacuum to give the pure title
compound. (85 mg, 88%); Mp 263ꢀ265 oC; 1H NMR (DMSOꢀd6):
J = 9.0 Hz, 2H), 7.86 (t, J = 7.9 Hz, 1H), 8.38 (d, J = 7.4 Hz, 1H), 8.71 (d, J = 8.2 Hz, 1H), 9.77 (s,
δ 6.86 (d, J = 9.0 Hz, 2H), 7.41 (d,
1H); 13C NMR (DMSOꢀd6):
δ
115.9, 127.1, 128.3, 128.8, 129.3, 130.3, 134.8, 135.8, 142.0, 156.6,
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163.6; 77Se NMR (DMSOꢀd6):
335.9649; found: 335.9642.
δ
923; HRMS (TOF MS EI+) m/z calcd for C13H8N2O4Se [M]+:
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X-ray Crystallographic Analysis. Xꢀray crystallographic studies for obtaining the structures of
compounds 7b and 8b were carried out using graphiteꢀmonochromatized Mo Kα radiation (λ =
0.7107 Å). The structures were solved by direct methods (SHELXSꢀ2013) and refined by a fullꢀ
matrix leastꢀsquares procedure on F2 for all reflections using SHELXLꢀ2013 software.28 Hydrogen
atoms were localized by geometrical means. A riding model was chosen for refinement. The
isotropic thermal parameters of hydrogen atoms were fixed at 1.5 times and 1.2 times U(eq) of the
corresponding carbon atoms for sp3 CꢀH and sp2 CꢀH bonds, respectively. Crystallographic data for
the structures reported in this paper, containing supplementary crystallographic data for this paper,
have been deposited with the Cambridge Crystallographic Data Centre (CCDC) as supplementary
publications. CCDC 1047473 (for compound 7b), and CCDC 1047474 (for compound 8b). These
data can be obtained free of charge from the Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif
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Crystal data for 7b. C30H22Cl6N4O2Se2, Mr = 841.14, Monoclinic, space group C2/c, a = 22.883(7)
Å, b = 12.0629(12) Å, c = 15.901(5) Å, α = γ = 90o, β = 132.43(5)o, V = 3239.5(15) Å3, λ = 0.71073
Å, Z = 4, T = 123(2) K, ρcalcd = 1.725 Mg/m3, GOF = 1.049, R1 = 0.0707, wR2 = 0.1422 [I>2σ (I)];
R1 = 0.1291, wR2 = 0.1754 (all data). Of the 18727 reflections that were collected, 6174 were
unique (Rint = 0.1199).
Crystal data for 8b. C28H26N4O2Se2, Mr = 608.45, Monoclinic, space group P 1 2 / c 1, a =
8.9772(2) Å, b = 26.5501(5) Å, c = 10.9101(3) Å, α = γ = 90o, β = 96.200(2)o, V = 2585.16(10) Å3,
λ = 0.71073 Å, Z = 4, T = 123(2) K, ρcalcd = 1.563 Mg/m3, GOF = 1.069, R1 = 0.0543, wR2 = 0.0813
[I>2σ (I)]; R1 = 0.0981, wR2 = 0.0921 (all data). Of the 59472 reflections that were collected, 16750
were unique (Rint = 0.0599).
Computational details. Computational calculations for compounds 6b-c, 7a-b, 7d, 11b were
executed by using the Gaussian 09 suite of quantum chemical programs.29 The hybrid B3LYP
exchange correlation functional was implemented for density functional theory (DFT) calculations.30
The geometry optimizations were carried out at the B3LYP level of DFT by using the 6ꢀ311+G(d)
basis sets. The quantifications of orbital interaction were done by natural bond orbital (NBO)
77
analysis at B3LYP/6–311+G(d,p) level.31 The Se NMR calculations were performed at B3LYP/6ꢀ
311+G (d,p) level on B3LYP/6–311+G(d)ꢀlevelꢀoptimized geometries by using the gaugeꢀincluding
atomic orbital (GIAO) method (referenced with respect to the peak of Me2Se).32 Atoms in molecules
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(AIM)33 calculations have also been used to confirm distinct bond critical point between the two
interacting atoms.
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Coupled Reductase Assay. The glutathione peroxidaseꢀlike activity of compounds prepared was
determined by UVꢀspectroscopy following the protocol by Wilson34 with slight modifications. The
test mixture contained GSH (1 mM), ethylene diamine tetra acetate (EDTA, 1 mM), glutathione
reductase (GR) (1.3 unit⋅mLꢀ1), and βꢀnicotinamide adenine dinucleotide phosphate (NADPH, 0.2
mM) in potassium phosphate buffer (100 mM), pH 7.5. Catalysts (20 µM) were added to the test
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o
mixture at 21 C and the reaction was initiated by addition of H2O2 (0.8 mM). Initial reaction rates
were based on the consumption of NADPH as assessed by UVꢀspectroscopy at 340 nm. The initial
reduction rates were triplicated and calculated from the first 10 seconds of reaction by using 6.22
mMꢀ1cmꢀ1 as the extinction coefficient for NADPH. GPxꢀdata reported in Table 2 are means ± SD.
Biological Studies. Blood buffy coats were diluted in a 1:1 ratio with 1 x phosphate buffered saline
(PBS) and mononuclear cells were segregated using FicollꢀPaque plus density gradient
centrifugation. Briefly, the diluted blood was gently placed on top of FicollꢀPaque and then
centrifuged at 400 g for 30 minutes. After removing the plasma layer, the mononuclear (MNC) layer
was collected, washed with PBS, and then spun at 100 g for 15 min. This washing procedure was
repeated for a total of 3 times. Total cell number was counted using a hemocytometer and trypan
blue exclusion method. The blood pellet was then suspended in 3% dextran/0.9% saline for 20 min.
The supernatant was then collected and the cell pellet collected after centrifugation at 250 g for 10
min. To remove erythrocyte, the pellet was subjected to 0.2% saline solution for 20 s and then an
equal volume of 1.6% saline was added. Then the cells were spun at 250 g for 10 min and the pellet
was suspended in PBS.
Chemiluminescence Assay. To measure release of ROS from monocytes, a luminol amplified
chemiluminescence assay was conducted. All measurements were performed in white 96 well plates
at 37°C in 1 x PBS with 50 mM of luminol, 0.1M NaOH, 2 ꢁg/mL of horse radish peroxidase,
different concentrations of ebselen, 3, 6b-c, 7a-b, 7d, 8b, 8d and 11b and Trolox initially diluted in
DMSO. Approximately 2 x 105 cells were plated in each well and then stimulated to generate ROS
using 500 nM of 1 ꢁM phorbol myristate acetate (PMA). Luminescence readings were taken using a
multiꢀplate reader every 2 min for 2 h. Total ROS was quantified by determining the area under the
chemiluminescence kinetic curve. Experiments were repeated 4ꢀ5 times with n = 3 per group per
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experiment. For statistical analysis, ANOVA was performed with Scheffe postꢀhoc tests. P values
below 0.05 were considered significant.
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Cell proliferation and toxicity assay. MC3T3 (plating density: 5 x 103 and 20 x 103 per well
respectively in a 96 well plate) was let adhered after plating for 24 h in αꢀMEM supplemented with
10% FBS and 1% pen/strep. Then the cells were exposed to antioxidants 3, 6b-c, 7a-b, 7d, 8b, 8d
and 11b (25 ꢁM) for 24 h, washed and then fed with fresh αꢀMEM every other day. To assess cell
viability, the Alamar Blue assay was conducted on day 1 and 3 after treatment. Briefly, cells were
washed with 1 x PBS and then incubated with Alamar Blue (1:20 dilution in phenol red free αꢀMEM
for 1.5 h and then fluorescence was read with a plate reader at 560 nm excitation and 590 nm
emissions. Experiments were repeated twice with n = 6 for each group.
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Mechanistic studies. A solution of H2O2 (3.7 ꢁL, 0.033 mmol) was added to an NMR tube
containing diselenide 8b (20 mg, 0.033 mml) in 400 ꢁL of DMSOꢀd6 and the 77Se NMR spectrum
was recorded after 30ꢀ60 min. Then another equivalent of H2O2 was added and the 77Se NMR
spectrum recorded again. Several portions (1+2 eqivalents) of GSH (10 mg, 0.033 mmol) in 100 ꢁL
of H2O were then syringed into the NMR tube. After every new addition of thiol the 77Se NMR
spectrum was recorded.
ASSOCIATED CONTENT
Supporting Information
1H, 13C and 77Se NMR data for all new compounds prepared. Results from kinetic studies of
compounds prepared and coordinates of optimized geometries. Xꢀray crystallographic data (CIF
files). This material is available free of charge via the Internet at http://pubs.acs.org
AUTHOR INFORMATION
Corresponding Authors
*Eꢀmail: vijaypalchem@gmail.com
*Eꢀmail: lars.engman@kemi.uu.se
Notes
The authors declare no competing financial interest
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ACKNOWLEDGEMENTS
Stiftelsen ÅꢀForsk (16ꢀ364) and Stiftelsen Olle Engkvist Byggmästare (2016/159) and Carl Tryggers
Stiftelse för Vetenskaplig Forskning (CTS 13:346) are gratefully acknowledged for financial support.
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29. Gaussian 09, Revision c.01 & d.01, Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria,
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J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V.
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