Amide-based glutathione peroxidase mimics: Effect of secondary and amide substituents on antioxidant activity

Article abstract of DOI:10.1002/asia.200800483

A series of secondary and tertiary amide-substituted diselenides were synthesized and studied for their GPx-like antioxidant activities using H ₂O₂, Cum-OOH, and tBuOOH as substrates and PhSH as thiol co-substrate. The effect of subs

Full text of DOI:10.1002/asia.200800483

FULL PAPERS
DOI: 10.1002/asia.200800483
Amide-Based Glutathione Peroxidase Mimics: Effect of Secondary and
Tertiary Amide Substituents on Antioxidant Activity
Krishna P. Bhabak and Govindasamy Mugesh*[a]
Dedicated to Professor C. N. R. Rao on the occasion of his 75th birthday
Abstract: A series of secondary and
tertiary amide-substituted diselenides
were synthesized and studied for their
GPx-like antioxidant activities using
H2O2, Cum-OOH, and tBuOOH as
substrates and PhSH as thiol co-sub-
strate. The effect of substitution at the
the GPx-like activities of the sec-
amide-based diselenides, mainly by re-
ducing the Se···O nonbonded interac-
tions. The reduction in strength of the
Se···O interaction upon introduction of
N,N-dialkyl substituents not only pre-
vents the undesired thiol exchange re-
actions, but also reduces the stability of
selenenyl sulfide intermediates. This
leads to a facile disproportionation of
the selenenyl sulfide to the correspond-
ing diselenide, which enhances the cat-
alytic activity. The mechanistic investi-
gations indicate that the reactivity of
diselenides having sec- or tert-amide
moieties with PhSH is extremely slow;
indicating that the first step of the cata-
lytic cycle involves the reaction be-
tween the diselenides and peroxide to
produce the corresponding selenenic
and seleninic acids.
À
free NH group of the amide moiety
in the sec-amide-based diselenides on
GPx activity was analyzed by detailed
experimental and theoretical methods.
It is observed that substitution at the
Keywords: antioxidants · catalysis ·
redox chemistry · selenium · thiol
exchange
À
free NH group significantly enhances
Introduction
Glutathione peroxidase (GPx) is a mammalian antioxidant
enzyme which contains selenocycteine, the 21st amino acid,
in the active site. GPx catalyzes the reduction of harmful
peroxides in the presence of glutathione (GSH).[1] The GPx
cycle involves oxidation of the catalytically active selenol
(E-SeH) moiety by peroxides to produce the corresponding
selenenic acid (E-SeOH), which upon reaction with the
thiol cofactor GSH generates the key intermediate selenenyl
sulfide (E-SeSG). The subsequent attack of the second GSH
Scheme 1. Proposed mechanism for the antioxidant activity of GPx.
À
À À
at the Se S bond in the selenenyl sulfide regenerates the
active site selenol (E-SeH) with a release of the thiol cofac-
tor in its oxidized form, GSSG (Scheme 1).[1e–g] When the
thiol is depleted, the selenenic acid (E-SeOH) produced
may undergo further oxidation to produce seleninic (E-
SeO2H) or selenonic (E-SeO3H) acids which interfere with
the main catalytic pathway. In the catalytic cycle, the rapid
reactions of selenenic acid with GSH and the resulting sele-
nenyl sulfide (E-SeSG) with the second GSH to produce se-
lenol are very important as these reactions ensure that the
selenium moiety in the enzyme is not irreversibly inactivat-
ed.
As selenium redox chemistry at the active site of GPx
plays an important role in oxidative stress,[2] several groups
have worked toward design and synthesis of small-molecule
organoselenium compounds that mimic the peroxide-reduc-
[a] K. P. Bhabak, Prof. G. Mugesh
Department of Inorganic and Physical Chemistry
Indian Institute of Science
Bangalore 560 012 (India)
Fax : (+91)80-2360 1552/2360 0683
E-mail: mugesh@ipc.iisc.ernet.in
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.200800483.
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Chem. Asian J. 2009, 4, 974 – 983

ing ability of GPx in the presence of thiols. These com-
pounds include the well-known anti-inflammatory com-
pound ebselen (1),[3] and related selenamides (2, 3),[4] disele-
nides (4–11),[5] and allyl-3-hydroxypropyl selenides (12)[6]
(Figure 1). Particularly, the diselenides having heteroatom in
Results and Discussion
Glutathione Peroxidase (GPx)-like Antioxidant Activities
The sec-amide-based diselenides (13–16) used in the present
study were synthesized in nearly quantitative yield from the
corresponding cyclic selenenyl amides by treating the com-
pounds with triphenyl phosphine. The tert-amide substituted
diselenides (17–20) were synthesized by treating the appro-
priate secondary amines with 2,2’-diselenobis(benzoyl chlo-
ride). The antioxidant activity of these synthetic compounds
was studied using PhSH as a GSH alternative and H2O2,
cumene hydroperoxide (Cum-OOH), and tert-butyl hydro-
peroxide (tBuOOH) as the substrates. The catalytic reduc-
tion of peroxides by PhSH in the presence of various seleni-
um compounds was studied by an HPLC method.[4b,10a,b] To
obtain some mechanistic insights into the catalytic reduction
of peroxides, we have performed detailed kinetic studies by
varying the concentration of the thiol co-substrate. The for-
mation of PhSSPh in the reaction was studied by a reverse-
phase HPLC method and the time required for 50% conver-
sion of PhSH to PhSSPh (t1/2) was calculated by determining
the peak areas at different time intervals. A calibration plot,
obtained from known concentrations of PhSSPh, was used
to calculate the amount of PhSSPh formed during the
course of reactions.
Figure 1. Some representative examples of synthetic GPx mimics that use
GSH or other thiols as cofactors for their catalytic activity.
the close proximity to selenium (4, 8, 9, and 11) have been
studied extensively owing to the involvement of Se···N non-
bonded interactions.[5c,e,h] It has been shown that two amino
acids, glutamine and tryptophan, which are located in close
proximity to the selenocysteine residue in the active site of
GPx play an important role in modulating the redox proper-
ties of selenium by Se···N/O nonbonded interactions.[1c,7]
While the tertiary amino group in compounds 4, 8, 9, and 11
can act as a general base during the catalytic cycle,[5c,e,h] the
redox chemistry at the selenium center in compounds 5–7
can be modulated by Se···O interactions.
Recent model studies on low molecular weight selenium
compounds show that the reduction of selenenyl sulfide to
selenol needs to overcome a large energy barrier (~21.5 kcal
molÀ1), and therefore the nucleophilic attack of thiol (or thi-
From Table 1 and Figure 2 it is clear that the tert-amide
substituted compounds 17–20 are more efficient catalysts
than the corresponding sec-amide-based diselenides 13–16.
For example, the t1/2 value obtained for compound 13
À
À À
olate) at the selenium center in the Se S bond is both ki-
netically as well as thermodynamically more favorable than
at the sulfur.[8] The thiol attack at the selenium center in se-
lenenyl sulfides can be further enhanced by nonbonded in-
teractions between selenium and other heteroatoms such as
O and N.[9] Ebselen (1) and its analogues exhibit poor cata-
lytic activities when aromatic thiols (PhSH or BnSH) are
used as cofactors. This is mainly owing to the presence of
Se···O non-covalent interactions which lead to the undesired
thiol exchange reaction in the selenenyl sulfide intermedi-
ates.[6,10] It has been shown previously that the catalytic ac-
tivity of tertiary amine-based compounds can be enhanced
when the amino substituent is weakly coordinated to seleni-
um in the selenenyl sulfide intermediate.[5c,e,h] However, it is
unknown whether the replacement of the secondary amide
group in compound 5 and its analogues by a tertiary amide
substituent would enhance the catalytic activity. In view of
this, we have synthesized various diselenides having tertiary
amide substituents and compared their GPx-like activities to
that of secondary amide-based diselenides.
Table 1. Reduction of peroxides by PhSH in the absence and presence of
compounds 13–20.
Compd t1/2 values [min][a]
Relative activity[b]
H2O2 tBu-OOH Cum-OOH H2O2 tBu-OOH Cum-OOH
Control 2905 1130
798
437
508
478
467
65
75
85
67
1.00 1.00
1.73 1.79
1.77 1.68
1.69 1.74
1.53 1.75
35.43 6.42
21.36 5.02
16.60 3.34
17.29 3.64
1.00
1.82
1.57
1.67
1.70
12.27
10.64
9.39
13
14
15
16
17
18
19
20
1675 629
1640 669
1721 649
1892 644
82
176
225
338
310
136
175
168
11.91
[a] Assay conditions: The reactions were carried out in MeOH at 228C.
Selenium compounds: 10.0 mm; PhSH: 1.0 mm; peroxide: 2.0 mm. The
control reactions were performed under identical condition in the ab-
sence of selenium compounds. [b] Relative activities are given with re-
spect to the corresponding control values.
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975

K. P. Bhabak and G. Mugesh.
FULL PAPERS
Previous studies on the GPx activity of tert-amine-substi-
tuted diselenides have shown that the nonbonded interac-
Figure 2. Catalytic reduction of H2O2 by PhSH in the presence of various
selenium compounds. The formation of PhSSPh was followed by a re-
verse-phase HPLC and the% conversion was calculated from calibration
plot. a) Control; b) 16; c) 13; d) 20; e) 17. Assay condition: catalyst:
10.0 mm; PhSH: 1.0 mm; H2O2: 2.0 mm in MeOH at 228C.
tions between the tert-amino group and selenium activate
[5c,e,h]
À
À À
the Se Se bond toward reactions with thiols.
There-
fore, we have performed single crystal X-ray diffraction
studies on compounds 17 and 20 to understand the nature of
Se···O/N interactions in these compounds (see Figure 3).
(1675 min) in H2O2 assay is much higher than that of 17
(82 min), indicating that the tert-amide substituted com-
pound 17 is almost 20 times more active than the sec-amide
derivative 13. Although the tert-amide-based compounds
(17–20) were found to be much better catalysts than the cor-
responding sec-amide derivatives (13–16), the relatively low
t1/2 values in organic peroxide (tBuOOH, Cum-OOH) assays
as compared to the H2O2 assay can be ascribed to the facile
oxidation of PhSH by tBuOOH and Cum-OOH in the ab-
sence of selenium compounds (Table 1, Control). However,
the relative activities (Table 1) indicate that the catalytic ac-
tivities of the tert-amide-based diselenides depend on the
nature of the peroxide, which is in contrast to the sec-
amide-based compounds that exhibit almost identical activi-
ties in all three peroxide systems. It has been shown previ-
ously that the nature of peroxide has very little effect on the
GPx-like activity of ebselen and related compounds.[10b]
Figure 3. X-ray crystal structures of compounds 17 and 20. ORTEP dia-
grams with the ellipsoids representing 50% probability. Hydrogen atoms
are omitted in 20 for clarity. Significant bond lengths: 17: Se1···O1=
À
2.793 ꢁ; Se2···O2=2.852 ꢁ; Se1 Se2=2.326 ꢁ. 20: Se1···O1=3.264 ꢁ;
À
Se2···O2=3.053 ꢁ; Se1 Se2=2.318 ꢁ.
The X-ray structures of 17 and 20 reveal that the carbonyl
oxygen atoms interact with selenium leading to the forma-
tion of O···Se···Se···O arrangements. Although the Se···O dis-
tances in compound 17 are shorter than those of 20, these
À
À À
interactions are not sufficient to cleave the Se Se bonds
by PhSH. These observations suggest that the tert-amino
group in 4, 8, 9, and 11 may serve as a general base to ab-
stract the proton from thiol to generate a more reactive thi-
olate anion for a nucleophilic attack at the selenium center.
In contrast, the amide groups in compounds 13–20 are rela-
tively weak bases and the basicity is further decreased by
the Se···O nonbonded interactions. Therefore, the incoming
Reactivity of Compounds 13–20 Toward Thiols
It has been observed that the reductive cleavage of the dise-
lenide bonds by thiols is required for the catalytic activity of
the amine-based diselenides 4, 8, 9 and 11.[5c,e,h] Therefore,
we have carried out reactions of compounds 13–20 with
PhSH to understand the effect of sec- and tert-amide sub-
À
À À
thiol is not sufficiently nucleophilic to cleave the Se Se
bond.
À
À À
stituents in the activation of the Se Se bond toward a re-
ductive cleavage. The reactions of 13–16 with PhSH did not
produce the expected selenenyl sulfides (21–24) or selenols
(29–32) even when two equivalents of PhSH were used. The
reactivity of compounds 17–20 toward PhSH was found to
be identical to that of the sec-amide-based diselenides 13–
16. However, when a large excess of PhSH (20 equiv) was
added and the reaction mixture was stirred for two days, the
corresponding selenenyl sulfides (25–28) were detected in
trace amounts by 77Se NMR spectroscopy. These observa-
Reaction of Diselenides 13–20 with Peroxides
The unusually low reactivity of diselenides 13–20 toward
PhSH led us to investigate their reactions with peroxides.
We have recently shown that the catalytic cycle of ebselen
(1) involves the formation of diselenide (5), which under-
goes a rapid reaction with H2O2 to produce the correspond-
ing selenenic and seleninic acids.[10d] When compounds 13–
16 were treated with H2O2, the 77Se NMR signals arising
from the diselenides disappeared completely and two new
signals were detected in the downfield region with respect
to the peaks for the diselenides. The isolation and character-
ization of products from these reactions revealed that the
À
À À
tions indicate that the reactivity of Se Se bonds in com-
pounds 13–20 toward thiols is significantly different from
those of compounds 4, 8, 9, and 11, which readily produce
the corresponding selenenyl sulfides and selenols even with
one equivalent of PhSH.[5c,e,h]
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Chem. Asian J. 2009, 4, 974 – 983

Effect of Amide Substituents on the Antioxidant Behaviour of Glutathione Peroxidase Mimics
À
À À
cleavage of Se Se bonds in 13–16 by H2O2 produces a
mixture of the corresponding seleninic acids (45–48) and se-
lenenyl amides (57–60). As previously shown for compound
5,[10d] the reactions of diselenides 13–16 with H2O2 produce
the selenenic acids (37–40) and seleninic acids (45–48).
Whereas the seleninic acids are stable to be isolated, the se-
lenenic acids 37–40 undergo cyclization reactions to produce
the corresponding selenenyl amides 57–60 (Scheme 2).
When an excess amount of H2O2 (10 equiv) was added, the
selenenyl amides 57–60 were converted to the seleninic
acids 45–48 via the formation of the corresponding selenox-
ides followed by hydrolysis.
was observed.[11] However, such over-oxidized species may
not be produced during the catalytic cycle in the presence of
thiol.
DFT calculations on compounds 37–52 indicate that both
the selenenic acids 37–40 and seleninic acids 45–48 based on
sec-amide exhibit strong Se···O interactions, although the in-
teraction energies (ESe···O) for the seleninic acids 45–48 are
almost two times lower than those of the corresponding se-
lenenic acids 37–40 (Table 2). Similarly, the tert-amide-based
selenenic acids 41–44 also exhibit strong Se···O interactions
and the interaction energies are almost identical to those of
Table 2. Theoretical data for 37–52 obtained by DFT calculations at
B3LYP/6-31+G(d)//B3LYP/6-311+GACTHUNRTGNE(GNU d,p) level.
Compd
rSe···O
[ꢁ]
ESe···O
Compd
rSe···O
[ꢁ]
ESe···O
[kcalmolÀ1
]
[kcalmolÀ1
]
37
38
39
40
41
42
43
44
2.353
2.345
2.345
2.340
2.340
2.329
2.337
2.300
27.05
27.61
27.62
28.10
25.42
26.06
24.95
29.04
45
46
47
48
49
50
51
52
2.500
2.462
2.490
2.507
2.923
2.899
2.930
3.012
14.61
17.27
14.66
13.69
0.70
0.66
0.00
0.00
compounds 37–40. In contrast, the seleninic acids 49–52 do
not exhibit any significant interactions. These observations
can be further supported by the experimental data. Al-
though the selenenic acids 37–44 were unstable and could
not be isolated, the corresponding seleninic acids 45–52
were obtained from the reactions of diselenides 13–20 with
an excess amount of H2O2 (5–10 equiv). The single crystal
X-ray structures of compounds 48 and 52 reveal that the
Se···O interaction in compound 48 is much stronger than
Scheme 2. Reactions of diselenides 13–16 with H2O2 to produce the sele-
ninic acids 45–48 and selenenyl amides 57–60.
The reactions of tert-amide-based diselenides 17–20 with
H2O2 (3 equiv) produced the seleninic acids 49–52 as major
products along with some unreacted diselenides (Scheme 3).
This is in contrast to the reactions of 13–16 with H2O2 in
which the diselenides were completely converted to the cor-
responding seleninic acids and selenenyl amides. This is
probably owing to the cyclization of selenenic acids 37–40 to
the selenenyl amides 57–60, which drives the conversion of
diselenides 13–16 to the oxidized products. As there is no
À
that of 52, indicating that the substitution of the free N H
group by an N-alkyl substituent reduces the strength of
Se···O interactions in the seleninic acid intermediates.
(Figure 4).
À
À
free NH group in compounds 41–44 for a possible cycli-
zation, the selenenic acids were further oxidized by H2O2 to
produce the seleninic acids 49–52 as the major oxidized
products. When a large excess of H2O2 (25 equiv) was
added, the formation of over-oxidized selenonic acids 53–56
Figure 4. Single crystal X-ray structures of compounds 48 and 52.
ORTEP diagrams with the ellipsoids representing 50% probability. Sig-
À
nificant bond lengths and bond angles: 48: Se1···O4=2.472 ꢁ; Se1···O4
À
O2=167.88. 52: Se1···O3=3.011 ꢁ; Se1···O3 O1=167.98.
Reactions of Selenenic and Seleninic Acids with Thiols
The poor reactivity of diselenides 13–20 toward PhSH and
the facile reactions of these compounds with H2O2 suggest
Scheme 3. Reaction of the diselenides 17–20 with H2O2 to produce the
seleninic acids 49–52 and selenonic acids 53–56.
À
À À
that the oxidative cleavage of Se Se bonds must occur
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977

K. P. Bhabak and G. Mugesh.
FULL PAPERS
before the thiol reactions (Scheme 4). Therefore, the disele-
nides 13–20 were first oxidized by H2O2 to produce the cor-
responding selenenic and seleninic acids and then treated
with PhSH to obtain the selenenyl sulfides 21–28. As expect-
Scheme 4. Reaction of diselenides 13–20 with H2O2/PhSH and selenenyl
amides 57–60 with PhSH to produce the selenenyl sulfides 21–28.
ed, the reactions of 13–20 with H2O2 (10 equiv) followed by
addition of PhSH (15 equiv) afforded the selenenyl sulfides
21–28, which were isolated and purified by column chroma-
tography. The sec-amide-based selenenyl sulfides can also be
obtained by treating the corresponding selenenyl amides 57–
60 with PhSH (see Experimental Section).
It has been shown that selenenyl sulfides are key inter-
mediates in the catalytic cycles of GPx and its synthetic
mimics.[1,5,6,10] The nucleophilic attack of thiol at the sulfur
center in the selenenyl sulfides generally produces the cata-
lytically active selenol as shown in Scheme 1. We have re-
cently shown that strong Se···O/N interactions in selenenyl
sulfides increase the possibility of nucleophilic attack of the
thiol at the selenium center, which leads to a thiol exchange
reaction rather than selenol formation. For example, the se-
lenenyl sulfide derived from ebselen does not produce any
selenol during the catalytic cycle because of the strong
Se···O interactions.[10b,d] On the other hand, the selenenyl
sulfide derived from 9 undergoes a facile reaction with
thiols to produce the corresponding selenol. In this particu-
Figure 5. The energy optimized geometries of the selenenyl sulfide inter-
mediates 21–28.
distances in compounds 21–24 (2.470–2.479 ꢁ) are slightly
weaker than those observed in the corresponding selenenic
acids 37–40 (2.340–2.353 ꢁ). In contrast, the tert-amide de-
rivatives 25–28 exhibit much weaker interactions as com-
pared to the corresponding selenenic acids 41–44. NBO
À
lar case, the OMe substituent has been shown to enhance
the nucleophilic attack of the thiol at the sulfur center by
preventing Se···N interactions.[5h] Therefore, it was of partic-
ular interest to understand the strength of Se···O interac-
tions in compounds 21–28.
analysis shows that the Se···O interaction energies (ESe···O
)
for compounds 21–24 are significantly higher than those of
À
25–28, indicating that the substitution of the free N H
The 77Se NMR chemical shifts and DFT calculations indi-
cate that the Se···O interactions in the sec-amide-based sele-
nenyl sulfides 21–24 are much stronger than those of the
tert-amide derivatives 25–28 (Table 3, Figure 5). The Se···O
group by an N-alkyl substituent reduces the strength of
Se···O interactions in selenenyl sulfides. This probably arises
from the steric hindrance of the second N-alkyl substituent
which prevents the planar conformation of the amido func-
À
tionality. Particularly, compound 28, having a
NACHTUNGTRENNUNG(iPr)2
group, does not exhibit any Se···O interaction (Table 3). It is
also observed that the positive charge on sulfur (qS) increas-
es with a decrease in the strength of the Se···O interactions.
The 77Se NMR experiments on compounds 21–28 show
that the chemical shifts for the selenenyl sulfides having sec-
amide moieties (21–24) are shifted downfield as compared
to those of 25–28, indicating that the Se···O interactions in
21–24 are retained in solution. This is in accordance with
the previous reports that Se···O/N interactions generally
lead to a downfield shift in the 77Se NMR studies.[5c,h,10] The
relatively strong Se···O interactions in 21–24 and weak inter-
actions in 25–28 suggest that the possibility of a nucleophilic
Table 3. Theoretical data for 21–28 obtained by DFT calculations at
B3LYP/6-31+G(d)//B3LYP/6-311+GACTHNUTRGNEUNG
(d,p) level and experimental 77Se
NMR chemical shifts.
Compd
rSe···O [ꢁ]
qSe
qS
ESe···O [kcalmolÀ1
]
77Se [ppm][a]
21
22
23
24
25
26
27
28
2.479
2.472
2.469
2.470
2.714
2.756
2.720
2.929
0.403
0.403
0.403
0.403
0.355
0.347
0.350
0.313
0.024
0.021
0.020
0.021
0.055
0.056
0.055
0.079
18.37
18.79
19.02
19.02
5.03
5.03
4.49
0.00
587
588
586
587
527
520
525
512
[a] The experimentally observed values are cited with respect to Me2Se.
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Chem. Asian J. 2009, 4, 974 – 983

Effect of Amide Substituents on the Antioxidant Behaviour of Glutathione Peroxidase Mimics
attack by thiol at the selenium center in compounds 25–28 is
expected to be considerably less as compared to that of
compounds 21–24. To test this hypothesis, we have studied
the catalytic activity of 13–20 and 57–60 at various concen-
trations of thiol and plotted the initial rate against the con-
centration of PhSH. When compound 16 was treated with
PhSH in the presence of Cum-OOH, only a minor change in
the reaction rate with increasing PhSH concentration (up to
~2 mm) was observed. However, when the concentration of
PhSH was increased further, a rapid enhancement in the ini-
tial rate was observed (Figure 6). A similar trend in the re-
Figure 7. Lineweaver–Burk pots obtained for compounds 17–20 (5.0 mm)
at various concentrations of PhSH (0.0–15.0 mm) at a fixed concentration
of Cum-OOH (2.0 mm). Line a 17; Line b 18; Line c 19; Line d): 20.
Table 4. Catalytic parameters [maximum velocities (Vmax), Michaelis con-
stants (KM), catalytic constants (kcat) and catalytic efficiencies (h)] for the
reduction of Cum-OOH by PhSH in the presence of diselenides 17–20.[a]
Compd
Vmax [mMminÀ1
]
KM [mM]
kcat [minÀ1
]
h [MÀ1 minÀ1
]
17
18
19
20
2.45
1.72
1.69
1.34
0.66
0.44
1.20
1.12
0.49
0.34
0.34
0.27
0.74ꢂ103
0.78ꢂ103
0.30ꢂ103
0.24ꢂ103
[a] Assay conditions: PhSH (0.0–15.0 mm), Cum-OOH (2.0 mm) and sele-
nium compounds (5.0 mm) in MeOH at 228C.
Figure 6. The effect of thiol co-substrate on the initial reduction rate of
Cum-OOH (2.0 mm) by PhSH (0.0–15.0 mm) in the presence of 5 mm of
selenium compounds [a 20; b 60; c 16]. The reactions were carried out in
methanol at 228C.
non-polar iPr groups may reduce the activity. However, the
relatively low KM values for compounds 17–20 compared to
those of the tert-amine-based compound 8,[5h] indicate that
the thiol exchange reactions in the selenenyl sulfides 25–28
are less favored as compared to the thiol exchange in the se-
lenenyl sulfide derived from 8.
action rate was observed for the corresponding selenenyl
amide 60.[10d] In contrast, compound 20 exhibited completely
different kinetics in the presence of various concentrations
of thiol at a fixed concentration of Cum-OOH.
In this case, the reaction rate increased rapidly at the be-
ginning with an increase in the PhSH concentration. When
the thiol concentration was further increased, compound 20
followed typical saturation kinetics generally observed for
enzymatic reactions.[12] The other tert-amide-based com-
pounds 17–19 also exhibited a similar kinetic behaviour. The
change in kinetic pattern can be attributed to the various
degrees of thiol exchange reactions in the selenenyl sulfide
intermediates. These observations suggest that a very high
concentration of thiol is required to overcome the thiol ex-
change reaction in compounds 21–24, whereas a relatively
low concentration of thiol is sufficient for the catalytic activ-
ity of compounds 25–28.
Mechanism for the Catalytic Activity
Based on our experimental and theoretical data, we propose
a mechanism for the catalytic activity of compounds 13–16
as shown in Figure 8. According to this mechanism, the dise-
lenides first react with peroxides to produce the correspond-
ing selenenic acids (37–40) and seleninic acids (45–48). In
The saturation kinetics allowed us to get the catalytic pa-
rameters for compounds 17–20 by using Lineweaver–Burk
plots. When the reciprocal of the initial rates (1/V0) were
plotted against the reciprocal of PhSH concentrations, a
linear relationship was obtained (Figure 7). The catalytic pa-
rameters Vmax, KM, Kcat, and catalytic efficiencies (h) were
obtained from the Lineweaver–Burk plots. This data indi-
cates that the catalytic efficiencies of methyl- and ethyl-sub-
stituted compounds 17 and 18 are almost twice that of their
nPr and iPr analogues (19, 20) (Table 4). Although the
Se···O interactions in the selenenyl sulfide derived from 20
is much weaker than that of 17, the sterically bulky and
Figure 8. The proposed catalytic mechanism for the reduction of perox-
ides by 13–16 in the presence of thiols.
Chem. Asian J. 2009, 4, 974 – 983
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
979

K. P. Bhabak and G. Mugesh.
FULL PAPERS
Table 5. t1/2 values for the reduction of peroxides by compound 57–60 in
the presence of PhSH at 228C.
the presence of PhSH, both selenenic and seleninic acids are
converted to the corresponding selenenyl sulfides (21–24),
which undergo disproportionation reactions to regenerate
the diselenides. As previously mentioned, the strong Se···O
interactions in the selenenyl sulfides (21–24) lead to the
thiol exchange reaction, which prevents the formation of the
corresponding selenols. As we have shown for ebselen
(1),[10d] the conversion of the selenenyl sulfides to the disele-
nides is the rate-determining step. The strong Se···O interac-
tions in the sec-amide-based compounds increase the stabili-
ty of selenenyl sulfides (21–24) in solution, which considera-
bly reduces the rate of disproportionation. On the other
hand, the tert-amide-based selenenyl sulfides 25–28 having
weak Se···O interactions are relatively less stable, and there-
fore, these compounds undergo a facile disproportionation
reaction to produce the corresponding diselenides (17–20)
and PhSSPh (Figure 9).
Compd t1/2 values [min][a]
Relative activity[b]
H2O2 tBu-OOH Cum-OOH H2O2 tBu-OOH Cum-OOH
Control 2905 1130
798
409
465
497
470
1.00 1.00
1.75 1.39
1.67 1.37
1.29 1.37
1.13 1.36
1.00
1.95
1.71
1.60
1.69
57
58
59
60
1660 808
1738 824
2242 825
2560 828
[a] Assay conditions: The reactions were carried out in MeOH at 228C.
Selenium compounds: 10.0 mm; PhSH: 1.0 mm; peroxide: 2.0 mm. The
control reactions were performed under identical condition in the ab-
sence of selenium compounds. [b] Relative activities are given with re-
spect to the corresponding control values.
The catalytic mechanism of the tert-amide-based disele-
nides 17–20 is very similar to that of the corresponding sec-
amide analogues 13–16, except the formation of cyclic sele-
nenyl amides (Figure 9). Although the replacement of the
sec-amide moiety by tert-amide considerably reduces the
thiol exchange reactions in the selenenyl sulfides by reduc-
ing the Se···O interactions, the selenenyl sulfides 25–28 do
not produce any selenol during the catalytic cycle. On the
other hand, the disproportionation of 25–28 to the disele-
nides 17–20 was found to be much faster than that of com-
pounds 13–16. This indicates that the facile disproportiona-
tion of 25–28 may account for the higher activity of com-
pounds 17–20 as compared to that of 13–16.
Conclusions
In this study, we have shown that diaryl diselenides having
tert-amide substitutents are much better catalysts than the
corresponding sec-amide-based diselenides in the reduction
of peroxides by thiols. The reactivity of tert-amide-based dis-
elenides toward thiols is extremely poor, which is in contrast
to tert-amine-based diselenides that readily react with thiols
to produce the corresponding selelenyl sulfides and selenols.
Therefore, the reaction of these amide-based compounds
with peroxide is important for their catalytic activity. Fur-
thermore, the presence of tert-amide moiety considerably re-
duces the thiol exchange reactions at the selenenyl sulfide
intermediates by preventing strong Se···O interactions. A
comparison of the catalytic cycles of sec- and tert-amide-sub-
stituted diselenides reveals that the mechanisms are very
similar for these two classes of compounds except that the
selenenic acids, having sec-amide substituents, undergo a
cyclization reaction to produce the corresponding selenenyl
amides.
Figure 9. The proposed catalytic mechanism for the reduction of perox-
ides by 17–20 in the presence of thiols.
Interestingly, when the concentration of PhSH is low, the
selenenic acids 37–40 undergo a cyclization to produce the
selenenyl amides 57–60, which upon treatment with PhSH
afford the selenenyl sulfides 21–24. As this catalytic mecha-
nism is similar to that of ebselen, we have synthesized the
selenenyl amides 57–60 independently by treating 2-chloro-
seleno benzoyl chloride with appropriate primary amines
and studied the GPx-like activities of 57–60 to find out
whether the cyclic compounds form a part of the catalytic
cycle. The catalytic activity of 57–60 was studied by using
PhSH as thiol co-substrate and H2O2, tBuOOH, and Cum-
OOH as substrates. The t1/2 values obtained for 57–60 are
comparable to those of the corresponding diselenides, indi-
cating that compounds 57–60 are produced during the cata-
lytic cycle (Table 5). The small changes in the t1/2 values can
be ascribed to the differences in the reactivity toward thiol
and peroxide. While the cyclic compounds 57–60 first react
with PhSH to produce the selenenyl sulfides 21–24, the dise-
lenides 13–16 do not react with PhSH, but they react readily
with peroxides to produce the oxidized compounds (37–40,
45–48) (Figure 8).
Experimental Section
General Procedure
n-Butyllithium (nBuLi) was purchased from Acros Chemical Co. (Bel-
gium). Methanol was obtained from Merck and dried before use. All
other chemicals were of the highest purity available. Most of the reac-
980
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ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2009, 4, 974 – 983

Effect of Amide Substituents on the Antioxidant Behaviour of Glutathione Peroxidase Mimics
tions were carried out under nitrogen atmosphere using standard
vacuum-line techniques. Owing to unpleasant odors of several of the re-
action mixtures involved, most manipulations were carried out in a well-
ventilated fume hood. Thin-layer chromatography analyses were carried
out on pre-coated silica gel plates (Merck) and spots were visualized by
UV irradiation. Column chromatography was performed on glass col-
umns loaded with silica gel or on automated flash chromatography
system (Biotage) by using preloaded silica cartridges. 1H (400 MHz), 13C
(100.56 MHz), and 77Se (76.29 MHz) NMR spectra were obtained on a
Bruker 400 MHz NMR spectrometer. Chemical shifts are cited with re-
spect to SiMe4 as internal (1H and 13C) and Me2Se as external (77Se)
standards. Mass spectral studies were carried out on a Q-TOF micro
mass spectrometer with ESI MS mode analysis. The melting point of the
solid compounds was determined in open capillary using ANALAB
Melting Point Apparatus.
18:[14] Yield: 0.65 g (51%). 1H NMR (CDCl3): d=1.07–1.11 (br, 3H),
1.24–1.29 (br, 3H), 3.18 (br, 2H), 3.56–3.57 (br, 2H), 7.18–7.29 (m, 3H),
7.78–7.80 ppm (d, 1H, J=8.0 Hz); 13C NMR (CDCl3): d=13.26, 14.62,
39.72, 43.68, 126.37, 127.59, 129.54, 130.35, 132.74, 138.23, 170.01 ppm;
77Se NMR (CDCl3): d=425 ppm. HRMS (TOF MS): m/z calcd for
C22H28N2O2Se2: 535.0379 [M+Na]+; found: 535.0370.
19: Yield: 0.69 g (49%). 1H NMR (CDCl3) d=0.66 (br, 3H), 0.93 (br,
3H), 1.44–1.45 (br, 2H), 1.67 (br, 2H), 3.05 (br, 2H), 3.41 (br, 2H), 7.10–
7.20 (m, 3H), 7.70–7.72 ppm (d, 1H, J=8.0 Hz); 13C NMR (CDCl3) d=
10.16, 10.61, 19.62, 20.81, 45.53, 49.68, 125.33, 125.88, 128.18, 128.84,
131.02, 136.54, 169.00 ppm; 77Se NMR (CDCl3) d=423 ppm. HRMS
(TOF MS): m/z calcd for C26H36N2O2Se2: 591.1005 [M+Na]+; found:
591.1000.
1
20:[13] Yield: 0.80 g (57%). M.p.: 154–1568C. H NMR (CDCl3): d=0.87–
1.17 (br, 6H), 1.42–1.65ACTHNUTRGENUG(N br, 6H), 3.61 (br, 1H), 3.78–3.83 (br, 1H), 7.09–
7.11 (d, 1H, J=8 Hz), 7.18–7.23 (m, 2H), and 7.75–7.77 ppm (d, 1H, J=
8 Hz); 13C NMR (CDCl3): d=20.81, 46.29, 51.35, 125.07, 126.87, 128.80,
129.49, 129.99, 131.73, 138.97, 169.30 ppm; 77Se NMR (CDCl3): d=
412 ppm. HRMS (TOF MS): m/z calcd for C26H40N2Se2: 541.1600
[M+H]+; found: 541.1613.
Synthesis of Compounds 13–16
Triphenylphosphine (0.265 mmol) was added to a CH2Cl2 solution of the
ebselen derivatives (57–60) (0.133 mmol), and the reaction mixture was
stirred at room temperature for 2 h to obtain a white precipitate of the
corresponding diselenide compounds. The solvent was evaporated and
the solid was washed 3–4 times with petroleum ether to remove the un-
reacted triphenylphosphine and its bi-products. The product, obtained in
quantitative yield was then recrystallized from dimethyl sulphoxide.
13: M.p.:>2508C. 1H NMR ([D6]DMSO): d=2.83 (d, 3H, J=4.0 Hz),
7.30–7.40 (m, 2H), 7.66–7.68 (d, 1H, J=8.0 Hz), 7.76–7.78 (d, 1H, J=
8.0 Hz), 8.72–8.73 ppm (d, 1H, J=4.0 Hz); 13C NMR ([D6]DMSO): d=
27.34, 127.16, 128.71, 130.75, 132.53, 132.72, 133.77, 168.71 ppm; 77Se
NMR ([D6]DMSO): d=439 ppm HRMS (TOF MS): m/z calcd for
C16H16N2O2Se2: 450.9440 [M+Na]+; found: 450.9447.
14: M.p.: 206–2088C. 1H NMR ([D6]DMSO): d=1.14–1.18 (t, 3H, J=
8.0 Hz), 3.29–3.36 (q, 2H, J=8.0 Hz), 7.30–7.40 (m, 2H), 7.67–7.68 (d,
1H, J=4.0 Hz), 7.77–7.79 (d, 1H, J=4.0 Hz), 8.75 ppm (br, 1H);
13C NMR ([D6]DMSO): d=15.55, 35.23, 127.11, 128.72, 130.77, 132.47,
132.75, 134.05, 168.06 ppm; 77Se NMR ([D6]DMSO): d=440 ppm. HRMS
(TOF MS): m/z calcd for C18H20N2O2Se2: 478.9753 [M+Na]+; found:
478.9762.
15: M.p.: 212–2148C. 1H NMR ([D6]DMSO): d=0.79–0.83 (t, 3H, J=
8.0 Hz), 1.41–1.50 (m, 2H), 3.12–3.17 (q, 2H, J=8.0 Hz), 7.19–7.28 (m,
2H), 7.66–7.58 (d, 1H, J=8.0 Hz), 7.66–7.67 (d, 1H, J=4.0 Hz), 8.64–
8.65 ppm (br, 1H); 13C NMR ([D6]DMSO): d=12.35, 23.20, 42.08,
127.13, 128.72, 130.77, 132.43, 132.72, 134.21, 168.27 ppm; 77Se NMR
([D6]DMSO): d=439 ppm. HRMS (TOF MS): m/z calcd for
C20H24N2O2Se2: 507.0066 [M+Na]+; found: 507.0041.
Synthesis of Compounds 21–24
Thiophenol (13.7 mm, 0.125 mmol) was added to a CH2Cl2 (5 mL) solution
of ebselen derivatives (57–60) (0.125 mmol), at room temperature and
the reaction mixture was stirred for 30 min. The solvent was evaporated
and the solid obtained was washed with petroleum ether (4ꢂ20 mL) to
remove the unreacted PhSH and the corresponding disulfide impurities.
The residue was then dried in vacuum to obain the corresponding sele-
nenyl sulfide in quantitative yield.
21: 1H NMR (CDCl3): d=2.98–3.00 (d, 3H, J=8.0 Hz), 6.56 (br, 1H),
7.10–7.14 (t, 1H, J=4.0 Hz), 7.18–7.22 (t, 3H, J=8.0 Hz), 7.38–7.47 (t,
1H, J=8.0 Hz), 7.49–7.52 (m, 3H), 8.14–8.16 ppm (d, 1H, J=8.0 Hz);
13C NMR (CDCl3): d=27.51, 126.51, 126.88, 127.04, 128.86, 129.35,
131.10, 132.53, 137.17, 137.42, 169.11 ppm; 77Se NMR (CDCl3): d=
587 ppm. HRMS (TOF MS): m/z calcd for C14H13NOSSe: 345.9781
[M+Na]+; found: 345.9769.
22: 1H NMR (CDCl3): d=1.15–1.18 (t, 3H, J=8.0 Hz), 3.37–3.44 (m,
2H), 6.42 (br, 1H), 7.02–7.06 (t, 1H, J=8.0 Hz), 7.11–7.14 (t, 3H,
8.0 Hz), 7.30–7.34 (t, 1H, J=8.0 Hz), 7.40–7.47 (m, 3H), 8.07–8.09 ppm
(d, 1H, J=8.0 Hz); 13C NMR (CDCl3): d=15.32, 35.73, 126.46, 126.83,
126.97, 128.87, 129.33, 131.23, 132.49, 137.21, 137.52, 168.33 ppm; 77Se
NMR (CDCl3): d=588 HRMS (TOF MS): m/z calcd for C15H15NOSSe:
359.9937 [M+Na]+; found: 359.9937.
23: 1H NMR (CDCl3): d=0.95–0.98 (t, 3H, J=8.0 Hz), 1.61–1.67 (m,
2H), 3.38–3.44 (q, 2H, J=8.0 Hz), 6.51 (br, 1H), 7.12–7.14 (t, 1H, J=
4.0 Hz), 7.19–7.25 (m, 3H), 7.39–7.43 (t, 1H, J=8.0 Hz), 7.48–7.55 (m,
3H), 8.14–8.17 (d, 1H, J=8.0 Hz); 13C NMR (CDCl3): d=11.95, 23.35,
42.52, 126.47, 126.82, 126.92, 128.89, 129.29, 129.35, 131.28, 132.50, 137.19,
137.53, 168.43; 77Se NMR (CDCl3): d=586 HRMS (TOF MS): m/z calcd
for C16H17NOSSe: 374.0091 [M+Na]+; found: 374.0102.
16: M.p.: 244–2468C. 1H NMR ([D6]DMSO): d=1.06–1.07 (d, 6H, J=
4.0 Hz), 3.94–4.02 (m, 1H), 7.16–7.24 (m, 2H), 7.53–7.55 (d, 1H, J=
8.0 Hz), 7.64–7.66 (d, 1H, J=8.0 Hz), 8.39–8.41 ppm (d, 1H, J=8.0 Hz);
13C NMR ([D6]DMSO): d=23.14, 42.31, 127.02, 128.85, 130.77, 132.37,
132.75, 134.37, 167.45 ppm; 77Se NMR ([D6]DMSO): d=440 ppm. HRMS
(TOF MS): m/z calcd for C20H24N2O2Se2 507.0066 [M+Na]+; found:
507.0066.
24: 1H NMR (CDCl3): d=1.27–1.28 (d, 6H, J=4.0 Hz), 4.24–4.32 (m,
1H), 6.22–6.23 (br, 1H), 7.12–7.14 (t, 1H, J=8.0 Hz), 7.19–7.25 (m, 3H),
7.39–7.43 (t, 1H, J=8.0 Hz), 7.48–7.51 (m, 3H), 8.14–8.16 ppm (d, 1H,
J=8.0 Hz); 13C NMR (CDCl3): d=23.29, 42.88, 126.40, 126.79, 126.84,
128.89, 129.27, 129.34, 131.33, 132.47, 137.21, 137.58, 167.57 ppm; 77Se
NMR (CDCl3): d=587 ppm. HRMS (TOF MS): m/z calcd for
C16H17NOSSe: 374.0094 [M+Na]+; found: 374.0102.
Synthesis of Compounds 17–20
Freshly distilled thionyl chloride (0.55 mL, 7.500 mmol) was added drop-
wise to a suspension of 2-selenosalicylic acid (1.0 g, 2.500 mmol) in ben-
zene and the mixture was refluxed for 3 h. The solvent was evaporated
under reduced pressure. Secondary amines (7.500 mmol) were added and
stirred for another 5 h at room temperature. The desired amide based
diselenides were obtained after purification by flash chromatography
using silica gel column with ethyl acetate and petroleum ether as eluents.
17:[13] Yield: 0.60 g (53%). M.p.: 150–1528C. 1H NMR (CDCl3): d=2.85–
2.90 (br, 3H), 3.10–3.11 (br, 3H), 7.20–7.30 (m, 3H), 7.79–7.81 ppm (d,
1H, J=8.0 Hz); 13C NMR (CDCl3): d=35.54, 39.63, 127.22, 127.62,
130.00, 130.61, 132.87, 137.54, 170.57 ppm; 77Se NMR (CDCl3): d=
433 ppm. HRMS (TOF MS): m/z calcd for C18H20N2O2Se2 [M+Na]+,
478.9758 found 478.9758.
Synthesis of Compounds 25–28
Hydrogen peroxide (30% solution, 0.70 mL, 7.00 mmol) was added to a
MeOH (5 mL) solution of the tertiary amide based diselenides (17–20)
(0.700 mmol) at room temperature and the reaction mixture was stirred
for 20 min followed by the addition of thiophenol (1.15 mL, 10.5 mmol).
After 20 min, the solvent was evaporated and the residue was dissolved
in CH2Cl2 and purified by flash chromatography using silica gel column
with petroleum ether and ethyl acetate as eluents.
Chem. Asian J. 2009, 4, 974 – 983
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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981

K. P. Bhabak and G. Mugesh.
FULL PAPERS
25: Yield: 0.13 g (55%). 1H NMR (CDCl3): d=2.98–3.03 (br, 6H), 7.17–
7.35 (m, 6H), 7.50–7.53 (m, 2H), 7.87–7.89 ppm (d, 1H, J=8.0 Hz).
13C NMR (CDCl3): d=30.18, 127.20, 127.54, 127.67, 129.45, 129.78,
130.80, 133.27, 136.27, 137.00, 170.59 ppm; 77Se NMR (CDCl3): d=
527 ppm. HRMS (TOF MS): m/z calcd for C15H15NOSSe: 359.9937
[M+Na]+; found: 359.9949.
26: Yield: 0.13 g (52%). 1H NMR (CDCl3): d=1.15–1.28 (br, 6H), 3.16–
3.20 (br, 2H), 3.47–3.52 (br, 2H), 7.16–7.34 (m, 6H), 7.50–7.53 (m, 2H),
7.84–7.86 ppm (d, 1H, J=8.0 Hz). 13C NMR (CDCl3): d=14.61, 39.98,
43.62, 126.64, 127.29, 127.61, 129.44, 129.91, 130.45, 130.79, 132.49, 136.99,
137.35, 170.02 ppm; 77Se NMR (CDCl3): d=520 ppm. HRMS (TOF MS):
m/z calcd for C17H19NOSSe; 388.0250 [M+Na]+; found: 388.0234.
27: Yield: 0.13 g (48%); 1H NMR (CDCl3): d=0.71 (br, 3H), 0.96–0.98
(br, 3H), 1.47–1.49 (br, 2H), 1.73 (br, 2H), 3.10 (br, 2H), 3.45 (br, 2H),
7.16–7.33 (m, 6H), 7.49–7.52 (m, 2H), 7.86–7.88 ppm (d, 1H, J=8.0 Hz);
13C NMR (CDCl3): d=11.71, 21.13, 22.27, 47.14, 51.17, 127.08, 127.62,
129.42, 130.01, 130.42, 130.51, 132.66, 137.00, 137.16, 170.46 ppm; 77Se
NMR (CDCl3): d=525 ppm; HRMS (TOF MS): m/z calcd for
C19H23NOSSe: 416.0563 [M+Na]+; found: 416.0565.
28: Yield: 0.14 g (51%); 1H NMR (CDCl3): d=1.18–1.35 (br, 12H), 3.61
(br, 2H), 7.06–7.23 (m, 6H), 7.44–7.47 (m, 2H), 7.75–7.77 ppm (d, 1H,
J=8.0 Hz); 13C NMR (CDCl3): d=19.72, 45.53, 49.86, 124.24, 125.63,
126.01, 127.92, 128.19, 128.19, 128.47, 128.80, 130.58, 135.50, 137.30,
168.15 ppm; 77Se NMR (CDCl3): d=512 ppm. HRMS (TOF MS): m/z
calcd for C19H23NOSSe: 394.1 [M+H]+; found: 394.4.
d=876 ppm; HRMS (TOF MS) m/z calcd for C9H9NOSe: 249.9747
[M+Na]+; found: 249.9753.
1
59: Yield: 0.167 g (59%). M.p.: 70–728C. H NMR (CDCl3): d=0.98–1.01
(t, 3H, J=8.0 Hz), 1.71–1.80 (m, 2H), 3.81–3.85 (t, 2H, J=8.0 Hz), 7.40–
7.42 (t, 1H, J=8.0 Hz), 7.56–7.60 (t, 1H, J=8.0 Hz), 7.65–7.67 (d, 1H,
J=8.0 Hz), 8.04–8.06 ppm (d, 1H, J=8.0 Hz); 13C NMR (CDCl3): d=
11.64, 24.36, 46.92, 124.48, 126.63, 128.10, 129.27, 132.32, 138.23,
167.72 ppm. 77Se NMR (CDCl3): d=883 ppm; HRMS (TOF MS) m/z
calcd for C10H11NOSe: 263.9904 [M+Na]+; found: 263.9901.
1
60: Yield: 0.178 g (69%). M.p.: 94–968C. H NMR (CDCl3): d=1.22–1.23
(d, 6H, J=4.0 Hz), 4.73–4.79 (m, 1H), 7.24–7.28 (t, 1H, J=8.0 Hz), 7.39–
7.43 (t, 1H, J=8.0 Hz), 7.62–7.64 (d, 1H, J=8.0 Hz), 7.91–7.93 (d, 1H,
J=8.0 Hz). 13C NMR (CDCl3): d=23.85, 47.03, 124.43, 126.59, 129.09,
132.13, 138.05, 167.08; 77Se NMR (CDCl3): d=820; HRMS (TOF MS) m/
z calcd for C10H11NOSe: 263.9904 [M+Na]+; found: 263.9917.
GPx Activity
HPLC assay. The GPx-like activity was measured using High Perfor-
mance Liquid Chromatography (HPLC) consisting of a 2695 separation
module and a 2996 photodiode-array detector and fraction collector. The
assays were performed in 1.8 mL sample vials and a built-in autosampler
was used for sample injection. In this assay, we employed a mixture con-
taining a 1:2 molar ratio of PhSH and peroxide in methanol at room tem-
perature as our model system. Runs with and without catalyst were car-
ried out under the same conditions. Periodically, aliquots were injected
into the reverse phase column (Lichrosphere 60, RP-select B, 5 mm) and
eluted with methanol and water (90:10), and the concentrations of the
product diphenyl disulfide (PhSSPh) were determined at 254 nm using
pure PhSSPh as an external standard. The amount of disulfide formed
during the course of the reaction was calculated from the calibration plot
for the standard (PhSSPh).
Synthesis of Compounds 48 and 52
Aqueous solution of H2O2 (30% aq solution (w/v), 118 mL, 1.037 mmol)
was added to a methanolic (5 mL) solution of 16 or 20 (0.207 mmol), and
the reaction mixture was stirred for 1 h at room temperature. The solvent
was evaporated to obtain a white solid which was recrystallized from
methanol to obtain colourless crystals.
48: Yield 0.02 g (38%). 1H NMR (CDCl3): d=1.19–1.21 (d, 6H, J=
8.0 Hz), 4.17–4.20 (m, 1H), 7.61–7.65 (t, 1H, J=8.0 Hz), 7.72–7.75 (t, 1H,
J=8.0 Hz), 7.95–8.00 (d, 1H, J=8.0 Hz), 8.20–8.22 ppm (d, 1H, J=
8.0 Hz); 13C NMR (CDCl3): d=21.58, 48.08, 126.73, 128.56, 131.68,
133.41, 134.54, 145.06, 168.40 ppm; 77Se NMR (CDCl3): d=1074 ppm.
52: Yield: 0.03 g (46%). 1H NMR ([D4]MeOH): d=1.12–1.43 (br, 12H),
3.63 (br, 1H), 3.91 (br, 1H), 7.43–7.45 (d, 1H, J=8.0 Hz), 7.54–7.58 (t,
1H, J=8.0 Hz), 7.60–7.64 (t, 1H, J=8.0 Hz), 8.01–8.03 ppm (d, 1H, J=
8.0 Hz); 13C NMR ([D4]MeOH): d=20.02, 52.51, 126.54, 126.90, 130.23,
131.96, 137.74, 145.01, 169.18 ppm; 77Se NMR ([D4]MeOH): d=
1222 ppm.
Computational Methods
All calculations were performed using Gaussian98 suite[16] of quantum
chemical program. The hybrid Becke 3-Lee–Yang–Parr (B3LYP) ex-
change correlation functional was applied for DFT calculations.[17] Geo-
metries were fully optimized at B3LYP level of theory using the 6-
31+G(d) basis sets. Orbital interactions were analyzed using natural
bond orbital (NBO)[18] method at B3LYP/6-311+G
ACTHNUTRGNEUNG(d,p) level and charges
were calculated from natural population analysis (NPA).[19]
X-ray Crystallography
X-ray crystallographic studies were carried out on a Bruker CCD diffrac-
tometer with graphite-monochromatized MoKa radiation (l=0.71073 ꢁ)
controlled by a Pentium-based PC running on the SMART software
package.[20] Single crystals were mounted at room temperature on the
ends of glass fibers and data were collected at room temperature. The
structures were solved by direct methods and refined using the
SHELXTL software package.[21] All non-hydrogen atoms were refined
anisotropically and hydrogen atoms were assigned idealized locations.
Empirical absorption corrections were applied to all structures using
SADABS.[22] The structures were solved by direct method (SIR-92) and
refined by full-matrix least-squares procedure on F2 for all reflections
(SHELXL-97).[23]
Synthesis of Compounds 57–60
The ebselen derivatives were synthesized by following the literature pro-
cedure with minor modifications.[10b] A solution of 2-(chloroseleno)benzo-
yl chloride (0.100 g, 0.39 mmol) in dry acetonitrile (5 mL) was added
dropwise with stirring to a solution of the primary amine (0.47 mmol) in
dry acetonitrile (5 mL), over a period of 10 min followed by addition of
triethyl amine (0.10 mL, 0.78 mmol). The reaction mixture was stirred at
room temperature for about 12 h and the solvent was evaporated in
vacuum. Water (30 mL) was added and stirring was continued for anoth-
er 12 h. The precipitate obtained was filtered off and dried to obtain a
white solid. The compound was then purified by flash chromatography
using silica gel column with ethyl acetate and petroleum ether as eluents.
Crystal data for 17: C18H20N2O2Se2, Mr =454.30, monoclinic, space group
P21/c, a=14.252(15), b=12.168(10), c=11.459(9) ꢁ; V=1823(9) ꢁ3, Z=
4, 1calcd =1.65 gcmÀ3, MoKa radiation (l=0.71073 ꢁ), T=291(2) K; R1 =
0.035, wR2 =0.084 (I>2s (I)); R1 =0.060, wR2 =0.094 (all data).
1
57:[15] Yield: 0.118 g (66%). M.p.: 144–1468C; H NMR (CDCl3): d=3.42
(s, 3H), 7.39–7.43 (t, 1H, J=8.0 Hz), 7.56–7.68 (t, 1H, J=8.0 Hz), 8.04–
8.06 (d, 1H, J=8.0 Hz), 8.04–8.06 ppm (d, 1H, J=8.0 Hz); 13C NMR
(CDCl3): d=32.05, 124.56, 126.70, 127.49, 129.20, 132.44, 138.11,
168.02 ppm. 77Se NMR (CDCl3): d=913 ppm. HRMS (TOF MS) m/z
calcd for C8H7NOSe: 235.9591 [M+Na]+; found: 235.9598.
Crystal data for 20: C26H36N2O2Se2, Mr =566.49, orthorhombic, space
group Pbca, a=19.238(5), b=9.326(2), c=30.988(7) ꢁ; V=5560(2) ꢁ3,
Z=8, 1calcd =1.354 gcmÀ3, MoKa radiation (l=0.71073 ꢁ), T=291(2) K;
R1 =0.055, wR2 =0.108 (I>2s (I)); R1 =0.146, wR2 =0.138 (all data).
58: Yield: 0.168 g (63%). M.p.: 92–948C; 1H NMR (CDCl3): d=1.32–
1.35 (t, 3H, J=8.0 Hz), 3.87–3.93 (q, 2H, J=8.0 Hz), 7.39–7.43(t, 1H, J=
8.0 Hz), 7.55–7.59 (t, 1H, J=8.0 Hz), 7.64–7.66(d, 1H, J=8.0 Hz), 8.03–
8.05 ppm (d, 1H, J=8.0 Hz). 13C NMR (CDCl3): d=16.15, 40.32, 124.53,
126.65, 128.17, 129.20, 132.31, 138.11, 167.40 ppm; 77Se NMR (CDCl3):
Crystal data for 48: C10H13NO3Se; Mr =274.2, Monoclinic, space group
P21/c, a=9.616(15), b=8.034(13), c=14.722(2) ꢁ, b=99.086(3), V=
1123.2(3) ꢁ3, Z=4, 1calcd =1.62 gcmÀ1, MoKa radiation (l=0.71073 ꢁ),
T=291(2) K; R1 =0.04, wR2 =0.07 (I>2s(I)); R1 =0.08, wR2 =0.083 (all
data).
982
www.chemasianj.org
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2009, 4, 974 – 983

Effect of Amide Substituents on the Antioxidant Behaviour of Glutathione Peroxidase Mimics
Crystal data for 52: C13H19NO3Se; Mr =316.2, Monoclinic, space group
P21/n, a=13.327(2), b=8.193(15), c=13.669(2) ꢁ, b=104.772(3), V=
1443.1(4) ꢁ3, Z=4, 1calcd =1.45 gcmÀ1, MoKa radiation (l=0.71073 ꢁ),
T=291 (2) K; R1 =0.04, wR2 =0.08 (I>2s(I)); R1 =0.07, wR2 =0.09 (all
data).
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Acknowledgements
[11] The identification of selenonic acids by 77Se NMR is based on litera-
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This study was supported by the Department of Science and Technology
(DST), New Delhi, India. We are grateful to the Alexander von Hum-
boldt Foundation, Bonn, Germany for the donation of an automated
flash chromatography system. G.M. acknowledges DST for the award of
Ramanna fellowship and K.P.B. thanks Council of Scientific and Industri-
al Research (CSIR) for the research fellowship.
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Received: December 28, 2008
Revised: February 17, 2009
Published online: April 17, 2009
Chem. Asian J. 2009, 4, 974 – 983
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
983