Tertiary amine-based glutathione peroxidase mimics: Some insights into the role of steric and electronic effects on antioxidant activity

Article abstract of DOI:10.1016/j.tet.2012.09.020

In this work, several tertiary amine-based diaryl diselenides were synthesized and evaluated for their glutathione peroxidase (GPx)-like antioxidant activities using hydrogen peroxide, tert-butyl hydroperoxide and cumene hydroperoxide as substrates and thiophenol (PhSH) and glutathione (GSH) as co-substrates. A comparison of the GPx-like activity of 4-methoxy-substituted N,N-dialkylbenzylamine-based diselenides with that of the corresponding 6-methoxy-substituted compounds indicates that the activity highly depends on the position of the methoxy substituent. Although the methoxy group at 4- and 6-position alters the electronic properties of selenium, the substitution at the 6-position provides the required steric protection for some of the key intermediates in the catalytic cycle. A detailed experimental and theoretical investigation reveals that the 6-methoxy substituent prevents the undesired thiol exchange reactions at the selenium centers in the selenenyl sulfide intermediates. The 6-methoxy substituent also prevents the formation of seleninic and selenonic acids. When PhSH is used as the thiol co-substrate, the 4-methoxy-substituted diselenides exhibit GPx-like activity similar to that of the parent compounds as the 4-methoxy substituent does not block the selenium center in the selenenyl sulfide intermediates from thiol exchange reactions. In contrast, the 4-methoxy substituent significantly enhances the GPx-like activity of the diselenides when glutathione (GSH) is used as the co-substrate.

Full text of DOI:10.1016/j.tet.2012.09.020

Tetrahedron 68 (2012) 10550e10560
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Tertiary amine-based glutathione peroxidase mimics: some insights into the role
of steric and electronic effects on antioxidant activity
*
Debasish Bhowmick, Govindasamy Mugesh
Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560 012, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 July 2012
Received in revised form 29 August 2012
Accepted 4 September 2012
Available online 10 September 2012
In this work, several tertiary amine-based diaryl diselenides were synthesized and evaluated for their
glutathione peroxidase (GPx)-like antioxidant activities using hydrogen peroxide, tert-butyl hydroper-
oxide and cumene hydroperoxide as substrates and thiophenol (PhSH) and glutathione (GSH) as co-
substrates. A comparison of the GPx-like activity of 4-methoxy-substituted N,N-dialkylbenzylamine-
based diselenides with that of the corresponding 6-methoxy-substituted compounds indicates that the
activity highly depends on the position of the methoxy substituent. Although the methoxy group at 4-
and 6-position alters the electronic properties of selenium, the substitution at the 6-position provides
the required steric protection for some of the key intermediates in the catalytic cycle. A detailed ex-
perimental and theoretical investigation reveals that the 6-methoxy substituent prevents the undesired
thiol exchange reactions at the selenium centers in the selenenyl sulfide intermediates. The 6-methoxy
substituent also prevents the formation of seleninic and selenonic acids. When PhSH is used as the thiol
co-substrate, the 4-methoxy-substituted diselenides exhibit GPx-like activity similar to that of the parent
compounds as the 4-methoxy substituent does not block the selenium center in the selenenyl sulfide
intermediates from thiol exchange reactions. In contrast, the 4-methoxy substituent significantly en-
hances the GPx-like activity of the diselenides when glutathione (GSH) is used as the co-substrate.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Antioxidant activity
GPx mimics
Selenoenzymes
Selenium
Thiol exchange
1. Introduction
Glutathione peroxidase (GPx) is a selenoenzyme that functions
as antioxidant by catalyzing the reduction of harmful peroxides by
using glutathione (GSH) as co-substrate.1 During the catalysis, the
selenol group present in the selenocysteine (Sec) residue of the
enzyme (E-SeH) reacts with peroxides to produce the corre-
sponding selenenic acid (E-SeOH) with the elimination of a water
molecule. The selenenic acid then undergoes reaction with GSH to
generate the enzyme-bound selenenyl sulfide (E-Se-SG). The nu-
cleophilic attack of GSH at the selenenyl sulfide linkage regenerates
the active site selenol with the release of glutathione disulfide,
GSSG (Scheme 1).1eeg At high concentrations of peroxide (oxidative
stress conditions) and low concentrations of GSH (depletion of
GSH), the selenenic acid may undergo further reaction with per-
oxides to produce the corresponding seleninic acid (E-SeO2H). The
rapid reactions of the selenenic acid with GSH and of the resulting
selenenyl sulfide (E-Se-SG) with a second GSH to produce the
selenol (E-SeH) are important for the catalytic activity.
Scheme 1. Proposed catalytic cycle for the reduction of H2O2 by GPx. At high con-
centrations of peroxides (oxidative stress conditions) and low concentrations of GSH
(GSH depletion), the formation of seleninic acid (E-SeO2H) may be formed.
The interesting redox chemistry at the active site of GPx2 led to
the design and development of simple organoselenium compounds
that can reduce peroxides catalytically in the presence of aliphatic
and aromatic thiols.3 These GPx model compounds include the
well-known anti-inflammatory compound ebselen (1)4 and its
analogues,5,6 camphor-based selenenyl amide (2),7 tert-amino
substituted diselenides (3e6),6e,8 diselenides 7e8 having Se/O
interactions,9 allyl and alkyl selenides (9e10),10 cyclodextrin-based
diselenide (11),11 3,3-diselenopropionic acid (12),12 nicotinoyl
* Corresponding author. Tel.: þ91 80 2360 2566; fax: þ91 80 2360 1552; e-mail
address: mugesh@ipc.iisc.ernet.in (G. Mugesh).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.09.020

D. Bhowmick, G. Mugesh / Tetrahedron 68 (2012) 10550e10560
10551
based diselenide (13),13 selenocysteine derivative (14),14 the cyclic
selenelate ester (15),15 and the spirodiazaselenuranes (16) (Fig. 1).16
Diaryl diselenides, such as 3, 4 and 6 having tert-amino substituents
attracted significant attention as the amino groups play several
positive roles in the catalytic cycle. Iwaoka and Tomoda proposed
that the tert-amino groups in the close proximity of selenium ab-
stract the proton from selenols to produce selenolates, which are
more reactive than the undissociated selenols.8b These amino
substituents may also facilitate the reduction of eSeeSee bond by
thiols to produce the selenenyl sulfide intermediates. However, the
strong Se/N non-bonded interactions between the amino groups
and selenium atom in the selenenyl sulfide intermediates signifi-
cantly reduce the GPx-like activity. In selenenyl sulfides having tert-
amine-substituents, the nucleophilic attack of thiol at selenium is
more favored than at sulfur, leading to thiol exchange reactions8
(Fig. 1).
moderate yield. The GPx-like activities of these diselenides were
studied using three different peroxidesdhydrogen peroxide
(H2O2), cumene hydroperoxide (Cum-OOH), and tert-butyl hydro-
peroxide (t-BuOOH)das the substrate. Glutathione (GSH) and thi-
ophenol (PhSH) were used as the thiol co-substrate. The catalytic
reduction of peroxides by GSH in the presence of various selenium
compounds were monitored by UVevis method using glutathione
reductase (GR) and NADPH. The decrease in concentration of
NADPH was monitored at 340 nm. The reduction of peroxides by
PhSH in the presence of selenium compounds was studied using
a HPLC method. The amount of PhSSPh produced in each reaction
was determined and the time required for 50% conversion of PhSH
into PhSSPh (t1/2 values) was calculated from the peak areas at
different time intervals.
Reactions of selenenyl amides, such as 1 or diselenides, such as 4
with thiols produce the corresponding selenenyl sulfides. In gen-
eral, the reduction of selenenyl sulfides to selenols need to over-
come substantial energy barriers.18 Therefore, the nucleophilic
attack of aromatic thiols at the selenium centers of the selenenyl
sulfides are energetically more favorable than at sulfur. The thiol
attack at the selenium center can be further enhanced by increasing
the electrophilic reactivity of selenium. It is known that ebselen (1)
exhibits a very weak GPx-like activity in the presence of PhSH. This
is due to the Se/O non-bonded interactions in the selenenyl sul-
fide intermediate, which favors the nucleophilic attack of PhSH at
the selenium center.6a Previous studies have shown that the N,N-
dialkylbenzylamine-based diselenides 4, 17, 18 exhibit much better
catalytic activity than ebselen in the presence of aromatic thi-
ols.8b,d,f The higher activity of these compounds is due to the
presence of a basic amino group, which can deprotonate the selenol
to generate a more reactive selenolate.
From Table 1 and Fig. 2, it is clear that the GPx-like activity of all
the amine-based diselenides in the presence of PhSH is much
higher than that of ebselen in all three peroxide systems. Wirth and
co-workers had previously shown that the replacement of aryl
protons with methoxy substituents in diselenides improves the
stereoselectivity in asymmetric selenenylation reactions.19 We re-
ported that simple replacement of hydrogen atoms by methoxy
substituents in N,N-dialkylbenzylamine-based diselenides en-
hances the catalytic activity.8f Compounds 19e21 having 6-
methoxy substituents were found to be much better catalysts
than the diselenides 4, 17 and 18, respectively. The protection of the
selenium moieties from overoxidation by peroxides and the pre-
vention of thiol exchange reactions at the selenium centers in the
selenenyl sulfide intermediates upon introduction of the methoxy
substituents have been shown to be the important factors for the
improvement in the catalytic activity.8f In the present study, all the
6-methoxy-substituted diselenides (19e21 and 26) exhibited much
better activity than the corresponding parent compounds 4, 17, 18
and 25, respectively. However, the increase in activity upon
Fig. 1. Some small-molecule GPx mimics reported in the literature.3e16
Recently, we have shown that the substitution of hydrogen atom
at 6-position in N,N-dialkylbenzylamine-based diselenides by
a methoxy group increases the catalytic activity.8f For example, the
GPx-like activity of the methoxy-substituted compounds 19e21
has been shown to be much higher than that of 4, 17 and 18.8f It has
been suggested that the electronic and steric effects of the 6-
methoxy group play key roles in the enhancement of catalytic ac-
tivity. However, it is not clear whether it is the electronic effect or
the steric effect that is responsible for the increase in the GPx-like
activity. In this paper, we report the synthesis and characterization
of a series of tert-amine-based diselenides having 4-methoxy group
and a comparison of their GPx-like activity with that of the corre-
sponding 6-methoxy-substituted compounds.
2. Results and discussion
2.1. Synthesis of diselenides
Amino-substituted diselenides 4, 17e27 used in this study were
synthesized from 2-bromo-N,N-dialkylbenzylamines, 3-methoxy-
N,N-dialkylbenzylamines or 2-bromo-5-methoxy-N,N-dilkylben-
zylamines via the ortho-lithiation method.17 The treatment of
amines with n-BuLi afforded the corresponding ortho-lithiated
compounds, which upon reaction with selenium powder followed
by oxidative work up afforded the corresponding diselenides in

10552
D. Bhowmick, G. Mugesh / Tetrahedron 68 (2012) 10550e10560
Table 1
Reduction of peroxides by PhSH in the absence of and the presence of compounds 1,
4, 17e31
Compd
t1/2 (min)a
H2O2
t-BuOOH
Cum-OOH
Control
1
4
512.0ꢀ20.2
422.0ꢀ10.0
18.3ꢀ0.1
19.8ꢀ0.7
27.1ꢀ0.5
5.8ꢀ0.1
781.0ꢀ41.0
329.0ꢀ2.0
23.9ꢀ3.4
35.5ꢀ4.6
47.3ꢀ11.2
12.5ꢀ1.2
37.8ꢀ5.1
40.3ꢀ2.9
12.5ꢀ0.8
16.9ꢀ0.6
17.3ꢀ0.9
60.4ꢀ5.1
45.0ꢀ1.5
28.1ꢀ3.7
592.0ꢀ29.4
699.0ꢀ46.8
471.0ꢀ33.7
375.0ꢀ9.2
22.4ꢀ1.5
24.3ꢀ0.8
23.8ꢀ2.2
4.9ꢀ0.2
17
18
19b
20
21
22
23
24
25
26
27
28
29
13.2ꢀ0.3
15.7ꢀ0.6
14.5ꢀ1.2
16.8ꢀ1.7
23.9ꢀ2.1
53.7ꢀ3.8
39.1ꢀ4.3
24.6ꢀ3.1
365.0ꢀ23.4
413.0ꢀ18.5
11.9ꢀ1.1
14.1ꢀ0.9
11.6ꢀ0.1
12.9ꢀ0.6
15.0ꢀ1.3
46.5ꢀ2.1
13.0ꢀ1.4
16.9ꢀ3.1
213.0ꢀ11.8
246.5ꢀ23.0
Fig. 3. X-ray crystal structure of compound 25. ORTEP diagram with the ellipsoids
representing 50% probability. ꢁSignificant bond lengths and bond angles: Se1eSe2:
ꢁ
ꢀ
2.355 A; Se1eSe2eN2: 168.86 . Se2eSe1eN1: 168.86 .
a
The reactions were carried out in MeOH at 22 ꢁC. Catalyst: 10.0
mM [except
compounds 4,17 and 18 marginally enhances the GPx-like activity. A
slight increase in the activity was observed for compound 22 as
compared to 4 when H2O2 was used as substrate. In contrast, a sig-
nificant enhancement in the activity was observed upon in-
troduction of a methoxy group at the 4-position of compound 25.
Compound 27 was found to be almost twotimes more active than 25
in all three peroxide systems. Interestingly, compound 27 was found
to be more active than compound 26, which contains a methoxy
group in the 6-position. The lower activity of compound 26 as
compared to that of 27 is probably due to the presence of N-cyclo-
hexyl and methoxy groups, which increases the steric hindrance
around selenium, and hence blocks the eSeeSee bond cleavage by
thiols. However, in both 4- and 6-methoxy-substituted diselenides,
the presence of tert-amino group is important for the catalytic ac-
tivity. Compounds 28 and 29, which lack the amino groups, were
found to be poor catalysts in all three peroxide assays. The 2-
methoxy-substituted diselenides 28 was found to be marginally
better than the 4-methoxy derivative 29.
compound 19]; PhSH: 1.0 mM; peroxide: 2.0 mM. The control reactions were per-
formed under identical condition in the absence of selenium compounds.
b
A lower concentration of compound 19 (5
m
M) was used as the conversion was
too fast to be measured at 10 mM concentration.
100
80
60
40
20
0
19
22
4
27
26
25
1
The catalytic reduction of peroxides by diselenides was also
studied by using GSH as the substrate. The initial rates (vo) for the
reactions were determined by following the oxidation of NADPH at
340 nm using GSHeGSSG coupled assay in phosphate buffer.3-
Control
0
20
40
60
80
100
time (min)
a,6a,b,8a
The initial rates, summarized in Table 2, indicate that most
Fig. 2. Catalytic reduction of H2O2 by PhSH in the presence of various selenium
compounds. The formation of PhSSPh was followed by a reverse-phase HPLC and the %
Table 2
Initial rates for the reduction of peroxides by GSH in the presence of compounds 1, 4
and 17e29
conversion was calculated from calibration plot. Assay condition: catalyst: 10.0
mM
(except compound 19, [catalyst 19]¼5.0
mM); PhSH: 1.0 mM; H2O2: 2.0 mM in MeOH at
22 ꢁC.
a
Compd
Initial rate (vo) [m ]
M minꢂ1
H2O2
t-BuOOH
Cum-OOH
introduction of methoxy groups appears to be dependent upon the
nature of amino groups. While compound 19 having an N,N-
dimethylamino substituent exhibits the highest activity, the in-
troduction of a sterically bulky N-cyclohexylamine significantly
reduces the catalytic activity. The X-ray crystal structure of com-
pound 25 indicates the existence of non-covalent Se/N in-
teractions (Fig. 3).
To understand whether it is the steric effect or the electronic
effect that plays an important role in the enhancement of catalytic
activity, we have determined the GPx-like activity of compounds
22e24, and 27 having 4-methoxy substituents. Back and co-workers
have reported the effect of substituents on the activity of aromatic
cyclic seleninate esters and spirodioxyselenuranes.20 They have
concluded that the introduction of methoxygroups at the 4-position
enhances the GPx-like activity. In these compounds, the electronic
effects of the substituents play an important role in modulating the
redox properties of selenium. From Table 1 and Fig. 2, it is observed
that the introduction of a methoxy substituent at the 4-position of
1
4
137.1ꢀ5.5
509ꢀ12.1
28.8ꢀ1.1
192.6ꢀ8.6
179.9ꢀ5.9
47.9ꢀ2.5
199.8ꢀ11.7
84.0ꢀ1.5
74.6ꢀ1.6
244.3ꢀ9.2
207.7ꢀ8.1
104.2ꢀ3.0
26.9ꢀ0.5
61.1ꢀ3.2
132.3ꢀ1.4
43.0ꢀ1.1
27.5ꢀ1.7
42.8ꢀ1.5
480.3ꢀ11.5
334.1ꢀ12.1
127.3ꢀ6.1
472.5ꢀ4.6
195.1ꢀ8.4
112.3ꢀ2.3
595.3ꢀ7.5
535.0ꢀ5.1
357.7ꢀ7.1
96.5ꢀ1.7
17
18
19
20
21
22
23
24
25
26
27
28
29
496.8ꢀ14.3
255.1ꢀ7.7
537.4ꢀ14.3
410.2ꢀ9.5
372.2ꢀ2.7
562.6ꢀ14.3
558.0ꢀ11.2
537.2ꢀ16.4
144.08ꢀ6.3
205.3ꢀ6.6
561.8ꢀ10.7
178.0ꢀ6.9
151.9ꢀ5.1
110.0ꢀ6.8
481.8ꢀ7.2
75.9ꢀ8.6
45.8ꢀ3.1
a
Assay condition: the reactions were carried out in phosphate buffer (100 mM,
M; GSH: 2.0 mM; NADPH: 0.4 mM; EDTA: 1 mM;
pH 7.5) at 23 ꢁC. Catalyst: 80.0
m
glutathione disulfide reductase: 1.7 units/ml; peroxide: 1.6 mM. The control re-
actions were performed under identical condition in the absence of selenium
compounds. For the peroxidase activity, rates were corrected for the background
reaction between thiol and peroxide.

D. Bhowmick, G. Mugesh / Tetrahedron 68 (2012) 10550e10560
10553
of the diselenides exhibit much better catalytic activity than
ebselen, which is in agreement with the previous studies on amine-
based diaryl diselenides.8b,d,f Similar to the effect of 6-methoxy
group on the GPx-like activity of compounds 19e21 and 26 in the
presence of PhSH, a significant enhancement in the catalytic ac-
tivity in the presence of GSH was observed upon introduction of 6-
methoxy substituent except for compound 17. On the other hand,
compounds 22e24 and 27 having 4-methoxy substituents were
found to be more active than the corresponding 6-methoxy-
substituted diselenides. This is in contrast to the PhSH assay in
which the 6-methoxy derivatives (19e21) were found to be more
active than the corresponding 4-methoxy-substituted compounds
(22e24). The lower activity of 19e21 and 26 as compared to that of
22e24 and 27 can be ascribed to the larger size of thiol co-substrate
(GSH). The 6-methoxy substituent may inhibit the nucleophilic
attack of GSH at the selenium center in compounds 19e21 and 26,
although this substituent may play an important role in preventing
the thiol exchange reactions at the selenium centers in the sele-
nenyl sulfide intermediates.
to produce selenenic acids. (iii) Reactions of selenenic acids with
thiols to generate the corresponding selenenyl sulfides. (iv) Re-
generation of the catalytically active selenols from the selenenyl
sulfide intermediates by reaction with thiols. To understand the
effect of methoxy substituents on the reactivity of various in-
termediates in the catalytic mechanism, we have studied the re-
activity of diselenides toward thiol (PhSH) and peroxide (H2O2). The
reactions were followed by 77Se NMR spectroscopy. In some cases,
the intermediates were isolated and fully characterized.
Scheme 2. Proposed mechanism for the reduction of H2O2 by diaryl diselenides in the
presence of a thiol.
To further understand the catalytic behavior of compounds 4,
17e27, we have carried out detailed kinetic experiments. The cat-
alytic parameters, such as maximum velocity (Vmax), Michaelis
2.2. Reactivity of diselenides toward thiol
constant (Km), catalytic constant (kcat) and catalytic efficiency (
for the reduction of H2O2 by PhSH in the presence of compounds 4,
17e27, were obtained by plotting the reciprocal of initial rates (1/no
against the reciprocal of substrate concentration (1/[S]).6b,e,8d,f It is
clear from Table 3 that the catalytic efficiencies ( ) of compound
h)
Reactions of diselenides with thiols generally produce a mixture
of selenenyl sulfides and selenols, and the relative amount of
selenols produced in these reactions depends on the nature and
concentration of the thiols. Although both the 6-methoxy- and 4-
methoxy-substituted compounds react readily with PhSH to pro-
duce the corresponding selenenyl sulfides and selenols, the con-
version of selenenyl sulfides to selenols is more favorable by the 6-
methoxy substituent. Treatment of diselenides 19e21 and 26 with
PhSH (1 equiv in each case) rapidly generated the selenols 30e32
and 36, respectively, with the formation of only trace amount of the
corresponding selenenyl sulfides (33e35, 37). As 1 equiv of PhSH in
each case was not sufficient for quantitative conversion of 19e21
and 26 into the selenols, some unreacted diselenides were ob-
served in the reaction mixtures. When 2 equiv of PhSH was added
in each case, the diselenides and selenenyl sulfides were converted
quantitatively into the corresponding selenols (30e32, 36).
)
h
22e24 are around 1.2e1.5 times higher than that of compound 4,
17e18, but significantly lower than that of 19e21. The higher cat-
alytic efficiencies of compounds 19e21 as compared to that of
compounds 4, 17e18 and 22e24 is mainly due to the difference in
their Km values. The relatively lower Km values of compound 19e21
and 26 indicate that the selenenyl sulfides formed in the reaction
from 19e21 and 26 are readily converted to the corresponding
selenols by reaction with PhSH. The higher Km values for other
diselenides (4, 17e18, 22e24, 25 and 27) indicate the extensive
thiol exchange reactions due to strong Se/N interactions. These
compounds, therefore, follow the nonsaturation kinetics. When the
initial rates were measured with increasing concentration of PhSH
at constant catalyst and peroxide concentrations, a rapid increase in
the rate was observed at the beginning, but after certain PhSH
concentration, the rate became constant. This indicates that com-
pounds 22e27 follow saturation kinetics.
Table 3
Catalytic parameters [maximum velocities (Vmax), Michaelis constants (Km for
PhSH), catalytic constants (kcat) and catalytic efficiencies (h)] for the reduction of
H2O2 by PhSH in the presence of diselenides 4, 17e27
To understand the effect of 4-methoxy substituent on the re-
activity of diselenides toward thiols, compounds 22e24 and 27
were treated with PhSH (1 equiv in each case) under identical ex-
perimental conditions. While these reactions produced the corre-
sponding selenols (38e40, 44) and the selenenyl sulfides (41e43,
45) in nearly 1:1 ratios, the addition of 2 equiv of PhSH in each case
did not lead to complete conversion of the selenenyl sulfides into
the corresponding selenols. This is due to the thiol exchange re-
actions that take place at the selenium centers in the selenenyl
sulfide intermediates.6a As previously shown for related com-
pounds, the extent of thiol exchange reactions depend on the na-
ture of substituents in the close proximity of selenium.8f The
reactions of 41e43, 45 with a different thiol (4-MeeC6H4SH) pro-
duced new selenenyl sulfide, confirming the thiol exchange re-
actions (Fig. S65, Supplementary data). In this respect, the 4-
methoxy-substituted selenenyl sulfides (41e43, 45) behave some-
what similar to the parent selenenyl sulfides (49e51, 53). We have
shown previously that treatment of compounds 4, 17 and 18 pro-
duces the corresponding selenols 46e48, together with the
Compd
Vmax
(
m
M minꢂ1
)
Km (mM)
kcat (minꢂ1
)
h
(Mꢂ1 minꢂ1
)
4
50.8
24.1
11.3
54.9
10.5
11.2
126.6
52.3
31.4
17.8
4.6
2.85
1.40
0.79
0.35
0.12
0.17
6.98
2.05
1.46
2.93
0.10
5.34
5.08
2.41
1.13
5.49
1.05
1.12
12.66
5.23
3.14
1.78
0.46
6.38
1.78ꢃ103
1.71ꢃ103
1.42ꢃ103
1.58ꢃ104
8.87ꢃ103
6.58ꢃ103
1.81ꢃ103
2.54ꢃ103
2.15ꢃ103
0.60ꢃ103
4.63ꢃ103
1.19ꢃ103
17
18
19
20
21
22
23
24
25
26
27
63.8
Assay condition: PhSH (0.0e4.0 mM), H2O2 (1.0 mM), and catalyst (10
at 22 ꢁC.
m
M) in MeOH
The mechanism for the GPx-like activity of diaryl diselenides
generally involves four major steps (Scheme 2). (i) Reductive
cleavage of eSeeSee bond by thiols, leading to the formation of the
corresponding selenols. (ii) Reaction of the selenols with peroxides

10554
D. Bhowmick, G. Mugesh / Tetrahedron 68 (2012) 10550e10560
selenenyl sulfides 49e51, respectively, even in the presence of
2 equiv of PhSH.8f However, the 4-methoxy substituents in com-
pounds 41e43 and 45 appear to enhance the formation of selenols,
although the introduction of a methoxy group at the 6-position was
found to be more effective than at the 4-position in suppressing the
thiol exchange reactions.
interactions (Fig. 4 and Table 5). The interaction energies between
the selenium and nitrogen atoms calculated by natural bond orbital
(NBO) analyses, indicate that the incorporation of methoxy group in
the 6-position decreases the ESe/N by almost 50% for each amino-
The 77Se NMR chemical shifts of the three sets of selenenyl
sulfides show some interesting features (Table 4). The 77Se NMR
chemical shifts for compounds 49e51 and 53, which lack methoxy
substituents, are found to be in the range 549e564 ppm. These
values are marginally shifted downfield as compared to that of
PhSeSPh (526 ppm), indicating the presence of Se/N non-covalent
interactions. The strength of these interactions depends on the
nature of amino substituent. Sterically bulky amino groups appear
to decrease the strength of Se/N interactions. For example, the
77Se NMR chemical shift of compound 53 having a N-cyclohexyl
amino group is shifted upfield (549 ppm) as compared to that of
N,N-dimethylamino-substituted selenenyl sulfide 49 (564 ppm).
Interestingly, the methoxy group at 4- and 6-position shows dif-
ferent effect on the 77Se NMR chemical shifts. The peaks for com-
pounds 33e35, 37 having the methoxy group in the 6-position are
shifted 85e100 ppm upfield with respect to compounds 49e51, 53,
respectively. On the other hand, the signals for compounds 41e43,
45 having the methoxy substituents in the 4-position do not show
significant shift in the 77Se NMR spectra. While the 77Se NMR
chemical shifts for compounds 41e43 are shifted w15 ppm upfield
with respect to the parent selenenyl sulfides (49e51), the N-cy-
clohexylamine-based compounds 45 and 53 exhibit almost iden-
tical chemical shift values. These observations indicate that the
selenium centers in compounds 33e35 and 37 are significantly less
electrophilic than that of compounds 41e43 and 45. Therefore, the
methoxy group in the 6-position is more effective than in the 4-
position in preventing the undesired thiol exchange reactions.
Fig. 4. Energy-optimized geometries of the selenenyl sulfide intermediates 33e35, 37,
41e43 and 45. The optimizations of the geometries were performed at the B3LYP/6-
31þG(d) level of theory.
Table 4
Experimentally obtained 77Se NMR chemical shifts and theoretically calculated
substituted selenenyl sulfide. In contrast, the Se/N interactions
in the 4-methoxy-substituted compounds are much stronger than
that of the corresponding 6-methoxy-substituted selenenyl sul-
ESe/N for the selenenyl sulfides
Compd
77Se NMR
chemical
ESe/N
(kcal
Compd
77Se NMR
chemical
ESe/N
(kcal
ꢀ
fides. For example, the Se/N distance in compound 41 (2.687 A) is
b
b
shift (ppm)a
molꢂ1
)
shift (ppm)a
molꢂ1
)
33
34
35
37
41
42
470
461
451
464
548
542
5.78
7.02
6.59
7.04
10.96
9.00
43
45
49
50
51
53
540
553
564
558
555
549
10.25
9.16
11.20
11.23
12.57
9.66
Table 5
Experimentally determined 77Se NMR chemical shifts and the theoretically calcu-
lated charge on selenium atom in the selenols
77Se NMR
chemical
qSe
Compd
77Se NMR
chemical
qSe
b
b
Compd
shift (ppm)a
shift (ppm)a
a
The values are cited with respect to Me2Se.
The optimizations of the geometries were performed at the B3LYP/631þG(d)//
30
31
32
36
38
39
ꢂ58
ꢂ42
ꢂ37
ꢂ44
19
ꢂ0.246
ꢂ0.279
ꢂ0.295
0.102
40
44
46
47
48
52
17
13
35
54
56
67
ꢂ0.337
0.067
ꢂ0.289
ꢂ0.312
ꢂ0.314
0.069
b
B3LYP/6-311þG(d,p) levels.
The density functional theory (DFT) calculations indicate that
the strength of Se/N non-covalent interactions depend not only on
the nature of amino substituents, but also on the location of the
methoxy group. The introduction of methoxy groups in the 6-
positions dramatically decreases the strength of Se/N
ꢂ0.294
16
ꢂ0.322
a
The values are cited with respect to Me2Se.
The charge on selenium calculated at the B3LYP/631þG(d)//B3LYP/6-
b
311þG(d,p) levels.

D. Bhowmick, G. Mugesh / Tetrahedron 68 (2012) 10550e10560
10555
ꢀ
much shorter than that in compound 33 (2.870 A). Similarly, the
ESe/N values for compounds 41e43 and 45 (9.00e10.96 kcal molꢂ1
)
are significantly higher than that of compounds 33e35 and 37
(5.78e7.02 kcal molꢂ1). However, the strength of Se/N in-
teractions in the 4-methoxy-substituted compounds are slightly
weaker than that of the parent selenenyl sulfides 49e51 and 53
(Table 4). This is in agreement with the minor shifts in the 77Se
NMR chemical shift values.
According to Scheme 2, the reactions of diselenides with 1 equiv
of PhSH should produce a 1:1 mixture of the corresponding sele-
nenyl sulfides and selenols. However, the conversion of the sele-
nenyl sulfides into the selenols in the presence of two or more
equiv of PhSH depends on the nature of the substituents and the
rate of thiol exchange reactions. In addition to the amount of
selenol produced in the reactions, the high reactivity of selenol
toward H2O2 is crucial for the GPx-like activity. Therefore, we have
carried out experimental and theoretical investigations to un-
derstand the effect of methoxy substituents on the reactivity of
selenols. In agreement with our previous study,8f treatment of the
diselenides 19e21 and 26 with PhSH (2 equiv in each case) quan-
titatively generated the corresponding selenols (30e32 and 36). A
comparison of the 77Se NMR chemical shifts of the 6-methoxy-
substituted compounds 30 (ꢂ58 ppm), 31 (ꢂ42 ppm) 32
(ꢂ37 ppm) and 36 (ꢂ44 ppm) with that of parent selenols 46
(35 ppm), 47 (54 ppm), 48 (56 ppm) and 52 (67 ppm) indicates that
the introduction of a methoxy substituent at the 6-position leads to
a remarkable upfield shift in the 77Se NMR signals. Particularly, the
chemical shifts for the 6-methoxy-substituted selenol having a N-
cyclohexyl amino group is shifted almost 100 ppm upfield relative
to that for the unsubstituted selenol. These observations indicate
that the introduction of a methoxy group in the 6-position dra-
matically increases the negative charge on selenium (Fig. 5). The
selenium moiety in compounds 30e32 and 36 are, therefore, sig-
nificantly more nucleophilic than that in compounds 46e48 and
52. However, the prevention of thiol exchange reactions in the
selenenyl sulfide intermediates appears to be more important than
the increase in the nucleophilic character of the selenol for the
enhancement in the GPx-like activity. This is in agreement with the
theoretical investigations of Haverly-Coulson and Boyd, which in-
dicate that the introduction of substituents in the ortho, meta or
para positions has little effect on the energy barrier for the peroxide
reduction reaction.21
Fig. 5. Energy-optimized geometries of the selenol species 30e32, 36, 38e40 and 44.
The optimizations of the geometries were performed at the B3LYP/6-31þG(d) level of
theory.
In contrast to the effect of 6-methoxy substituent, the in-
troduction of a methoxy group at the 4-position does not lead to
a drastic change in the 77Se NMR chemical shifts. The chemical
shifts for compounds 38 (19 ppm), 39 (16 ppm), 40 (17 ppm) and 44
(13 ppm) are shifted 16e54 ppm upfield relative to those for
compounds 46e48 and 52, which lack the methoxy substituent.
However, the moderate change in the chemical shift values in-
dicates that the incorporation of a methoxy substituents at the 4-
position does increase the negative charge on selenium. There-
fore, the more amount of selenols produced in the reactions of
compounds 19e21 and 26 with PhSH relative to those of 22e24 and
27 may account for the higher activity of the 6-methoxy-
substituted diselenides as compared to that of the 4-methoxy-
substituted compounds. It should be noted that a quantitative
conversion of compounds 22e24 and 27 into the corresponding
selenols were observed only in the presence of a large excess of
PhSH (60 equiv in each case). The quantitative conversion at this
concentration of PhSH was confirmed by treating the selenols with
iodoacetic acid to get the corresponding alkylated products 54e56
(Scheme 3). Although the DFT calculations indicate that the in-
troduction of a 4-methoxy substituent can increase the negative
charge on selenium in most of the selenols (Table 5), the solvents
may have a significant effect on the zwitterionic character of the
selenols in solution.
Scheme 3. Reactions of diselenides 22e24 with an excess amount of PhSH (60 equiv).
The selenols produced in these reactions could be trapped by treating with iodoacetic
acid.
2.3. Reactions of diselenides with peroxides
In addition to the experiments to understand the role of
methoxy substituents in the selenols and selenenyl sulfides, we
have also studied the effect of Se/N interactions in the selenenic
acids that are considered to be a crucial type of intermediates in the
GPx catalytic cycle. It is known that the amine-based diselenides
readily react with peroxides to produce selenenic/seleninic acids.
Treatment of compounds 22e24 with H2O2 produced a mixture of
the corresponding selenenic acids (57e59) and seleninic acids
(60e62). When compound 22 was treated with H2O2, two 77Se
peaks were observed at 1163 ppm and 1189 ppm, which could be
assigned to the selenenic acid 57 and seleninic acid 60, respectively.
In the presence of an excess amount (10 equiv) of H2O2, these two
compounds were converted to the selenonic acid 63, which
exhibited a signal in 77Se NMR at 1018 ppm. The signal is

10556
D. Bhowmick, G. Mugesh / Tetrahedron 68 (2012) 10550e10560
significantly shifted upfield as compared to that of the seleninic
acid 60 (1189 ppm). This is in agreement with the report of Iwaoka
and Tomoda that the selenonic acid derived from compound 4
exhibits an upfield shift in 77Se NMR with respect to the corre-
sponding selenenic and seleninic acids,8b which is probably due to
conversion of 66 into 72.8f The higher stability of the 6-methoxy-
substituted selenenic acids 75e77 as compared to that of 57e59
or 66e68 may contribute to the enhanced GPx-like activity of
compounds 19e21.
The optimized geometries (Fig. 6) and NBO analysis (Table 6)
indicate that both the 4-methoxy- and 6-methoxy-substituted
selenenic acids exhibit strong Se/N interactions, which help
nucleophilic attack by incoming thiols at the selenium centers.
Although the Se/N distances in the 6-methoxy-substituted
compounds 75e77 and 86 are slightly longer than that observed
in the corresponding 4-methoxy-substituted compounds (57e59
and 84) (Fig. 6), they exhibit similar reactivity toward thiols.
Therefore, the oxidation of selenenic acids to the corresponding
seleninic and selenonic acids may partly be responsible for the
lower activity of compounds 22e24 as compared to that of 19e21.
Haverly-Coulson and Boyd have shown that ortho-substituents
generally alter the environment around the selenium atom such
a way that it becomes less favorable for the H2O2 to form a close
association with the selenium center.21 In agreement with this,
the 6-methoxy substituents in compounds 75e77 may disfavor
the formation of seleninic and selenonic acids by preventing
the interactions between the selenium atom and peroxide
molecule.
a
decrease in the strength of Se/N interactions upon
overoxidation.
Similarly to compounds 4 and 22, the other 4-methoxy-
substituted compounds 23 and 24 invariably produced the sele-
nonic acids 64 (1017 ppm) and 65 (1015 ppm), respectively, upon
treatment with an excess amount (10 equiv) of H2O2 (Scheme 4).
The introduction of a sterically bulky cyclohexyl group on the
nitrogen atom does not appear to block the overoxidation as the
reaction of compound 27 with H2O2 produced the selenonic acid
89 (1018 ppm) as the major oxidized product. Therefore, the
electronic effects introduced by the 4-methoxy substitution in
compounds 22e24 and 27 are not sufficient to stabilize the
selenenic and seleninic acids against overoxidation. In this
regard, the reactivity of compounds 22e24 and 27 toward H2O2
very similar to that of their parent compounds 4, 17, 18 and 25,
which readily produce the selenonic acids 72 (1019 ppm), 73
(1019 ppm), 74 (1017 ppm) and 90 (1018 ppm), respectively. In-
terestingly, the introduction of methoxy substituents at their 6-
positions prevents the overoxidation of the selenenic acids to
the corresponding seleninic acids. For example, the energy dif-
ference between 75 and 81 has been shown to be almost
4.0 kcal molꢂ1 higher than that between 66 and 72, indicating
that the conversion of 75 into 81 is less favored than the
Fig. 6. Energy-optimized geometries of the selenenic acid intermediates 57e59,
75e77, 84 and 86. The optimizations of the geometries were performed at the B3LYP/
6-31þG(d) level of theory.
Scheme 4. Reaction of diselenides 22e24 and 27 with H2O2 to produce corresponding
selenenic acids (57e59); seleninic acids (60e62); and selenonic acids (63e65).

D. Bhowmick, G. Mugesh / Tetrahedron 68 (2012) 10550e10560
10557
Table 6
6-position prevent the formation of seleninic and selenonic acids
by disfavoring the interactions between selenium center and per-
oxides. When PhSH is used as the thiol co-substrate, the 4-methoxy
substituent neither blocks the selenium center in the selenenyl
sulfide intermediates from thiol exchange reactions nor prevents
the formation of seleninic and selenonic acids. On the other hand,
when larger thiols, such as glutathione (GSH) are used in assay, the
introduction of steric hindrance decreases the catalytic activity by
blocking the nucleophilic attack of GSH at the selenium center in
the selenenic acids. In such cases, the substitution at the 4-position
significantly enhances GPx-like activity of the diselenides.
Experimentally obtained 77Se NMR chemical shifts and theoretically calculated
ESe/N for the selenenic acids
Compd
77Se NMR
chemical
ESe/N
(kcal
Compd
77Se NMR
chemical
ESe/N
(kcal
b
b
shift (ppm)a
molꢂ1
)
shift (ppm)a
molꢂ1
)
57
58
59
66
67
68
1163
1165
1168
1168
1171
n.dc
19.92
15.29
15.42
20.47
16.02
16.24
75
76
77
84
85
86
1170
1174
1172
1160
1163
1172
12.84
12.41
12.04
18.43
18.94
13.40
a
The values are cited with respect to Me2Se.
The optimizations of the geometries were performed at the B3LYP/631þG(d)//
b
B3LYP/6-311þG(d,p) levels.
4. Experimental
c
Not detected.
4.1. General procedure
2.4. Catalytic mechanism
n-Butyllithium (n-BuLi) was purchased from Acros Chemical Co.
(Belgium). Methanol was obtained from Merck and dried before
use. All other chemicals were of the highest purity available. All the
reactions were carried out under nitrogen with use of standard
vacuum-line techniques. Because of the unpleasant odors and toxic
nature of several of the reaction mixtures involved, most manipu-
lations were carried out in a well-ventilated fume hood. Et2O was
dried over sodium metal with benzophenone. Thin-layer chroma-
tography analyses were carried out on pre-coated silica gel plates
(Merck), and spots were visualized with UV radiation. Column
chromatography was performed on glass columns loaded with
silica gel or on automated flash chromatography systems (Biotage)
with use of 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 respect to SiMe4 as internal (1H and 13C) and Me2Se as external
From the experimental and theoretical observations, we pro-
pose a catalytic mechanism for the reduction of peroxides by PhSH
in the presence of diselenides 22e24 and 27 as shown in Scheme 3.
The initial reductive cleavage of eSeeSee bond in the diselenides
by PhSH produces the corresponding selenenyl sulfides (41e43 and
45) and selenols (38e40 and 44). The selenols thus produced un-
dergo reaction with H2O2 to produce the corresponding selenenic
acids (57e59 and 84), which upon reaction with 1 equiv of PhSH
generates the selenenyl sulfide intermediates. Addition of an excess
amount of thiol regenerates the selenols with the formation of
PhSSPh. When the concentration of H2O2 is higher than that of
thiol, the selenenic acids generated from the selenols undergo
further oxidation to produce the corresponding seleninic acids
(60e62 and 87) and selenonic acid (63e65 and 89). However, in the
presence of an excess amount of PhSH, the selenenic, seleninic, and
selenonic acids are converted to the corresponding selenenyl sul-
fides. This catalytic cycle is very similar to that of diselenide 4, 17, 18
and 25, but slightly different from that of compounds 19e21 and 26
in which no overoxidized product (78e83) is generated in the
presence of an excess amount of peroxides (Scheme 5).
(
77Se) standard. A PerkineElmer Lambda 5 UV/Vis spectropho-
tometer was used to measure the GPx-like activities. Mass spectral
studies were carried out on a Bruker Daltonics Esquire 6000 plus
mass spectrometer with ESI-MS mode analysis. Compound 1, 4,
17e21 were synthesized by the literature method.4,8a,f
4.2. General procedure for the synthesis of compound 22e25
n-BuLi (2.5 equiv of a 1.6 M solution in hexane) was added
with stirring at ꢂ78 ꢁC to a solution of 2-bromo-5-methoxy-N,N-
dialkylbenzylamine in dry THF and the mixture was allowed to
stir at the same temperature. After 1.5 h finely ground selenium
powder (1.1 equiv) was added to the mixture at 0 ꢁC. After the
addition of the selenium powder the solution turned to brownish
color and the reaction mixture was stirred for 12 h at room
temperature. The solution was poured to the saturated solution of
ammonium chloride (NH4Cl) and stirred for 10 min. Then it was
extracted with diethyl ether three times. It was dried with an-
hydrous Na2SO4, filtered and the solvent was evaporated under
reduced pressure to obtain yellow-colored liquid. Diselenides
were purified in flash chromatography using petroleum ether and
ethyl acetate as the solvent. The compounds were characterized
by NMR spectroscopy.
Scheme 5. The proposed catalytic mechanism for the reduction of peroxides by the
diselenides 22e24, and 27 in the presence of thiol.
3. Conclusion
In this study we have compared the glutathione peroxidase-like
activity of a series of 4-methoxy-substituted N,N-dialkylbenzyl-
amine-based diselenides with that of the corresponding 6-
methoxy-substituted compounds. While both 4- and 6-methoxy
substituents can alter the electronic properties of the selenium
center, the substitution at the 6-position provides the required
steric protection for some of the catalytically active species. Par-
ticularly, the 6-methoxy group prevents the thiol exchange re-
actions at the selenium atoms in the selenenyl sulfide
intermediates. Furthermore, the introduction of a substituent at the
4.2.1. Compound 22. Yield: 0.388 g (41%), 1H NMR (CDCl3),
d (ppm):
2.25 (s, 12H, NCH3), 3.50 (s, 4H, ArCH2N), 3.61 (s, 6H, OCH3),
6.71e6.73 (d, 2H, ArH), 6.77 (s, 2H, ArH), 7.69e7.71 (d, 2H, ArH); 13C
NMR (CDCl3),
d (ppm): 44.9, 55.8, 65.0, 113.8, 115.0, 124.8, 133.6,
141.0, 158.9; 77Se NMR (CDCl3),
d
(ppm): 420. ESI-MS: m/z: calcd for
C20H28N2O2Se2 [MþH]þ: 489.0559; found: 489.0557.
4.2.2. Compound 23. Yield: 0.41 g (38%), 1H NMR (CDCl3),
d
(ppm):
1.00e1.04 (t, 12H, NCH2CH3), 2.54e2.58 (m, 8H, NCH2CH3), 3.64 (s,

10558
D. Bhowmick, G. Mugesh / Tetrahedron 68 (2012) 10550e10560
4H, ArCH2N), 3.77 (s, 6H, OCH3), 6.68e6.70 (d, 2H, ArH), 6.80 (s, 2H,
ArH), 7.65e7.68 (d, 2H, ArH); 13C NMR (CDCl3),
(ppm): 11.2, 45.5,
55.7, 59.5, 113.4, 114.7, 124.2, 133.3, 134.9, 141.9, 158.8; 77Se NMR
141.1, 158.7; 77Se NMR (CDCl3),
d (ppm): 418. ESI-MS: m/z: calcd for
d
C26H36N2O2Se2 [MþH]þ: 569.1185; found: 569.1508.
(CDCl3),
d
(ppm): 417. ESI-MS: m/z: calcd for C24H38N2O2Se2
4.2.7. Synthesis of 28. n-BuLi (3.5 ml of a 1.6 M solution in hexane)
was added with stirring at 0 ꢁC to a solution of 2-bromoanisole
(1.0 g, 5.34 mmol) in dry hexane (10 ml) and the mixture was
allowed to stir at room temperature. After 2 h stirring the n-butyl
bromide formed in the reaction mixture was evaporated under
reduced pressure without introduction of air. Dry THF (10 ml) was
added followed by the addition of the finely ground selenium
powder (0.42 g, 5.34 mmol) at 0 ꢁC. It was stirred for 4 h. It was
poured to the ice-cooled saturated NaHCO3 solution and O2 gas was
passed to the mixture in a moderate rate for 15 min. The compound
was extracted with diethyl ether and dried with anhydrous Na2SO4.
The solvent was evaporated to obtain yellow-colored solid. The
compound was purified in flash chromatography using petroleum
ether and ethyl acetate as solvent. It was characterized by NMR
[MþH]þ: 546.1264; found: 545.1079.
4.2.3. Compound 24. Yield: 0.47 g (39%), 1H NMR (CDCl3),
d (ppm):
0.82e0.85 (t, 12H, NCH2CH2CH3), 1.47e1.57 (m, 8H, NCH2CH2CH3),
2.39e2.43 (t, 8H, NCH2CH2CH3), 3.63 (s, 4H, ArCH2N), 3.77 (s, 6H,
OCH3), 6.67e6.70 (d, 2H, ArH), 6.83 (s, 2H, ArH), 7.64e7.66 (d, 2H,
ArH); 13C NMR (CDCl3),
d (ppm): 12.5, 19.8, 55.3, 55.7, 60.7, 113.4,
114.9,124.1,133.5,142.2, 158.9; 77Se NMR (CDCl3),
d (ppm): 414. ESI-
MS: m/z: calcd for C28H46N2O2Se2 [MþH]þ: 602.1890; found:
601.2214.
4.2.4. Compound 25. It was crystallized in CHCl3/ether mixture.
Yield: 0.75 g (47%), 1H NMR (CDCl3),
d (ppm): 1.43e1.46 (br, 4H,
CylHa), 1.56e1.61 (m, 8H, CylHb), 2.42 (br, 8H, CylHc), 3.55 (s, 4H,
NCH2N), 7.08e7.13 (br, 4H, ArH), 7.79e7.81 (d, 2H, ArH), 7.20e7.22
spectroscopy. Yield: 0.58 g (31%), 1H NMR (CDCl3),
d (ppm): 3.91 (s,
6H, OCH3), 6.80e6.83 (d, 2H, ArH), 6.85e6.89 (m, 2H, ArH),
(t, 2H, ArH); 13C NMR (CDCl3),
d (ppm): 25.0, 26.5, 54.1, 64.5, 126.0,
7.19e7.23 (m, 2H, ArH), 7.53e7.55 (d, 2H, ArH); 13C NMR (CDCl3),
128.5, 128.9, 131.8, 134.8, 139.8; 77Se NMR (CDCl3),
d
(ppm): 425.
d
(ppm): 56.4, 110.6, 119.1, 122.4, 128.6, 131.0, 157.3; 77Se NMR
ESI-MS: m/z: calcd for C24H32N2Se2 [MþH]þ: 508.0896; found:
(CDCl3),
396.9222; found: 397.0495.
d
(ppm): 328. ESI-MS: m/z: calcd for C14H13O2Se2 [MþNa]þ:
508.9519.
4.2.5. Synthesis of 26. n-BuLi (2.1 ml of a 1.6 M solution in hexane)
was added with stirring at 0 ꢁC to a solution of 1-(3-methox-
ybenzyl)piperidine (0.60 g, 2.92 mmol) in dry THF (15 ml) and the
mixture was allowed to stir at the same temperature. After 2 h
finely ground selenium powder (0.125 g, 3.21 mmol) was added
to the mixture at 0 ꢁC. After the addition of the selenium powder
the solution turned to brownish color and the reaction mixture
was stirred for 12 h. The solution was poured to the saturated
solution of ammonium chloride (NH4Cl) and stirred for 10 min.
Then it was extracted with diethyl ether (20 ml) three times. It
was dried with anhydrous Na2SO4, filtered and the solvent was
evaporated under reduced pressure to obtain yellow-colored
liquid, which was purified in flash chromatography using petro-
leum ether and ethyl acetate as the solvent. The compound was
characterized by NMR spectroscopy. Yield: 0.68 g (43%), 1H NMR
4.2.8. Synthesis of 41. Thiophenol (2 mL, 0.04 mmol) was added at
room temperature to the stirred solution of 22 (10 mg, 0.02 mmol)
in dichloromethane (1 ml). The reaction mixture was stirred for
30 min, and the solvent was then evaporated. The expected com-
pound was purified by flash chromatography on a silica gel column
with ethyl acetate and petroleum ether as eluents to provide pale
yellow-colored oil. 1H NMR (CDCl3),
d (ppm): 2.29 (s, 6H, NCH3),
3.54 (s, 2H, ArCH2N), 3.77 (s, 3H, OCH3) 6.70 (s, 1H, ArH), 6.75e6.78
(d, 1H, ArH), 7.11e7.13 (m, 1H, ArH), 7.19e7.23 (t, 2H, ArH),
7.48e7.50 (d, 2H, ArH), 7.78e7.80 (d, 1H, ArH); 13C NMR (CDCl3),
d
(ppm): 44.3, 55.8, 64.6,113.7,114.5,126.2,129.1,129.3,130.4,138.9,
140.1, 158.3; 77Se NMR (CDCl3),
d (ppm): 548 ppm; ESI-MS: m/z:
calcd for C16H18NOSSe [MþH]þ: 353.0353; found: 353.9982.
4.2.9. Synthesis of 63. Hydrogen peroxide (200 ml, 1.845 mmol,
30% aqueous solution) was added dropwise to a solution of dis-
elenide 22 (50 mg, 0.092 mmol) in methanol/chloroform (4:1). The
reaction mixture was stirred for 1 h at room temperature. After 1 h,
the yellow color of the diselenide disappeared completely and the
solution became colorless. The solvent was removed under reduced
(CDCl3),
d (ppm): 1.43e1.46 (br, 4H, CylHa), 1.56e1.61 (m, 8H,
CylHb), 2.38 (br, 8H, CylHc), 3.46 (s, 4H, ArCH2N), 3.81 (s, 6H,
OCH3), 6.78e6.81 (d, 2H, ArH), 6.90e6.92 (m, 2H, ArH), 7.20e7.22
(t, 2H, ArH); 13C NMR (CDCl3),
d (ppm): 24.9, 26.5, 54.9, 55.6, 64.4,
112.7, 115.0, 121.9, 129.4, 140.8, 160.0; 77Se NMR (CDCl3),
d (ppm):
379. ESI-MS: m/z: calcd for C26H36N2O2Se2 [MþH]þ: 569.1185;
pressure to obtain a white solid. 1H NMR (MeOH-d4),
d (ppm): 3.46
found: 569.0675.
(s, 6H, NCH3), 3.81 (s, 3H, OCH3), 5.25 (s, 2H, ArCH2N), 7.22e7.25 (d,
1H, ArH), 7.27e7.28 (s, 1H, ArH), 8.05e8.07 (d, 1H, ArH); 13C NMR
4.2.6. Synthesis of 27. n-BuLi (3.5 ml of a 1.6 M solution in hexane)
was added with stirring at ꢂ78 ꢁC to a solution of 1-(2-bromo-5-
methoxybenzyl)piperidine (0.64 g, 2.24 mmol) in dry THF (20 ml)
and the mixture was allowed to stir at the same temperature. After
2 h finely ground selenium powder (0.195 g, 2.47 mmol) was added
to the mixture at ꢂ20 ꢁC. After the addition of the selenium powder
the solution turned to brownish color and the reaction mixture was
stirred for 12 h. The solution was poured to the saturated solution of
ammonium chloride (NH4Cl) and stirred for 10 min. Then it was
extracted with diethyl ether (20 ml) three times. It was dried with
anhydrous Na2SO4, filtered and the solvent was evaporated under
reduced pressure to obtain yellow-colored liquid, which was pu-
rified in flash chromatography using petroleum ether and ethyl
acetate as the solvent. The compound was characterized by NMR
(MeOH-d4), d (ppm): 55.7, 56.3, 68.1, 115.9, 121.8, 127.7, 130.5, 138.8,
163.5; 77Se NMR (MeOH-d4),
d (ppm): 1018; ESI-MS: m/z calcd for
C10H14NO4Se [MþNa]þ: 316.0064; found: 315.9739.
4.3. GPx-like activity-HPLC assay
GPx-like activity was carried out by high-performance liquid
chromatography (HPLC) with use of a 2695 separation module and
a 2996 photodiode-array detector and a fraction collector. The as-
says were performed in 1.8 ml sample vials and a built-in auto-
sampler was used for sample injection. In this assay, we used
mixtures containing a 1:2 molar ratio of PhSH and peroxide in
methanol at room temperature (22 ꢁC) as our model system. Runs
with and without catalyst were carried out under the same con-
ditions. Periodically, aliquots were injected onto the reversed-
spectroscopy. Yield: 0.40 g (32%), 1H NMR (CDCl3),
d (ppm):
1.42e1.43 (br, 4H, CylHa), 1.57e1.60 (m, 8H, CylHb), 2.42 (br, 8H,
CylHc), 3.52 (s, 4H, ArCH2N), 3.78 (s, 6H, OCH3), 6.69e6.72 (d, 2H,
ArH), 6.77 (s, 2H, ArH), 7.68e7.70 (d, 2H, ArH); 13C NMR (CDCl3),
phase column (Princeton C18 column, 4.6ꢃ150 mm, 5
mm) and
eluted with methanol and water (85:15), and the concentrations of
the diphenyl disulfide (PhSSPh) produced in the reaction were
determined at 254 nm with the aid of pure PhSSPh as an external
d
(ppm): 24.8, 26.3, 30.2, 54.1, 55.8, 64.3, 113.7, 115.1, 124.9, 133.4,

D. Bhowmick, G. Mugesh / Tetrahedron 68 (2012) 10550e10560
10559
standard. The amount of disulfide formed during the course of the
Supplementary data
reaction was calculated from the calibration plot for the standard
(PhSSPh). The plots for kinetic parameters were obtained by use
either of linear or of sigmoidal curve fitting.
This data includes NMR and mass spectra, plots, mechanistic
study by NMR, coordinates for optimized structures. Supplemen-
tary data related to this article can be found at http://dx.doi.org/
10.1016/j.tet.2012.09.020.
4.4. GPx-like activity: GSHeGSSG coupled assay
The GPx-like activity was followed spectrophotometrically. The
test mixture contained GSH (2.0 mM), EDTA (1 mM), glutathione
disulfide reductase (1.7 units/ml), and NADPH (0.4 mM) in 0.1 M
References and notes
ꢁ
€
1. (a) Flohe, L.; Gunzler, E. A.; Schock, H. H. FEBS Lett. 1973, 32, 132e134; (b)
Rotruck, J. T.; Pope, A. L.; Ganther, H. E.; Swanson, A. B.; Hafeman, D. G.;
Hoekstra, W. G. Science 1973, 179, 588e590; (c) Epp, O.; Ladenstein, R.;
Wendel, A. Eur. J. Biochem. 1983, 133, 51e69; (d) Selenium in Biology and Hu-
man Health; Burk, R. F., Ed.; Springer: New York, NY, 1994; (e) Mugesh, G.; du
Mont, W.-W. Chem.dEur. J. 2001, 7, 1365e1370; (f) Jacob, C.; Giles, G. I.; Giles,
N. M.; Sies, H. Angew. Chem. 2003, 115, 4890e4907; Angew. Chem., Int. Ed.
2003, 42, 4742e4758; (g) Roy, G.; Sarma, B. K.; Phadnis, P. P.; Mugesh, G. J.
Chem. Sci. 2005, 117, 287e303.
potassium phosphate buffer of pH 7.5. GPx samples (80 mM) were
added to the test mixture at 23 ꢁC temperature and the reaction was
started by the addition of peroxide (1.6 mM). The initial reduction
rates were calculated from the rate of NADPH oxidation at 340 nm
in GSH assay. Each initial rate was measured at least three times
and calculated from the first 5e10% of the reaction by using
6.22 mMꢂ1 cmꢂ1 as the molar extinction coefficient for NADPH. For
the peroxidase activity, the rates were corrected for background
reaction between peroxide and thiol.
2. (a) Sies, H. Oxidative Stress: Introductory Remarks In Oxidative Stress; Sies, H.,
Ed.; Academic: London, 1985; pp 1e8; (b) Sies, H. Angew. Chem. 1986, 98,
1061e1075; Angew. Chem., Int. Ed. Engl. 1986, 25, 1058e1071.
3. (a) Mugesh, G.; Singh, H. B. Chem. Soc. Rev. 2000, 29, 347e357; (b) Mugesh, G.;
du Mont, W.-W.; Sies, H. Chem. Rev. 2001, 101, 2125e2179.
€
4. (a) Muller, A.; Cadenas, E.; Graf, P.; Sies, H. Biochem. Pharmacol. 1984, 33,
4.5. Single-crystal X-ray structure determination
3235e3240; (b) Wendel, A.; Fausel, M.; Safayhi, H.; Tiegs, G.; Otter, R. Biochem.
Pharmacol. 1984, 33, 3241e3246.
5. (a) Reich, H. J.; Jasperse, C. P. J. Am. Chem. Soc. 1987, 109, 5549e5551; (b) Jac-
quemin, P. V.; Christiaens, L. E.; Renson, M. J.; Evers, M. J.; Dereu, N. Tetrahedron
Lett. 1992, 33, 3863e3866.
6. (a) Sarma, B. K.; Mugesh, G. J. Am. Chem. Soc. 2005, 127, 11477e11485; (b)
Bhabak, K. P.; Mugesh, G. Chem.dEur. J. 2007, 13, 4594e4601; (c) Sarma, B. K.;
Mugesh, G. Org. Biomol. Chem. 2008, 6, 965e974; (d) Sarma, B. K.; Mugesh, G.
Chem.dEur. J. 2008, 14, 10603e10614; (e) Bhabak, K. P.; Mugesh, G. Chem.
dAsian. J. 2009, 4, 974e983.
7. Back, T. G.; Dyck, B. P. J. Am. Chem. Soc. 1997, 119, 2079e2083.
8. (a) Wilson, S. R.; Zucker, P. A.; Huang, R. R. C.; Spector, A. J. Am. Chem. Soc. 1989,
111, 5936e5939; (b) Iwaoka, M.; Tomoda, S. J. Am. Chem. Soc. 1994, 116,
2557e2561; (c) Mugesh, G.; Panda, A.; Singh, H. B.; Punekar, N. S.; Butcher, R. J.
Chem. Commun. 1998, 2227e2228; (d) Mugesh, G.; Panda, A.; Singh, H. B.;
Punekar, N. S.; Butcher, R. J. J. Am. Chem. Soc. 2001, 123, 839e850; (e) Sarma, B.
K.; Mugesh, G. Inorg. Chem. 2006, 45, 5307e5314; (f) Bhabak, K. P.; Mugesh, G.
Chem.dEur. J. 2008, 14, 8640e8651; (g) Bhabak, K. P.; Mugesh, G. Inorg. Chem.
2009, 48, 2449e2455.
X-ray crystallographic studies were carried out using a Bruker
CCD diffractometer with graphite-monochromatized Mo Ka radi-
ꢀ
ation (
l
¼0.71073 A) controlled by a Pentium-based PC running the
SMART software package.22 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.23 All non-
hydrogen atoms were refined anisotropically and hydrogen atoms
were assigned idealized locations. Empirical absorption corrections
were applied to all structures using SADABS.24 The structures were
solved by direct method (SIR-92) and refined by full-matrix least-
squares procedure on F2 for all reflections (SHELXL-97).25
9. (a) Wirth, T. Molecules 1998, 3, 164e166; (b) Jauslin, M. L.; Wirth, T.; Meier, T.;
Schoumacher, F. Hum. Mol. Genet. 2002, 11, 3055e3063.
4.6. Computational methods
10. (a) Back, T. G.; Moussa, Z. J. Am. Chem. Soc. 2002, 124, 12104e12105; (b) Back, T.
G.; Moussa, Z.; Parvez, M. Angew. Chem. 2004, 116, 1288e1290; Angew. Chem.,
Int. Ed. 2004, 43, 1268e1270.
All calculations were performed by use of the Gaussian98
suite26 of quantum chemical programs. The hybrid Becke 3-Lee-
eYangeParr (B3LYP) exchange correlation functional was applied
for DFT calculations.27 Geometries were fully optimized at the
B3LYP level of theory with use of the 6-31þG(d) basis sets. Orbital
interactions were analyzed by the natural bond orbital (NBO)
method at the B3LYP/6-311þG(d,p) level, and charges were calcu-
lated by natural population analysis (NPA).28
11. Sun, Y.; Mu, Y.; Ma, S.; Gong, P.; Yan, G.; Liu, J.; Shen, J.; Luo, G. Biochim. Biophys.
Acta 2005, 1743, 199e204.
12. (a) Kunwar, A.; Bansal, P.; Kumar, S. J.; Bag, P. P.; Paul, P.; Reddy, N. D.; Kumb-
hare, L. B.; Jain, V. K.; Chaubey, R. C.; Unnikrishnan, M. K.; Priyadarsini, K. I. Free
Radical Biol. Med. 2010, 48, 399e410; (b) Kunwar, A.; Mishra, B.; Barik, A.;
Kumbhare, L. B.; Pandey, R.; Jain, V. K.; Priyadarsini, K. I. Chem. Res. Toxicol.
2007, 20, 1482e1487.
13. Prabhu, C. P.; Phadnis, P. P.; Wadawale, A. P.; Priyadarsini, K. I.; Jain, V. K. J.
Organomet. Chem. 2012, 713, 42e50.
14. Phadnis, P. P.; Mugesh, G. Org. Biomol. Chem. 2005, 3, 2476e2481.
15. Zade, S. S.; Singh, H. B.; Butcher, R. J. Angew. Chem. 2004, 116, 4613e4615;
Angew. Chem., Int. Ed. 2004, 43, 4513e4515.
4.7. Crystal data for 25
16. Sarma, B. K.; Manna, D.; Minoura, M.; Mugesh, G. J. Am. Chem. Soc. 2010, 132,
C24H32N2Se2; Mr¼506.4; monoclinic; space group
C
2/c;
¼100.318(3),
radiation
(I));
5364e5374.
ꢀ
17. Mugesh, G.; Singh, H. B. Acc. Chem. Res. 2002, 35, 226e236.
a¼16.2254(9), b¼11.3658(6), c¼12.6999(8) A;
b
3
ꢁꢂ
18. (a) Benkova, S.; Kona, J.; Gann, G.; Fabian, W. M. F. Int. J. Quantum Chem. 2002,
ꢀ
V¼2304.2(2) A ; Z¼4; rcalcd¼1.46
g ; Mo Ka
cmꢂ3
90, 555e565; (b) Bachrach, S.; Demoin, D. W.; Luk, M.; Miller, J. V., Jr. J. Phys.
Chem. 2004, 108, 4040e4046 and references therein; (c) Bayse, C. A. J. Phys.
Chem. A 2007, 111, 9070e9075.
ꢀ
(l
¼0.71073 A); T¼293(2) K; R1¼0.0282, wR2¼0.0678 (I>2
s
R1¼0.0479, wR2¼0.0616 (all data). CCDC-847710 contains the
supplementary crystallographic data for this paper. These data can
be obtained free of charge from The Cambridge Crystallographic
Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
19. (a) Wirth, T. Angew. Chem. 1995, 107, 1872e1873; Angew. Chem., Int. Ed. Engl.
1995, 34, 1726e1728; (b) Wirth, T.; Fragale, G. Chem.dEur. J. 1997, 3,
1894e1902; (c) Wirth, T. Liebigs Ann. 1997, 2189e2196; (d) Fragale, G.;
Neuburger, M.; Wirth, T. Chem. Commun. 1998, 1867e1868; (e) Fragale, G.;
Wirth, T. Eur. J. Org. Chem. 1998, 1361e1369; (f) Uehlin, L.; Fragale, G.; Wirth,
T. Chem.dEur. J. 2002, 8, 1125e1133.
Acknowledgements
20. Press, D. J.; Mercier, E. A.; Kuzma, D.; Back, T. G. J. Org. Chem. 2008, 73,
4252e4255.
21. Heverly-Coulson, G. S.; Boyd, R. J. J. Phys. Chem. A 2010, 114, 10706e10711.
22. SMART, Version 5.05; Bruker AXS: Madison, WI, 1998.
This study was supported by the Department of Science and
Technology (DST), New Delhi, India. We are grateful to the Alex-
ander von Humboldt Foundation, Bonn, Germany for the donation
of an automated flash chromatography system. G.M. acknowledges
the DST for the award of Swarnajayanti fellowship and D.B. thanks
the CSIR for a research fellowship.
23. (a) Sheldric, G. M. Acta Crystallogr., Sect. A 1990, 46, 467e473; (b) Altomare, A.;
Cascarano, G.; Giacovazzo, C.; Gualardi, A. J. Appl. Crystallogr. 1993, 26, 343e350.
24. Sheldrick, G.M.SADABS,aProgramfor AdsorptionCorrectionwiththeSiemensSMART
€
€
Area Detaction System; University of Gottingen: Gottingen, (Germany), 1996.
25. Sheldrick, G. M. SHELX-97, Program for Refinement of Crystal Structures; Uni-
€
€
versity of Gottingen: Gottingen, (Germany), 1997.

10560
D. Bhowmick, G. Mugesh / Tetrahedron 68 (2012) 10550e10560
26. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheese-
man, J. R.; Zakrzewski, V. G.; Montgomery, J. A.; Stratmann, R. E.; Burant, J. C.;
Dapprich, S.; Millam, J. M.; Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.;
Tomasi, J.; Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.;
Clifford, S.; Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.;
Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.;
Ortiz, J. V.; Baboul, A. G.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Ko-
maromi, I.; Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng,
C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe, M.; Gill, P. M. W.; Johnson, B.;
Chen, W.; Wong, M. W.; Andres, J. L.; Gonzalez, C.; Head-Gordon, M.; Replogle,
E. J.; Pople, A. J. Gaussian 98; Gaussian: Pittsburgh, PA, 1998.
27. (a) Lee, C.; Yang, W.; Parr, R. G. Phys. Rev. B: Condens. Matter 1988, 37, 785e789;
(b) Becke, A. D. J. Chem. Phys. 1993, 98, 5648e5652.
28. (a) Reed, A. E.; Curtiss, L. A.; Weinhold, F. Chem. Rev. 1988, 88, 899e926; (b)
Glendening, E. D.; Reed, J. E.; Carpenter, J. E.; Weinhold, F. Natural Bond Orbital
(NBO), Version 3.1.