Spirodiazaselenuranes: Synthesis, structure and antioxidant activity

Article abstract of DOI:10.1039/c2ob26156a

In this paper, the synthesis, characterization and glutathione peroxidase and peroxynitrite scavenging activities of a series of stable spirodiazaselenuranes are described. The spiro compounds were synthesized in good yields by oxidative cyclization of diaryl selenides bearing amide moieties. All the selenides and spiro derivatives were characterized by ¹H, ¹³C and ⁷⁷Se NMR spectroscopy, mass spectral techniques and the structures of some of the spirodiazaselenuranes were confirmed by single crystal X-ray crystallography. The structures reveal that the selenium atom occupies the center of a distorted trigonal bipyramid core with two nitrogen atoms occupying the apical positions and two carbon atoms and the selenium lone pair occupying the equatorial positions. Mechanistic investigations indicate that the spirocyclization occurs via the formation of selenoxide intermediates. The new compounds were evaluated for their glutathione peroxidase (GPx) mimetic activity by using H₂O₂ as a substrate and glutathione (GSH) as a co-substrate. It was found that the substituents attached to the nitrogen atom of the selenazole ring have a significant effect on the GPx activity. While the introduction of electron withdrawing groups such as -Cl, -Br etc. to the phenyl ring decreases the activity, the introduction of electron donating groups such as -OH, -OMe significantly enhances the GPx activity of both diaryl selenides and spirodiazaselenuranes. In addition to GPx activity, the selenides and spiro derivatives were studied for their ability to inhibit peroxynitrite (PN)-mediated nitration of bovine serum albumin (BSA) and oxidation of dihydrorhodamine 123. These studies indicate that the diarylselenides effectively inhibit the PN-mediated nitration and oxidation reactions by reacting with PN to produce the corresponding spirodiazaselenuranes.

Full text of DOI:10.1039/c2ob26156a

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PAPER
Spirodiazaselenuranes: synthesis, structure and antioxidant activity†
Devappa S. Lamani, Debasish Bhowmick and Govindasamy Mugesh*
Received 16th June 2012, Accepted 13th August 2012
DOI: 10.1039/c2ob26156a
In this paper, the synthesis, characterization and glutathione peroxidase and peroxynitrite scavenging
activities of a series of stable spirodiazaselenuranes are described. The spiro compounds were synthesized
in good yields by oxidative cyclization of diaryl selenides bearing amide moieties. All the selenides and
spiro derivatives were characterized by 1H, 13C and 77Se NMR spectroscopy, mass spectral techniques
and the structures of some of the spirodiazaselenuranes were confirmed by single crystal X-ray
crystallography. The structures reveal that the selenium atom occupies the center of a distorted trigonal
bipyramid core with two nitrogen atoms occupying the apical positions and two carbon atoms and the
selenium lone pair occupying the equatorial positions. Mechanistic investigations indicate that the
spirocyclization occurs via the formation of selenoxide intermediates. The new compounds were
evaluated for their glutathione peroxidase (GPx) mimetic activity by using H2O2 as a substrate and
glutathione (GSH) as a co-substrate. It was found that the substituents attached to the nitrogen atom of the
selenazole ring have a significant effect on the GPx activity. While the introduction of electron
withdrawing groups such as –Cl, –Br etc. to the phenyl ring decreases the activity, the introduction of
electron donating groups such as –OH, –OMe significantly enhances the GPx activity of both diaryl
selenides and spirodiazaselenuranes. In addition to GPx activity, the selenides and spiro derivatives were
studied for their ability to inhibit peroxynitrite (PN)-mediated nitration of bovine serum albumin (BSA)
and oxidation of dihydrorhodamine 123. These studies indicate that the diarylselenides effectively inhibit
the PN-mediated nitration and oxidation reactions by reacting with PN to produce the corresponding
spirodiazaselenuranes.
selenium center in 2,2′-selenobis(benzamide) by H2O2 does not
Introduction
produce the expected spirodiaza derivative 8, but it results in the
formation of azaselenonium hydroxide 9.5 The azaselenonium
The first example of a spirodioxyselenurane (1) was reported in
1914 by Lesser and Weiss.1 After this initial study, several spiro-
cation contains one Se–N bond and the compound is stabilized
dioxyselenuranes such as 2–7 have been reported in the litera-
ture.2,3 This type of hypervalent selenium compounds attracted
significant attention in recent years due to their interesting struc-
tural and stereochemical properties.4 The general structure of
spirodioxyselenuranes shows that the geometry around the sel-
enium center is trigonal bipyramidal with the lone pair lying in
the equatorial plane and the electronegative oxygen atoms occu-
pying the apical positions.1,4 In contrast to the well-studied spir-
odioxyselenuranes, spirodiazaselenuranes that contain two
nitrogen substituents are extremely rare. A few years ago, Back
and co-workers5 demonstrated the relative instability of
spirodiazaselenuranes. They reported that the oxidation of the
by a noncovalent interaction between the selenium atom and the
carbonyl oxygen atom of the other amide moiety (Fig. 1).
Very recently, we reported the first example of a hydrolytically
stable spirodiazaselenurane (10) and its tellurium analogue (11)
bearing two nitrogen substituents in the apical positions.6 This
study indicated that the substituents attached to the amide nitro-
gen atoms play an important role in the stability of spirodiazase-
lenurane and the replacement of the hydrogen atoms in
compound 8 by phenyl rings dramatically enhances the stability
of Se–N bonds. A detailed mechanistic study indicated that the
oxidation of selenide by H2O2 produces the corresponding Se–
oxide, which undergoes a spirocyclization via a trigonal-bipyra-
midal intermediate to produce the spirodiazaselenurane 10.6 The
cyclization occurs in a stepwise manner, involving a hydroxy
selenurane intermediate. Furthermore, the activation energy for
the spirocyclization of selenoxide to the spirodiazaselenurane
was found to be much higher than that of telluroxide to the spiro-
diazatellurane, but considerably lower than that of the sulfoxide
to sulfurane conversion.
Department of Inorganic and Physical Chemistry, Indian Institute of
Science, Bangalore 560 012, India. E-mail: mugesh@ipc.iisc.ernet.in;
Fax: +91-80-2360 1552/2360 0683
†Electronic supplementary information (ESI) available: PN assay plots,
mass and NMR spectra. CCDC 869457 (13), 869456 (16), 869454 (20),
869455 (22), 869458 (25). For ESI and crystallographic data in CIF or
other electronic format see DOI: 10.1039/c2ob26156a
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Fig. 2 Diaryl selenides and spirodiazaselenuranes used in this study.
synthesize the corresponding diaryl tellurides were unsuccessful.
In contrast to 2,2′-selenobis(benzoic acid), the tellurium ana-
logue (2,2′-tellurobis(benzoic acid)) underwent a facile spiro-
cyclization during workup/purification to produce the
corresponding spirodioxytellurane (the tellurium analogue of 1,
Fig. 1) as a stable product. Therefore, 2,2′-tellurobis(benzoyl
chloride) required for the synthesis of diaryl tellurides and the
corresponding spirodiazatelluranes could not be synthesized.
Treatment of the selenides 12–18 with hydrogen peroxide pro-
duced the spirodiazaselenuranes 19–25, respectively, in excellent
yields. The compounds were purified by column chromato-
graphy methods and all the new compounds were characterized
Fig. 1 Some representative examples of stable spirochalcogenuranes.
Although spirodioxyselenuranes have been studied extensively
for their structural and stereochemical properties, the biological
activities of these compounds have received little attention. Back
and co-workers3 reported an interesting observation that the spiro-
dioxyselenurane 6 and its homologues act as efficient mimetics
of glutathione peroxidase (GPx), a selenoenzyme that protects
various organisms from oxidative stress by catalyzing the
reduction of harmful peroxides such as H2O2 in the presence of
glutathione (GSH).7 They also reported that the tellurium ana-
logue of 6 exhibits much better GPx activity than 6, indicating
that the antioxidant activity of spiro compounds depends on the
redox properties of the chalcogen atom.8 In agreement with this,
the GPx activity of the spirodiazatellurane 11 was found to be
much higher than that of the selenium analogue 10.6 To under-
stand the effect of different substituents attached to the nitrogen
atom on the stability and antioxidant activity, we have syn-
thesized a series of spirodiazaselenuranes. In this paper, we
report a systematic study on the synthesis, characterization and
GPx-like antioxidant activity of several diaryl selenides and spir-
odiazaselenuranes. We also describe the ability of these com-
pounds to inhibit peroxynitrite-mediated nitration and oxidation
reactions.
1
by H, 13C and 77Se NMR spectroscopy and mass spectral tech-
niques. The 77Se NMR signals observed for the spiro com-
pounds 19–25 (554–648 ppm) were significantly shifted
downfield as compared to that of the selenides 12–18
(407–438 ppm), indicating the hypervalent nature of the sel-
enium in the spiro derivatives. In addition to the spectroscopic
characterization, compounds 13, 16, 20, 22 and 25 were studied
by single crystal X-ray diffraction. We reported previously that
spirodiazatellurane can be produced from the corresponding tell-
uride even in the absence of any oxidizing agent.6 However, the
selenides 12–18 are quite stable in their pure forms for several
days and no such aerial oxidation of the selenium center was
observed for these compounds.
The mechanism for the formation of spirodiazaselenuranes
involves the initial oxidation of the selenium center in the diaryl
selenides 12–18 to produce the corresponding selenoxides
(26–32), followed by a spirocyclization with elimination of a
water molecule (Scheme 1). Although the diaryl selenides 12–18
Results and discussion
Synthesis of spirodiazaselenuranes
All the spirodiazaselenuranes were synthesized from the corre-
sponding diaryl selenides by oxidation. The key compound
diaryl selenide, 2,2′-selenobis(benzoic acid), was synthesized
from 2,2′-diselenobis(benzoic acid)9 and 2-iodobenzoic acid by
refluxing with copper powder in DMF.10 The reaction of 2,2′-
selenobis(benzoic acid) with thionyl chloride afforded 2,2′-
selenobis(benzoyl chloride), which was treated with various
substituted anilines to obtain the corresponding diaryl selenides
bearing amide moieties (compounds 12–18, Fig. 2). Attempts to
Scheme 1 Mechanism for the formation of spirodiazaselenuranes from
the corresponding diaryl selenides.
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forming a characteristic envelope-type conformation. However,
the two Se⋯O distances are not identical. The Se⋯O2
(2.703 Å) interaction is much stronger than the Se⋯O1
(3.379 Å) interaction. However, the Se⋯O2 distance is much
shorter than the sum of the van der Waals radii of Se and O
(3.42 Å). The Se⋯O2 distance is significantly shorter than the
Se⋯O distance observed in bis(o-formylphenyl)selenide
(2.806 Å),12 but longer than that observed for (phenylseleno)
imino-quinone (2.476 Å).13 The Se–C1 and Se–C15 lengths of
1.924(7) and 1.917(7) Å, respectively, are in agreement with the
value of 1.93 Å suggested by Pauling14 and typical values for
other related diaryl selenides.15 Interestingly, the structure of
compound 16 having a 4-methylphenyl substituent attached to
the nitrogen is significantly different from that of 13. In contrast
to the unequal Se⋯O distances in 13, the Se⋯O1 and Se⋯O2
distances (3.229 Å) in compound 16 are identical. These Se⋯O
distances are slightly shorter than the sum of van der Waals radii,
indicating only weak interactions. The C1–Se–C15 bond angle
of 93.72(13)° is significantly lower than that of 13 (101.9(3)°).
In contrast, the Se–C1 and Se–C15 bond lengths in compound
16 are identical (1.924(2) Å) and are similar to the Se–C1 dis-
tance observed for compound 13.
The noncovalent Se⋯O interactions appear to modulate the
reactivity of compounds 12–18 toward H2O2. Furthermore, the
strength of such interactions in the selenoxides 26–32 may
influence the spirocyclization. We have reported that the carbo-
nyl oxygen can interact with selenium in the selenoxide form
and such interactions need to be broken in the first step during
spirocyclization.6 The 77Se NMR spectrum of compound 34
indicates that at least one of the oxygen atoms in this compound
strongly interacts with the selenium. It is known that
Se⋯O/Se⋯N noncovalent interactions increase the electrophilic
reactivity of selenium. Therefore, the Se⋯O interactions in com-
pound 34 may facilitate the initial attack of water at the selenium
center. Furthermore, such interaction in compound 35 may
Scheme 2 Formation of spirodioxyselenurane 1 from diaryl selenide
33 by an oxidation–elimination mechanism.
are quite stable in their pure forms, compounds 16 and 18
having electron donating substituents attached to the phenyl ring
underwent spontaneous spirocyclization during the column
purification. On the other hand, compounds 14 and 15 bearing
electron withdrawing substituents produced the spiro compounds
21 and 22, respectively, only in the presence of H2O2. In contrast
to spirodiazasulfuranes, which have been shown to be hydrolyti-
cally unstable, the spirodiazaselenuranes 19–25 were found to be
very stable in both solid state and solution. No hydrolysis of the
1
spiro-ring in these compounds was observed in the H and 77Se
NMR spectra when recorded in CD3OD in the presence of 50 μL
of water.
The cyclization of the selenides to the corresponding spiro
compounds was too fast to detect any intermediates at room
temperature (25 °C). However, the formation of selenoxide could
be observed when the cyclization is blocked by replacing the
N–H moiety in compound 13 with an N–Et group. Therefore,
the oxidation of selenide 33 by H2O2 produced the selenoxide
34 in good yield. However, when a solution of compound 34 in
acetonitrile was kept for a week, formation of the corresponding
spirodioxyselenurane (1) was observed. The mechanism for the
formation of 1 may proceed via the initial attack of a water mole-
cule at the selenium center in compound 34 followed by a
nucleophilic attack of the selenium-bound oxygen atom at the
carbonyl carbon of one of the amide moieties, leading to the for-
mation of an intermediate (35) (Scheme 2). A nucleophilic
attack of the selenium-bound hydroxyl group in compound 35
then generates the spirodioxyselenurane with elimination of
N-ethyl-3-methoxyaniline. Although the formation of a spiro-
dioxyselenurane has been proposed for a selenide having an
N-methyl-N-phenylamide moiety,6 the conversion of 33 into 1
suggests that such a mechanism can be generalized for diaryl
selenides having different substituents on the amide nitrogen
atom.
Single crystal X-ray structures
The structures of diaryl selenides 13 and 16 and the spirodiaza-
selenuranes 20, 22 and 25 were studied by the single crystal
X-ray diffraction method. The molecular structure of compound
13 and selected bond lengths and angles are given in Fig. 3. The
geometry around the selenium atom is V-shaped, and the
C1–Se–C15 bond angle is 101.9(3)°. As previously noted for
several amide-based diaryl diselenides,11 there is a possibility of
noncovalent interactions between the selenium and carbonyl
oxygen atoms. An analysis of the structure indicates that the car-
bonyl oxygen atoms noncovalently interact with selenium,
Fig. 3 Single crystal X-ray structures of compounds 13 and 16. The
thermal ellipsoids were drawn at 50% probability level.
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facilitate the attack of the selenium-bound hydroxyl group at the
carbonyl carbon, leading to the cleavage of the C–N bond
(Scheme 2).
The crystal structure of compound 25 shows some interesting
features. While the difference between the two Se–N bond
lengths in compounds 10, 20 and 22 is marginal, the Se–N1
bond length (2.018 (3) Å) is significantly different from the
Se–N2 bond length (2.131(3) Å) in compound 25. This is prob-
ably due to the presence of noncovalent interactions between the
selenium atom and the methoxy substituents. The Se⋯O3 dis-
tance (2.664 Å) is remarkably shorter than the sum of the van
der Waals radii of Se and O (3.42 Å). The second methoxy
group, on the other hand, does not involve any strong inter-
actions with the selenium as the Se⋯O4 distance (3.606 Å) is
found to be considerably longer than the sum of the van der
Waals radii of selenium and oxygen atoms. The strong Se⋯O3
interaction is probably responsible for the elongation of the
Se–N2 bond. The Se–C1 and Se–C21 bond lengths of
1.942(3) Å and 1.933(3) Å, respectively, in compound 25 are
longer than that observed in the diaryl selenides 13 and 16
(1.917–1.924 Å). It should be noted that compound 25 rep-
resents the first example of a spirodiazaselenurane that exhibits
noncovalent selenium–heteroatom interactions.
The structures of the spirodiazaselenuranes 20, 22 and 25 indi-
cate that the selenium center in these compounds adopts a dis-
torted trigonal bipyramidal (TBP) geometry with the two
nitrogen atoms occupying the apical positions and the two
carbon atoms and the lone pair of selenium occupying the equa-
torial positions (Fig. 4). A comparison of the significant bond
lengths and angles (Table 1) indicates that the TBP geometry
around the selenium in compounds 20, 22 and 25 is comparable
to that in compound 10, but it is less distorted than that observed
for the tellurium analogue 11. While the Se–N2 bond lengths are
almost identical in 10, 20 and 22, the Se–N1 bond lengths in
compound 20 (2.075(5) Å) and 22 (2.066(4) Å) are significantly
longer than that of 10 (2.024(2) Å). This indicates that the intro-
duction of a methoxy group, which is known to act as an elec-
tron-withdrawing group in the meta position, and a bromo
substituent, which also acts as an electron-withdrawing group in
the para position, considerably weakens one of the Se–N bonds.
This effect is also reflected in the C1–Se–N1 bond angles in
compounds 20 (80.81(2)°) and 22 (82.03(2)°), which are signifi-
cantly lower than that observed in compound 10 (94.73(11)°).
Glutathione peroxidase (GPx) activity
One of the objectives of this study was to understand the effect
of different substituents attached to the nitrogen atom on the anti-
oxidant activity of selenides and spirodiazaselenuranes. There-
fore, the GPx-like catalytic activity of compounds 12–25 was
studied using glutathione (GSH) as the thiol cofactor and hydro-
gen peroxide (H2O2) as the substrate.16 Initial rates (v0) for the
reduction of H2O2 were determined at 2 mM concentrations of
the selenium compounds. For a comparison, the initial rate for
the reduction of H2O2 by compound 10 in the presence of GSH
was also determined. From Table 2, it is evident that the GPx
activity of the diaryl selenides 12–18 highly depends on the
nature of the substituents present on the phenyl ring attached to
the nitrogen atom. The activities of compound 12 having a
m-OH group and compound 17 having a p-CO2H group were
found to be much higher than that of compounds 13, 15 and 18,
which have m-OMe, p-Br and o-OMe groups, respectively. On
Table 2 Initial rates (v0) for the reduction of hydrogen peroxide by
GSH in the presence of diaryl selenides and spirodiazaselenuranes
Fig. 4 Single crystal X-ray structures of compounds 20, 22 and 25.
The thermal ellipsoids were drawn at 50% probability level.
Compd
v0 a (μM min−1
)
Compd
v0 a (μM min−1
)
10
12
13
14
15
16
17
18
36.81 1.86
55.02 1.82
19.05 1.55
33.20 0.80
11.07 0.27
30.24 2.88
52.85 1.81
21.84 1.15
19
20
21
22
23
24
25
70.36 1.15
41.47 0.27
24.08 4.90
51.21 0.24
10.11 1.34
59.08 1.86
14.02 1.15
Table 1 A comparison of significant bond lengths (Å) and bond
angles (°) in the spirodiazaselenuranes 10, 20, 22 and 25
Spirodiazaselenuranes
Bond lengths
(Å)/angles (°)
10
20
22
25
Se–N1
Se–N2
Se–C1
Se–C21
N1–Se–N2
C1–Se–C21
C1–Se–N1
C21–Se–N2
2.024(2)
2.058(2)
1.938(3)
1.929(3)
174.33(9)
101.34(13)
94.73(11)
81.70(10)
2.075(5)
2.066(5)
1.917(7)
1.957(5)
172.64(2)
98.13(3)
80.81(2)
82.76(2)
2.066(4)
2.066(4)
1.942(6)
1.942(6)
173.11(3)
99.33(3)
82.03(2)
82.03(2)
2.018(3)
2.131(3)
1.942(3)
1.933(3)
174.00(10)
96.04(13)
81.97(13)
82.61(13)
a Assay condition: The reactions were carried out in phosphate buffer
(100 mM, pH 7.5) at 23 °C. Catalyst: 80.0 μM; GSH: 2.0 mM;
NADPH: 0.4 mM; EDTA: 1 mM; glutathione disulfide reductase: 1.7
units per mL; hydrogen peroxide: 1.6 mM. The control reactions were
performed under identical conditions in the absence of selenium
compounds. The initial rates were corrected for the background reaction
between thiol and peroxide.
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the other hand, compounds 14 and 16 having p-Cl and p-Me
groups exhibited moderate activities. A similar trend in the
activity was observed for the spirodiazaselenuranes. Compound
19 having a m-OH group exhibited the highest activity in this
series. This was followed by compounds 22 and 24 having p-Br
and p-CO2H substituents, respectively. Similar to the effect of
substituents on the GPx activity of diaryl selenides, the introduc-
tion of p-Cl, p-Me or o-OMe substituents significantly decreases
the activity. Compounds 21, 23 and 25 were found to be poor
catalysts as compared to the parent compound 10.
processes, cells produce high levels of nitric oxide (NO˙) and the
superoxide radical,19 and consequently the formation of PN is
expected to be rapid during the inflammation. It is known that
PN can induce DNA damage20 and initiate lipid peroxidation in
biomembranes or low-density lipoproteins.21 In addition, PN can
inactivate a variety of enzymes/hormones by tyrosine nitration.22
Therefore, the inhibition of PN-mediated nitration and oxidation
is important. In addition to the naturally occurring compounds
such as GSH, N-acetylcysteine, ascorbate etc., several synthetic
selenium compounds have been shown to inhibit PN-mediated
reactions.23
The mechanism for the reduction of H2O2 by the selenides
may involve a redox shuttle between the selenides and spiro-
diazaselenuranes via the corresponding selenoxides (Scheme 3).
As previously described, the reactions of compounds 12–18 with
H2O2 produce the corresponding selenoxides 26–32, which upon
elimination of a water molecule generate the spirodiazasele-
nuranes 19–25. The reductive cleavage of the Se–N bonds in
compounds 19–25 by GSH then regenerates the selenides 12–18
(Path A, Scheme 3). This pathway is particularly favoured at
higher concentrations of peroxide. However, the reduction of
compounds 21, 23 and 25 by GSH is very slow, which may
account for the poor GPx activity of these compounds as com-
pared to that of 19. At higher concentrations of GSH, the nucleo-
philic attack of the thiol at the selenium center may produce the
intermediates 36–42, which upon reaction with GSH can regen-
erate the selenides. As shown in Scheme 2, the noncovalent
interaction between the selenium and one of the carbonyl
oxygens is expected to favour the nucleophilic attack of GSH at
the selenium center. It should be noted that the mechanism
shown in Scheme 3 is different from that of GPx and other di-
selenide-based mimetics that utilize a selenol moiety for the
reduction of peroxides.
The good GPx activity of many of the diaryl selenides and
spirodiazaselenuranes prompted us to investigate the effect of
these compounds on PN-mediated nitration and oxidation reac-
tions. It is known that diaryl selenides inhibit PN-mediated
nitration and oxidation reactions and these compounds undergo
oxidation to produce the corresponding selenoxides.23,24 To
understand the effect of different substituents on the PN-scaven-
ging antioxidant activity, we have studied the nitration of tyrosyl
residues in bovine serum albumin (BSA) by PN.25 The inhi-
bition of tyrosine nitration was followed by immunoblotting
methods using an antibody against 3-nitro-L-tyrosine. In a
typical experiment, BSA (100 mM) was mixed with PN
(1.2 mM) and an inhibitor (166 μM), and the reaction was incu-
bated at 20 °C for 30 min. At 166 μM concentration, the relative
inhibitory activities of diaryl selenides on protein tyrosine
nitration are summarized in Fig. 5. A comparison of the percen-
tage of nitration in the presence of diaryl selenides with that of
control indicates that compounds 12–18 significantly inhibit the
PN-mediated nitration reaction. In contrast to the GPx activity,
the PN-scavenging activity of compound 12 was much lower
than that of other selenides, indicating that the facile cleavage of
the Se–N bond in the corresponding spirodiazaselenurane plays
an important role in the GPx activity. Similarly, compound 16,
which exhibited moderate GPx activity (v0 = 30.24 2.88 μM
min−1), was found to be the most effective inhibitor of the
PN-mediated nitration. This indicates that an electron donating
substituent in the para-position of the phenyl ring facilitates oxi-
dation of the selenium center.
Inhibition of peroxynitrite-mediated nitration and oxidation
Peroxynitrite (PN, ONOO−) is a powerful in vivo oxidant, which
is capable of nitrating proteins and can cause serious damage to
biomolecules by oxidation.17 PN is generated by a diffusion-
controlled reaction of nitric oxide (NO˙) and the superoxide
radical (O2˙−).18 Reaction of PN with proteins/enzymes gener-
ally leads to the formation of 3-nitrotyrosine (3-NT), an indicator
of cell damage and inflammation. During the inflammatory
To understand the effect of spirodiazaselenuranes on
PN-mediated nitration, we have studied the inhibition of BSA
nitration in the presence of compounds 19–25 (Fig. 6). As
Fig. 5 Immunoblots for the inhibition of PN mediated nitration of
BSA by diaryl selenides 12–18. BSA (100 mM) was incubated with an
inhibitor (166 μM) and PN (1.2 mM) for 30 min at 20 °C and then sub-
jected to gel electrophoresis.
Scheme 3 Proposed mechanism for GPx activity of compounds
12–25.
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Table 3 Inhibition of PN-mediated oxidation of dihydrorhodamine
123 by ebselen and spirodiazaselenurane and its analoguesa
Compd
IC50 (μM)
Compd
IC50 (μM)
Ebselen
10
12
13
14
15
16
17
0.90 0.02
71.96 1.68
17.62 0.39
22.50 0.39
52.09 0.07
22.54 0.27
2.99 0.23
2.39 0.18
18
19
20
21
22
23
24
25
1.82 0.05
32.60 0.70
19.52 0.49
119.47 0.96
20.69 0.15
13.36 0.16
27.83 0.30
13.92 0.70
a Assay conditions: dihydrorhodamine 123 (DHR; 0.1 mM),
peroxynitrate (0.69 mM), and inhibitors (variable) in sodium phosphate
buffer (100 mM, pH 7.5) with DTPA (100 mM) at 23 °C.
Fig. 6 Immunoblots for the inhibition of PN mediated nitration of
BSA by spirodiazaselenuranes 19–25. BSA (100 mM) was incubated
with an inhibitor (166 μM) and PN (1.2 mM) for 30 min at 20 °C and
then subjected to gel electrophoresis.
expected, all the spirodiazaselenuranes were found to be less
active in inhibiting the nitration than the corresponding diaryl
selenides. However, the significant inhibition of PN-mediated
nitration (>30% in some cases) indicates that the selenium
centers in compounds 19–25 may undergo further oxidation by
PN to produce the corresponding selenurane oxides
(vide infra).26 Similar to compound 16, the methyl group facili-
tates oxidation of the selenium center in compound 23. On the
other hand, the electron withdrawing chloro or bromo substituent
appears to reduce the PN-scavenging activity as compounds 21
and 22 were found to be less active than 23.
ebselen, a much lower activity was observed for compounds
12–15. These observations suggest that electron donating substi-
tuents in the o- and p-positions generally favor the oxidation of
the selenium center. Interestingly, most of the spirodiazasele-
nuranes inhibited the PN-mediated oxidation and some of them
were found to be more active than the corresponding diaryl sele-
nides. For example, the IC50 values obtained for compounds 22
and 23 are almost two times lower than that of 14 and 15, indi-
cating that the oxidation of the selenium center in 22 and 23 by
PN is more favoured than that in compounds 10, 14 and 15.
These observations support the assumption that the spirodiazase-
lenuranes can undergo further oxidation to the corresponding
selenurane oxides.
The mechanism for the PN scavenging activity of compounds
12–18 involves an initial oxidation of the selenides by PN to
produce the corresponding selenoxides. A spirocyclization of the
selenoxides generates the spirodiazaselenuranes, which then
react with a second equivalent of PN to produce the correspond-
ing selenurane oxides (Scheme 5). The formation of selenurane
oxides was confirmed by 77Se NMR spectroscopy and mass
spectral analysis. Interestingly, when the reaction mixture was
kept for 2–3 h, the regeneration of spirodiazaselenuranes was
observed. On the other hand, the formation of diaryl selenides
was not observed. These observations indicate that compounds
19–25 maintain a redox shuttle between the selenuranes and
their oxides in the presence of PN and nitrite, and therefore can
catalyze the isomerisation of PN to nitrate as shown in
Scheme 5. Recently, we reported that selenenyl amides can cata-
lyze the decomposition of peroxynitrite.30 We have shown that
In addition to the nitration of tyrosyl residues in proteins/
peptides, PN is known to oxidize several biomolecules. PN
mediates the oxidation of the thiol group of glutathione and
cysteine residues in proteins/enzymes. It has been shown that PN
can exert cytotoxic effects in part by oxidizing tissue thiol
groups.27 Selenium compounds such as ebselen (2-phenyl-1,2-
benzisoselenazol-3(2H)-one) act as efficient scavengers of PN
and provide defense against PN-mediated toxicity.28 Ebselen
rapidly reacts with PN and the rate constant for this reaction has
been shown to be in the order of 106 M−1 s−1 29
Therefore, it
.
was thought worthwhile to study the effect of diaryl selenides
and spirodiazaselenuranes on PN-mediated oxidation of di-
hydrorhodamine 123 (DHR) to rhodamine 123 (Scheme 4). The
oxidation reaction was studied by fluorescence spectroscopy and
the concentrations of selenium compounds required to inhibit
50% oxidation are represented as IC50 values.
As reported earlier, ebselen was found to be a very effective
scavenger of PN. Ebselen inhibited the oxidation of DHR with
an IC50 value of 0.90 0.02 μM (Table 3). For the diaryl sele-
nides, the IC50 values highly depend on the nature of substitu-
ents attached to the nitrogen atom. While the activity of
compounds 16–18 was found to be comparable to that of
Scheme 4 Peroxynitrite-mediated oxidation of dihydrorhodamine 123
Scheme 5 Proposed mechanism for the reduction and isomerization of
(DHR) to rhodamine 123.
peroxynitrite by the diaryl selenides and spirodiazaselenuranes.
7938 | Org. Biomol. Chem., 2012, 10, 7933–7943
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Synthesis of 12. A solution of m-hydroxyaniline (0.92 g,
5.85 mmol) was added dropwise to a solution of 2,2′-selenobis-
(benzoyl chloride) (1.00 g, 5.32 mmol) in dry acetonitrile
(10 mL). The reaction mixture was stirred for 2 h at room temp-
erature. The solvent was removed under vacuum to obtain a light
yellow solid, which was purified by column chromatography
using petroleum ether–ethyl acetate as an eluent to give 12 as a
ebselen and its analogues react with PN to generate the corre-
sponding selenoxides, which then interact with nitrite produced
in the reaction to regenerate the selenenyl amides with the for-
mation of nitrate. It should be noted that the reaction of com-
pounds 19–25 with H2O2 does not produce the selenurane
oxides.
1
yellow solid. Yield: 1.25 g, 89%; m.p.: 122–125 °C. H NMR
(DMSO-d6, ppm): δ 6.45–6.46 (d, J = 6.43 Hz, 2H), 7.04–7.06
(d, J = 7.02 Hz, 2H), 7.29–7.32 (d, J = 7.27 Hz, 4H), 7.32–7.39
(m, 4H), 7.64–7.66 (d, J = 7.63 Hz, 2H), 7.85 (d, 2H), 9.41 (d,
2H), 10.30 (s, 2H). 13C NMR (DMSO-d6, ppm): δ 105.9, 107.9,
111.6, 112.0, 127.6, 128.1, 129.8, 131.4, 134.9, 139.1, 143.0,
157.8, 168.7. 77Se NMR (DMSO-d6): 410. ESI-MS (m/z) calcd
C26H20N2O4Se: 503.46, found: 527.10 [M + Na]+.
Conclusions
In this article, we have described the synthesis, structure and
antioxidant activity of several stable spirodiazaselenuranes. The
X-ray structures of the spiro compounds indicate that the
selenium atom occupies the center of a distorted trigonal bipyra-
mid core with two nitrogen atoms occupying the apical positions
and two carbon atoms and the selenium lone pair occupying the
equatorial positions. A detailed mechanistic study suggests that
the spirocyclization occurs via the formation of selenoxide inter-
mediates. The glutathione peroxidase (GPx) mimetic activity of
the selenides and the spirodiazaselenuranes indicates that the
substituents attached to the nitrogen atom have a significant
effect on the activity. It is observed that the introduction of elec-
tron withdrawing groups generally decreases the activity. On the
other hand, the introduction of electron donating groups signifi-
cantly enhances the GPx activity of both diaryl selenides and
spirodiazaselenuranes. In addition to GPx mimetic activity, the
peroxynitrite-scavenging activity of the selenium compounds
was evaluated by using nitration of tyrosyl residues in bovine
serum albumin and oxidation of dihydrorhodamine 123. These
results indicate that the diarylselenides and some of the spiro
compounds effectively inhibit the peroxynitrite-mediated
nitration and oxidation reactions.
Synthesis of 13. This compound was prepared by following
the method given for 12 by using 2,2′-selenobis-(benzoyl chlor-
ide) and m-methoxyaniline. Yield: 1.44 g, 97%; m.p.:
1
110–112 °C. H NMR (DMSO-d6, ppm): δ 6.66–6.67 (d, J =
6.64 Hz, 2H), 6.81–6.83 (d, J = 6.66 Hz, 2H), 7.12–7.16
(t, J = 7.10 Hz, 4H), 7.33–7.36 (m, J = 7.31 Hz, 4H), 7.50–7.52
(t, J = 7.48 Hz, 2H), 7.61 (d, 2H), 10.45 (s, 2H), 3.74 (6H).
13C NMR (CDCl3, ppm): δ 55.7, 105.8, 111.0, 112.6, 128.5,
129.4, 130.0, 131.2, 132.0, 135.0, 138.2, 139.2, 160.4, 166.8.
77Se NMR (CDCl3): 412. ESI-MS (m/z) calcd C28H24N2O2Se:
531.9, found: 550.89 [M + Na]+.
Synthesis of 14. This compound was prepared by following
the method given for 12 by using 2,2′-selenobis-(benzoyl chlor-
ide) and p-chloroaniline. Yield: 1.20 g, 80%; m.p.: 152–154 °C.
1H NMR (DMSO-d6, ppm): δ 7.07 (d, J = 7.05 Hz, 2H),
7.31–7.33 (d, 2H), 7.37–7.39 (t, J = 7.35 Hz, 2H), 7.43–7.45
(d, 4H), 7.49 (d, J = 7.47 Hz, 2H), 7.59–7.66 (m, J = 7.57 Hz,
2H), 7.67 (d, J = 7.65 Hz, 2H), 10.61 (s, 2H). 13C NMR
(DMSO-d6, ppm): 126.2, 126.6, 126.9, 129.5, 129.6, 129.6,
133.6, 134.9, 136.0, 137.1, 142.0, 165.5. 77Se NMR (DMSO-
d6): 415. ESI-MS (m/z) calcd C26H18N2O2Cl2Se: 540.3, found:
563.09 [M + Na]+.
Experimental section
General procedure
Selenium powder was purchased from Aldrich Chemical Co.
Acetonitrile and dichloromethane were dried over P2O5. All
syntheses were carried out under anhydrous and anaerobic con-
ditions using standard Schlenk techniques. Due to unpleasant
odors of several of the reaction mixtures involved, all the
manipulations were carried out in a well-ventilated fume hood.
1H (400 MHz), 13C (100.56 MHz), and 77Se (76.29 MHz) NMR
spectra were obtained at room temperature on a 400 MHz NMR
spectrometer (Bruker Corporation, Germany). Chemical shifts
are quoted with respect to SiMe4 as internal (1H and 13C), and
Me2Se as external (77Se)31 standards. A Perkin-Elmer Lambda
5 UV/Vis spectrophotometer was used to measure GPx activity.
The melting points were determined in an open capillary with a
B-540 Melting Point apparatus (Büchi Labortechnik AG, Swit-
zerland). Mass spectral studies were carried out on a Q-TOF
micro mass spectrometer (Waters Inc., USA) with ESI-MS mode
analysis. Thin-layer chromatography analyses were carried out
on precoated silica gel plates (Merck), and spots were visualized
by UV irradiation. Column chromatography was performed on
glass columns loaded with silica gel or on an automated flash
chromatography system (Biotage) by using preloaded silica
cartridges.
Synthesis of 15. This compound was prepared by following
the method given for 12 by using 2,2′-selenobis-(benzoyl chlor-
ide) and p-bromoaniline. Yield: 1.65 g, 95%; m.p.: 128–129 °C.
1H NMR (DMSO-d6, ppm): δ 6.95–6.97 (d, J = 6.93 Hz, 2H),
7.23–7.32 (t, J = 7.25 Hz, 2H), 7.34–7.38 (t, J = 7.32 Hz, 4H),
7.47 (d, 2H), 7.56–7.58 (m, J = 7.54 Hz, 2H), 7.70–7.90
(d, 2H), 7.91 (d, 2H), 10.57 (s, 2H). 13C NMR (DMSO-d6,
ppm): δ 117.2, 126.8, 129.7, 132.5, 133.6, 134.9, 136.1, 137.1,
136.2, 135.7, 140.6, 165.5. 77Se NMR (DMSO-d6): 438.
ESI-MS (m/z) calcd C26H18N2O2Br2Se: 629.2, found:
629.9 [M+].
Synthesis of 16. This compound was prepared by following
the method given for 12 by using 2,2′-selenobis-(benzoyl chlor-
ide) and p-methylaniline. Yield: 1.26 g, 90%; m.p.: 123–124 °C.
1H NMR (DMSO-d6, ppm): δ 7.10–7.12 (d, J = 7.27 Hz, 2H),
7.29 (d, J = 7.27 Hz, 2H), 7.43 (d, 2H), 7.48–7.50 (t, J = 7.46
Hz, 2H), 7.52–7.56 (t, J = 7.50 Hz, 2H), 7.55–7.59 (d, 2H), 7.68
(d, J = 7.66 Hz, 2H), 7.80–7.85 (t, J = 7.78 Hz, 2H), 10.53
(s, 2H), 2.50 (s, 6H). 13C NMR (DMSO-d6, ppm): δ 21.4, 120.8,
This journal is © The Royal Society of Chemistry 2012
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123.9, 128.0, 129.9, 129.5, 130.1, 131.0, 131.7, 134.9, 137.3,
138.5, 166.2. 77Se NMR (DMSO-d6): 407. ESI-MS (m/z) calcd
C28H24N2O2Se: 500.6, found: 523.09 [M + Na]+.
(d, 2H). 13C NMR (CDCl3, ppm): δ 126.4, 126.7, 129.1, 129.6,
129.7, 133.6, 134.9, 136.0, 137.1, 141.0, 165.5. 77Se NMR
(DMSO-d6): 591. ESI-MS (m/z) calcd C26H16N2O2Cl2Se:
537.09, found: 560.6 [M + Na]+.
Synthesis of 17. This compound was prepared by following
the method given for 12 by using 2,2′-selenobis-(benzoyl chlor-
ide) and p-aminobenzoic acid. Yield: 1.11 g, 73%; m.p.:
Synthesis of 22. This compound was prepared by following
the method given for 19 by using compound 15 and hydrogen
peroxide. Yield: 0.24 g, 98%; m.p.: 193–194 °C. 1H NMR
(DMSO-d6, ppm): δ 7.50 (d, 2H), 7.65–7.67 (t, J = 7.63 Hz,
2H), 7.68–7.70 (t, J = 7.66 Hz, 4H), 7.71 (d, J = 7.46 Hz, 4H),
7.73 (d, 2H), 7.80 (d, J = 7.78 Hz, 2H), 8.03–8.04 (d, 2H).
13C NMR (DMSO-d6, ppm): δ 116.3, 125.9, 126.7, 128.8,
131.6, 132.7, 134.2, 135.7, 140.6, 162.3, 164.6. 77Se NMR
(DMSO-d6): 562. ESI-MS (m/z) calcd C26H18N2O2Br2Se: 627.2,
found: 629.8 [M+].
1
183–184 °C. H NMR (DMSO-d6, ppm): δ 6.53–6.55 (d, J =
6.51 Hz, 2H), 7.32–7.39 (d, 2H), 7.41–7.42 (t, J = 7.39 Hz, 4H),
7.57–7.59 (d, J = 7.55 Hz, 2H), 7.73–7.75 (d, J = 7.71 Hz, 4H),
7.79–7.81 (d, J = 7.77 Hz, 2H), 7.88–7.90 (d, 2H), 10.81
(s, 2H). 13C NMR (DMSO-d6, ppm): 113.4, 117.8, 120.8, 128.2,
129.1, 131.0, 132.0, 132.7, 134.0, 139.4, 154.0, 168.8.
77Se NMR (DMSO-d6): 418. ESI-MS (m/z) calcd
C28H20N2O6Se: 582.2, found: 580.99 [M+].
Synthesis of 18. This compound was prepared by following
the method given for 12 by using 2,2′-selenobis-(benzoyl chlor-
ide) and o-methoxyaniline. Yield: 1.38 g, 93%; m.p.:
Synthesis of 23. This compound was prepared by following
the method given for 19 by using compound 16 and hydrogen
peroxide. Yield: 0.23 g, 93%; m.p.: 177–178 °C. 1H NMR
(CDCl3, ppm): δ 7.04 (d, J = 7.02 Hz, 2H), 7.21 (d, J = 7.19 Hz,
2H), 7.33–7.36 (t, J = 7.31 Hz, 4H), 7.50–7.52 (t, J = 7.48 Hz,
2H), 7.62 (d, 2H), 7.80 (d, 2H), 8.07–8.1 (d, J = 8.04 Hz, 2H),
2.31 (s, 6H). 13C NMR (DMSO-d6, ppm): δ 21.4, 120.4, 128.5,
129.4, 129.8, 130.6, 131.2, 131.8, 134.0, 134.6, 135.3, 135.5,
138.4, 166.7. 77Se NMR (DMSO-d6, ppm): 562. ESI-MS (m/z)
calcd C28H22N2O2Se: 497.93, found: 521.00 [M + Na]+.
1
138–140 °C. H NMR (CDCl3, ppm): δ 6.73–6.75 (d, J = 6.71
Hz, 2H), 6.77 (d, 2H), 6.92–6.94 (m, J = 6.90 Hz, 2H),
6.95–6.99 (t, 4H), 7.33–7.36 (m, J = 7.33 Hz, 4H), 7.48 (d, J =
7.46 Hz, 2H), 7.50–7.52 (d, 2H), 3.78 (s, 6H). 13C NMR
(CDCl3, ppm): δ 56.0, 110.2, 120.2, 121.3, 124.3, 128.9, 129.0,
130.6, 132.4, 135.2, 135.2, 138.2, 148.6, 166.3. 77Se NMR
(DMSO-d6): 417.9. ESI-MS (m/z) calcd C28H24N2O2Se: 531.2,
found: 553.8 [M + Na]+.
Synthesis of 24. This compound was prepared by following
the method given for 19 by using compound 17 and hydrogen
peroxide. Yield: 0.22 g, 89%; m.p.: 232–235 °C. 1H NMR
(DMSO-d6, ppm): δ 7.67–7.68 (d, J = 7.65 Hz, 2H), 7.72–7.75
(m, J = 7.70 Hz, 4H), 7.89–7.90 (d, J = 7.87 Hz, 2H), 7.90–7.91
(d, J = 7.88 Hz, 4H), 8.00–8.02 (d, 7.97 Hz, 2H), 8.07–8.09 (d,
J = 8.04 Hz, 4H). 13C NMR (DMSO-d6, ppm): 123.8, 124.8,
126.8, 127.1, 129.4, 131.2, 133.7, 137.0, 146.4, 165.8, 168.0.
77Se NMR (DMSO-d6): 648. ESI-MS (m/z) calcd
C28H18N2O6Se: 557.4, found: 578.9 [M + Na]+.
Synthesis of 19. To a stirred solution of 12 (0.25 g,
0.53 mmol) in dry CH2Cl2 (5 mL) was added 30% hydrogen
peroxide (0.018 mL, 0.53 mmol). The reaction mixture was
stirred for 30 min at room temperature and the solvent was evap-
orated under vacuum. A white solid was obtained. The com-
pound was purified by column chromatography on silica gel
using petroleum ether and ethyl acetate (2 : 1) as an eluent.
Yield: 0.22 g, 89%; m.p.: 196–197 °C. 1H NMR (CD3OD,
ppm): δ 6.58 (d, J = 6.56 Hz, 2H), 6.63 (d, J = 6.61 Hz, 2H),
7.03–7.09 (t, J = 7.03 Hz, 4H), 7.50 (d, J = 7.40 Hz, 4H),
7.78–7.86 (m, 2H), 8.12 (d, 2H), 8.18–8.20 (d, J = 8.17 Hz,
2H). 13C NMR (DMSO-d6, ppm): δ 111.5, 112.4, 115.4, 126.8,
129.6, 130.4, 133.6, 135.7, 136.5, 137.1, 141.0, 158.5, 165.6.
77Se NMR (DMSO-d6): 580. ESI-MS (m/z) calcd
C26H18N2O4Se: 501.56, found: 524.9 5 [M + Na]+.
Synthesis of 25. This compound was prepared by following
the method given for 19 by using compound 18 and hydrogen
peroxide. Yield: 0.24 g, 98%; m.p.: 179–182 °C. 1H NMR
(CDCl3, ppm): δ 7.00 (d, 2H), 7.05–7.09 (d, J = 7.07 Hz, 2H),
7.22–7.24 (m, J = 7.20 Hz, 2H), 7.27–7.28 (t, 4H), 7.28–7.37
(m, J = 7.26 Hz, 2H), 7.52 (d, 2H), 7.55 (d, J = 7.53 Hz, 2H),
3.78 (s, 6H). 13C NMR (CDCl3, ppm): δ 56.0, 112.1, 120.3,
124.3, 127.9, 128.1, 128.9, 129.8, 132.4, 134.0, 135.2, 138.2,
148.6, 166.3. 77Se NMR (DMSO-d6, ppm): 595. ESI-MS (m/z)
calcd C28H22N2O2Se: 530.7, found: 550.89 [M + Na]+.
Synthesis of 20. This compound was prepared by following
the method given for 19 by using compound 13 and hydrogen
peroxide. Yield: 0.24 g, 98%; m.p.: 163–164 °C. 1H NMR
(CDCl3, ppm): δ 6.72–6.73 (d, J = 6.70 Hz, 2H), 6.74 (d, 2H),
7.16–7.19 (t, J = 7.14 Hz, 2H), 7.27–7.29 (d, 2H), 7.40–7.44
(t, J = 7.38 Hz, 4H), 7.58 (d, 2H), 7.63 (d, J = 7.61 Hz, 2H),
3.81 (s, 6H). 13C NMR (DMSO-d6, ppm): δ 55.7, 109.7, 111.3,
115.8, 126.9, 130.2, 130.6, 133.5, 134.4, 135.8, 136.0, 160.9,
165.0. 77Se NMR (CDCl3): 554. ESI-MS (m/z) calcd
C28H22N2O2Se: 529.4, found: 553.09 [M + Na]+.
Synthesis of 33. A solution of iodoethane (0.37 mmol) in dry
acetonitrile (1 mL) was added dropwise to a solution of 13
(0.88 mmol) and NaH (0.39 mmol) in acetonitrile (5 mL). The
reaction mixture was stirred for 12 h at room temperature and the
solvent was evaporated under vacuum. The yellow solid obtained
was purified by column chromatography on silica gel using pet-
roleum ether and ethyl acetate (3 : 1) as an eluent to give a
Synthesis of 21. This compound was prepared by following
the method given for 19 by using compound 14 and hydrogen
peroxide. Yield: 0.22 g, 88%; m.p.: 173–176 °C. 1H NMR
(CDCl3, ppm): δ 7.35 (d, J = 7.33 Hz, 2H), 7.37 (d, 2H),
7.63–7.65 (t, J = 7.61 Hz, 2H), 7.65 (d, 2H), 7.71–7.72 (d, 2H),
7.73 (t, J = 7.71 Hz, 2H), 7.78–7.80 (d, J = 7.76 Hz, 2H), 8.26
1
yellow crystalline solid. Yield: 1.15 g, 82%; H NMR (CDCl3
ppm): δ 4.06–4.09 (m, 4H), 3.66 (s, 6H), 1.22–1.28 (m, 6H),
6.72–6.73 (d, J = 6.70 Hz, 2H), 6.75–6.82 (t, J = 6.73 Hz, 2H),
6.84–6.86 (t, J = 6.82 Hz, 4H), 7.05–7.71 (d, J = 7.03 Hz, 2H),
7.15–7.33 (m, 2H), 7.34–7.36 (d, J = 7.32 Hz, 2H), 7.82–7.84
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(d, J = 7.80 Hz, 2H). 13C NMR (CDCl3, ppm): δ 13.22, 30.21,
46.55, 55.9, 113.2, 114.1, 120.4, 128.3, 129.6, 130.4, 131.1,
135.4, 144.4, 146.4, 160.6, 168.1. 77Se NMR (CDCl3): 380.
ESI-MS (m/z) calcd C32H32N2O4Se: 588.2, found: 611.0
[M + Na]+.
11.8836(18) Å; α = 90.00°, β = 121.836(7)°, γ = 90.00°; V =
2791.66 Å3; Z = 4; ρcalcd = 1.67 g cm−3; MoKα radiation
λ = 0.71073 Å; T = 293(2) K; R1 = 0.1612, wR2 = 0.1843
(I > 2σ(I)); R1 = 0.0638, wR2 = 0.1485.
Crystal data for 25. C28H22N2O4Se; Mr = 531.2; monoclinic;
space group P21/c; a = 15.0038(7), b = 9.2872(4), c =
17.4994(7) Å; α = 90.00°, β = 105.640(2)°, γ = 90.00°;
V = 2348.14 Å3; Z = 4; ρcalcd = 1.49 g cm−3; MoKα radiation
(λ = 0.71073 Å); T = 296(2) K; R1 = 0.0401, wR2 = 0.0651
(I > 2σ(I)); R1 = 0.0263, wR2 = 0.0592 (all data).
Synthesis of 34. To a solution of 33 (0.13 mmol) in dry
CH2Cl2 (5 mL) was added 30% hydrogen peroxide
(0.13 mmol). The reaction mixture was stirred for 4 h at room
temperature and the solvent was evaporated under vacuum to
give a dark brown solid. The compound was purified by column
chromatography using silica gel with (5 : 3) petroleum ether and
ethyl acetate as an eluent to give a crystalline solid. Yield:
1.06 g, 76%; 1H NMR (CDCl3, ppm): δ 1.24–1.28 (m, 6H), 3.66
(m, 4H), 3.94–3.40 (m, 6H), 6.72–6.74 (d, J = 6.70 Hz, 2H),
6.76–6.85 (t, 2H), 7.05–7.07 (d, J = 7.03 Hz, 2H), 7.13–7.17
(t, 4H), 7.32–7.36 (m, 4H), 7.82–7.84 (d, J = 7.80 Hz, 2H).
13C NMR (CDCl3, ppm): δ 13.20, 30.1, 46.5, 55.8, 113.2,
114.1, 120.4, 128.2, 129.6, 130.4, 131.2, 135.4, 144.3, 146.3,
160.6, 168.1. 77Se NMR (CDCl3): 863. ESI-MS (m/z) calcd
C32H32N2O4Se: 603.6, found: 627.0 [M + Na]+.
GSH–GSSG coupled assay
The GPx activity was followed spectrophotometrically by using
a
glutathione/glutathione reductase/NADPH coupled assay
system. The initial rates for the reduction of H2O2 by GSH in the
presence and absence of selenium compounds were calculated
from the rate of NADPH oxidation at 340 nm.16 Each initial rate
was measured at least three times and calculated from the first
5–10% of the reaction by using 6.22 mM−1 cm−1 as the molar
extinction coefficient for NADPH. For the peroxidase activity,
the rates were corrected for the background reaction between per-
oxide and thiol.
Single crystal X-ray diffraction
X-ray crystallographic studies were carried out on a Bruker CCD
diffractometer with graphite-monochromatized MoKα radiation
(λ = 0.71073 Å) controlled by a Pentium-based PC running on
the SMART software package.32 Single crystals were mounted at
room temperature on the ends of glass fibers, and data were col-
lected at room temperature. The structures were solved by direct
methods and refined by using the SHELXTL software
package.33 All non-hydrogen atoms were refined anisotropically,
and hydrogen atoms were assigned idealized locations. Empirical
absorption corrections were applied to all structures by using
SADABS software.34 The structures were solved by direct
methods (SIR-92) and refined by a full-matrix least-squares pro-
cedure on F2 for all reflections (SHELXL-97).35
Peroxynitrite-mediated oxidation assay
Peroxynitrite-mediated oxidation of dihydrorhodamine 123
(DHR) was followed as suggested in the literature with minor
modifications.37 Fluorescence intensity was measured using a
Perkin-Elmer LS 50B luminescence spectrometer with excitation
and emission wavelengths of 500 and 526 nm, respectively. The
stock solution of DHR in dimethylformamide (DMF) was
purged with nitrogen and stored at −20 °C. The working solu-
tions of DHR and peroxynitrite were kept on an ice bath. The
assay mixture contained DHR (0.50 μM) and peroxynitrite
(1.0 μM) in 100 mM sodium phosphate buffer of pH 7.4 with
100 μM DTPA and variable inhibitor concentrations. The fluor-
escence intensity from the reaction of DHR with PN was set as
100%, and the intensity after the addition of various inhibitor
amounts was expressed as the percentage of that observed in the
absence of inhibitors. The final fluorescence intensities were cor-
rected for background reactions. The inhibition plots were
obtained using Origin 6.1 software, and these plots were used
for calculating the IC50 values of different inhibitors.
Crystal data for 13.36 C28H24N2O4Se; Mr = 532.2; monocli-
nic; space group P21/c; a = 10.1344(3), b = 10.3521(3), c =
12.1801(4) Å; α = 82.512(2)°, β = 78.896(2)°, γ = 79.956(2)°,
V = 1228.5 Å3; Z = 2; ρcalcd = 1.37 g cm−3; MoKα radiation
(λ = 0.72073 Å); T = 296(2) K; R1 = 0.0761, wR2 = 0.1155
(I > 2σ(I)); R1 = 0.0429, wR2 = 0.1011.
Crystal data for 16. C28H24N2O2Se; Mr = 499.45; monocli-
nic; space group C12/c1; a = 27.425(6), b = 5.1133(9), c =
17.468(4) Å; α = 90.00°, β = 109.987(13)°, γ = 90.00°;
V = 2302.04 Å3; Z = 4; ρcalcd = 1.441 g cm−3; MoKα radiation
(λ = 0.71073 Å); T = 296(2) K; R1 = 0.1427, wR2 = 0.1565
(I > 2σ(I)); R1 = 0.0513, wR2 = 0.1194.
Inhibition of protein nitration of BSA
For bovine serum albumin (BSA), the nitration was performed
by the addition of PN (1.2 mm) to BSA (100 mm) in 0.5 mM
phosphate buffer of pH 7.0 with DTPA (0.1 mm) at 20 °C. After
the addition of PN, the final pH was maintained below 7.5. The
reaction mixture was incubated for 20 min at 20 °C. Similarly,
the reactions of BSA with PN were performed in the presence of
different selenium compounds (133 mM) as inhibitors. Upon
performing the reactions, the mixture was denatured by boiling
at 100 °C for 5 min in the presence of sample loading dye and
Crystal data for 20. C28H22N2O4Se; Mr = 531.9; monoclinic;
space group P1; a = 10.6590(14), b = 11.1175(8), c = 11.4796(8)
Å; α = 115.030(3)°, β = 100.751(4)°, γ = 101.470(4)°, V =
1150.49 Å3; Z = 2; ρcalcd = 1.49 g cm−3; MoKα radiation
(λ = 0.71073 Å); T = 296(2) K; R1 = 0.0413, wR2 = 0.0599
(I > 2σ(I)); R1 = 0.1114, wR2 = 0.1230.
Crystal data for 22. C27H16Br2N2O2Se; Mr = 703.2; monocli-
nic; space group C2/c; a = 21.023(2), b = 13.153(2), c =
This journal is © The Royal Society of Chemistry 2012
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Acknowledgements
This study was supported by a grant from AstraZeneca
Pharmaceuticals. G. M. acknowledges the DST for the award of
a Swarnajayanti fellowship and D. S. L. thanks the UGC for the
award of a Dr D. S. Kothari postdoctoral research fellowship.
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Org. Biomol. Chem., 2012, 10, 7933–7943 | 7943