Protection against peroxynitrite-mediated nitration reaction by intramolecularly coordinated diorganoselenides

Article abstract of DOI:10.1021/om050353c

A series of intramolecularly Se...X (X = N, O) coordinated diorganoselenides and -sulfides are synthesized by using the heteroatom-directed lithiation route and characterized by multinuclear (¹H, ¹³C, ⁷⁷Se) NMR, IR spectroscopy, and electrospray mass spectrometry (ES-MS). The intramolecular Se...O interactions in diorganoselenides 26-31 have been studied by multinuclear NMR studies in solution and in the solid state by single-crystal X-ray crystallography. The protection against peroxynitrite-mediated nitration reaction (PN assay) by diorganoselenides/-sulfides (with and without intramolecular coordination) has been evaluated. The PN assay data of diorganoselenides reveal that the selenides 20-22, having a basic amino group (sp³-N donor) in close proximity of selenium, are more active compared to the diorganoselenides 16-19, having an imino group (sp²-N donor), and also show much higher protective action than the unsubstituted diorganoselenides 14 and 15. The diorganoselenides 16-31 were oxidized to corresponding selenoxides 44-59. The redox properties of the selenoxides 13 and 44-59 have been investigated by cyclic voltammetry and potentiometric titration experiments. Two redox potentials for in situ-generated ferrocenyl selenoxides (50, 54-59) were observed. The reduction of selenoxides 13 and 48 to ebselen (2) and selenide 20 with benzenethiol (PhSH) was monitored by ES-MS.

Full text of DOI:10.1021/om050353c

382
Organometallics 2006, 25, 382-393
Protection against Peroxynitrite-Mediated Nitration Reaction by
Intramolecularly Coordinated Diorganoselenides
Sangit Kumar,† Harkesh B. Singh,*,† and Gotthelf Wolmersha¨user‡
Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai 400 076, India, and
Fachbereich Chemie, UniVersita¨t Kaiserslautern, Postfach 3049, Kaiserslautern 67653, Germany
ReceiVed May 5, 2005
A series of intramolecularly Se‚‚‚X (X ) N, O) coordinated diorganoselenides and -sulfides are
synthesized by using the heteroatom-directed lithiation route and characterized by multinuclear (1H, 13C,
77Se) NMR, IR spectroscopy, and electrospray mass spectrometry (ES-MS). The intramolecular Se‚‚‚O
interactions in diorganoselenides 26-31 have been studied by multinuclear NMR studies in solution and
in the solid state by single-crystal X-ray crystallography. The protection against peroxynitrite-mediated
nitration reaction (PN assay) by diorganoselenides/-sulfides (with and without intramolecular coordination)
has been evaluated. The PN assay data of diorganoselenides reveal that the selenides 20-22, having a
basic amino group (sp3-N donor) in close proximity of selenium, are more active compared to the
diorganoselenides 16-19, having an imino group (sp2-N donor), and also show much higher protective
action than the unsubstituted diorganoselenides 14 and 15. The diorganoselenides 16-31 were oxidized
to corresponding selenoxides 44-59. The redox properties of the selenoxides 13 and 44-59 have been
investigated by cyclic voltammetry and potentiometric titration experiments. Two redox potentials for in
situ-generated ferrocenyl selenoxides (50, 54-59) were observed. The reduction of selenoxides 13 and
48 to ebselen (2) and selenide 20 with benzenethiol (PhSH) was monitored by ES-MS.
Low molecular mass biologically occurring compounds such
as CO2, ascorbate, cysteine, and methionine or tryptophan have
been shown to react with PN and protect biomolecules against
PN-mediated damage.5 In addition to these small molecules,
some water-soluble manganese and iron porphyrins as well as
other macrocyclic metal complexes and heme-containing pro-
teins have been shown to act against PN-mediated reactions.6
Selenoproteins such as glutathione peroxidase (GPx) and
selenoprotein P and synthetic organoselenium compounds such
as 1-12 have also been shown to reduce PN species (Chart
1).7,8 It has also been demonstrated that some diorganotellurides
might provide a defense system against PN.7k,o
Sies et al. have reported that selenomethionine (1) protects
against PN more effectively than its sulfur analogue, methionine,
because the selenium atom in 1 can be easily oxidized to the
corresponding selenoxide and the oxidized species can be
effectively and rapidly reduced to 1 by glutathione (GSH).7b,p
They have also shown that the synthetic organoselenium
compound, ebselen (2), which has been the subject of clinical
trials to evaluate its anti-inflammatory properties,9 is oxidized
to the corresponding selenoxide 13 by the reaction with PN.
The selenoxide is reduced back to ebselen at the expense of
GSH, thus permitting a catalytic cycle as shown in Scheme 1.7d
Introduction
Peroxynitrite (ONOO-/ONOOH), formed by the combination
of nitric oxide (•NO) and superoxide (O2-•) anion radicals, is a
potent oxidizing agent. The radicals are generated by the
endothelial cells.1 The reactivity of peroxynitrite (PN) can be
considered beneficial at the level of the whole organism because
of its cytotoxicity to bacteria and other invading organisms.2
However, excessive production of PN can damage normal tissue
and induce DNA damage as well as initiate lipid peroxidation
in biomembranes or low-density lipoproteins.3 PN also causes
tyrosine nitration of proteins and is known to deactivate a variety
of enzymes.4 Thus, its high reactivity with biological molecules
and its high mobility even in the presence of biological
membranes implicate its many disease states.1,3
* To whom correspondence should be addressed. E-mail: chhbsia@
chem.iitb.ac.in.
† Indian Institute of Technology Bombay.
‡ Universita¨t Kaiserslautern.
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10.1021/om050353c CCC: $33.50 © 2006 American Chemical Society
Publication on Web 12/15/2005

Peroxynitrite-Mediated Nitration Reaction
Organometallics, Vol. 25, No. 2, 2006 383
Chart 1. Organoselenium Compounds 1-12 with Protective Action against PN
Scheme 1. Redox Cycle of Ebselen (2) with PN
the synthetic diorganoselenides (6-12) can protect against PN-
induced DNA damage effectively in a catalytic fashion at the
expense of GSH or ascorbate via a thiolselenurane intermediate.8b
These studies have provided evidence that the selenium redox
cycling can enhance the protective effects of organoselenium
compounds against oxidant-induced DNA damage.
Intramolecularly coordinated organoselenium compounds
have attracted considerable interest as glutathione peroxidase
(GPx) enzyme mimetics.11,12 Our group has recently reported
the antioxidant property (thiol peroxidase-like activity) of a
series of intramolecularly coordinated diorganodiselenides and
ditellurides and found that diselenides having a ferrocenylamine
redox active group show excellent thiol peroxidase activity.12b-f
These observations prompted us to evaluate the protective action
of intramolecularly coordinated organoselenium compounds
against PN-mediated nitration reaction, because the intramo-
lecular interaction in diorganoselenium compounds can (a) tune
the electronic environment of selenium; (b) stabilize the selen-
oxide which is the intermediate in the PN assay; and (c) play a
crucial role in the reduction of selenoxide to selenide. In this
paper we report, for the first time, a systematic investigation
on the use of intramolecularly coordinated diorganoselenides
in PN-mediated nitration reactions (Chart 2).
In view of the excellent protective action against PN of
ebselen (2) and selenides 3 and 6-12, having amide and amino
functional groups against PN, we contemplated incorporating
intramolecularly coordinating amine and amide functional
groups and the redox active ferrocenyl group in the desired
selenides for potent protective action against PN-mediated
nitration reaction. The protective effects of the intramolecularly
coordinated ferrocenyl selenides/sulfides 22 and 26-34 incor-
porating tert-amine and amide functional groups have been
studied to delineate the role of a ferrocene-like redox active
Furthermore, it has been reported that 1 and 2 can pro-
tect DNA from single-strand break formation caused by PN and
the rate of the reaction is about 3 orders of magnitude faster
than that of biologically occurring small molecules, such as
ascorbate, cysteine, and methionine.7d,e Musaev et al.10 have
reported theoretical studies on the reaction of 2 and related
derivatives with PN and found that the ebselen derivatives
with weak Se-N bonds are active toward coordination with
PN and peroxide (O-O) bond cleavage. Recent theoretical
studies on the mechanism of the reaction of PN with dimethyl
selenide have shown that the reaction proceeds via an O atom
transfer mechanism and produces the corresponding selenoxide
and NO2- anion. More recently May et al.8b have reported that
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384 Organometallics, Vol. 25, No. 2, 2006
Kumar et al.
Chart 2. Diorganoselenides/Sulfides 14-34 with Protective
with minor modification. N,N-Dimethylaminomethyl-2-(ben-
zylseleno)naphthalene (21) was prepared by the standard
protocol shown in Scheme 2. The reaction of 36 with n-BuLi
gave a lithiated intermediate (37). The reaction of 37 with
selenium powder, followed by quenching of the resulting lithium
selenolate 38 with benzyl chloride yielded the desired selenide
21.
Action against PN-Mediated Nitration Reaction
It is worth noting that the lithium halogen exchange reaction
proceeded smoothly only at -78 °C and not at room temper-
ature. On the other hand the synthesis of (R,S)-[1-(N,N-
dimethylamino)ethyl]-2-(benzylseleno)ferrocene (22) by direct
lithiation of (R)-(N,N-dimethylamino)ethylferrocene followed
by selenium insertion22 and then quenching with benzyl chloride
was unsuccessful. Alternatively, the novel selenide 22 was
prepared by the reduction of (RR,SS)-bis-2-[1-(N,N-dimethy-
lamino)ethyl]ferrocenyl diselenide22 followed by quenching of
the resulting selenolate with benzyl chloride. Synthesis of
N-phenyl-2-(benzylseleno)benzamide (25) was achieved by the
direct metalation of benzanilide, followed by the addition of
selenium powder and then quenching with benzyl chloride in
one pot. The synthesis of 25 has been reported earlier by a
multistep route. Schiesser et al.20 have reported the synthesis
of 25 from 2-benzylselenobenzoic acid (which itself requires
several steps)20c,23 by the addition of phosgene and subsequent
quenching with aniline.
Ferrocenyl selenides/sulfides 26-34 were prepared by depro-
tonation of N,N-diisopropyl ferrocenecarboxamide (39) followed
by quenching of the resulting arenelithium (40) with the
corresponding diselenides/disulfides (Scheme 3). Reaction of
39 with the diselenides/disulfides (in 1:1 ratio) afforded the
ferrocenyl selenides/sulfides 26-34 in 35-45% yield. Interest-
ingly, deprotonation of 39 using 3.2 equiv of n-BuLi and
TMEDA, followed by quenching with 4 equiv of corresponding
diselenides, gave the ferrocenyl selenides in excellent yield
(80-96%), and no deprotonation of the second Cp ring of 39
was noticed. All attempts to synthesize the benzylic compound
(RSeCH2Ph, R ) N,N-diisopropyl ferrocenylcarboxamide) were
unsuccessful.
The synthesis of ferrocene-ebselen (35) was first approached
by the lithiation of 41 and cyclization of lithium selenolate
dianion 42, as shown in Scheme 4, and the reaction gave the
corresponding diselenide 43.24 Also, the reaction of the dilithi-
ated intermediate with SeCl2 did not afford 35, but led to
decomposition. The synthesis of 35 from diselenide 43 following
the procedures reported for the synthesis of 2 (radical
cyclization,20b bromination, and then cyclization by NEt320c) was
unsuccessful.
group in the PN assay. To incorporate the ebselen moiety in
ferrocene, the synthesis of ferrocene-ebselen (35) was also
attempted.
The reduction of selenoxides to selenides has been investi-
gated by potentiometric titrations, and the reaction intermediates
involved in the reduction of ebselen oxide (13) and selenoxide
48 by PhSH have been characterized by ES-MS. The redox
properties of selenides/sulfides 22 and 26-34 and selenoxides/
sulfoxides 13 and 44-62 were also evaluated by cyclic
voltammetry in addition to the detailed synthesis and spectro-
scopic studies of the diorganoselenides. To gain insight into
the intramolecular interactions, the crystal structures of 26-28
and 33-34 are determined by single-crystal X-ray crystal-
lography.
Results and Discussion
Synthesis. Compounds 2,13 15,14 16,15 18,12d 17 and 20,16
19,17 23,18 and 2419 were synthesized by literature methods, and
2520 and 26, 32, and 3921 were synthesized by literature methods
Selenoxides/sulfoxides 13 and 44-62 were generated in situ
by the oxidation of selenides and sulfides by using H2O2 as an
alternate to PN (Scheme 5)10a and were used directly for further
investigations.
(13) Engman, L.; Hallberg, A. J. Org. Chem. 1989, 54, 2964.
(14) Burgess, M. R.; Morley, C. P. J. Organomet. Chem. 2001, 623,
101.
1
Spectroscopic Studies. The H NMR chemical shifts for
-CH2NMe2 and -CH2N(CH3)2 protons of 21 exhibit consider-
able downfield shifts (∼1 ppm) as compared to naphthylamine
ligand (36), which indicates the presence of an intramolecular
Se‚‚‚N interaction in 21. The 77Se NMR chemical shifts of
organoselenium compounds are quite informative about the
electronic environment on the selenium nucleus. The 77Se NMR
chemical shifts of diorganyl selenides are listed in Table 1. The
(15) Kumar. S.; Kandasamy, K.; Singh, H. B.; Butcher, R. J. New J.
Chem. 2004, 28, 640.
(16) Mugesh, G.; Panda, A.; Singh, H. B.; Butcher, R. J. Chem Eur. J.
1999, 5, 1411.
(17) Apte, S. D.; Singh, H. B.; Butcher, R. J. J. Chem. Res., Synop. 2000,
160. J. Chem. Res., Minipr. 2000, 559.
(18) (a) Perkins, C. W.; Martin, J. C.; Arduengo, A. J.; Lau, W.; Alegria,
A.; Kochi, J. K. J. Am. Chem. Soc. 1980, 102, 7753. (b) Panda, A.; Menon,
S. C.; Singh, H. B.; Butcher, R. J. J. Organomet. Chem. 2001, 623, 81.
(19) Piette J. L.; Renson, M. Bull. Soc. Chem. Belges. 1970, 79, 367.
(20) (a) Fong, M. C.; Laws, M. J.; Schiesser, C. H. Aus. J. Chem. 1995,
48, 1221. (b) Fong, M. C.; Schiesser, C. H. J. Org. Chem. 1997, 62, 3103.
(c) Evers, M.; Fischer, H.; Biedermann, J.; Terlinden, R.; Leyck, S. European
Patent, 042715, 1991.
(22) Nishibayashi, Y.; Segawa, K.; Singh, J. D.; Fukuzawa, S.-i.; Ohe,
K.; Uemura, S. Organometallics 1996, 15, 370.
(23) Syper, L.; Mlochowski, J. Tetrahedron 1988, 44, 6119.
(24) The details of the synthesis of compound 43 will be reported
elsewhere. Here we present only the reaction scheme for the attempted
synthesis of ferrocene-ebselen (36).
(21) Tsukazaki, M.; Roglans, A.; Chapell, B. J.; Taylor, N. J.; Snieckus,
V. J. Am. Chem. Soc. 1996, 118, 685.

Peroxynitrite-Mediated Nitration Reaction
Organometallics, Vol. 25, No. 2, 2006 385
Scheme 2. Synthesis of Selenide 21a
a Reagents and conditions: (i) n-BuLi, THF, -78 °C; (ii) Se powder, 5 h, 0 °C; (iii) C6H5CH2Cl.
Scheme 3. Synthesis of Selenides/Sulfides (26-34)a
a Reagents and conditions: (i) n-BuLi, TMEDA, -78 °C; (ii) diphenyl diselenide, THF, -78 to 0 °C, (iii) dimethyl diselenide, -78 °C; (iv)
di(n-butyl) diselenide, -78 °C; (v) di(tert-butyl) diselenide, -78 °C; (vi) di(2,6-dimethyl-4-tert-butylphenyl) diselenide, THF, -78 °C; (vii) di(2-
methylphenyl) diselenide, THF, -78 to 0 °C; (viii) diphenyl disulfide; (iv) di(2,6-dimethyl-4-tert-butylphenyl) disulfide; (ix) di(2-methylphenyl)
disulfide.
Scheme 4. Attempted Synthesis of Ferrocene-Ebselen (35)a
alkyl selenides 29-31 showed optical rotation (vide infra,
Experimental Section).21,25
Protection against PN-Mediated Nitration of 4-Hydroxy-
phenylacetate (4-HPA). Protection by diorganoselenides and
sulfides against nitration of 4-HPA was studied by following
the method reported by Sies et al.7b Figure 1 shows the
protection by selected diorganoselenides against PN-mediated
nitration of 4-HPA.
The half-maximal inhibitory concentrations (IC50 values)
obtained for the selenides/sulfides 14-34 are summarized in
Table 1 along with their protective action against PN-mediated
nitration reaction at 20 µM of selenides/sulfides 14-34 [%
concentration of 4-hydroxy-3-nitrophenylacetate (NO2-HPA)
formed in the presence of 20 µM of testing compounds]. Most
of the selenides exhibited some protective action in the PN assay.
The result obtained for 2 is also included for a comparison.7b
The concentration of NO2-HPA in the absence of any test
compound was ∼50 µM, and this value was set as 100%. The
IC50 value for 2, which has been previously used, was 63
µM (entry a, Table 1) and is in good agreement with the value
(IC50 ) 60 µM) reported in the literature.7b The IC50 value of
simple selenide 14 is 134 µM (entry b, Table 1), and a slight
enhancement in the inhibition was observed when the phenyl
group in 14 was replaced by a redox active group, ferrocene
(compound 15; IC50 ) 120 µM; entry b, Table 1). As expected,
ferrocene alone did not have any effect on the formation of
NO2-HPA in the PN assay. Intramolecularly Se‚‚‚N coordinated
diorganoselenides 16 and 18, having a sp2-N donor atom in close
proximity of selenium, exhibited lower protective action (IC50
) 157, 149 µM; entries d, f; Table 1) as compared with selenide
14. This is probably due to the presence of an imino-nitrogen
in 16-19, which is not basic enough to enhance the nucleo-
philicity of selenium.12e On the other hand, the intramolecularly
a Reagent and conditions: (i) n-BuLi, Se; (ii) CuBr2, (iii) Br2, CuI,
NEt3, DMF; (iv) tert-butyl peroxide; (v) n-BuLi, SeCl2.
77Se NMR chemical shifts for naphthylamine selenide 21 and
ferrocenylamine selenide 22 are observed at 262 and 260 ppm,
respectively, and are upfield shifted as compared to the
benzylamine selenide 20 (321 ppm).16 The 77Se NMR chemical
shift of 25 (376 ppm) is comparable with that of related selenides
[(2-benzylseleno)benzamides (>N-R ) hexyl, Bu, benzyl)]
(361-371 ppm)20b and indicates a stronger Se‚‚‚O interaction
than ferrocenecarboxamide-based selenides (vide infra). The
77Se NMR chemical shifts of 26-31 are in the order (ppm)
phenyl > 2-methylphenyl > 2,6-dimethyl-4-tert-butylphenyl >
tert-butyl > n-butyl . methyl and can be correlated by the
electron-donating nature of the -Se-R group (except methyl).
The significant upfield 77Se NMR chemical shift for selenide
28 (184 ppm) compared with 26 and 27 (294 and 242 ppm)
indicates the strong electron donation of the 2,6-dimethyl-4-
tert-butylphenyl group. Selenide 31 exhibits a signal at 85 ppm,
which is significantly upfield shifted as compared to other
related selenides. Ferrocenyl aryl selenides/sulfides 26-28 and
32-34 did not show any optical rotation, whereas ferrocenyl
(25) Slocum, D. W.; Tucker, S. P.; Engelmann, T. R. Tetrahedron Lett.
1970, 621. Roman, E.; Astruc, D.; des Abbayes, H. J. Organomet. Chem.
1981, 219, 211.

386 Organometallics, Vol. 25, No. 2, 2006
Scheme 5. In Situ Preparation of Selenoxides and Sulfoxides (44-62)
Kumar et al.
Table 1. IC50 Values of Testing Compounds; Nitration in
4-HPA at 20 µM of Testing Compounds, E1/2
77Se NMR
Chemical Shifts of Diorganoselenides
;
IC50
nitration of
E
1/2 (mV vs Fc 77Se NMR
entry compd (µM)a 4-HPA (20 µM)b
in CH3CN)c
(ppm)d
942
a
b
c
d
e
f
2
63
134
120
157
ND
149
ND
43
34
28
130
54
176
80
78 ( 12
89 ( 5
87 ( 8
61 ( 7
ND
85 ( 13
ND
83 ( 7
79 ( 6
65 ( 6
95 ( 11
87 ( 13
81 ( 9
85 ( 14
86 ( 7
77 ( 8
86 ( 5
88 ( 9
96 ( 12
97 ( 14
96 ( 19
95 ( 15
98 ( 17
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
39
65
287
376
420
362
420
321
262
260
393
321
377
294
242
184
172
162
86
ND
g
h
i
j
k
l
+52 ( 3
Figure 1. Effect of selenides [14 (2), 20 (b), 21 (×), and 22 (9)]
on the nitration of 4-HPA caused by PN. Results are given as means
( SD (n ) 5-7).
m
n
o
p
q
r
+193 ( 4
+189 ( 2
+139 ( 6
+124 ( 7
+126 ( 4
+128 ( 5
+191 ( 2
+190 ( 3
+128 ( 6
+125 ( 1
72
52
84
90
effect of the aldehyde group. This is in agreement with the
reports of Sies et al.7k and May et al.8 that aryl selenides with
electron-withdrawing substituents show very poor activity. It
is worth noting that selenide 25, incorporating an amide
functionality of ebselen, led to a decrease in the protective action
(176 µM; entry m, Table 1) as compared to 2, which is sim-
ilar to the observation7c that the methyl derivative of 2 essen-
tially reduces the protective effect. Further studies show that
the aryl selenides 26-28 (80, 72, 52 µM; entries n, o, p) are
considerably more active than the corresponding alkyl selenides
29-31 (84, 90, 102 µM; entries q, r, s, Table 1). The aryl
selenide 28, which contains an electron-donating substituent in
the para position and two methyl substituents in the ortho
positions, was found to be an efficient scavenger of PN (IC50
) 52 µM).7k,8 In contrast to the selenium compounds, the sul-
fides 32-34 are found to be essentially inactive under iden-
tical experimental conditions. The very high IC50 values
observed for these compounds indicate that there is no signifi-
cant inhibition on the control activities (entries t, u, v, Table
1). The high reactivity of ferrocenyl selenides 26-28 as
compared with their sulfur analogues may be ascribed to the
higher nucleophilic character of selenium compared with
sulfur.26 Interestingly, diphenyl diselenide and bis[2-phenyl-2-
oxazolinyl]diselenide are devoid of any protective action in the
PN assay. Ebselen oxide (13) and selenoxide 48, which are
s
t
u
v
w
102
>250
>250
>250
a Concentration of compound causing 50% inhibition of PN-mediated
reaction (see also Figure 1). b Concentration of NO2-HPA (%) when 20
c
µM of testing compound was used in PN assay. E1/2 oxidation potentials
values for ferrocenyl selenides/sulfides and Fc ) ferrocene. d Chemical shifts
measured in CDCl3 at RT and chemical shifts δ relative to Me2Se; ND,
not determined.
Se‚‚‚N coordinated diorganoselenide 20, having a sp3-N donor
atom, shows much better protective action (IC50 ) 43 µM; entry
h, Table 1) than the unsubstituted diorganoselenides 14 and 15.
Interestingly, very effective protective action was observed when
the phenyl group in selenide 20 was replaced by a naphthyl or
a ferrocenyl with the same functionality (21 and 22). The IC50
values (34, 28 µM, entries i, j, Table 1) observed for these
compounds are much lower than that of all other compounds
in the present study. This is probably due to the presence of a
tert-amino group, because the presence of a tert-amino group
in close proximity of selenium may enhance the nucleophilicity
of selenium. The IC50 values of the intramolecularly Se‚‚‚O
coordinated selenides 23 and 24 are observed to be 130 and 54
µM, respectively (entries k, l, Table 1). The lower activity of
these compounds may be ascribed to the electron-withdrawing
(26) Wada, M.; Nobuki, S.; Tenkyuu, Y.; Natsume, S.; Asahara, M.;
Erabi, T. J. Organomet. Chem. 1999, 580, 282.

Peroxynitrite-Mediated Nitration Reaction
Organometallics, Vol. 25, No. 2, 2006 387
Table 2. Redox Potentials Obtained from Potentiometric
Titrations and Reduction Potentials (Epc) Obtained from
Cyclic Voltammetry for Selenoxides and Sulfoxides
entry
compd
E
1/2, Epc(mV)a vs NHE
E(mV)b vs NHE
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
13
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
-852 ( 7
-949 ( 10
-968 ( 12
-924 ( 6
ND
e
e
e
e
-1176 ( 5
-1200 ( 4
+150, -1295 ( 6
-896 ( 3
-1168 ( 6
-907 ( 11
+680, -1205
+638, -1270
+618, -1350
+622, -1361
+654, -1362
+646, -1359
+656
+423
+385
+380
+490
+415
+435
+420
+405
+398
+385
+384
+395
e
Figure 3. Potentiometric titration of selenoxide 54 (1 mM) by using
10 mM sodium dithionite as the titrant in 100 mM sodium acetate,
pH 5.5.
in the range from -852 to -1362 mV (Table 2). The Epc values
are in good agreement with the related compounds reported in
the literature, which generally range from -892 to -1160 mV.8b
Ferrocenyl alkyl selenoxides 57-59 exhibit Epc values (-1361,
-1362, and -1359 mV; entries o, p, q, Table 2) that are close
to or slightly lower than those observed for the ferrocenyl aryl
selenoxides 54-56 (Epc ) -1205, -1270, -1350 mV; entries
m, n, o, Table 2). Selenoxide 56 with an electron-donating tert-
butyl group at the para position shows a considerably lower
Epc value (-1350 mV) than the selenoxides 54 and 55, and
this is in accordance with the report of May et al.8b that electron-
donating para substituents in benzene result in a decrease in
Epc. It is also evident from Table 1 that the Epc values of
selenoxides 51 and 52 are influenced by the presence of electron-
withdrawing and -donating ortho groups in the aromatic rings
(entries i, j, Table 2).
s
t
+658
+645
e
e
a E1/2 values correspond to ferrocene in ferrocenyl selenoxide/sulfoxides;
E
pc values correspond to selenoxides/sulfoxides. b Redox potentials obtained
from potentiometric titration. e A well-defined titration curve could not be
obtained using sodium dithionite.
Potentiometric Titrations. To further understand the redox
properties of the selenoxides, potentiometric titrations have been
carried out by following the method reported by May et al.8b
The results from the potentiometric titrations of selenoxides 13
and 44-59 were compared with their redox potentials (E) and
related potentiometric values for some reported selenoxides
(Table 2). A representative plot for potentiometric titration of
54 is shown in Figure 3.
The salient features of the experimental results are (a)
electron-withdrawing groups in the aromatic ring in 51 and 53
result in the increase in E (+490, +435 mV; entries i, k, Table
2); (b) naphthyl and ferrocenyl selenoxides 49 and 50 show
lower E values (+385, +380 mV, entries g, h, Table 2) than
the phenyl analogues, 48 (+423 mV; entry f, Table 2); and (c)
ferrocenyl aryl selenoxides 54-56 exhibit higher E (+418,
+405, +398 mV; entries m, n, o, Table 2) than the ferrocenyl
alkyl selenoxides, 57-59 (E ) +385, +384, +395 mV). It is
worth mentioning that the selenoxides that exhibited lower
reduction potentials in the cyclic voltammetry experiments show
lower redox potentials (E) also in the potentiometric titrations
(Table 2).
Electrospray Mass Spectrometry (ES-MS) Study. It has
been well documented that diorganoselenides can be oxidized
to selenoxides by PN and reduced back to diorganoselenides
by reducing agents such as GSH or ascorbate, via the formation
of a thiolselenurane intermediate.8b To understand the nature
of the intermediates in the reduction of selenoxides to selenides
in detail, the reduction of selenoxides 13 and 48 was monitored
by ES-MS by using benzenethiol (PhSH) as an alternate to GSH.
The electrospray mass spectra of organoselenium compounds
gave distinct molecular ion peaks with characteristic isotopic
patterns (illustrated for 13, 29-31, 48, 54, 65, 67-69, and 72
in Figures S50, S54, and S58-71 in the Supporting Informa-
Figure 2. Representative cyclic voltammogram of selenoxide (54).
already in the oxidized form, also do not show any protective
action in the PN assay.
Cyclic Voltammetry Studies. Peak potentials (Epc) for
selenoxides were for the first time reported by May et al.8b For
comparison purposes, the Epc values for in situ-generated
selenoxides 13 and 44-62 were obtained under similar condi-
tions by cyclic voltammetry. Cyclic voltammetry experiments
were carried out in acetonitrile for selenides/sulfides 22 and
26-34 and in aqueous solutions for selenoxides/sulfoxides 13
and 44-62 at pH 5.5. Half-wave potentials (E1/2) and peak
potentials (Epc) for reductive waves are given in Tables 1 and
2. In the case of selenides 22 and 26-31 the wave (which is
not well defined) due to the selenium atom was observed at
1.60-1.90 V. These species exhibit electrochemically irrevers-
ible voltammograms, as evidenced by the absence of reduction
waves. The additional feature may be due to the selenium acting
as a redox center.14 The selenoxides 13, 44-49, and 51-53
show a single redox wave, whereas ferrocene selenoxides 50
and 54-59 show two redox waves. A representative cyclic
voltammogram for 54 is given in Figure 2.
In ferrocene selenoxides 54-59, the redox potentials (E1/2
)
due to the ferrocenyl moiety are observed in the range from
+622 to +680 mV. In contrast, a significantly low E1/2 value
was observed for the ferrocenylamine selenoxide 50 (+150 mV;
entry h, Table 2), which may be due to electron donation of
the tert-aminoethyl group to the -Cp ring. The redox waves
due to the selenoxide moiety are irreversible for 44-46 and
48-53 and quasi-reversible for 13 and 54-59 as evident from
a large separation of the cathodic and anodic peak potentials.
The peak potentials for the reductive waves (Epc) are observed

388 Organometallics, Vol. 25, No. 2, 2006
Kumar et al.
Scheme 6. Proposed Mechanism for the Reduction of
Ebselen Oxide (13) to Ebselen (2)
Figure 4. Molecular structure of 26.
Scheme 7. Proposed Mechanism for the Reduction of
Selenoxide 48 to Selenide 20
Table 3. Bond Lengths [Å] and Angles [deg] for 26-28, 33,
and 34
26
27
28
Se(1)-C(7)
1.885(3)
1.914(4)
1.473(4)
101.1(15)
104.1(9)
100.9(4)
1.891(4)
1.925(5)
1.472(6)
100.5(2)
78.2(3)
1.898(3)
1.927(3)
1.488(3)
99.9(10)
151.5(2)
164.6(7)
Se(1)-C(18)
N(1)-C(12)
C(7 or 6)-Se(1)-C(18)
N(1)‚‚‚Se(1)-C(18)
O(1)‚‚‚Se(1)-C(18)
109.2(5)
33
34
S(1)-C(7)
1.744(3)
1.780(3)
1.470(4)
102.4(14)
78.7(11)
1.776(7)
S(1)-C(18)
1.780(8)
1.49(9)
105.3(4)
69.4(3)
N(1)-C(12)
C(7)-S(1)-C(18)
N(1)‚‚‚S(1)-C(18)
the presence of an ortho N-donor atom near selenium.28 It
appears that nitrogen donates its lone pair of electrons to
selenium and increases the basic nature of selenoxide oxygen
in 48 via resonance (48A and 48B). The 1:1 molar reaction of
48 with PhSH shows the formation of 72 (m/z, 412), but does
not show the formation of the thioseleneurane intermediate (71).
This suggests that the reduction of selenoxide 48 takes place
via 71. The formation of 72 may also be possible directly by
the reaction of selenoxide 48 with thiol. A similar mechanism
has been proposed for the tellurium analogue of 48 by Detty et
al.29 It is worth mentioning that the oxidized selenomethionine
residue in proteins can be reduced back to 1 rapidly by GSH
through the formation of a three-electron Se‚‚‚N species.7p Thus
this Se‚‚‚N interaction may also play a crucial role in the overall
reduction of selenoxide 48 as shown in Scheme 7. The ES-MS
experiments on the reaction of selenoxides 13 and 48 with PhSH
suggest that the selenoxide 48 is reduced back to selenide 20
via a different reaction intermediate.
tion). The ES-MS of ebselen oxide (13) shows the molecular
ion peak at 292 (m/z), and this compound may be in equilibrium
with dihydroxy- and hydroxymethoxy-selenuranes; 63 and 64
(Scheme 6).
Although the ES-MS of 13 was somewhat complicated, it
produced thiolseleninate (65, m/z, 400), when treated with 1
equiv of PhSH. When the 1:1 reaction mixture of 13 and PhSH
was treated with an additional equivalent of PhSH, the molec-
ular ion peaks for 2 and selenenyl sulfide 67 (m/z, 382) were
detected in the ES-MS. This suggests that ebselen is regen-
erated from thiolseleninate (65) via the formation of intermediate
66, although the formation of 66 has not been observed in the
ES-MS, presumably due to its high reactivity. The conversion
of selenenic acid 66 into selenenyl sulfide 67 also takes place
as shown in Scheme 6.27 In the case of intramolecularly Se‚‚‚N
coordinated selenoxide 48, the ES-MS shows the molecular ion
peaks for dihydroxy- (68, m/z 336), hydroxymethoxy- (69, m/z,
352), and dimethoxyselenuranes (70, m/z, 365) in addition to
the selenoxide 48 (m/z, 322) (Scheme 7).
X-ray Crystallographic Study. The selenides 26, 27, and
28 are isostructural. The molecular structure of a representative
selenide (26) is shown in Figure 4. Significant bond angles and
bond lengths are given in Table 3. The distance between carbon
and selenium (-Se-C-) and angle (-C-Se-C-) are well
within the range of reported values for other diorganose-
lenides.16,30
It seems that the solvation of 48 is more favorable than
ebselen oxide (13), and this may be due the more basic nature
of the selenoxide oxygen in 48, which may in turn be due to
(28) (a) Goodman, M. A.; Detty, M. R. Organometallics 2004, 23, 3016.
(b) Shimizu, T.; Enomoto, M.; Takai, H.; Kamigata, N. J. Org. Chem. 1999,
64, 8242.
(29) You, Y.; Ahsan, K.; Detty, M. R. J. Am. Chem. Soc. 2003, 125,
4918.
(27) (a) Glass, R. S.; Farooqui, F.; Sabahi, M.; Ehler, K. W.J. Org. Chem.
1989, 54, 1092. (b) Haenen, G. R. M.; De Rooij, B. M.; Vermeulen, N. P.
E.; Bast, A. Mol. Pharmacol. 1990, 37, 412.

Peroxynitrite-Mediated Nitration Reaction
Organometallics, Vol. 25, No. 2, 2006 389
Chart 3. Resonance in 39 Based on the Related
The Se‚‚‚O intramolecular distances in selenides based on 39
are in contrast to oxazoline-based ferrocenyl selenide30 and
oxazoline-based phenyl selenides,16 in which selenium atoms
are in contact with nitrogen. However, in both the ligands
(oxazoline and carboxamide) both heteroatoms (O and N) are
capable of forming a five-membered ring. The Se‚‚‚O distances
in 26-28 and Se‚‚‚N distances in oxazoline-based selenides
indicate that resonance plays a crucial role in Se‚‚‚X (X ) O,
N) interamolecular interactions. Similarly the nature of S‚‚‚O
and S‚‚‚N distances can be explained in 33 and 34. However,
there is no significant S‚‚‚O interaction between sulfur and
oxygen in 33 and 34.
N,N-Dimethylferrocenecarboxamide35
The intramolecular Se‚‚‚N distance (4.0741 Å) in selenide
26 is longer than the sum of van der Waals radii (3.45 Å)31 of
nitrogen and selenium, whereas the Se‚‚‚O distance (3.5069 Å)
is close to the van der Waals radius (3.40 Å). This crucial
observation can be rationalized by resonance (vide infra, Chart
3). Compound 27 is isostructural to 26. In this structure the
Se‚‚‚N distance is 4.102 Å, which is slightly larger than that
for compound 26. The Se‚‚‚O distance is 4.1012 Å. The
Se‚‚‚N distance (4.4728 Å) in selenide 28 is longer than that
observed for 26 and 27. Surprisingly the Se‚‚‚O distance (2.9425
Å) in 28 is shorter than the sum of van der Waals radii (Figure
5). Compared to the selenides 26 and 27, the shorter distance
between the selenium and oxygen in selenide 28 indicates that
steric effects may play an obvious role.
The diorganosulfur molecules 33 and 34 are isostructural to
27 and 28, respectively. The molecular structure of a representa-
tive sulfide (33) is shown in Figure 6. The carbon-sulfur bond
distances and bond angles are comparable with the reported
diorganosulfides.15,32,33 The intramolecular S(1)‚‚‚O(1) and
S(1)‚‚‚N(1) distances in 33 and 34 are longer than their van
der Waals radii (O; S; 3.30 Å; N; S; 3.35 Å) and indicate that
there is no significant intramolecular interaction between oxygen
and sulfur.
The C-O bond lengths for compounds 26 and 27 (1.212-
1.220 Å) are longer than CdO bond lengths (1.17-1.20 Å),
while the C(11)-N bond distances (1.347-1.363 Å) for
26-28 are significantly shorter than the C(22)-N bond (1.488-
1.463 Å) (Table 4). These indicate the double-bond character
in C(11)-N as reflected in the resonance structures (39A and
39B) shown in Chart 3.34 The Se‚‚‚O interactions in selenides
26-28 may also be correlated with the C(11)-N distances. The
Se‚‚‚O, Se‚‚‚N, C(11)-O, and C(11)-N bond distances are
shown in Table 4 for comparison. For selenide 28, where the
Se‚‚‚O interaction is strong, the C(11)-N bond length [1.351-
(4) Å] is significantly shorter than the C-N [C(12)-N 1.488-
(3); C(15)-N 1.465(4) Å] single bond. The same is true for 26
and 27. These distances suggest the existence of resonating
structures in selenides 26 and 27. Owing to the charged nature
of the second resonance structures, 39A and 39B, one may
expect O to be an electron donor. This diminishes the possibility
of any intramolecular interaction of the nitrogen atom with
selenium.
Conclusion
This study reveals that the selenides having basic amino
groups with weak intramolecular Se‚‚‚N interaction (20-22)
are more active than the selenides having imino groups with
strong Se‚‚‚N interaction (16-19) against PN-mediated nitration
reactions. The selenoxides of diorganoselenides, which show
better protective action in the PN assay, can also be reduced
back to selenides. Intramolecularly coordinated diaryl selenox-
ides (47, 51, 52, and 54-56), aryl benzyl selenoxides (44-46,
48-50, and 53), and aryl alkyl selenoxide (59), lacking a
â-hydrogen, do not undergo any selenoxide elimination reaction.
The absence of selenoxide elimination reactions28,35 may help
in recycling the selenoxides back to selenides without loss of
any activity. The present study will enhance our understanding
of the mechanism and protective action of the model compounds
and may ultimately yield insight that results in improved
protective action of selenides against PN-mediated nitration
reactions.
Experimental Section
General Procedures. All reactions were carried out under
nitrogen or argon using standard vacuum-line techniques. Solvents
were purified by standard procedures36 and were freshly distilled
prior to use. Melting points were recorded in capillary tubes and
are uncorrected. H and 13C NMR spectra were obtained at 300
1
1
MHz in CDCl3 on a Varian VXR 300S spectrometer. H NMR
chemical shifts are cited with respect to SiMe4 as internal standard.
IR spectra were recorded as KBr pellets on a Nicolet Impact 400
FTIR spectrometer. The 77Se NMR spectra were obtained at 95.35
in CDCl3 on a Bruker AMX500 spectrometer using diphenyl
diselenide as external standard. Chemical shifts are reported rela-
tive to dimethyl selenide (77Se) (0 ppm) by assuming that the reso-
nance of the standard is at 461 ppm. Elemental analyses were
performed on a Carlo-Erba model 1106 elemental analyzer. Optical
rotations were measured by a JASCO Model DIP 370 digital
polarimeter.
N,N-Dimethylaminomethyl-2-(benzylseleno)naphtha-
lene (21). To a THF (35 mL) solution of 1-bromo-2-(N,N-dimeth-
ylaminomethyl)naphthalene (36)37 (0.66 g, 2.5 mmol) was added
dropwise n-BuLi (1.7 mL, 3 mmol, 1.6 M solution in pentane) at
-78 °C. The solution was stirred for 20 min, and then Se powder
(0.198 g, 2.5 mmol) was added in portions to the solution at 0 °C.
After the disappearance of selenium powder, benzyl chloride (0.33
mL, 2.8 mmol) was added to the solution and the stirring was con-
tinued for 1 h at 0 °C and then at room temperature for 8 h. The
solution was washed with a saturated NH4Cl solution, and the
Steric crowding by two isopropyl groups, which are bonded
to nitrogen, may be an additional factor to diminish the
possibility of a selenium and nitrogen intramolecular interaction.
(30) Nishibayashi, Y.; Uemura, S. Synlett 1995, 79.
(31) Pauling, L. The Nature of the Chemical Bond, 3rd ed.; Cornell
University Press: Ithaca, NY, 1960; p 224.
(32) (a) Mugesh, G.; Singh, H. B.; Butcher, R. J. J. Chem. Res., Synop.
1999, 472. J. Chem. Res., Minipr. 1999, 2020.
(33) You, S.-L.; Hou, X.-L.; Dai, L.-X.; Yu, Y.-H.; Xia, W. J. Org. Chem.
2002, 67, 4684.
(35) Drake, M. D.; Bright, F. V.; Detty, M. R. J. Am. Chem. Soc. 2003,
125, 12558.
(36) Perrin, D. D.; Armarego, W. L. F; Perrin, D. R. Purification of
Laboratory Chemicals, 2nd ed.; Pergamon: Oxford, 1980.
(37) Gay, L. R.; Hauser, C. R. J. Am. Chem. Soc. 1967, 89, 2297.
(34) Petter, R. C.; Rao, S. J. J. Org. Chem. 1991, 56, 2932.

390 Organometallics, Vol. 25, No. 2, 2006
Kumar et al.
Figure 5. Molecular structure of 28.
N-Phenyl-2-(benzylseleno)benzamide (25). To a stirred solution
of benzanilide (2.0 g, 10.0 mmol) in dry THF (50 mL) was added
n-BuLi (12.8 mL, 20 mmol, 1.6 M solution in hexane) at 0 °C
under N2. To the dark red-colored solution of the dilithiated product
formed after 30 min, Se powder (0.8 g, 10 mmol) was added under
a brisk flow of N2 and the stirring was continued further for 30
min. Benzyl chloride (1.2 mL, 10 mmol) was added to the
homogeneous dark orange solution via syringe. After 1 h, the
reaction mixture was poured in water and extracted with CHCl3.
The organic layer was washed with 2 × 50 mL of water, dried
over Na2SO4, and concentrated in vacuo to give a white-colored
solid. Recrystallization from EtOH afforded a white crystalline
powder. Yield: 2.04 g (56%); mp 192-194 °C; 1H NMR (CDCl3)
δ 4.12 (s, 2H), 7.19-7.89 (many signals, 15H); 77Se NMR (CDCl3)
δ 377. Anal. Calcd (%) for C20H17NOSe (366.2): C, 65.59; H,
4.67; N, 3.82. Found: C, 65.38; H, 5.13; N, 4.64. All other data
are in accordance with the literature values.20a
Preparation of (R,S)-1-[1-(N,N-Dimethylamino)ethyl]-2-(ben-
zylseleno)ferrocene (22). In a two-necked 50 mL round-bottomed
flask containing a magnetic stirring bar was placed (RR,SS)-bis-2-
[1-(N,N-dimethylamino)ethyl]ferrocenyl diselenide22 (0.1 g, 0.32
mmol) in THF (30 mL) under N2. Lithium superhydride (0.7 mL,
0.7 mmol, 1 M solution in THF) was added to the flask at 0 °C,
and the reaction mixture was stirred for an additional 30 min. Benzyl
chloride (0.1 mL, 0.7 mmol) in 10 mL of THF was added to the
resulting solution, and the reaction mixture was stirred at room
temperature overnight. The reaction mixture was treated with brine
(100 mL) and then extracted with CH2Cl2 (3 × 25 mL). The extract
was dried over Na2SO4 and evaporated to leave a red oil of 22,
which was purified by column chromatography on alumina with
hexane/ethyl acetate (9:3) as an eluent to get pure 22 as an orange
Figure 6. Molecular structure of 33.
Table 4. Structural Comparison of Selenides 26-28 and
Sulfides 33 and 34
compound Se/S‚‚‚O (Å) Se/S‚‚‚N (Å) O-C(11) (Å) N-C(11) (Å)
1
oil. Yield: 0.16 g, (57%); [R]25 58.0 (c 0.034, CHCl3); H NMR
26
27
28
33
34a
3.506(9)
3.760(2)
2.950(2)
3.703(3)
3.543(7)
4.074(1)
4.101(2)
4.493(4)
4.023(2)
4.254(7)
1.212(4)
1.220(6)
1.216(3)
1.222(4)
1.242(9)
1.363(4)S
1.347(5)
1.353(3)
1.351(4)
1.246(9)
(CDCl3) δ 1.32 (d, 3H), 2.18 (s, 6H), 3.63-3.78 (m, 1H)
4.14-4.49 (m, 3H), 4.17 (s, 5H), 4.42 (s, 2H), 6.84-7.29 (m, 5H);
13C NMR (CDCl3) δ 15.73, 19.44, 23.85, 29.36, 31.86, 53.90, 60.56,
68.51, 69.72, 70.18, 70.52, 71.10, 85.38, 115.73, 119.88, 129.54,
156.83, 211.09; IR (neat) ν 2897, 2961, 2845, 1468, 1228, 1118,
82 cm-1; ES-MS m/z 413.
a Average distances from two asymmetric molecules.
product was extracted with ether and dried over Na2SO4. The
solvent was removed in vacuo. To the residue was added hexane/
CH2Cl2 (2:3), and it was kept for crystallization to obtain a white-
colored compound. Yield: 0.75 g (84%); mp 186-189 °C; 1H NMR
(CDCl3) δ 3.18 (s, 6H), 5.18 (s, 2H), 5.34 (s, 2H), 7.45-7.55 (m,
6H), 7.65-7.73 (m, 2H), 7.84-7.89 (m, 2H), 8.17-8.20(d, 1H);
13C NMR (CDCl3) δ 48.08, 48.86, 67.50, 69.00, 124.91, 126.62,
127.38, 127.44, 127.54, 128.30, 128.62, 128.86, 129.43, 130.25,
132.66, 133.28, 133.54, 133.75; 77Se NMR (CDCl3) δ 262. Anal.
Calcd (%) for C20H21NSe (354.2): C, 67.81; H, 5.97; N, 3.95.
Found: C, 67.62; H, 5.76; N, 3.46. ES-MS: m/z 355.
Preparation of N,N-Diisopropylferrocenecarboxamide (39).
A stirred solution of ferrocenecarboxylic acid (2.30 g, 10 mmol)
in dry CH2Cl2 (15 mL) was treated dropwise with oxalyl chloride
(1.74 mL, 20 mmol) under N2 at room temperature, and the
stirring was continued for 30 min at this temperature to obtain a
red-colored solution. Excess oxalyl chloride was removed under
vacuo, CH2Cl2 was added, and the solution was cooled to 0 °C. To
this were added dry diisoprylamine (1.01 g, 10 mmol) in CH2Cl2
(25 mL) and triethylamine (1.4 mL, 10 mmol), and the stirring
was continued overnight. The reaction mixture was treated with a
saturated NH4Cl solution and extracted with CH2Cl2 (2 × 50 mL).

Peroxynitrite-Mediated Nitration Reaction
Organometallics, Vol. 25, No. 2, 2006 391
The combined organic layer was washed with water (100 mL), dried
over Na2SO4, filtered, and concentrated under vacuo to give a dark
red-colored solid. Purification was accomplished by column chro-
matography using SiO2 (60-120 mass) and petroleum ether
(60-80 °C)/ethyl acetate (10:2). Crystallization from pentane and
hexane (3:2) provided orange-colored needles. Yield: 2.89 g, (92%);
mp 86-88 (lit. 88-89 °C);21 1H NMR (CDCl3) δ 1.23-1.48 (b,
12H), 3.18-3.69 (b, 2H), 4.27 (s, 5H) 4.26 (m, 2H), 4.56 (m, 2H);
IR (KBr) ν 2980, 2945, 1628, 1580, 1462, 1373, 1206, 1034, 818
cm-1. Anal. Calcd (%) for C17H23FeNO (313.2): C, 65.21; H, 7.40;
N, 4.47. Found: C, 64.97; H, 7.52; N, 4.51. All other data are
consistent with the values reported in the literature.21
Synthesis of N,N-Diisopropyl-2-(phenylseleno)ferrocenecar-
boxamide (26). Under a N2 atmosphere n-BuLi (2.07 mL, 3.7
mmol, 1.6 M solution in hexane) was added to a stirred solution of
TMEDA (0.48 mL, 3.6 mmol) in dry Et2O (10 mL) at -78 °C,
and the stirring was continued further for 10 min. A solution of 39
(0.313 g, 1.0 mmol) in dry Et2O (15 mL) was added dropwise via
cannula, and the resulting red-colored reaction mixture was stirred
for 45 min at -78 °C. Diphenyl diselenide (1.18 g, 3.8 mmol) in
dry THF (8 mL) was added by syringe, and the stirring was
continued for an additional 30 min at -78 °C and 1 h at room
temperature. The reaction mixture was quenched with a saturated
NH4Cl solution and the organic layer separated. The aqueous layer
was extracted with 2 × 50 mL of Et2O, dried over Na2SO4, and
concentrated in vacuo. Purification by column chromatography
using petroleum ether (60-80 °C)/ethyl acetate (10:2) gave 26 as
an orange solid. Recrystallization from pentane/hexane (3:1)
provided orange crystals. Yield: 0.44 g (94%), mp 136-138 (lit.
133-135 °C);21 1H NMR (CDCl3) δ 0.52 (b, 3H), 1.03 (b, 3H),
1.32-163 (b, 6H); 3.22-3.42 (b, 1H); 3.62-3.85 (b, 1H); 4.29
(m, 1H); 4.38 (m, 1H); 4.41 (s, 5H); 4.51 (m, 1H); 7.12-7.24 (m,
3H); 7.32-7.40 (m, 2H); 77Se NMR (CDCl3) δ 294; IR (KBr) ν
2943, 2924, 1632, 1580, 1460, 1206, 818 cm-1. Anal. Calcd (%)
for C23H27FeNOSe (468.2): C, 58.99; H, 5.81; N, 2.99. Found:
C, 58.89; H, 5.68; N, 2.82. All other data are consistent with the
values reported in the literature.21
1.26 (s, 9H), 1.35-1.57 (b, 6H), 2.52 (s, 6H), 3.28-3.62 (m, 1H),
3.42-3.68 (b, 1H), 4.09(t, 1H), 4.14 (m, 1H), 4.26 (m, 1H), 4.41
(s, 5H), 7.02 (s, 2H); 13C NMR (CDCl3) δ 21.02, 24.98, 31.29,
34.315, 46.05, 50.24, 66.30, 66.87, 71.73, 73.05, 76.78, 77.10,
77.42, 80.56, 86.18, 124.74, 129.88, 142.29, 151.11, 167.66; 77Se
NMR (CDCl3) δ 184; IR (KBr) ν 2973, 2927, 2868, 1643, 1446,
1321, 1032, 828, cm-1. Anal. Calcd (%) for C29H39FeNOSe
(552.3): C, 63.07; H, 7.11; N, 2.53. Found: C, 62.83; H, 7.10; N,
2.45.
Preparation of N,N-Diisopropyl-2-(tert-butylseleno)ferrocen-
ecaboxamide (29). To a stirred lithiated product 40 (prepared as
described for compound 26) was added bis(tert-butyl) diselenide
(2.2 g, 8.0 mmol) via syringe over 15 min at -78 °C. The reaction
mixture was further stirred for 45 min at this temperature and 2 h
at room temperature. The reaction mixture was worked up and
purified by chromatography using petroleum ether (60-80 °C) and
ethyl acetate (30:2) to afford 29 as an orange colored-liquid.
1
Yield: 0.78 g (87%); [R]25 -0.89 (c 0.025, CHCl3); H NMR
(CDCl3) δ 0.08-1.68 (many signal, 12H), 2.68 (m, 9H), 4.20 (t,
1H), 4.30 (m, 1H), 4.32 (s, 5H), 4.40 (t, 1H); 13C NMR (CDCl3) δ
13.76, 21.09, 23.15, 24.19, 28.53, 29.21, 31.64, 32.73, 44.81, 46.03,
50.43, 67.39, 67.55, 71.37, 72.17, 71.38, 72.71, 73.03, 75.29, 76.88,
77.20, 77.52, 90.04, 167.31; 77Se NMR (CDCl3) δ 172; IR (neat)
ν 3098, 2960, 2921, 1644, 1460, 1374, 1321, 1038, 821 cm-1
ES-MS m/z 450.
;
Preparation of N,N-Diisoprpyl-2-(n-butylseleno)ferrocene-
caboxamide (30). Compound 30 was synthesized in a similar
manner as described for 26, from 39 (0.876 g, 2.8 mmol) and di-
(n-butyl) diselenide (2.75 g, 10 mmol). Purification by column
chromatography using petroleum ether (60-80 °C) and ethyl acetate
(32:2) afforded 30 as an orange-colored liquid. Yield: 0.1 g (86%);
[R]25 1.78 (c 0.025, CHCl3); 1H NMR (CDCl3) δ 0.80 (t, 3H), 1.08
(b, 6H), 1.21-1.82 (many signal, 10H), 3.22 (b, 1H), 3.82 (b, 1H),
2.71 (m, 2H), 4.20 (t, 1H), 4.26 (b, 1H), 4.32 (s, 5H), 4.37 (b, 1H);
13C NMR (CDCl3) δ 13.77, 21.09, 23.16, 28.53, 29.84, 32.73, 46.08,
50.43, 67.39, 67.53, 71.37, 73.05, 75.10, 76.92, 90.06, 167.3; 77Se
NMR (CDCl3) δ 162; IR (neat) ν 3098, 2960, 2868, 1644, 1460,
1374, 1321, 834 cm-1; ES-MS m/z 450.
Synthesis of N,N-Diisopropyl-2-{(2-methyphenyl)seleno}-
ferrocenecarboxamide (27). A solution of 39 (0.626 g, 2.0 mmol)
in Et2O (10 mL) was added to a mixture of n-BuLi (3.7 mL, 6.7
mmol) and TMEDA (1.05 mL 6.6 mmol) in Et2O (15 mL). Bis-
(2-methylphenyl) diselenide38 (2.26 g, 7 mmol) was added to the
reaction mixture. Standard workup and purification by column
chromatography using petroleum ether (60-80 °C) and ethyl acetate
(18:2) afforded 27 as an orange solid. Recrystallization from
CH2Cl2/hexane (1:5) provided orange crystals. Yield: 0.93 g (96%);
Preparation of N,N-Diisopropyl-2-(methylseleno)ferrocen-
ecaboxamide (31). Selenide 31 was synthesized by a similar
method as that described for 30 except the addition of dimethyl
diselenide in place of di(n-butyl) diselenide. The crude was purified
by column chromatography using petroleum ether/Et2O (20:5) to
afford 31 as an orange-colored liquid. Yield: 0.8 g (87%); [R]25
-16.66 (c 0.025, CHCl3); 1H NMR (CDCl3) δ 0.89-1.25 (b, 6H),
1.38-1.66 (b, 6H), 2.21 (s, 3H), 3.51 (b, 1H), 4.07 (b, 1H), 4.20
(t, 1H), 4.29 (b, 1H), 4.31 (b, 1H), 4.32 (s, 5H); 13C NMR (CDCl3)
δ 8.48, 21.10, 29.85, 46.11, 50.47, 67.48, 67.69, 71.29, 71.59, 89.18,
167.54; 77Se NMR (CDCl3) δ 85; IR (neat) ν 3105, 2973, 2920,
1644, 1460, 1374, 1321, 821 cm-1; ES-MS m/z 406.
1
mp 126-129 °C; H NMR (CDCl3) δ 0.47 (b, 3H), 0.97 (b, 3H),
1.25-1.47 (b, 6H); 2.36 (s, 3H), 3.25 (b, 1H); 3.70 (b, 1H); 4.32
(m, 1H); 4.39 (s, 5H); 4.47 (m, 2H); 6.93-7.00 (m, 2H); 7.06-
7.26 (m, 2H); 13C NMR (CDCl3) δ 20.19, 20.86, 21.67, 29.78,
45.88, 50.35, 68.32, 68.53, 71.53, 75.72, 92.40, 125.79, 126.36,
128.92, 129.81, 135.31, 137.05, 166.42; 77Se NMR (CDCl3) δ 242;
IR (KBr) ν 2973, 2927, 1637, 1466, 1374, 1314, 1209, 828, 744
cm-1. Anal. Calcd (%) for C24H29FeNOSe (482.2): C, 59.78; H,
6.06; N, 2.90. Found: C, 59.16; H, 6.84; N, 2.65.
Synthesis of N,N-Diisopropyl-2-(phenylthio)ferrocenecarbox-
amide (32). Sulfide 32 was prepared by a similar method as that
described for 26 except the addition of diphenyl disulfide. Standard
workup and purification by column chromatography using petro-
leum ether (60-80 °C)/ethyl acetate afforded 32 as an orange solid.
Recrystallization from pentane/hexane (3:1) provided orange crys-
tals. Yield: 0.39 g (92%), mp 150-153 (154-155 °C);21 1H NMR
(CDCl3) δ 0.44 (b, 3H), 0.97 (b, 3H), 1.37-1.59 (b, 6H); 3.25 (b,
1H); 3.66 (b, 1H); 4.31 (m, 1H); 4.38 (m, 2H); 4.40 (s, 5H); 4.53
(m, 2H); 7.03-7.13 (m, 3H); 7.14-7.26 (m, 2H); IR (KBr) ν 2941,
2919, 1637, 1586, 1465, 1216, 821 cm-1. Anal. Calcd (%) for
C23H27FeNOS (421.4): C, 65.56; H, 6.46; N, 3.32; S, 7.61.
Found: C, 65.37; H, 6.25; N, 3.54; S, 7.43. All other data are
consistent with the values reported in the literature.21
Preparation of N,N-Diisopropyl-2-{(2,5-dimethyl-4-tert-bu-
tylphenyl)seleno}ferrocenecaboxamide (28). To a stirred solution
of the lithiated derivative 40 was added bis[2,5-dimethyl-4-tert-
butylphenyl] diselenide12c (4.82 g, 10 mmol) in 8 mL of dry THF
by syringe over 30 min at -78 °C. The reaction mixture was further
stirred for 45 min at this temperature and 2 h at 60 °C. The reaction
mixture on usual workup and purification by column chromatog-
raphy using petroleum ether (60-80 °C) and ethyl acetate (19:2)
afforded the desired compound as an orange solid. Recrystallization
from toluene at -30 °C provided red pellets. Yield: 1.3 g (98%),
1
Synthesis of N,N-Diisopropyl-2-[(2-methyl-phenyl)thio]fer-
rocenecarboxamide (33). Synthesis of 33 was achevied by a similar
method as that reported for 27, except the addition of bis(2-
mp 110-113 °C; H NMR (CDCl3) δ 0.73 (b, 3H), 1.10 (b, 3H),
(38) Zhang, Y.; Jia, X.; Zhou, X. Synth. Commun. 1994, 24, 1247.

392 Organometallics, Vol. 25, No. 2, 2006
Kumar et al.
methylphenyl) disulfide39 in place of bis(2-methylphenyl)diselenide.
Standard workup and purification by column chromatography using
petroleum ether (60-80 °C) and ethyl acetate (18:2) afforded 33
as an orange solid. Recrystallizion from CH2Cl2/hexane (1:5)
provided orange crystals. Yield: 0.70 g (80%), mp 135-137 °C;
1H NMR (CDCl3) δ 0.44 (b, 3H), 0.97 (b, 3H), 1.26-1.59 (b, 6H);
2.37 (s, 3H), 3.25 (b, 1H); 3.70 (b, 1H); 4.33 (m, 1H); 4.43 (s,
5H); 4.52 (m, 2H); 6.94-6.95 (m, 2H); 7.05-7.26 (m, 2H); IR
Table 5. Crystal Data and Structure Refinement for 26-28
26
27
28
empirical formula
fw
C23H27FeNOSe C24H29FeNOSe C29H39FeNOSe
468.27
482.29
552.42
cryst syst
space group
a (Å)
b (Å)
c (Å)
triclinic
P1h
triclinic
P1h
triclinic
P1h
7.3377(7)
11.6860(13)
13.4351(16)
66.420(12)
85.705(13)
86.313(12)
1052.1(2)
2
7.4339(7)
10.4051(12)
14.8043(16)
83.841(13)
81.238(12)
78.814(12)
1106.7(2)
2
7.6049(6)
12.2227(10)
15.1327(12)
90.729(10)
93.247(10)
104.268(10)
1360.53(19)
2
(KBr) ν 2978, 2934, 1643, 1469, 1371, 1308, 1204, 821, 743 cm-1
.
R (deg)
Anal. Calcd (%) for C24H29FeNOS (435.4): C, 66.20; H, 6.71; N,
3.21; S, 7.36. Found: C, 65.87; H, 6.51; N, 3.57; S, 7.31.
Preparation of N,N-Diisopropyl-2-[(2,5-dimethyl-4-tert-butyl-
phenyl)thio]ferrocenecaboxamide (34). To a stirred solution of
arenelithium (39) was added bis(2,5-dimethyl-4-tert-butylphenyl)
disulfide40 (3.86 g, 10 mmol in 8 mL of dry THF) via syringe over
30 min at -78 °C. The reaction mixture was worked up and purified
by column chromatography using petroleum ether (60-80 °C) and
ethyl acetate (19:2) to afford the desired compound as an orange
solid. Recrystallization from toluene at -30 °C provided red pellets.
Yield: 1.1 g (87%), mp 127-129 °C; 1H NMR (CDCl3) δ 0.0.68
(b, 3H), 1.05 (b, 3H), 1.25 (s, 9H), 1.28-1.58 (b, 6H), 2.45 (s,
6H), 3.21-3.53 (m, 1H), 3.33-3.57 (b, 1H), 4.01 (t, 1H), 4.16
(m, 1H), 4.21 (m, 1H), 4.30 (s, 5H), 7.02 (s, 2H); 13C NMR (CDCl3)
δ 20.90, 22.61, 29.72, 31.26, 34.31, 65.62, 66.05, 71.62, 71.75,
86.47, 87.73, 125.26, 126.80, 131.64, 141.83, 150.96, 166.9; IR
(KBr) ν 2977, 2931, 2873, 1631, 1453, 1327, 1027, 825 cm-1. Anal.
Calcd (%) for C29H39FeNOS (505.5): C, 68.90; H, 7.78; N, 2.77;
S, 6.34. Found: C, 68.78; H, 7.23; N, 2.81; S, 6.27.
Peroxynitrite Synthesis. PN synthesis was carried out by
modifying the method described by Beckman et al.41 Sodium nitrite
(0.6 M), hydrogen peroxide (0.6 M in 0.7 M HCl), and sodium
hydroxide (1.2 M) were mixed by quenched flow reactor at 0 °C.
Excess hydrogen peroxide was removed by MnO2 treatment. The
concentration of PN was conveniently assayed by diluting the PN
stock solution to 100-fold in 1.2 M NaOH and measuring the
absorbance at 302 nm (ꢀ ) 1670 M-1 cm-1).
General Procedure for the Determination of IC50 Values of
Diorganselenides in PN Assay. Experiments were conducted at
room temperature in a 0.1 M sodium phosphate buffer (pH 7.4)
containing Fe(III)/EDTA (0.5 mM) [Fe(III)/EDTA complex was
prepared by mixing equimolar solutions of Fe(III) chloride and
sodium EDTA], 4-HPA (1 mM), and various concentrations of the
testing compounds in methanol. PN (50 µM) was added to the above
reaction mixture under constant stirring. Samples were mixed for
30 min at room temperature. The pH was adjusted to 10-11 with
1 M NaOH before absorbance was measured at 430 nm. The yield
of the nitrated product (4-hydroxy-3-nitrophenyl acetate) was
calculated by using ꢀ ) 4400 M-1 cm-1. Alternatively, as a control,
the experiment was carried out without PN. The result of the
reaction of 4-HPA with PN and methanol alone was set equal to
100%.
Preparation of Selenoxides and Sulfoxides 44-62. The sele-
noxides were prepared by the reaction of corresponding diorga-
noselenides/sulfides 16-34 with 1.5 equiv of H2O2 in methanol at
0 °C. The progress of the reaction was monitored by TLC. Once
the reaction was complete, MnO2 powder was added to the reaction
mixture to remove the excess of H2O2, and the reaction mixture
was further stirred for 1 h. Then MnO2 powder was removed by
filtration and the filtrate concentrated in vacuo to give the
selenoxides/sulfoxides 44-62, which were directly used for cyclic
voltammetry, potentiometric titrations, and ES-MS study. Selenox-
ide 48 was also prepared by the reported method by using
â (deg)
γ (deg)
V (Å3)
Z
D(calcd) (Mg/m3)
1.478
2.459
1.447
2.340
1.348
1.912
abs coeff (mm-1
)
no. of obsd reflns
14 830
15 535
21 303
[I > 2σ(I)]
final R(F) [I > 2σ(I)]a 0.0343
0.0412
0.0876
3923/0/258
0.0339
0.0758
5386/0/307
wR(F2) [I > 2σ(I)]
0.0709
no. of data/restraints/ 3754/0/248
params
goodness fit on F2
0.772
0.824
0.912
2
2
a Definitions: R(F0) ) ∑||Fo| - |Fc||/∑|Fo| and wR(Fo ) ) {∑[w(Fo
-
Fc )2]/∑[w(Fc )2}1/2
.
2
2
Table 6. Crystal Data and Structure Refinement for 33 and
34
33
34
empirical formula
fw
C24H29FeNOS
435.39
C29H39FeNOS
505.52
cryst syst
space group
a (Å)
b (Å)
c (Å)
triclinic
P1h
triclinic
P1h
7.4118(8)
10.4149(12)
14.6040(17)
83.639(14)
81.266(13)
79.564(12)
1091.8(2)
2
8.0669(7)
17.1460(18)
19.849(2)
91.560(13)
90.312(12)
98.494(12)
2714.2(5)
4
R (deg)
â (deg)
γ (deg)
V (Å3)
Z
D(calcd) (Mg/m3)
1.324
0.800
1.237
0.653
abs coeff (mm-1
)
no. of obsd reflns [I > 2σ(I)]
final R(F) [I > 2σ(I)]a
wR(F2) [I > 2σ(I)]
16 825
0.0509
0.1327
4298/0/258
1.052
17 041
0.0577
0.1095
6512/0/613
0.752
no. of data/restraints/params
goodness fit on F2
2
2
a Definitions: R(Fo) ) ∑||Fo| - |Fc||/∑|Fo| and wR(Fo ) ) {∑[w(Fo
-
Fc )2]/∑[w(Fc )2}1/2
.
2
2
m-chloroperbenzoic acid oxidation of selenide 20.18 Selenoxide 48
obtained from this method and the above method (H2O2) exhibited
the same cyclic voltammetry and potentiometric titration data.
Cyclic Voltammetry Study. Cyclic voltammetry was performed
with a CH-1660 scanning potentiostat/galvanostat interfaced to a
base PC using the EG&G model 270 software package. Cyclic
voltammograms of selenoxides/sulfoxides 13 and 44-62 were
obtained by using a glassy carbon working electrode, a platinum
coil counter electrode, and a calomel reference electrode in 100
mM sodium acetate buffer (pH ) 5.5) under argon. The scan rate
was 100 mV/s, and full IR compensation was employed in all
measurements. The fresh solutions of in situ-generated selenoxides/
sulfoxides 13 and 44-62 in CH3OH were used for cyclic volta-
mmetry experiments. A blank experiment was carried out in sodium
acetate aqueous buffer under argon (for more information, see
Supporting Information, pp 19-29, Figures S13-S23).
(39) Boerzel, H.; Koeckert, M.; Bu, W.; Spingler, B.; Lippard, S. J. Inorg.
Chem. 2003, 42, 1604.
(40) Barton, D. H. R.; Britten-Kelly, R. M.; Ferreira, D. J. Chem. Soc.,
Perkin Trans. 1 1978, 12, 1682.
(41) Koppenol, W. H.; Kissner, R.; Beckman, J. S. Methods Enzymol.
1996, 269, 296.
Potentiometric Titrations. The cell reduction potentials (E) for
the selenoxides were obtained by potentiometric titration with the
reducing agent sodium dithionite as a titrant. Measurements were
performed in a two-electrode-cell system with a single compartment
containing a platinum spiral wire indicator electrode and an aqueous

Peroxynitrite-Mediated Nitration Reaction
Organometallics, Vol. 25, No. 2, 2006 393
saturated calomel electrode (SCE) connected to a millivoltmeter.
Measurements were carried out at 25 °C under argon in 100 mM
sodium acetate, pH 5.5, with an initial concentration of selenoxide
of 1 mM. Sodium dithionite (10 mM) was titrated into the cell
with a 30 min equilibration period between each additional aliquot.
All reported potentials were corrected to potentials versus the
normal hydrogen electrode (NHE). A stock solution of selenoxides
13 and 44-62 was prepared in methanol. In the case of selenoxides
and sulfoxides 44-47 and 60-62 no significant changes in voltage
were observed.
ES-MS Experiment. ES-MS were performed at room temper-
ature on a Q-Tof micro (YA-105) mass spectrometer. Mass spectra
were obtained with a Platform II single quadrupole mass spec-
trometer (Micromass, Altrincham, UK) using a CH3OH mobile
phase. A solution of PhSH (11 mg, 0.10 mmol) in CH3OH/H2O
(1:1) was added to a solution of ebselen oxide (13) (29 mg, 0.10
mmol) in MeOH. The resulting solution was stirred for 15 min,
and then ES-MS of the reaction mixture was obtained. Similarly
ES-MS of the reaction of PhSH and selenoxide 48 was obtained
(for more information, see Supporting Information, pp 68-80,
Figures S59-S69).
by full-matrix least squares with the non-hydrogen atoms anisotropic
and hydrogen with fixed isotropic thermal parameters of 0.07 Å2
by means of the SHELEXL-97 program.43 Hydrogens were partially
located from difference electron-density maps, and the rest were
fixed at predetermined positions. Scattering factors were from
common sources. Some details of the structural refinement are given
in Tables 5 and 6.
Acknowledgment. We heartily thank Dr. G. Mugesh (Indian
Institute of Science, Bangalore, India) for helpful discussions.
This work was supported by the Department of Science and
Technology, New Delhi (DST grant to H.B.S.), and Council of
Scientific and Industrial Research (CSIR), Government of India,
New Delhi (Senior Research Fellowship to S.K.).
Supporting Information Available: The synthesis of 41 and
43 and details of the PN assay (for compounds 2, 14, 20-22, 24,
and 26-31), 77Se NMR (for compounds 21, 22, and 25-31), ES-
MS spectra (for compounds 13, 29-31, 48, 54, 65, 67-69, and
72), colored molecular structures of 26-28, 33, and 34, and CIF
data for 26-28, 33, and 34. This material is available free of charge
via the Internet at http://pubs.acs.org.
X-ray Structure Determination of Compounds 26-28 and
33-34. The diffraction measurements were performed on a STOE
(Darmstadt, Germany) IPDS imaging plate single-crystal diffrac-
tometer at room temperature with graphite-monochromated Mo KR
radiation (λ ) 0.7107 Å). The structures were determined by routine
heavy-atom using SHELXS-9042 and Fourier methods and refined
OM050353C
(42) Sheldrick, G. M. SHELXS-90, Program for the Solution of Crystal
Structures; University of Go¨ttingen: Germany, 1990.
(43) Sheldrick, G. M. SHELXL-97, A Program for Refining Crystal
Structures; University of Go¨ttingen: Germany, 1997.