The antioxidant profile of 2,3-dihydrobenzo[b]furan-5-ol and its 1-thio, 1-seleno, and 1-telluro analogues

Article abstract of DOI:10.1021/ja0035811

A novel synthesis of 2,3-dihydrobenzo[b]thiophene-5-ol based on intramolecular homolytic substitution on sulfur was reported. The "antioxidant profile" of the series of 2,3-dihydrobenzo[b]furan-5-ol (2a) its 1-thio (2b), 1-seleno (2c) and 1-telluro (2d) a

Full text of DOI:10.1021/ja0035811

3434
J. Am. Chem. Soc. 2001, 123, 3434-3440
The Antioxidant Profile of 2,3-Dihydrobenzo[b]furan-5-ol and Its
1-Thio, 1-Seleno, and 1-Telluro Analogues
Jonas Malmstro1m,† Mats Jonsson,*,‡ Ian A. Cotgreave,| Leif Hammarstro1m,§
Martin Sjo1din,§ and Lars Engman*,†
Contribution from the Department of Chemistry, Nuclear Chemistry, Royal Institute of Technologyy, S-100
44 Stockholm, Sweden, DiVision of Biochemical Toxicology, Institute of EnVironmental Medicine,
Karolinska Institute, Box 210, S-171 77 Stockholm, Sweden, Department of Physical Chemistry,
Uppsala UniVersity, Box 532, S-751 21 Uppsala, Sweden, and Uppsala UniVersity, Institute of Chemistry,
Department of Organic Chemistry, Box 531, S-751 21 Uppsala, Sweden
ReceiVed October 3, 2000
Abstract: A novel synthesis of 2,3-dihydrobenzo[b]thiophene-5-ol based on intramolecular homolytic
substitution on sulfur was reported. The “antioxidant profile” of the series of 2,3-dihydrobenzo[b]furan-5-ol
(2a) its 1-thio (2b), 1-seleno (2c) and 1-telluro (2d) analogues was determined by studies of redox properties,
the capacity to inhibit stimulated lipid peroxidation, the reactivity toward tert-butoxyl radicals, the ability to
catalyze decomposition of hydrogen peroxide in the presence of glutathione, and the inhibiting effect on
stimulated peroxidation in liver microsomes. The one-electron reduction potentials of the aroxyl radicals
corresponding to compounds 2a-2d, E° (ArO•/ArO-) were 0.49, 0.49, 0.49, and 0.52 V vs NHE, respectively,
as determined by pulse radiolysis. With increasing chalcogen substitution the compounds become slightly
more acidic (pKa ) 10.6, 10.0, 9.9, and 9.5, respectively, for compounds 2a-2d). By using Hess’ law, the
homolytic O-H bond dissociation enthalpies of compounds 2a-2d (340, 337, 336, and 337 kJ mol-1
,
respectively) were calculated. The reduction potentials for the proton coupled oxidation of compounds 2a-2d
(ArOH f ArO• + H+) as determined by cyclic voltammetry in acetonitrile were 1.35 (irreversible), 1.35
(quasireversible) 1.13 (reversible), and 0.74 (reversible) V vs NHE, respectively. As judged by the inhibited
rates of peroxidation, Rinh, in a water/chlorobenzene two-phase lipid peroxidation system containing
N-acetylcysteine as a thiol-reducing agent in the aqueous phase, the antioxidant capacity increases (2d > 2c
) 2b > 2a) as one traverses the group of chalcogens. Whereas the times of inhibition, Tinh, were slightly
reduced for the oxygen (2a) and sulfur (2b) derivatives in the absence of the thiol-reducing agent, they were
drastically reduced for the selenium (2c) and tellurium (2d) derivatives. This seems to indicate that the
organochalcogen compounds are continuously regenerated at the lipid aqueous interphase and that regeneration
is much more efficient for the selenium and tellurium compounds. The absolute rate constants for the oxidation
of compounds 2a-2b by the tert-butoxyl radical in acetonitrile/di-tert-butyl peroxide (10/1) were the sames2
× 108 M-1 s-1. Whereas the oxygen, sulfur, and selenium derivatives 2a-2c were essentially void of any
glutathione peroxidase-like activity, the organotellurium compound 2d accelerated the initial reduction of
hydrogen peroxide, tert-butyl hydroperoxide, and cumene hydroperoxide in the presence of glutathione 100,
333, and 213 times, respectively, as compared to the spontaneous reaction. Compounds 2a-2d were assessed
for their capacity to inhibit lipid peroxidation in liver microsomes stimulated by Fe(II)/ADP/ascorbate. Whereas
the oxygen, sulfur, and selenium compounds showed weak inhibiting activity (IC50 values of ∼250, 25, and
13 µM, respectively), the organotellurium compound 2d was a potent inhibitor with an IC50 value of 0.13 µM.
Introduction
substituent para to the phenolic oxygen) to a stereoelectronic
effect,2 involving the p-type lone pair orbital of the nonphenolic
oxygen. Sulfur is considered to be more effective than oxygen
at stabilizing a neighboring radical center.3 It was therefore
surprising that 1-thio-R-tocopherol (1b) and related 6-hydrox-
ythiochromanes were found slightly less reactive toward peroxyl
radicals than their corresponding chromane derivatives.4 1-Se-
leno-R-tocopherol (1c) and 1-telluro-R-tocopherol (1d) have not
yet been prepared. Organoselenium and organotellurium com-
pounds show many interesting antioxidative properties. As one
traverses group 16 of the periodic table, the elements become
R-Tocopherol (1a), the main component of vitamin E, other
6-hydroxychromanes as well as the corresponding ring-
contracted analoguess2,3-dihydrobenzofuranssare some of the
most active peroxyl radical trapping agents known.1 Ingold and
co-workers attributed the improved antioxidant activity of these
compounds (as compared to other phenols lacking an oxygen
‡ Royal Institute of Technology.
| Karolinska Institute.
§ Department of Physical Chemistry, Uppsala University.
† Uppsala University, Institute of Chemistry.
(1) (a) Burton, G. W.; Ingold, K. U. Acc. Chem. Res. 1986, 19, 194. (b)
Burton, G. W.; Ingold, K. U. J. Am. Chem. Soc. 1981, 103, 6472. (c)
Barclay, L. R. C.; Ingold, K. U. J. Am. Chem. Soc. 1981, 103, 6478. (d)
Mukai, K.; Okabe, K.; Hosose, H. J. Org. Chem. 1989, 54, 557. (e) Gilbert,
J. C.; Pinto, M. J. Org. Chem. 1992, 57, 5271. (f) Barclay, L. R. C.; Vinqvist,
M. R.; Mukai, K.; Itoh, S.; Morimoto, H. J. Org. Chem. 1993, 58, 7416.
(g) Barclay, L. R. C.; Edwards, C. D.; Mukai, K.; Egawa, Y.; Nishi, T. J.
Org. Chem. 1995, 60, 2739.
(2) Burton, G. W.; Doba, T.; Gabe, E. J.; Hughes, L.; Lee, F. L.; Prasad,
L.; Ingold, K. U. J. Am. Chem. Soc. 1985, 107, 7053.
(3) Luedtke, A. E.; Timberlake, J. W. J. Org. Chem. 1985, 50, 268.
(4) Robillard, B.; Hughes, L.; Slaby, M.; Lindsay, D. A.; Ingold, K. U.
J. Org. Chem. 1986, 51, 1700. Zahalka, H. A.; Robillard, B.; Hughes, L.;
Lusztyk, J.; Burton, G. W.; Janzen, E. G.; Kotake, Y.; Ingold, K. U. J.
Org. Chem. 1988, 53, 3739.
10.1021/ja0035811 CCC: $20.00 © 2001 American Chemical Society
Published on Web 03/27/2001

Antioxidant Profile of 2,3-Dihydrobenzo[b]furan-5-ol
J. Am. Chem. Soc., Vol. 123, No. 15, 2001 3435
Scheme 1a
less electronegative. Thus, electron transfer to reactive alkoxyl
and peroxyl radicals becomes more likely to occur. In addition
to this chain-breaking antioxidative capacity, organochalcogens
are preventive antioxidants in the sense that they reduce organic
hydroperoxides to the corresponding alcohols. The selenium-
containing glutathione peroxidases5 catalyze the reduction of
hydrogen peroxide, fatty-acid hydroperoxides, and phospholipid
and cholesterol hydroperoxides using the tripeptide glutathione
and other thiols as stoichiometric reducing agents. We and others
have reported organoselenium and organotellurium compounds
with chain-breaking donating6,7 or thiol peroxidase activity.8
Recently, we described a tandem SRN1/SHi sequence for
radical-based synthesis of 5-hydroxy-2,3-dihydrobenzo[b]sele-
nophene (2c) and 5-hydroxy-2,3-dihydrobenzo[b]tellurophene.9
It occurred to us that these materials, together with the known
oxygen (2a) and sulfur (2b) analogues, constitute a nice series
a Conditions: (i.) Br2, KOAc, HOAc, 15-17 °C, 2.5 h, 97%. (ii.)
LiAlH4, Et2O, 0 °C, 70 min, 93%. (iii.) (PhS)2, Bu3P, C6H6, ambient
temperature, 8 h, 76%. (iv.) n-Bu3SnH, AIBN, C6H6, reflux, 44 h. (v.)
TBAF, THF, ambient temperature, 62% from 5.
of compounds for studying antioxidant capacity as a function
of the chalcogen substituent para to the phenolic group. In the
following we report the “antioxidant profile” of compounds 2
as determined by studies of their redox properties, their capacity
to inhibit stimulated lipid peroxidation, their reactivity toward
tert-butoxyl radicals, their ability to catalyze decomposition of
hydrogen peroxide in the presence of glutathione, and their
inhibiting effect on peroxidation in liver microsomes.
(5) Spallholz, J. E.; Boylan, L. M. In Peroxidases in Chemistry and
Biology; Everse, J., Everse, K. E., Grisham, M. B., Eds.; CRC Press: Boca
Raton, 1991; Vol. 1, Chapter 12. Sunde, R. A. In Handbook of Nutritionally
Essential Mineral Elements; O’Dell, B. L., Sunde, R. A., Eds.; Marcel
Dekker: New York, 1997, Chapter 18.
(6) Vessman, K.; Ekstro¨m, M.; Berglund, M.; Andersson, C.-M.; Engman,
L. J. Org. Chem. 1995, 60, 4461.
(7) Cotgreave, I. A.; Engman, L. In Handbook of Synthetic Antioxidants;
Packer, L.; Cadenas, E., Eds.; Marcel Dekker: New York, 1997. Andersson,
C.-M.; Hallberg, A.; Ho¨gberg, T. AdV. Drug Res. 1996, 28, 67.
Results and Discussion
Synthesis. 5-Hydroxy-2,3-dihydrobenzo[b]furan (2a) is readily
prepared according to the literature.10 However, for the known11
sulfur derivative 2b we developed an efficent alternative protocol
based on homolytic substitution at sulfur (Scheme 1). TBDMS-
protected ethyl 3-hydroxyphenylacetic acid was regioselectively
brominated in the 6-position and the ester 3 reduced to the
corresponding alcohol 4. The required radical precursor 5 formed
when the alcohol was treated with diphenyl disulfide in the
presence of tri-n-butyl phosphine. Homolytic substitution at
sulfur with expulsion of a phenyl radical occurred during heating
in benzene in the presence of tri-n butyltin hydride and AIBN.
Final deprotection using tetra-n-butylammonium fluoride af-
forded compound 2b.
(8) Mu¨ller, A.; Cadenas, E.; Graf, P.; Sies, H. Biochem. Pharmacol. 1984,
33, 3235. Sies, H. Free Rad. Biol. Med. 1993, 14, 313, Schewe, T. Gen.
Pharmacol. 1995, 26, 1153. Sies, H.; Masumoto, H. AdV. Pharmacol. 1997,
38, 229. Cotgreave, I. A.; Morgenstern, R.; Engman, L.; Ahokas, J. Chem.-
Biol. Interact. 1992, 84, 69. Morgenstern, R.; Cotgreave, I. A.; Engman,
L. Chem.-Biol. Interact. 1992, 84, 77. Jacquemin, P. V.; Christiaens, L. E.;
Renson, M. J. Tetrahedron Lett. 1992, 33, 3863. Reich, H. J.; Jasperse, C.
P. J. Am. Chem. Soc. 1987, 109, 5549. Back, T. G.; Dyck, B. P. J. Am.
Chem. Soc. 1997, 119, 2079. Moutet, M.; D’Alessio, P.; Malette, P.; Devaux,
V.; Chaudie`re, J. Free. Rad. Biol. Med. 1998, 25, 270. Erdelmeier, I.;
Tailhan-Lomont, C.; Moutet, M.; Chaudie`re, J.; Yadan, J.-C. PCT Int. Appl.
WO 98/29417, 1998. Wilson, S. R.; Zucker, P. A.; Huang, R.-R. C.; Spector,
A. J. Am. Chem. Soc. 1989, 111, 5936. Iwaoka, M.; Tomoda, S. J. Am.
Chem. Soc. 1994, 116, 2557. Galet, V.; Bernier, J.-L.; He´nichart, J.-P.;
Lesieur, D.; Abadie, C.; Rochette, L.; Lindenbaum, A.; Chalas, J.; Renaud
de la Faverie, J.-F.; Pfeiffer, B.; Renard, P. J. Med. Chem. 1994, 37, 2903.
Wirth, T. Molecules 1998, 3, 164. Mugesh, G.; Panda, A.; Singh, H. B.;
Punekar, N. S.; Butcher, R. J. J. Chem. Soc., Chem. Commun. 1998, 2227.
Tailhan-Lomont, C.; Erdelmeier, I.; Moutet, M.; Chaudie`re, J.; Yadan, J.-
C. Eur. Pat. Appl. EP 850,924. 1998. Engman, L. Andersson, C.;
Morgenstern, R.; Cotgreave, I. A.; Andersson, C.-M.; Hallberg, A.
Tetrahedron 1994, 50, 2929. Wu, Z.-P.; Hilvert, D. J. Am. Chem. Soc. 1990,
112, 5647. House, K. L.; Dunlap, R. B.; Odom, J. D.; Wu, Z.-P.; Hilvert,
D. J. Am. Chem. Soc. 1992, 114, 8573. Liu, J.-Q.; Jiang, M.-S.; Luo, G.-
M.; Yan, G.-L.; Shen, J.-C. Biotechnol. Lett. 1998, 20, 693. Ding, L.; Liu,
Z.; Zhu, Z.; Luo, G.; Zhao, D.; Ni, J. Biochem. J. 1998, 332, 251. Liu,
J.-Q.; Gao, S.-J.; Luo, G.-M.; Yan, G.-L.; Shen, J.-C. Biochem. Biophys.
Res. Commun. 1998, 247, 397. Engman, L.; Stern, D.; Cotgreave, I. A.;
Andersson, C.-M. J. Am. Chem. Soc. 1992, 114, 9737. Andersson, C.-M.;
Hallberg, A.; Brattsand, R.; Cotgreave, I. A.; Engman, L.; Persson, J. Bioorg.
Med. Chem. Lett. 1993, 3, 2553. Engman, L.; Stern, D.; Pelcman, M.
Andersson, C.-M. J. Org. Chem. 1994, 59, 1973. Detty, M. R.; Gibson, S.
L. Organometallics 1992, 11, 2147. Detty, M. R.; Friedman, A. E.; Oseroff,
A. R. J. Org. Chem. 1994, 59, 8245. Francavilla, C.; Bright, F. V.; Detty,
M. R. Org. Lett. 1999, 1, 1043. Kanda, T.; Engman, L.; Cotgreave, I. A.;
Powis, G. J. Org. Chem. 1999, 64, 8161. For a recent review on synthetic
organoselenium compounds with glutathione peroxidase-like properties
see: Mugesh, G.; Singh, H. B. Chem. Soc. ReV. 2000, 29, 347.
Acidity and Redox Properties. Data obtained from pKa and
redox studies of compounds 2 in aqueous solution are sum-
marized in Table 1. Primary oxidation of the phenolates ArO-
corresponding to compounds 2 (pH 12, NaOH) was achieved
by N3• produced in pulse radiolysis experiments by the reaction
of OH• with N3 (10-2 M NaN3). To determine one-electron
-
reduction potentials of the phenoxyl radicals, the redox equi-
librium between the radical of interest and a redox couple with
a known one-electron reduction potential was studied (eq 1).
ArO• + Ref a ArO- + Ref+•
(1)
The equilibrium constant can be derived from the rate constants
of the electron-transfer reaction and the back reaction and/or
(10) Alabaster, R. J.; Cottrell, I. F.; Marley, H.; Wright, S. H. B. Synthesis
1988, 950.
(11) (a) Clark, P. D.; Rahman, L. K. A.; Scrowston, R. M. J. Chem.
Soc., Perkin Trans. 1 1982, 815. (b) Zambias, R. A.; Hammond, M. L.
Synth. Commun. 1991, 21, 959.
(9) Engman, L.; Laws, M. J.; Malmstro¨m, J.; Schiesser, C. H.; Zugaro,
L. M. J. Org. Chem. 1999, 64, 6764.

3436 J. Am. Chem. Soc., Vol. 123, No. 15, 2001
Malmstro¨m et al.
BDE O-H
Table 1. Acidity and Redox Properties of Compounds 2 in Aqueous Solution
E° (ArO•/ArO-)
(V vs NHE)
compd
pKa (H2O
λ
max ArO• (nm)
reference substance
K
(kJ mol-1
)
2a
2b
2c
2d
10.6
10.0
9.9
450
560
630
800
2b
1
7.86
1
0.49
0.49
0.49
0.52
340
337
336
337
4-MeOC6H4OH
2a
4-MeOC6H4OH
9.5
2.53
the equilibrium concentrations of the two redox couples.12 The
one-electron reduction potential of interest is then calculated
from the equilibrium constant and the one-electron reduction
potential of the redox reference couple using Nernst’s equation
(∆E° ) 0.0591 log K).
The phenolic O-H bond dissociation enthalpies given in
Table 1 were calculated from eq 2, which is derived from Hess’
law (C ) 232 kJ mol-1 for phenols).13,14
Table 2. Oxidation Potentials as Determined by Cyclic
Voltammetry (Ep and E1/2) and Pulse Radiolysis (E°)
compd
E (V vs Fc)
E (V vs NHE)
solvent
2a
2b
2c
2d
Ph-O-Me
Ph-S-Me
Ph-Se-Me
Ph-Te-Me
PhOH
0.66a
0.66b
0.44b
0.05b
-
1.35a
MeCN
MeCN
MeCN
MeCN
H2O
H2O
H2O
H2O
H2O
H2O
1.35b
1.13b
0.74b
1.62c (from ref 18)
1.45c (from ref 19)
1.09c (from ref 19)
0.74c (from ref 19)
1.5c (from ref 13)
∼1.3c (from ref 13)
-
-
BDE (O-H) ) 96.48E° + 5.70pKa + C
(2)
-
-
As can be seen in Table 1, the pKa values and the one-electron
reduction potentials do not differ dramatically in the series of
compounds 2. Consequently, the O-H bond dissociation
enthalpies are also essentially independent of the chalcogen
(336-340 kJ mol-1) and significantly higher than that reported
for R-tocopherol (323 kJ mol-1).15 Interestingly, the experi-
mentally determined O-H bond dissociation enthalpy for
compound 2a (relative to phenol) is identical to the correspond-
ing relative value determined from DFT calculations by Wright
et al.16
The large differences in the absorption maxima of the aryloxyl
radicals corresponding to compounds 2a-d (Table 1) can
probably be attributed to the involvement of heavier atoms in
the delocalization of the radical, resulting in a red-shift. This
does not imply that the radical is more delocalized for heavier
substituents since this would result in considerably lower one-
electron reduction potentials. For the same reason, λmax for 4-I-
C6H4O• is considerably red-shifted as compared to other
phenoxyl radicals, for example, 4-Cl-C6H4O• and 4-Br-C6H4O•;
however, the one-electron reduction potentials are identical for
these three radicals.13
4-MeOC6H4OH
-
a Ep. E1/2
.
c E°.
b
σ- ) 0.06)17 we can conclude that the S-containing aryloxyl
radical should have a somewhat higher one-electron reduction
potential than the O-containing one and that the S-containing
phenol should be somewhat more acidic than the O-containing
one. Whereas the reduction potentials are identical for the O-
and S-containing aryloxyl radicals, there is a significant differ-
ence in the phenolic pKa values, pointing in the direction
expected from the substituent constants of -OCH3 and -SCH3.
The substituent constants for -SeCH3 and -TeCH3 are not
known, but it is reasonable to assume that σ+ and σ- will
increase somewhat with atomic number of the chalcogen. This
is also reflected in the experimental data.
The oxidation potentials determined by cyclic voltammetry
in acetonitrile are given in Table 2 along with the data for some
related compounds. It should be noted that, whereas the
oxidation potential for compound 2a is irreversible (peak
potential, Ep) and the one for compound 2b is quasireversible,
the potentials for compounds 2c and 2d are fully reversible.
The potentials given for the other compounds in Table 2 are
thermodynamically derived potentials.
If we compare the oxidation potentials of compounds 2a-
2d (recorded in acetonitrile) with those for the corresponding
aryl methyl chalcogenides (recorded in water), assuming the
solvent effect can be neglected,20 some interesting conclusions
can be drawn (Figure 1). For the selenium- and tellurium-
containing compounds 2c and 2d, the oxidation potentials are
essentially identical to those recorded for Ph-Se-Me and Ph-
Te-Me, respectively. For the O- and S-containing analogues 2a
and 2b, the potentials are lower than those recorded for Ph-O-
Me and Ph-S-Me, respectively. The oxidation potentials for
compounds 2a and 2b are approaching the value recorded for
4-MeOC6H4OH, though. Consequently, from a redox point of
view, these compounds behave like phenols while the Se- and
Te-containing compounds behave like methyl phenyl chalco-
genides. In the latter case, the heteroatom is likely to be the
redox center.
It has previously been found that the one-electron reduction
potentials of substituted phenoxyl radicals are linearly dependent
on the Brown substituent constant, σ+(eq 3).13
+
E° ) 0.82 + 0.38 σp
(3)
On the other hand, the phenolic pKa’s are linearly dependent
on σ-(eq 4).17
-
pKa ) 10.0 - σp
(4)
Unfortunately, substituent constants for chalcogens contained
in a five-membered ring are not available, and it is therefore
not possible to make direct quantitative comparisons with the
linear relationships derived for substituted phenols and phenoxyl
radicals. However, if we compare the substituent constants for
-OCH3 (σ+ ) -0.78, σ- ) -0.26)17 and -SCH3 (σ+ ) -0.60,
(12) Wardman, P. J. Phys. Chem. Ref. Data 1989, 18, 1637.
(13) Lind, J.; Shen, X.; Eriksen, T. E.; Mere´nyi, G. J. Am. Chem. Soc.
1990, 112, 479.
With the notable exception of strongly electron-donating
substituents, the one-electron reduction potentials of 4-substi-
(14) If we adopt 365.3 kJ mol-1 as the correct O-H bond dissociation
energy for phenol, the constant C is 232 kJ mol-1
.
(18) Jonsson, M.; Lind, J.; Reitberger, T.; Eriksen, T. E.; Mere´nyi, G. J.
Phys. Chem. 1993, 97, 11278.
(19) Jonsson, M.; Lind, J.; Mere´nyi, G.; Eriksen, T. E. J. Chem. Soc.,
Perkin Trans. 2 1995, 67.
(20) Jonsson, M.; Houmam, A.; Jocys, G.; Wayner, D. D. M. J. Chem.
Soc., Perkin Trans. 2 1999, 425.
(15) Wayner, D. D. M.; Lusztyk, E.; Ingold, K. U.; Mulder, P. J. Org.
Chem. 1996, 61, 6430.
(16) Wright, J. S.; Carpenter, D. J.; McKay, D. J.; Ingold, K. U. J. Am.
Chem. Soc. 1997, 119, 4245.
(17) Hansch, C.; Leo, A.; Taft, R. W. Chem. ReV. 1991, 91, 165

Antioxidant Profile of 2,3-Dihydrobenzo[b]furan-5-ol
J. Am. Chem. Soc., Vol. 123, No. 15, 2001 3437
luride (8). For comparison, Rinh and Tinh values for all
compounds were also recorded in the absence of thiol-reducing
agent in the aqueous phase. It is clear from the inhibited rates
of peroxidation in the absence of N-acetylcysteine that none of
the synthetic compounds are as good as R-tocopherol as an
antioxidant (Rinh ) 18 µM/h). In fact, organotellurium com-
pounds 2d and 8 do not inhibit peroxidation at all (Rinh ) 655
µM/h ) uninhibited rate), and the organoselenium compound
2c is a poor inhibitor (Rinh ) 113 µM/h). Probably, the
organotellurium compounds are rapidly deactivated (oxidized)
by residual hydroperoxide contained in the linoleic acid. It is
also noteworthy that introduction of methyl groups into the 3-
and aromatic positions (compound 7) significantly increases
antioxidant efficiency (Rinh ) 35 µM/h) as compared with the
parent (compound 2b). Under conditions where selenoxides and
telluroxides are reduced by a thiol-reducing agent in the aqueous
phase, the antioxidant efficiency increases in the series of
compounds 2 as one traverses the periodic table from oxygen
to tellurium (Rinh ) 79, 64, 64, and 30 µM/h, respectively, for
compounds 2a-2d). Except for R-tocopherol and compound
7, all antioxidants investigated inhibit peroxidation for signifi-
cantly longer times when N-acetylcysteine is present in the
aqueous phase. This seems to indicate that the thiol is capable
of regenerating the phenol at the aqueous lipid interphase.
Obviously, this process is much less efficient with the two
sterically hindered ortho-disubstituted phenolic compounds.
Whereas the relative increase in inhibition time is only moderate
for the oxygen and sulfur analogues 2a and 2b, it is substantial
for organoselenium derivative 2c and for tellurides 2d and 8
(see Table 3). This could be indicative of an additional
mechanism of regeneration for the Se- and Te-containing
antioxidants. Whereas compounds 2a-b are likely to be
regenerated by H-atom donation to their respective phenoxyl
radicals, compounds 2c-2d may also be regenerated by
reduction of their corresponding 1-oxides. These species are
conceivably formed by hydroperoxide oxidation of the divalent
chalcogenides or by further oxidation/hydrolysis of hypervalent
chalcogen-centered radicals 9. In contrast to the less efficient
Figure 1. Oxidation potentials of compounds 2a-2d (y-axis) plotted
against the oxidation potentials of the corresponding methyl phenyl
chalcogenides (PhOMe, PhSMe, PhSeMe, PhTeMe; x-axis).
Table 3. Inhibited Rate of Peroxidation, Rinh, and Time of
Inhibition, Tinh, for Antioxidants Tested
a Rate of peroxidation during the inhibited phase (uninhibited rate
ca. 655 µM/h). b Duration of the inhibited phase of peroxidation.
tuted aryl alkyl chalcogenides were found to be insensitive to
the nature of the substituent.19 The rationale for this is that a
strongly electron-donating substituent is required for the charge
of the radical cation to become delocalized on the phenyl ring
(to become an aromatic radical cation) rather than on the
chalcogen. Since -O- is an extremely strong electron-donating
substituent, this also explains why compounds 2a-2d seem to
behave as ordinary phenolates in alkaline aqueous solution.
Inhibition of Lipid Peroxidation. Azo-initiated peroxidation
of linoleic acid or derivatives thereof has been often used for
studying the antioxidative properties of synthetic and natural
compounds.21 Some time ago we reported a two-phase variant
of this method which allows regeneration of the antioxidant by
a water soluble thiol.6 In the procedure used, linoleic acid and
the antioxidant to be evaluated were vigorously stirred in
chlorobenzene at 42 °C with an aqueous solution of N-
acetylcysteine. 2,2′-Azobis(2,4-dimethylvaleronitrile) was added
as an initiator in the organic phase and the progress of
peroxidation monitored by HPLC with UV detection of con-
jugated diene at 234 nm. For comparison of catalyst efficiency,
the inhibited rate of peroxidation, Rinh, was determined by least-
squares methods from absorbance/time plots. The progress of
peroxidation was followed for 300 min and the duration of the
inhibited phase, Tinh, determined graphically as the cross-point
for the inhibited and the uninhibited lines. Rinh and Tinh values
for compounds 2a-2d are shown in Table 3 together with those
recorded for R-tocopherol, 3,3,4,6,7-pentamethyl-2,3-dihy-
drobenzo[b]thiophene-5-ol (7) and bis(4-hydroxyphenyl) tel-
organotellurium antioxidant bis(4-hydroxyphenyl) telluride (8),6
Tinh for compound 2d could not be extended by increasing the
amount of thiol-reducing agent in the aqueous phase. This could
indicate that compound 2d (or its corresponding telluroxide) is
somehow destroyed under the conditions of the peroxidation
assay, for example via telluroxide syn elimination.
Reactivity toward tert-Butoxyl Radicals. Photodecompo-
sition of di-tert-butyl peroxide by 355-nm laser pulses yields
tert-butoxyl radicals within the duration of the pulse. This radical
is known to abstract phenolic hydrogen atoms rapidly in
comparison with decay by â-scisson.22 The resulting phenoxyl
radicals can be directly monitored by their spectral absorption.
Buildup of the signal follows pseudo-first-order kinetics (eq 5)
kobs ) k0 + k[ArOH]
(5)
where kobs is the observed rate constant and k the absolute
second-order rate constant for reaction of the tert-butoxyl radical
(21) a) Niki, E.; Saito, T.; Kawakami, A.; Kamiya, Y. J. Biol. Chem.
1984, 259, 4177. (b) Cosgrove, J. P.; Church, D. F.; Pryor, W. A. Lipids
1987, 22, 299. (c) Braughler, J. M.; Pregenzer, J. F. Free Rad. Biol. Med.
1989, 7, 125. (d) Pryor, W. A.; Cornicelli, J. A.; Devall, L. J.; Tait, B.;
Trivedi, B. K.; Witiak, D. T.; Wu, M. J. Org. Chem. 1993, 58, 3521.
(22) Das, P. K.; Encinas, M. V.; Steenken, S.; Scaiano, J. C. J. Am.
Chem. Soc. 1981, 103, 4162.

3438 J. Am. Chem. Soc., Vol. 123, No. 15, 2001
Malmstro¨m et al.
Table 4. Glutathione Peroxidase-Like Activity of Compounds 2
with Hydroperoxide Substrates
with the phenolic compound (eq 6).
NADPH consumptiona
Me3CO• + ArOH
9
k8 Me3COH + ArO•
(6)
catalyst
hydroperoxide
(µM/min)
% catalysisb
-
2a
2b
2c
2d
-
H2O2
6.3 ( 0.1
6.1 ( 0.3
6.0 ( 0.2
6.5 ( 0.3
624 ( 56c
1.1 ( 0.2
1.4 ( 0.2
365 ( 17
2.4 ( 0.4
3.7 ( 0.4
511 ( 26
-
97
H2O2
Absolute rate constants for reaction of compounds 2 with
the tert-butoxyl radical were measured in acetonitrile/di-tert-
butyl peroxide (10/1, v/v). Plots according to eq 5 with
monitoring at the λmax of the corresponding phenoxyl radical
(Table 1) gave the absolute rate constants k. Within experimental
error, the rate constant for the oxygen (2a) and sulfur (2b)
derivatives were the same: 2 × 108 M-1 s-1 (due to laser
photoexitation of compounds 2c and 2d, assessment of the
corresponding rate constants was impossible). For comparison,
R-tocopherol was found to react under the same conditions with
a rate of 6 × 108 M-1 s-1 and compound 7 with a rate of 4 ×
108 M-1 s-1. With the limited rate data at hand, it seems that
the reactivity of the tert-butoxyl radical toward the series of
compounds 2 and R-tocopherol is reflected by the phenolic O-H
bond dissociation enthalpies given in Table 1 and consistent
with a H-atom transfer mechanism.
H2O2
95
H2O2
103
9900
-
H2O2
t-BuOOH
t-BuOOH
t-BuOOH
CuOOHd
CuOOHd
CuOOHd
2c
2d
-
127
33300
-
2c
2d
156
21300
a Calculated over a 20 s period of stable decline of absorption at
340 nm. b The catalyst percentage increase of the basal reaction rate
between GSH and H2O2 was calculated as rate of NADPH consumption
+ 5 mol % catalyst (µM/min)/rate of NADPH consumption + vehicle
(µM/min) × 100. c The following values [µM NADPH/min (%
catalysis)] were recorded after repeated additions of 250 µM NADPH,
250 µM H2O2 and 500 µM GSH: 628.1 (9968), 640.2 (10161), 635.3
(10079), 622.8 (9885), 612.9 (9728). d The incubation contained 20%
DMSO.
Hydroperoxide-Decomposing Capacity. Hydroperoxide de-
composition is maybe the most important duty for preventive
antioxidants. In biological systems, this task is fulfilled by the
Se-containing glutathione peroxidases and catalase. Third-row
or higher divalent organochalcogen compounds react with
hydrogen peroxide or organic hydroperoxides to form the
corresponding oxides. Reactivity toward hydroperoxides usually
increases as one traverses the group of chalcogens (sulfides <
selenides < tellurides). Regeneration of the divalent organoch-
alcogen compounds from their oxides by reducing agents also
occur in a similar order. Thus, telluroxides and selenoxides are
far more reactive than sulfoxides toward mild reducing agents
such as thiols or ascorbate. For obtaining compounds that could
decompose hydroperoxides in a catalytic fashion in the presence
of thiols (thiol peroxidase activity), it seems that incorporation
of selenium and tellurium rather than sulfur into the catalyst is
likely to be successful. Thiol peroxidase activity can be
conveniently assessed by using the coupled reductase method.23
In this assay, hydroperoxide, glutathione, and the catalyst to be
evaluated are allowed to react at pH 7.4 in the presence of
glutathione reductase and NADPH. As soon as glutathione is
oxidized to the corresponding disulfide, it will be enzymatically
reduced. The reaction can be conveniently followed spectro-
photometrically by observing the consumption of NADPH at
340 nm. The results with compounds 2 are shown in Table 4.
Not unexpectedly, the oxygen and sulfur derivatives 2a and 2b
were essentially void of any glutathione peroxidase activity.
Catalyst activity (% catalysis) as determined by the initial rate
increase (as compared with the uncatalyzed process) in the
reaction between glutathione and hydroperoxide was also
insignificant for the organoselenium derivative. In contrast, the
organotellurium compound 2d was a highly active catalyst.
When hydrogen peroxide was used as the oxidant, NADPH was
consumed almost 100 times faster than in the control experiment.
To be sure that the organotellurium compounds act in a truely
catalytic fashion under the conditions used, more NADPH,
hydrogen peroxide, and glutathione were added to the incuba-
tion, and the consumption of NADPH was recorded again. After
five repeated additions/recordings, no significant change in
catalyst efficiency was seen. When tert-butyl hydroperoxide and
cumene hydroperoxide were used as oxidants, the reaction was
Figure 2. Inhibition of TBARS formation in Fe(II)/ADP/ascorbate-
treated microsomes as a function of the concentration of compounds
2. For compound 2d, see inset.
accelerated even more (333 and 213 times, respectively, as
compared to the uncatalyzed reaction; Table 4).
The exceptional (as compared to the other chalcogen deriva-
tives) nucleophilicity of compound 2d toward hydroperoxide
substrates is also reflected in its substantially lower oxidation
potential (see Table 2).
Lipid Peroxidation in Microsomes. Microsomal fractions
prepared from animal tissue undergo lipid peroxidation when
incubated with Fe(II) salts or Fe(III) salts plus ascorbate or plus
NADPH.24 During peroxidation, cytochromes b5 and P450 are
attacked, and the heme groups are degraded. The progress of
peroxidation can be conveniently followed by assessment of
thiobarbituric acid-reactive substances (TBARS).25 Compounds
2a-2d were assessed for their capacity to inhibit lipid peroxi-
dation in liver microsomes stimulated by Fe(II)/ADP/ascorbate.
Whereas the oxygen, sulfur, and selenium compounds showed
weak inhibiting activity (IC50 values of ∼250, 25, and 13 µM,
respectively), the organotellurium compound 2d was a potent
inhibitor with an IC50 value of 0.13 µM. The concentration
dependency of the antioxidant activity of compounds 2a-2d
are shown in Figure 2. The outstanding performance of the
organotellurium compound 2d seems to indicate that peroxide-
decomposing capacity may be more important than chain-
breaking ability to inhibit stimulated lipid peroxidation in
(24) Sevanian, A.; Nordenbrand, K.; Kim, E.; Ernster, L.; Hochstein, P.
Free Rad. Biol. Med. 1990, 8, 145.
(25) Greenwald, R. A. Ed. CRC Handbook of Methods for Oxygen
Radical Research; CRC Press: Boca Raton, 1985.
(23) Cotgreave, I. A.; Molde´us, P.; Brattsand, R.; Hallberg, A.; Ander-
sson, C.-M.; Engman, L. Biochem. Pharmacol. 1992, 43, 793 and references
therein.

Antioxidant Profile of 2,3-Dihydrobenzo[b]furan-5-ol
J. Am. Chem. Soc., Vol. 123, No. 15, 2001 3439
used in the next step without further purification. 1H NMR δ 0.19 (6H,
s), 0.97 (9H, s), 1.26 (3H, t, J ) 7.1 Hz), 3.70 (2H, s), 4.18 (2H, q, J
) 7.1 Hz), 6.63 (1H, dd, J ) 8.6, 2.9 Hz), 6.79 (1H, d, J ) 2.9 Hz),
7.38 (1H, d, J ) 8.6 Hz). 13C NMR δ -4.5, 14.2, 18.2, 25.6, 41.8,
61.0, 116.1, 120.6, 123.2, 133.2, 135.2, 155.0, 170.4. Anal. Calcd for
C16H25BrO3Si: C, 51.47; H, 6.75. Found: C, 51.39; H, 6.68.
microsomes under the conditions used. Also, the stimulants
ascorbatesmay serve to amplify the protective effect by
continuously regenerating the organotellurium and organosele-
nium compounds from their corresponding oxides.
Conclusions
4-Bromo-3-(2-hydroxyethyl)phenyl tert-Butyldimethylsilyl Ether
(4). To a solution of compound 3 (3.15 g, 8.41 mmol) in dry diethyl
ether (95 mL) was added LiAlH4 (0.390 g, 10.3 mmol) in one portion
at -20 °C under an atmosphere of dry nitrogen. After 70 min of stirring
at 0 °C, the reaction was quenched by addition of aqueous HCl (1 M),
and the organic layer was separated. The aqueous layer was extracted
with diethyl ether (3×), and the combined organic phases were washed
with water and brine. The organic phase was dried (MgSO4), filtered,
and concentrated under reduced pressure to give the title compound
(2.59 g, 93%) as a pale yellow oil, which was used in the next step
without further purification. 1H NMR δ 0.19 (6H, s), 0.97 (9H, s), 1.41
(1H, br s), 2.95 (2H, t, J ) 6.7 Hz), 3.86 (2H, t, J ) 6.8 Hz), 6.60
(1H, dd, J ) 8.6, 2.9 Hz), 6.77 (1H, d, J ) 2.9 Hz), 7.37 (1H, d, J )
8.6 Hz). 13C NMR δ -4.4, 18.2, 25.6, 39.4, 62.1, 115.8, 120.0, 123.0,
133.4, 138.7, 155.0.
4-Bromo-3-[2-(phenylthio)ethyl]phenyl tert-Butyldimethylsilyl Ether
(5). To a solution of compound 4 (1.76 g, 5.33 mmol) in dry benzene
(45 mL) was added diphenyl disulfide (1.28 g, 5.86 mmol) and
tributylphosphine (1.45 mL, 5.86 mmol) under an atmosphere of dry
nitrogen. The reaction mixture was then stirred at ambient temperature
for 8 h. NaHCO3 (5% aq) was added, and the layers were separated.
The aqueous phase was extracted with diethyl ether (3×). The combined
organic phases were washed with NaHCO3 (5% aq), water, and brine.
After drying (MgSO4), filtration, and concentration in vacuo, the residue
was purified by flash chromatography (pentane:EtOAc, 99:1) to furnish
1.71 g (76%) of the title compound as a colorless oil. 1H NMR δ 0.19,
(6H, s), 0.97 (9H, s), 2.97 (2H, m), 3.15 (2H, m), 6.59 (1H, dd, J )
8.6, 2.9 Hz), 6.71 (1H, d, J ) 2.9 Hz), 7.19 (1H, m), 7.28-7.40 (5H,
m). 13C NMR δ -4.4, 18.2, 25.6, 33.2, 36.3, 115.4, 120.1, 122.6, 126.0,
128.9, 129.3, 133.4, 136.1, 140.3, 155.1. Anal. Calcd for C20H27-
BrOSSi: C, 56.72; H, 6.43. Found: C, 56.77; H, 6.41.
From experimental pKa and redox data, the homolytic O-H
bond dissociation enthalpies of compounds 2a-2d were esti-
mated to be very similar (336-340 kJ mol-1). These values
were also reflected in the similar absolute rate constants (k )
2 × 108 M-1 s-1) for reaction of two of the compounds with
the tert-butyloxyl radical generated in laser flash photolysis
experiments in acetonitrile/di-tert-butyl peroxide. As judged by
the inhibited rates of peroxidation, Rinh, in a two-phase lipid
peroxidation system containing a thiol-reducing agent in the
aqueous phase, the antioxidant capacity increases as one
traverses the group of chalcogens (2d > 2c ) 2b > 2a). This
is probably because of facile regeneration of the organoselenium
and organotellurium compounds at the lipid aqueous interphase.
Thus, because of increasingly facile redox cycling, substitution
with heavier chalcogens in the series of compounds 2 set the
scene for a catalytic mode of action. Also, the introduction of
tellurium (compound 2d) imposes another antioxidative capacity
on the moleculesthe ability to catalytically decompose hydro-
peroxides in the presence of a stoichiometric reducing agent.
As compared with the other chalcogen analogues, compound
2d showed a far superior glutathione peroxidase-like behavior
and an outstanding ability to protect liver microsomes subjected
to stimulated lipid peroxidation. With the perspective to obtain
antioxidants with similarly good H-atom donating capacity as
R-tocopherol, but with a catalytic mode of action in the presence
of mild reducing agents and glutathione peroxidase-like activity,
we have recently embarked on the synthesis of the “real”
selenium and tellurium analogues of R-tocopherol.
5-(tert-Butyldimethylsilyloxy)-2,3-dihydrobenzo[b]thiophene (6).
To a solution of compound 5 (0.300 g, 0.708 mmol) in dry degassed
benzene (75 mL) under nitrogen was added AIBN (8.7 mg, 0.053
mmol). The reaction mixture was then heated to reflux, and tributyltin
hydride (230 µL, 0.850 mmol) was added. Heating was then continued
at reflux for 17 h, and another 1.2 equiv of tin hydride was added
together with another 0.075 equiv of AIBN. The reaction was then
refluxed for 10 h and another 0.075 equiv of AIBN was added, and
the reaction was refluxed for 17 h. According to 1H NMR, the starting
material was now almost consumed. After cooling of the flask, the
sovent was removed in vacuo. Purification of the crude material by
flash chromatography (pentane) afforded the title compound as a
colorless oil together with an inseparable impurity. The crude material
Experimental Section
Melting points are uncorrected. 1H NMR and 13C NMR spectra were
recorded in CDCl3 at 400 and 100 MHz, respectively. For proton spectra
the residual peak of CHCl3 was used as the internal reference (7.26
ppm), while the central peak of CDCl3 (77.0 ppm) was used as the
reference for carbon spectra. Silica gel was used for flash column
chromatography. 5-hydroxy-2,3-dihydrobenzofuran,10 3,3,4,6,7-pen-
tamethyl-2,3-dihydrobenzo[b]thiophene-5-ol,26 bis(4-hydroxyphenyl)-
telluride,27 ethyl 3-(tert-butyldimethylsilyloxy)phenyl acetate, 5-hydroxy-
2,3-dihydrobenzo[b]selenophene, and 5-hydroxy-2,3-dihydrobenzo-
[b]tellurophene were prepared according to literature procedures.9
Tetrahydrofuran was distilled under nitrogen from sodium/benzophe-
none. Benzene and dichloromethane were distilled under nitrogen from
calcium hydride. Elemental analyses were performed by Analytical
Laboratories, Lindlar, Germany.
Ethyl 6-Bromo-3-(tert-butyldimethylsilyloxy)phenyl Acetate (3).
A solution of bromine (525 µL, 10.2 mmol) in acetic acid (30 mL)
was added dropwise to a magnetically stirred solution of ethyl 3-(tert-
butyldimethylsilyloxy)phenyl acetate (3.00 g, 10.2 mmol) and potassium
acetate (1.00 g, 10.2 mmol) in acetic acid (60 mL) maintained at 15
°C. The reaction was then stirred at 15-17 °C for 2.5 h. After filtration
of salt, water and diethyl ether were added, and the organic layer was
separated. The aqueous phase was then extracted with diethyl ether
(3×). The combined organic phases were washed with saturated
NaHCO3 solution (6×), water, and brine. The organic phase was then
dried (MgSO4), filtered, and concentrated under reduced pressure to
give the title compound (3.69 g, 97%) as a colorless oil, which was
1
was subjected to deprotection. H NMR δ 0.17 (6H, s), 0.97 (9H, s),
3.21 (2H, m), 3.35 (2H, m), 6.61 (1H, m), 6.70 (1H, m), 7.02 (1H, m).
13C NMR δ -4.5, 18.2, 25.7, 33.9, 36.5, 116.8, 119.0, 122.3, 132.8,
141.6, 153.0.
5-Hydroxy-2,3-dihydrobenzo[b]thiophene (2b). To a solution of
compound 6 (165 mg, 0.617 mmol) in dry THF (20 mL) was added
tetra-n-butylammonium fluoride (650 µL, 1.0 M in THF, 0.648 mmol)
under an atmosphere of dry nitrogen. The reaction mixture was then
stirred at ambient temperature for 1 h. Water was added, and the mixture
was extracted with diethyl ether (3×). The combined organic phases
were washed with water and brine and dried over MgSO4, and the
solvent was removed in vacuo. Flash chromatography (EtOAc/pentane
10:90) afforded the title compound (67 mg, 62% from compound 3)
as white crystals; mp 86-88 °C (lit. 87-89 °C).11b 13C NMR δ 33.8,
36.4, 112.3, 114.4, 122.6, 132.3, 141.9, 153.0.
pKa Values. The pKa’s of the phenols were determined by recording
the UV-vis spectra at a number of different pH values. The spectral
differences between the phenols and the corresponding phenolates were
used to generate pKa plots from which the pKa could be determined.
Pulse Radiolysis. Radiolysis of water results in the formation of
(26) Malmstro¨m, J.; Gupta, V.; Engman, L. J. Org. Chem. 1998, 63,
3318.
(27) Kanda, T.; Engman, L.; Cotgreave, I. A.; Powis, G. J. Org. Chem.
1999, 64, 8161.
OH•, eaq-, H2O2, H2, and H3O+, with OH• and eaq being the major
-

3440 J. Am. Chem. Soc., Vol. 123, No. 15, 2001
Malmstro¨m et al.
radical species with primary radiation chemical yields of 0.28 µmol/J
each above pH 3. N2O-saturated solutions were used throughout in order
to convert the reducing solvated electron into the oxidizing hydroxyl
radical (GOH ) 5.6 × 10-7 mol/J).28 Millipore Milli-Q filtered water
was used throughout. The pulse radiolysis equipment consists of a linear
accelerator delivering 3-MeV electrons and a computerized optical
detection system.29 The pulses were of 5-10 ns duration, giving doses
of 3-6 Gy. For dosimetry a N2O-saturated 10-2 KSCN solution was
used.30 The Gꢀ value of (SCN)2•- was taken to be 4.78 × 10-4 m2/J at
500 nm.
frequency tripled fundamental at 355 nm from a YAG-laser (Quantel
Brilliant) was used. The pulses had a duration of ∼6 ns and an energy
of ∼200 mJ/pulse. The pulses were defocused to somewhat reduce the
excitation intensity. A cross-beam overlap with the analyzing light (150
W pulsed Xe-lamp) was obtained using a cylendrical lens. The time
resolution of the instrument was <10 ns. tert-Butoxyl radicals were
generated by 355 nm LFP of di-tert-butyl peroxide in acetonitrile (1/
10, v/v) in the presence of the H-atom donating substrate. The reaction
was followed by monitoring the growth in the absorption of the
respective aryloxyl radical at the appropriate wavelength (440 nm for
R-tocopherol; for the other compounds see Table 1). Pseudo-first-order
rate constants (kobs) were determined at 298 °C using digitally averaged
growth curves from five to 10 laser flashes. Absolute second-order rate
constants (k) were calculated by least-squares fitting of kobs vs [ArOH]
for five different ArOH concentrations (0.3-5 mM).
Coupled Reductase Assay. The glutathione peroxidase-like activity
of the compounds under study was assessed by their ability to catalyze
the reaction between hydroperoxides and glutathione in an aqueous
buffer at physiological pH. The oxidation of GSH to GSSG was
measured indirectly by spectrophotometrically assessing the stimulated
oxidation of NADPH in the presence of glutathione reductase. Incuba-
tions were conducted at room temperature in a Shimadzu model 160
spectrophotometer recording at 340 nm with air as a reference. They
were constructed in the following manner: Incubations in quartz
cuvettes were with 50 mM potassium phosphate buffer pH 7.4
containing 20% DMSO where necessary (1 mL). Additions were made
in the order (all final concentrations): NADPH (250 µM), GSH (1
mM), test substance (50 µM), record baseline, GSSG reductase (1 unit),
record, hydroperoxide (1 mM), record the decline in absorbance. Rate
assessments were performed when the decline in absorbance was
constant for at least 20 s. Control experiments revealed that the observed
catalytic action of the compounds was not influenced by increasing
amounts of GSSG reductase (0.5, 1.0 and 2.0 units) in the incubation.
Controls also showed that none of the compounds directly interacted
with the reduction of GSSG (250 and 500µM) by the reductase.
Lipid Peroxidation in Microsomes. The livers of male Sprague-
Dawley rats were exsanguinated, excised, and homogenized in ice-
cold sucrose (250 mM)/phosphate (50 mM) buffer, pH 7.4, using a
polytron. The homogenate was centrifuged once at 12000g, at 4 °C
for 60 min. The pellets were resuspended and washed twice with 150
mM KCl before being used in the experiments. Microsome preparations
were always freshly prepared.
Cyclic Voltammetry. Cyclic voltammetry was performed with a
PAR 263A Potentiostat/Galvanostat interfaced to a base PC using the
EG&G model 270 software package. The cell was a standard three-
electrode setup using a 3 mm diameter glassy carbon working electrode,
a platinum coil counter electrode, and a calomel reference electrode.
Acetonitrile was of the purest grade available, and the supporting
electrolyte, 0.1 M tetrabutylammonium perchlorate (TBAP), was
recrystallized from 10% hexane in ethyl acetate. The scan rate was
500 mV/s, and full IR compensation was employed in all measurements.
Peroxidation Assay. An HPLC (Waters 600 E) equipped with an
autoinjector (5 µL samples were injected every 10 min) with a sample
holder at 42.0 °C (Gilson 233 XL with thermostated sample rack),
Photodiode Array Detector (Waters 996), and a Millenium 32 chro-
matography data system was used for the peroxidation studies. In a
typical experiment linoleic acid in chlorobenzene (7.5 mL, 36.2 mM)
was stirred (1100 rpm) in a 20 mL thermostated reaction vessel. To
this solution the inhibitor in ethanol (107 µL, 3.0 mM; 40 µM final
concentration) was added by syringe followed by an aqueous thermo-
stated solution of NAC (8.0 mL, 1.0 mM). After 25 min of stirring/
equilibration, a thermostated solution of AMVN in chlorobenzene (0.5
mL, 22.4 mM) was added. Samples were withdrawn (after interruption
of the stirring and phase separation during 30 s) from the lower
chlorobenzene layer and injected onto a Waters Resolve Silica 90 Å
column (5 µM, 3.9 × 150 mm) eluted with hexane/ethanol (90/10) at
a flow rate of 1.0 mL/min. After sampling, stirring was immediately
resumed. The formation of conjugated dienes (retention time 2.8-3.2
min) was monitored at 234 nm and the concentration determined by
integration using an experimentally determined response factor (a
weighted amount of linoleic acid containing a trace of linoleic acid
hydroperoxide was allowed to react in CDCl3 with bis[4-(dimethy-
lamino)phenyl]telluride, and the conversion to the corresponding
1
telluroxide was determined by integration in the H NMR spectrum).
Microsomal lipid peroxidation was performed in incubations con-
structed as follows: incubations (1 mL) in phosphate buffer (50 mM),
pH 7.4, containing microsomal protein (1 mg), ADP (200 µM), FeSO4
(1 mM), and DMSO vehicle/test substance were preincubated for 5
min at 37 °C before addition of the initiation stimulus ascorbate (50
µM). For screening experiments, the accumulation of thiobarbituric acid
reactive substances over 30 min of incubation in antioxidant-treated
samples was compared to control levels in microsomes treated with
DMSO vehicle only.32 The DMSO concentration of the incubations
never exceeded 0.5% (v/v). Aliquots, (0.5 mL) of incubation were mixed
with equal amounts of trichloroacetic acid (10% aq) containing 10 mM
butylated hydroxytoluene and then reacted with thiobarbituric acid at
95 °C for 15 min. The samples were then centrifuged (1000g, 5 min),
and the absorbance was determined at 535 nm. The concentration of
The inhibited rate of peroxidation, Rinh (Table 3), was calculated by
least-squares methods from plots of linoleic acid hydroperoxide
concentration versus time during the inhibited phase of peroxidation.
In a blank experiment, in the absence of inhibitor, the uninhibited rate
of peroxidation was ∼655 µM/h. Tinh values were determined graphi-
cally as the cross-point for the inhibited and the uninhibited lines.
Linoleic acid hydroperoxide concentration increased in a linear fashion
for 0 < t < ∼0.75 Tinh. Chain lengths, νt, in the beginning of the
inhibited reaction (t ) 0.1 × Tinh) were calculated as described in the
literature1b assuming n ) 2 for all antioxidants:
Antioxidant nr; νt in the presence of NAC (νt in the absence of
NAC): 2a; 2.6 (1.7); 2b; 2.7 (1.0), 2c; 4.4 (1.3), 2d; 0.42 (-),
R-tocopherol; 0.40 (0.33), 7; 0.60 (0.53), 8; 2.2 (-).
In some of these experiments νt < 1. This means the inhibited aut-
oxidation is not a chain reaction. However, since the rate constant for
the scavenging of peroxyl radical with R-tocopherol calculated using
a similar procedure21c to the one described in this paper is in satisfactory
agreement with earlier rate determinations,21a we believe that the obser-
ved Rinh values reflect antioxidant activities of the compounds studied.
Laser Flash Photolysis. The laser flash photolysis (LFP) experi-
ments were carried out using a modification of the procedure described
in ref 31. Briefly, experiments were carried out using a LKS60 flash
photolysis spectrometer from Applied Photophysics. For excitation, the
malondialdehyde was determined using ꢀ ) 1.56 × 105 M-1 cm-1
.
Individual 50% inhibition concentrations (IC50 values) were calculated
from best-fit curves of the inhibitory effect as a function of concentra-
tion. Controls demonstrated that the compounds did not react with
thiobarbituric acid reactive compounds in the system.
Acknowledgment. Financial support from the Swedish
Council for Engineering Sciences, the Swedish Natural Science
Research Council, and the Swedish Medical Research Council
is gratefully acknowledged.
(28) Spinks, J. W. T.; Woods, R. J. Introduction to Radiation Chemistry,
Wiley: New York, 1990.
(29) Eriksen, T. E.; Lind, J.; Reitberger, T. Chem. Scr. 1976, 10, 5.
(30) Fielden, E. M. In The Study of Fast Processes and Transient Species
by Pulse Radiolysi; Baxendale, J. H., Busi, F., Eds.; NATO Advanced Study
Institutes Series; Reidel: Dordrecht, 1982; pp 49-62.
JA0035811
(31) Valgimigli, L.; Ingold, K. U.; Lusztyk, J. J. Am. Chem. Soc. 1996,
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