New promising antioxidants based on 2,6-dimethylphenol

Article abstract of DOI:10.1134/S106816200804016X

Three new sulfur-containing derivatives of 2,6-dimethylphenol were synthesized. Their antioxidative activity, mutagenicity, and genotoxicity were examined by bacterial tests and by calculating the dominant lethal mutations in murine embryonic cells. It was shown that all the compounds synthesized have a marked antioxidative effect and have no genotoxic and mutagenic properties. One of the antioxidants, 4-(3-dodecylthiopropyl)-2,6-dimethylphenol, increases the survival of cells of both the wild-type Escherichia coli strain and bacterial strains defective in the genes of repair enzymes and has a more distinct antioxidative effect than the classic antioxidants α-tocopherol and trolox, increasing the survival of cells devoid of repair enzymes.

Full text of DOI:10.1134/S106816200804016X

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2008, Vol. 34, No. 4, pp. 499–509. © Pleiades Publishing, Ltd., 2008.
Original Russian Text © E.A. Kemeleva, E.A. Vasyunina, O.I. Sinitsina, A.S. Khomchenko, M.A. Gross, N.V. Kandalintseva, A.E. Prosenko, G.A. Nevinskii, 2008, published in
Bioorganicheskaya Khimiya, 2008, Vol. 34, No. 4, pp. 558–569.
New Promising Antioxidants Based on 2,6-Dimethylphenol
E. A. Kemelevaa,b, E. A. Vasyuninaa, O. I. Sinitsinaa, A. S. Khomchenkoc, M. A. Grossc,
N. V. Kandalintsevac, A. E. Prosenkoc, and G. A. Nevinskiia,b,1
a Institute of Cytology and Genetics, Siberian Division, Russian Academy of Sciences,
pr. Akademika Lavrent’eva 8, Novosibirsk, 630090 Russia
b Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences,
Novosibirsk, 630090 Russia
c Novosibirsk State Pedagogical University, Novosibirsk, 630090 Russia
Received July 4, 2007; in final form, October 22, 2007
Abstract—Three new sulfur-containing derivatives of 2,6-dimethylphenol were synthesized. Their antioxida-
tive activity, mutagenicity, and genotoxicity were examined by bacterial tests and by calculating the dominant
lethal mutations in murine embryonic cells. It was shown that all the compounds synthesized have a marked
antioxidative effect and have no genotoxic and mutagenic properties. One of the antioxidants, 4-(3-dodecylth-
iopropyl)-2,6-dimethylphenol, increases the survival of cells of both the wild-type Escherichia coli strain and
bacterial strains defective in the genes of repair enzymes and has a more distinct antioxidative effect than the
classic antioxidants α-tocopherol and trolox, increasing the survival of cells devoid of repair enzymes.
Key words: oxidative stress, phenol antioxidants, sulfur-containing antioxidants, 4-thiaalkyl-2,6-dimethylphe-
nols
DOI: 10.1134/S106816200804016X
INTRODUCTION
cesses can also occur in the water and lipid phases
where the major role in protection against ROS is
played by low-molecular-weight antioxidants. In addi-
tion, in OS, antioxidant enzymes are often less effective
than low-molecular antioxidants. The reasons for this
are the fast inactivation of the constitutive pool of
enzymes in cells (especially after they enter the blood
flow) and a great deal of time required for the induction
of their synthesis. Therefore, freely migrating endoge-
nous and exogenous low-molecular-weight antioxi-
dants in high concentrations play an important role in
the protection against OS [17]. At present, a great num-
ber of natural and synthetic compounds possessing
antioxidative and anticarcinogenic properties have
been obtained [18, 19]. Phenol antioxidants [18, 20],
including spatially restrained phenols [20], are effec-
tive scavengers of radicals.
Aerobic respiration involves the stage-by-stage
four-electron reduction of molecular oxygen to water.2
The intermediates of molecular oxygen, O•2–, ç2é2,
and çé•, are ROS by themselves, and they also gener-
ate other ROS when interacting with cell components.
One of the main reasons for the development of OS in
the organism is the accumulation of ROS, which arise
from the disturbance of the balance between the pro-
cesses of generation of free radicals and the mecha-
nisms of their utilization [1–4]. ROS oxidize biological
membrane lipids [5] and destroy proteins and enzymes
[6, 7], cellular DNA and RNA [8], and other compo-
nents of living cells.
A gradual accumulation of mutations in the genome
of somatic cells [9, 10] leads to the development of age-
related pathologies, which is the main cause of prema-
ture aging, mutagenesis, carcinogenesis, and a great
number of pathologies of old age [11, 12]. An effective
mechanism of protection of cells against ROS is the
functioning of the complex of specialized systems of
enzymic and nonenzymic antioxidants [2, 13–16].
Enzymes are important first of all for the intracellular
defense against ROS. However, radical oxidative pro-
The studies of the last few years have shown the
specificity of the protective action of antioxidants to
various organs, tissues, and cells of the man, the differ-
ences in the mechanisms of their functioning, as well as
the synergism or antagonism of their effects [2, 13, 21–
23]. Thus, a study of the effect of the known antioxi-
dants: an extract of the bilberry (Vaccinium myrtillus)
and Adrusen zinco (a complex of vitamin E with zinc,
selene, and ω3-polyunsaturated fatty acids) on the level
of oxidized proteins, as well as enzymes having antiox-
idative functions (superoxide dismutase, catalase, and
glutathione peroxidase) in Wistar and OXYS rats
1
Corresponding author; phone: +7 (383) 335-6226; fax: +7 (383)
333-3677; e-mail: nevinsky@niboch.nsc.ru
Abbreviations: ROS, reactive oxygen species; OS, oxidative
2
stress.
499

500
KEMELEVA et al.
showed that these antioxidants affect differently these [29, 30]. The Ames test estimates the ability of com-
indicators in different organs and tissues and differ in pounds to induce mutations in specially designed
their effects on the components of nuclei, mitochon- strains of Salmonella typhimurium (Ames strains) [31].
dria, and cytosol [24]. The use of the bilberry extract It is expedient to analyze the lesions of DNA, cell mem-
provides a maximal protection of eye tissues against branes, and whole cells, as well as some organs
ROS and prevents the formation of cataract, whereas exposed to ROS in the presence and absence of antiox-
the effect of Adrusen zinco on cataract formation in idants [33, 34] using model animals [32]. The protec-
OXYS rats with genetically determined enhanced sen- tive properties of antioxidants are also studied using
sitivity to ROX is substantially weaker; at the same special lines of animals with genetically determined
time, however, it produces a more pronounced protec- enhanced sensitivity to OS [35–39].
tive effect on other organs [24]. This selectivity of
Here we described the synthesis of three novel sul-
action of the antioxidants can be related to the differ-
fur-containing compounds based on 2,6-dimethylphe-
ences in their structures and ability to penetrate into
nol and showed that the three compounds compare well
mitochondria, nuclei, and other compartments of living
in their characteristics and are even more promising in
cells. In view of this, the synthesis of novel antioxidants
some aspects than the previously described antioxi-
that combine in their molecules several active frag-
dants α-tocopherol and trolox.
ments differing in structure and the mechanism of anti-
oxidant action appears to be a promising task. It is
known that sulfur-containing derivatives of alkyl phe-
nols exhibit a high antioxidative activity in vitro and in
RESULTS AND DISCUSSION
vivo owing to the synergism of the antiradical activity
of the phenol fragment and the antiperoxide effect of
sulfur-containing functional groups [25–28].
Synthesis of novel antioxidants. We examined sul-
fur-containing phenol compounds F1–F3, obtained on
the basis of 2,6-dimethylphenol (scheme). Compounds
The primary screening of antioxidants is most often F1 and F2 were synthesized in one stage by condensing
performed using the tests based on the assessment of 2,6-dimethylphenol with formalin and the correspond-
survival of cells of wild-type E. coli strains or oxidative ing thiols, and sulfide F3 was obtained through the
lesions of DNA of genes defective in repair enzymes in intermediate synthesis of hydroxy- and bromosubsti-
the absence or presence of oxidizers and antioxidants tuted derivatives.
a
d
HO
Br
HO
OH
HO
HO
25%
90%
3
3
71%
e
c
94%
81%
b
HO
S
OH
HO
S
S
C12H25
C12H25
2
3
F-1
F-2
F-3
The following reagents and conditions were used: a, Allyl
alcohol/NaOH; b, aqueous H CO,
2
C
H SH/aqueous EtOH; d, concentrated HBr; e,
C
12 25
c,
H
SH/KOH, EtOH.
12 25
A scheme of synthesis of sulfur-containing derivatives
based on 2,6-dimethylphenol.
It was shown by an example of the oxidation of
model hydrocarbons that compounds F1–F3 are highly
reactive towards peroxide radicals (the rate constants
for the corresponding reactions are 1.5 × 105 å–1 s–1)
and inhibit hydrocarbon autoxidation more effectively
than phenol antioxidants containing no sulfide groups
[40, 41].
Analysis of the Mutagenicity of Compounds
by the Ames Test
The mutagenic activity of the compounds synthe-
sized was revealed by the Ames test using the S. typh-
imurium strain TA102. TA102 is a histidine-dependent
tester strain developed by Ames [42], which carries an
ocher-mutation at the histidine operon hisG 428.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 4 2008

NEW PROMISING ANTIOXIDANTS
501
TA102 cells contain about 30 copies of plasmid pAQ1
100
80
60
40
20
0
carrying the mutant allele of hisG 428 and seven to
eight copies of plasmid pKM101 (R-factor), which
increases the sensitivity of the strain to mutagens. The
genome of strain TA102 also contains mutations com-
mon in all Ames strains: rfa-mutation, which increases
the cell membrane permeability for large molecules,
and a deletion in the uvrB gene, which causes a destruc-
tion of the excision repair system, which further
increases the sensitivity of tester strains to some
mutagens. The use of strain TA102 makes it possible to
reveal the mutagenic activity of hydroperoxides, hydro-
gen peroxide, quinones, phenylhydrazine, various alde-
hydes and agents inducing the damage to thymine [31].
The test for mutation induction in the Ames assay is
performed by calculating the back mutations from aux-
otrophicity to prototrophicity at histidine, which arise
in response to various mutagenic substances [31].
Strain TA102 is commonly used both to assess the
mutagenicity of compounds by analyzing the number
of mutants formed by the action of a potential mutagen
[31] and to determine the antioxidant activity of antiox-
idants both from the inhibition of spontaneous
mutagenesis in this strain [43] and the decrease in the
mutagenicity of oxidants (most often hydrogen perox-
ide) [44].
1 2 3 4 5 6 7
1 2 3 4 5 6 7
Fig. 1. Analysis of the mutagenic activity of compounds
F1–F3 and control antioxidants at a concentration of 20
(open columns) and 80 nM (dashed columns in the absence
of hydrogen peroxide; (1) control untreated cells; cells
treated with (2) DMSO, (3) trolox solution, (4) α-toco-
pherol, and compounds (5) F1, (6) F2, and (7) F3 in DMSO.
Average values of three separate experiments at each con-
centration and the standard deviations of these values are
given. Maximum values of mutagenicity in the series of
these values obtained in experiments are taken to be 100%.
effects of H2O2, this oxidant is often used in experi-
ments to estimate the antioxidant properties of various
compounds [49–52].
It was shown that the addition to TA102 cells of only
the compounds dissolved in DMSO at a final concen-
tration of 20 nM leads to the statistically significant for-
mation of a greater number of His+-revertants than in
the case of their spontaneous formation in the control
with the cells of this strain not treated with DMSO
(Fig. 1). The addition to cells of DMSA alone at the
same concentration increases the frequency of forma-
tion of revertants, which becomes comparable with,
and statistically indistinguishable from that for the con-
trol antioxidants α-tocopherol and trolox added in the
same amount of DMSO. The effect of compound F1 on
the formation of mutants does not differ statistically
from the effect of α-tocopherol, and the efficiency of
the formation of mutants in the presence of compounds
F2 and F3 is only by about 2–3% higher (Fig. 1).
Increasing the concentration of the test and reference
compounds to 80 nM leads to a slight decrease in the
revertant formation efficiency, which becomes statisti-
cally indistinguishable from that of control cells treated
with DMSO (Fig. 1). Taken together, these data indi-
cate that the three novel compounds (F1–F3), similar to
the control antioxidants α-tocopherol and trolox, do not
produce any pronounced mutagenic effect and do not
decrease the level of spontaneous mutations (Fig. 1).
In the Ames test, the optimal concentration of
hydrogen peroxide at which it induces mutations but
has a relatively low toxicity and does not induce sub-
stantial cell death varies within the limits of 3 mM [48].
Solutions of the test compounds in DMSO were added
to TA102 cells in different concentrations, and H2O2
was added to a final concentration of 3 mM. Cells
treated with ç2é2 were used as a control, and the effect
of the solvent was accounted for by treating the cells
with hydrogen peroxide in the presence of DMSO
(Fig. 2).
It was shown that DMSO does not markedly affect
the mutagenic oxidative activity of ç2é2. Trolox at a
concentration of 20 nM had a maximum antioxidative
activity (100 1.3%), and the protective properties of
α-tocopherol (98 1.2%) and compound F1 (99.4
0.6%) were practically the same. The activity of com-
pounds F2 (84.1 0.4%) and F3 (84.9 1.5%) was
somewhat lower. At a concentration of 80 nM, trolox
retained the maximum activity (100 0.5%); the effi-
ciency of the protective action of compounds F1
(92.1 0.7%) and α-tocopherol (91.8 0.5%) slightly
decreased. However, the antioxidative properties of
The main premutational oxidative DNA lesions compounds F2 (96.2 0.6%) and F3 (98.1 0.7%)
induced by H2O2, the mechanisms of their repair and increased markedly and approached those of trolox.
transformation in the mutation, the induction of the Interestingly, water-soluble trolox was more reactive as
SOS-response by hydrogen peroxide, and the influence antioxidant in this system than water-insoluble α-toco-
of enzymatic and chemical systems of cellular antioxi- pherol (Fig. 2). Because these compounds are structur-
dant defense on the genotoxicity of H2O2 have been ally close and their mechanisms of action are similar,
described in the literature [45–48]. Because some anti- this difference in activity may be related to different
oxidants effectively reduce the toxic and mutagenic efficiency of their penetration into cells.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 4 2008

502
KEMELEVA et al.
100
80
60
40
20
0
1
2 3
4
5 6
7
1 2 3 4 5 6 7
Fig. 2. Analysis of the antioxidative activity of compounds F1–F3 and control antioxidants at a concentration of 20 (open columns)
and 80 nM (dashed columns) in the presence of 3 mM H O ; (1) control untreated cells; cells treated with (2) DMSO, (3) trolox
2
2
solution, (4) α-tocopherol, and compounds (5) F1, (6) F2, and (7) F3 in DMSO. Average values of three separate experiments at
each concentration and the standard deviations of these values are given. Maximum values of the antioxidative activity in the series
of these values obtained in experiments are taken to be 100%.
Of the three compounds synthesized, only F2 is and corrects them, using the N-glycosylase activity to
readilt soluble in water; F1 and F3 are lipophilic like remove adenine [57]. 8-OxodGua is formed in 8-oxoG · C
α-tocopherol. The protective effect of compounds F2 pairs both during DNA replication and as a result of
and F3 at high concentrations is stronger than that of
α-tocopherol. Thus, theAmes test showed that the three
novel compounds possess no mutagenic activity, and
their antioxidative effect is comparable with that of
α-tocopherol and trolox.
Analysis of the antioxidant activity of the com-
pounds using special E. coli strains. E. coli strains
cc104mutT and cc104mutMY, which are deficient in the
system of oxidative DNA damage repair [53, 54] were
used. The original E. coli strain cc104 with the entirely
intact repair system served as a control.
modification of guanine incorporated into DNA. The
generation of the 8-oxoG-oxidative DNA damage is
one of the main reasons for the appearance of various
mutations, in particular, GC
TA and AT
CG
transversions. If the base 8-oxoGua would not be
removed from DNA by the MutM protein, DNA-poly-
merase III could “put” adenine in the next replication
round opposite 8-oxoGua. The double mutation mutM-
mutY increases the frequency of spontaneous GC
TA transversions 20 times compared with the sum of
frequencies of transversions due to mutations in genes
mutY and mutM taken separately [58].
The strain cc104mutT carries a mutation of the gene
mutT, which encodes 8-oxo-2'-deoxyguanosine-5'-
triphosphate pyrophosphohydrolase (8-oxodGTPase or
MutT). The protein MutT is not an excision repair
enzyme. It has a nucleosidetriphosphate-pyrophospho-
hydrolase activity and prevents the incorporation of 8-
oxodGTP to the DNA from the nucleotide pool during
replication, by hydrolyzing it to 8-oxodGMP and pyro-
phosphate. The inactivation of the mutT gene gives rise
We studied the antioxidant activity of the com-
pounds by their ability to increase the survival of bacte-
rial cells treated with hydrogen peroxide. The strains
defective in genes mutT, mutM, and mutY exhibited a
higher sensitivity to 3 mM ç2é2 compared with the
original strain. A pretreatment of cells of all E. coli
strains studied with antioxidants increased their sur-
vival after the treatment with 3 mM ç2é2. The addition
of 3 mM ç2é2 together with DMSO in different
amounts in control experiments led to different survival
of E. coli cells of strains cc104, cc104mutT, and
ÒÒ104mutMY (Table 1). α-Tocopherol only at a high
concentration of 80 nM was effective in decreasing the
toxicity of ç2é2 (increased the survival of cells treated
with ç2é2) in the original E. coli strain cc104 as com-
pared with the control (Table 1). At a concentration of
almost exclusively to AT
CG transversions; in this
case, the frequency of mutT mutations in the strain is
considerably higher due to a greater amount of
8-oxoGua in the nucleotide pool [55].
The strain cc104mutMY carries two mutations: of
gene fpg encoding DNA-8-oxoguanineglycosylase
(MutM protein), which has a DNA-glycosylase, apu-
rine/apyrimidine lyase and 5'-endodeoxyribophos-
phatase activities [56], and of gene mutY, which 8 and 20 nM, it produced a weak antioxidative effect,
encodes adenine-DNA-glycosylase (MutY protein), and the cell survival was only insignificantly higher
which recognizes G · A- and 8-oxoG · A mismatches than in the control in the presence of ç2é2 in DMSO.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 4 2008

NEW PROMISING ANTIOXIDANTS
503
Table 1. Effect of antioxidants on the cell survival of E. coli strains cc104, cc104mutT, and cc104mutMY treated with 3 mM H2O2
Cell survival, relative units
Strain
cc104
C, nM
α-Tocopherol
Trolox
F1
F2
F3
DMSO
8
20
80
8
8.0 0.5
6.1 2.6
31.6 1.5
2.5 1.1
2.0 3.2
26.9 0.8
1.6 3.7
4.4 1.3
3.4 0.8
8.7 0.4
22.2 2.7
10.4 0.9
1.3 1.5
6.3 2.2
19.3 1.9
5.0 4.3
6.0 1.2
11.6 1.9
4.6 1.2
5.4 1.9
3.3 1.1
7.9 3.6
4.3 0.2
16.2 2.2
11.0 1.3
7.4 1.4
25.9 3.8
14.7 1.8
10.4 3.0
3.9 1.6
2.0 0.1
3.6 0.7
4.9 3.1
10.4 1.1
4.1 0.6
3.7 1.6
14.2 1.8
0.3 1.0
1.8 0.2
0.1 0.3
–6.8 1.1** –3.9 1.8
cc104mutT
0.4 0.7
22.8 3.8
9.1 1.5
–0.6 1.4
4.1 1.6
22.9 2.6
3.6 0.7
10.8 2.9
3.9 0.8
9.9 1.9
2.0 0.4
10.3 1.5
20
80
8
cc104mutMY
20
80
Notes: * Effect of antioxidants was assessed by the difference in cell survival in the presence and absence of the corresponding antioxidant.
The average values of cell number and standard deviations of these values (n = 3 for each concentration) are given in relative
units calculated using the program Image Pro Plus. The statistical processing of the data showed that all differences were statistically
significant (P ≤ 0.05). The values of the maximum protecting effects for each compound are shown in boldface type.
** Negative values indicate that the compounds, which exhibit a prooxidant effect.
In the case of the cc104mutT strain defective in idative effect of trolox in the case of the cc104mutT
8-oxoGTPase, the survival of cells in the presence of strain was the lowest compared with the effects of other
8 and 20 nM α-tocopherol was approximately 1.6– compounds. At the same time, trolox substantially
1.9 times lower than in the control; i. e., the compound stimulated the survival of very badly growing cells of
exhibited a prooxidant effect. Increasing the concentra- the cc104mutMY strain: the survival of these cells in the
tion of α-tocopherol to 80 nM significantly increased presence of trolox at any concentration increased 3.3–
the cell survival; with regard for the effect of DMSO, it 116 times compared with the control (ç2é2 + DMSO).
was only 1.7 times lower compared with the starting Thus, despite the structural similarity of the antioxidant
strain cc104.
groups of fat-soluble α-tocopherol and its water-solu-
ble analogue trolox, the antioxidative effects of these
compounds in the case of the wild-type E. coli strain
and the strains defective in repair enzymes substantially
differ.
The survival of ÒÒ104mutMY cells in the control
experiment (in the presence of ç2é2 and DMSO) was
low. Increasing the concentration of DMSO first led to
an increase and then to a decrease in cell growth
(Table 1). No prooxidant effect (a decrease in the
Of interest are the data obtained for compound F1.
amount of grown cells compared with the control) was This substance at low concentrations (8–20 nM) does
observed. The addition of α-tocopherol (Table 1) not protect the cells of the cc104 strain and produces the
increased the cell growth 2.4–5.3 times at a concentra- prooxidant effect at a concentration of 80 nM, substan-
tion of 8 and 20 nM and 34 times at a concentration of tially reducing the rate of cell growth compared with
80 nM; however, in general, the survival of cells was the control. It is noteworthy that F1 has the prooxidant
low compared with the values obtained with other anti- effect at low and high concentrations (8 and 80 nM) but
oxidants (Table 1, see below).
protects well the cells of the cc104mutT strain at a con-
centration of 20 nM, providing the survival 3.6 and
11.4 times higher than with trolox and α-tocopherol.
In the case of trolox, complicated concentration
dependences were observed. On going from 8 to 20 nM
concentration (with due regard for the control values at
A somewhat different situation occurs with F1 in
these concentrations), the protective effect in the case experiments with the cc104mutMY strain: a prooxidant
of the wild-type strain cc104 increased approximately effect at a concentration of 8 nM and an increase in cell
3.4 times, reaching a maximum; then, at a concentra- survival 2.3 and 229 times at concentrations of 20 and
tion of 80 nM, the cell survival fell to control values 80 nM, respectively, compared with the control. The
(Table 1). A similar complicated concentration depen- increase in the survival of the cc104mutMY strain in the
dence for trolox was observed with the cc104mutT presence of 80 nM F1 is 1.2 and 6.7 times higher com-
strain: the cell growth slowed down at a concentration pared with the effects of trolox and α-tocopherol,
of 8 nM (the prooxidant effect), and the protecting respectively (Table 1). Presumably, in normally func-
effect at a concentration of 80 nM was lower than in the tioning cells where repair enzymes effectively remove
case of α-tocopherol; however, at a concentration of oxidized bases, this compound at low concentrations
20 nM, the cell growth was more intensive than in the can mainly act as a prooxidant, whereas at high concen-
case of the fat-soluble analogue. In general, the antiox- trations and particularly upon the disturbance of oxida-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 4 2008

504
KEMELEVA et al.
Table 2. Lethality of embryos and the level of dominant lethal mutations in embryonic cells of mice
Potential Embryos per one pregnant female
Number
fertility
(number of
yellow
Total
death of
embryos
Com- Stage of sper- of preg-
pound matogenesis nant fe-
males
Survival, Dominant
χ2
number of
implanta-
tions
%
lethals, %
alive
dead
bodies)
F3
Mature
42
34
38
29
36
30
8.19 0.21 7.45 0.24 7.00 0.29 0.45
7.79 0.23 6.79 0.29 6.70 0.30 0.18
7.23 0.15 6.42 0.22 6.00 0.22 0.42
8.13 0.16 6.86 0.27 6.58 0.25 0.28
7.58 0.18 6.75 0.21 6.36 0.22 0.39
7.40 0.25 6.80 0.23 6.46 0.24 0.33
14.53
12.83
17.09
19.07
16.11
12.61
85.47
87.13
82.91
80.63
83.88
87.39
1.7
1.3
0.003
0.002
0.250
0.023
0.080
0.047
spermatozoa
Late
spermatids
Early
spermatids
12.7
4.3
α-Toco- Mature
pherol spermatozoa
Late
spermatids
7.5
Early
spermatids
6.1
tion-reduction processes, it effectively deactivates free
radicals.
Two of the best preparations widely used currently
in the therapy of OS, the antioxidants α-tocopherol and
trolox, have very complicated concentration–antioxi-
dant effect dependences and can exhibit the prooxidant
effect at some concentrations (Table 1). At the same
time, the data obtained suggest that compound F3 may
be a more promising antioxidant since it has no proox-
idant properties in a wide concentration range.
Compound F2 (Table 2) either had a moderate
prooxidant effect or did not exhibit any marked protec-
tive effect in the case of two strains (cc104 and
c104mutT) at all concentration; only at a concentration
of 20 nM it substantially increased the survival of the
strain c104mutT. At concentrations of 8 and 80 nM,
compound F2 was effective in protecting the
cc104mutMY cells.
Determination of Dominant Lethal Mutations
in Embryonic Murine Cells
We showed that compound F3 significantly
increases the survival of cells of all strains examined,
but the concentration dependences have a complicated
dose-dependent shape for each strain. Interestingly, as
distinct from other compounds, including α-tocopherol
and trolox, this compound does not produce the proox-
idant effect at all concentrations and with all of the
E. coli strains examined. The rate of growth of cc104
cells at a concentration of F3 of 20 nM was approxi-
mately twofold higher than in the presence of the other
compounds studied and only ~1.3 times lower than in
the presence of trolox. In the case of the cc104mutT
strain, compound F3 at a concentration of 20 nM was
12.5 times more effective in protecting cells than α-
tocopherol and 4 times more effective than trolox. The
efficiency of F3 was 2.5 times higher than that of com-
pound F2 and practically comparable with that of F1.
At a concentration of 8 nM, compound F3 was approx-
imately six times more efficient in protecting
cc104mutMY cells than α-tocopherol and about two
times more efficient than trolox.
The mutagenic effect of compound F3 in the mam-
malian organism was studied by estimating the level of
dominant lethal mutations in embryonic murine cells
[59]. A great part of dominant lethals causes the death
of embryos during, or shortly after the implantation. An
indicator of the level of dominant lethal mutations is the
level of postimplantation losses, which is expressed as
the ratio of dead embryos to the sum of alive and dead
embryos [60]. Compound F3 and α-tocopherol were
injected to animals at a concentration of 52 µM, which
a fortiori exceeds the therapeutic dose for these com-
pounds. It was shown that the potential fertility of
females of all groups used is similar (Table 2). The val-
ues of embryonic lethality at all stages of pregnancy of
females of the experimental group were comparable
with those of the control group. Consequently, F3 does
not affect either the pre- or the postimplantation stage
of embryogenesis. The frequency of occurrence of
dominant lethals in females mated with males treated
with F3 did not depend on the spermatogenesis stage
Thus, of all control and test compounds examined (Table 3). In experiments with linear animals, the sig-
by this assay system, compound F3 had the highest nificance of the increase in the level of postimplantation
antioxidant activity; it increased the survival of cells of lethality compared with the control is estimated using
both wild-type strain and the strains defective in the the χ2 criterion for the ratio of the sums of alive and
genes of repair enzymes.
dead embryos. With this approach, the individual sensi-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 4 2008

NEW PROMISING ANTIOXIDANTS
505
Table 3. Level of dominant lethal mutations in mice treated with compound F3, α-tocopherol, and cyclophosphane*
Groups of comparison
F3/control
Spermatogenesis stages
Dominant lethals, %
χ2
Mature spermatozoa
Late spermatids
1.7
1.3
0.003
0.002
Early spermatids
Mature spermatozoa
Late spermatids
12.7
4.3
0.250
α-Tocopherol/control
F3/α-tocopherol
0.023
7.5
0.080
Early spermatids
Mature spermatozoa
Late spermatids
6.1
0.047
6.0
0.055
6.0
0.065
Early spermatids
Mature spermatozoa
Late spermatids
7.0
0.080
Cyclophosphane/control
21.79
37.12
8.35
15.43**
40.54**
4.37***
Early spermatids
Notes: * The level of dominant lethal mutations was estimated from the value of postimplantation losses (see the Experimental section).
Control animals were given olive oil.
2
The significance of differences between groups was estimated by the χ -criterion.
** The significance of differences between experimental and control groups of animals at P < 0.01.
*** The significance of differences between experimental and control groups of animals at P < 0.05.
tivity of mice is ignored. The absence of significant dif- pherol, trolox (Sigma, United States); cyclophosphane
ferences in the level of dominant lethals in experimen- (OAO Biokhimik, Russia), yeast extract, sodium chlo-
tal and control groups of animals indicates that F3 pro- ride, glucose (Helicon, Russia), 2,6-dimethylphenol
duces no mutagenic effect (Table 3).
(Merck, Germany); 2-mercaptoethanol-1, dodecaneth-
iol-1 (Aldrich, United States); refined olive oil
(Greece), bacterial strains of E. Òoli: cc104, cc104mutT
and cc104mutMY from the collection of J. Miller
(France), S. typhimurium TA102 from the collection of
B. Ames (United States).
For comparison, Table 3 presents the frequency of
occurrence of dominant lethal mutations at different
stages of spermatogenesis in mice treated with the very
potent mutagen cyclophosphamide (cyclophosphane)
[61]. The injection to animals of cyclophosphane, as
opposed to F3 and α-tocopherol, induces intensive
Synthesis of antioxidants. Target compounds
mutagenesis at all stages of spermatogenesis. The per- F1−F3 were obtained from 2,6-dimethylphenol accord-
cent of dominant lethal mutations at the stage of mature ing to the scheme. 2,6-Dimethylphenol, 2-mercaptoet-
spermatozoa after administration of cyclophosphane is hanol-1, and dodecanethiol-1 were used as initial syn-
1
12 and 5 times higher than after the administration of thons. H NMR spectra (δ, ppm) were recorded on a
F3 and α-tocopherol, respectively. At the stage of late Bruker DRX500 spectrometer (Bruker, Germany) at an
spermatids, the mutagenicity of cyclophosphane is 28 operating frequency of 500.13 MHz in ëDël3 (refer-
times higher than that of F3 and 5 times higher than that ence ëHël3). Melting temperatures were determined in
of α-tocopherol. Thus, these data suggest that com- a capillary on a PTP instrument (PO Khimlaborpribor,
pound F3 has no mutagenic effect on the organism of Russia).
model mice.
Taken together, our data indicate that two novel anti-
oxidants F1 and F3 at optimal concentrations are com-
parable in properties with α-tocopherol and trolox, and
compound F3 may appear a more efficient and promis-
ing antioxidant in the treatment of diseases caused
by OS.
Dodecyl-3,5-dimethyl-4-hydroxybenzylsulfide (F1).
2,6-Dimethylphenol (50 g, 0.40 mol), dodecanethiol-1
(81 g, 0.40 mol), KOH (8.87 g, 0.12 mol), and ethanol
(90 ml) were heated to boiling in an atmosphere of inert
gas, and aqueous formalin (81 ml, 0.80 mol H2CO) was
added dropwise. The mixture was boiled for 2 h, neu-
tralized by hydrochloric acid, and treated with hot
water. The organic layer was separated, 250 ml of hot
(60°ë) water was added, and the solution was cooled
under vigorous stirring to room temperature. Precipi-
EXPERIMENTAL
The following preparations were used: trypton, tated crystals were sucked off, dried, and recrystallized
DMSO, potassium dihydrophosphate, sodium hydro- from petroleum ether.Yield of the target product: 129 g
phosphate, ammonium chloride, magnesium sulfate, (94%), mp 57–58°ë. 1ç NMR: 0.86 (3 H, t,
calcium chloride, thiamine (Amerso, United States); (ëH2)11åÂ); 1.24 (18 H, m, (ëH2)9åÂ); 1.54 (2 H, m,
agar, (BactoTMAgar, “BD” (France); H2O2, α-toco- CH2ë10ç21); 2.21 (6 H, s, ArMe), 2.40 (2 H, t,
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 4 2008

506
KEMELEVA et al.
CH2ë11ç23); 3.58 (2 H, s, ArCH2S); 4.54 (1 H, s, OH); acid, and treated with toluene. The extract was washed
6.89 (2 H, s, Ar). Found, %: C 75.03, H 10.92, S 9.32. with water, dried over Na2SO4, and the solvent was dis-
ë21ç36éS. Calculated, %: C 74.94, H 10.78, S 9.53.
tilled off. After the crystallization of the residue from
methanol 2.59 g (71%) of the target product was
2-Hydroxyethyl-(3,5-dimethyl-4-hydroxyben-
zyl)sulfide (F2). Aqueous formaldehyde (152 ml,
1.64 mol H2CO) was added dropwise in an atmosphere
of inert gas at 80°ë to a solution of 2,6-dimethylphenol
(100 g, 0.82 mol) and KOH (29.4 g, 0.41 mol) in 2-mer-
captoethanol (73.4 g, 0.94 mol) and stirred for 2 h at
80°ë. The mixture was cooled, neutralized by hydro-
chloric acid, and treated with benzene. The extract was
washed with hot water, cooled, and precipitated crys-
tals were sucked off, dried, and recrystallized from ben-
zene. Yield of the target product: 141 g (81%); mp
1
obtained; mp 50–51.5°ë. ç NMR: 0.89 (3 H, t,
(ëH2)11åÂ); 1.26 (16 H, m, (ëH2)8åÂ); 1.36 (2 H, m,
CH2ë9ç19); 1.54 (2H, m, CH2ë10ç21); 1.80 (2 H, m,
ArCH2CH2), 2.19 (6 H, s, ArMe), 2.40 (4 H, m, SCH2),
2.54 (2 H, t, ArCH2), 4.30 (1 H, s, OH), 6.71 (2 H, s,
Ar). Found, %: C 75.61, H 10.92, S 8.97. C23H40OS.
Calculated, %: C 75.76, H 11.06, S 8.79.
Determination of the mutagenicity of com-
pounds. In the Ames test, the histidine-dependent
strain of S. typhimurium TA102 was used, which carries
a mutation at the histidine operon [31]. The mutagenic
activity of samples was assayed by the standard method
without metabolic activation [31]. A night culture of
strain OA102 was obtained by a 16-h incubation at
37°ë in LB medium with ampicillin. Then cells were
seeded on plates with minimal glucose agar, antibiotics,
and histidine in such a way as to obtain isolated colo-
nies. A separate bacterial colony was added to LB
medium (5 ml) containing ampicillin (50 µg/ml) and
tetracycline (2 µg/ml) and incubated on a shaker
(130 rpm) for 15 h at 37°C [55].
The Ames test was carried out using the method of
double-layer agar as described in [31]. A night culture
of S. typhimurium strain TA102 (100 µl), one of the
three compounds at a final concentration of 20–80 nM
(in 5 µl of DMSO), and H2O2 to a final concentration of
3 mM were added at 42°C to 2 ml of melted 0.6% upper
agar. The mixture was stirred and seeded onto plates
with minimal agarized medium A containing 0.2% glu-
cose [55] and 3% agar, distributing the reaction mixture
uniformly on the surface of the solidified agar. The
plates were incubated for 48 h at 37°ë, and the number
of His+-revertants was estimated (see below). Cells
incubated with ç2é2 in the absence of antioxidants
were used as positive controls, and bacterial cells
grown in the absence of peroxide and antioxidants
served as a negative control. The number of His+-rever-
tants on the plate was counted after a 48-h incubation at
37°ë using the program Image Pro PlusV. 4.1 (MEDIA
CYBERNETICS). In determinations of the mutagenic-
ity, a maximum amount of revertants, and in determina-
tions of the antioxidant activity, a minimal amount of
revertants (the average of three independent experi-
ments) was taken to be 100%. The mutagenicity and the
antioxidant activity for the rest of compounds were cal-
culated relative to these values. The statistical process-
ing of the data was carried out using the Student t-test
(Statistica 5.0). Differences at P ≤ 0.05 were considered
to be significant.
1
63.5–65°ë. ç NMR: 2.20 (6 H, s, Me), 2.32 (1 H, s,
br, CH2OH), 2.63 (2 H, t, SCH2CH2), 3.59 (2 H, s,
ArCH2S); 3.66 (2 H, m, CH2OH), 4.95 (1 H, s, ArOH),
6.89 (2 H, s, Ar). Found, %: C 62.34, H 7.51, S 15.25.
ë11ç16é2S. Calculated, %: C 62.23, H 7.60, S 15.10.
3-(3,5-Dimethyl-4-hydroxyphenyl)propanol-1.
2,6-Dimethylphenol (7.48 g, 61.2 mmol), allyl alcohol
(16.8 ml, 0.25 mol), and finely ground NaOH (2.52 g,
61.2 mmol) were placed in an ampoule of heat-stable
glass. The ampoule was sealed and kept in a thermostat
equipped with a shaker for 5 h at 160°ë. After cooling,
the mixture was diluted with water, neutralized with
HCl, and treated with benzene. The extract was washed
with water and dried over Na2SO4, and the solvent was
distilled off. The residue was distilled in a vacuum to
yield 2.76 g (25%) of the target alkanol, mp 69–70°ë
(from a mixture of toluene and hexane); 1H NMR: 1.50
(1 H, s, br, CH2OH), 1.83 (2 H, m, ÄrCH2CH2); 2.20
(6 H, s, Me), 2.56 (2 H, t, ArCH2), 3.65 (2 H, t,
CH2OH), 4.47 (1 H, s, br, ArOH), 6.79 (2 H, s, Ar).
Found, %: C 73.15, H 8.88. ë11ç16é2. Calculated, %:
C 73.30, H 8.95.
1-Bromo-3-(3,5-dimethyl-4-hydroxyphenyl)pro-
pane. A mixture of 3-(3,5-dimethyl-4-hydroxyphe-
nyl)propanol-1 (20 g, 0.11 mol) and hydrobromic acid
(97 ml, 0.67 mol HBr) was boiled in an atmosphere of
inert gas for 6 h using a Dean–Stark attachment, cooled,
and treated with toluene. The extract was washed with
water, dried over Na2SO4, and the solvent was distilled
off. The residue was distilled in a vacuum to yield
24.3 g (90%) of the target bromo derivative, mp 34–
35°ë (from hexane); 1H NMR: 2.19 (2 H, m,
ÄrCH2CH2), 2.30 (6 H, s, Me), 2.71 (2 H, t, ArCH2),
3.46 (2 H, t, CH2Br), 4.68 (1 H, s, OH), 6.87 (2 H, s,
Ar). Found, %: C 54.25, H 6.13, Br 32.75. C11H15BrO.
Calculated, %: C 54.34, H 6.22, Br 32.86.
Dodecyl-3-(3,5-dimethyl-4-hydroxyphenyl)pro-
pylsulfide (F3). 1-Bromo-3-(3,5-dimethyl-4-hydrox-
yphenyl)propane (2.43 g, 10 mmol) in ethanol (10 ml)
was added dropwise in an atmosphere of inert gas to a
Antioxidant activity of compounds was deter-
solution of dodecanethiol-1 (2.02 g, 10 mmol) and mined using special E. coli strains as described [29].
KOH (0.69 g, 10.5 mmol) in ethanol (15 ml), and the Bacterial cells were grown on a shaker (130 rpm) for
solution was heated and stirred for 4 h at 30°ë. The 15 h at 37°ë in LB medium containing no antibiotics in
reaction mass was cooled, neutralized by hydrochloric experiments with strain cc104 or containing kanamycin
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 4 2008

NEW PROMISING ANTIOXIDANTS
507
(12.5 µg/ml) in experiments with strain cc104mutT, or rence of mating; the day the plug was detected was con-
kanamycin (12.5 mg/ml) and tetracycline (50 µg/ml) in sidered to be the first day of pregnancy. Females set
experiments with strain cc104mutMY as described in away from males were autopsied on day 17–18 of preg-
[55]. An inoculate of the night culture was diluted nancy since the greater portion of dominant lethals
100 times, and cells were grown in LB medium until causes the death of embryos during, or shortly after the
the middle of the exponential growth phase. A solution implantation. The number of dead embryos (dark
of the antioxidant tested in DMSO (0.5, 1.25, and 5 µl) homogenous rounded bodies 2.5-3 mm in diameter)
was added to aliquots of cell cultures (0.9 ml) corre- and the number of alive and dead embryos in both
sponding to the middle of the exponential growth phase uterus horns were calculated. The lethality of embryos
(A405 ~ 0.26) to a final concentration of 8, 20, or 80 nM, in females that became pregnant in the first week after
and the corresponding volume of DMSO or medium the administration of the chemical compound reflects
was added to control samples. The mixture was incu- mutational events that occur in mature spermatozoa, the
bated for 20 min on a shaker at 37°ë (130 rpm). Then second week corresponds to late spermatids, and the
25 mM ç2é2 (100 µl) was added to all samples to a third week corresponds to early and middle spermatids.
final concentration of 3 mM, and cells were incubated The number of pregnant females and the percent of fer-
for an additional 20 min under the same conditions. The tility were determined from the number of yellow bod-
cell growth was arrested by a tenfold dilution of the ies in ovaries as described in [59]. The frequency of
incubation mixture with 0.15 M NaCl to a concentra- occurrence of dominant lethals was calculated by
tion of 2 × 103 colony-forming units/ml, and the cell postimplantation losses by the formula: FL% = [1 –
culture (100 µl) was seeded on dishes with 2% LB agar, number of alive embryos per female in the experimen-
three dishes for each oxidant concentration examined. tal group/number of alive embryos per female in the
The dishes were incubated for 24 h at 37°ë, and the control group] × 100 [60]. The data were processed by
number of grown colonies was calculated using the pro- two-factor dispersion analysis.
gram Image Pro PlusV. 4.1. Cells that were not exposed
to hydrogen peroxide and antioxidants and cells treated
with DMSO were used as controls. The survival was
ACKNOWLEDGMENTS
estimated by calculating the ratio of the mean number
of colonies in experiment to the mean number of colo-
nies in the control having regard to the average data of
three separate experiments. The protecting effect of
antioxidants was assessed from the difference in cell
survival in the presence and absence of the correspond-
ing antioxidant. The statistical processing of the data
was carried out using the Student t-test (Statistica 5.0).
Differences at P ≤ 0.05 were considered significant.
Determination of dominant lethal mutations in
embryonic cells of mice. Experiments were performed
with males and females of the genetically homoge-
neous line CBA/Lac. Each experimental and control
group included 20 males weighing 20–25 g. All ani-
mals were kept on a 12-h light regime with free access
to food and water. A compound being examined was
dissolved in olive oil at a concentration of 52 µM and
administered per os singly to the animal using a gastric
tube, 0.2 ml per animal. For comparison of the genotox-
icity of the compound with that of the known com-
pounds, α-tocopherol in the same amount was adminis-
tered to animals. Cyclophosphane, which has a strong
mutagenic effect [61] at a dose of 1 mg per animal, dis-
solved in the same amount of olive oil, served as a pos-
itive control. Control animals were given pure olive oil.
Immediately after the treatment, five Virgin females
were placed to the cage with a male from the control
and experimental groups. Then females were added
every week to analyze the dominant lethality according
to the stage of spermatogenesis of treated males.
The authors thank B.N. Ames (Biochemistry
Department, University of California, Berkeley, United
States) for kindly providing the strain of S. typhimu-
rium TA102.
This work was supported by the integration grant of
the Siberian Division of the Russian Academy of Sci-
ences, the Program of Basic Research of the Presidium
of the Russian Academy of Sciences “Molecular and
cell biology” (project no. 10.5), and the Russian Foun-
dation for Basic Research (project no. 05-04-48779).
REFERENCES
1. Sies, H., Oxidative Stress, London: Academic, 1985,
pp. 1–8.
2. Sies, H., Am. J. Med., 1991, vol. 91, pp. 313–323.
3. Seifulla, R.D. and Borisova, I.G., Farmakol. Toksikol.,
1990, vol. 6, pp. 3–10.
4. Men’shchikova, E.B., Lankin, V.Z., Zenkov, N.K.,
Bondar’, I.A., Krugovykh, N.F., and Trufakin, V.A.,
Okislitel’nyi stress. Prooksidanty i antioksidanty (Proxi-
dants and Antioxidants), Moscow: Slovo, 2006.
5. Vol’skii, N.N., Kashlakova, N.V., and Kozlov, V.A., Tsi-
tologiya, 1988, vol. 30, pp. 895–902.
6. Richards, D.M.S., Dean, R.T., and Jessup, W., Biochim.
Biophys. Acta, 1988, vol. 946, pp. 281–288.
7. Drapier, J.-C. and Hibbs, J.B., J. Immunol., 1988,
vol. 140, pp. 2829–2838.
8. Hoffman, M.E., Mello-Filho, A.C., and Meneghini, R.,
Biochim. Biophys. Acta, 1984, vol. 781, pp. 234–238.
The study was performed for 3 weeks at postmeiosis
stages of spermatogenesis. Females were chosen by the
presence of a vaginal plug, which indicates the occur-
9. Harman, D., Mutat. Res., 1992, vol. 275, pp. 257–266.
10. Beckman, K.B. and Ames, B.N., Physiol. Rev., 1998,
vol. 78, pp. 547–581.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 4 2008

508
KEMELEVA et al.
11. Grollman, A.P. and Moriya, M., Trends Genet., 1993, 33. Baraboi, V.A., Brekhman, I.I., Golotin, V.G., and Kudr-
vol. 9, pp. 246–249.
yashov, Yu.B., Perekisnoe okislenie i stress (Peroxide
Oxidation and Stress), St. Petersburg: Nauka, 1992.
12. Halliwell, B., Lancet, 1994, vol. 344, no. 8924, pp. 721–
34. Richard, M.J., Guiraud, P., Monjo, A.M., and Favier, A.,
724.
Free Radical. Res. Commun., 1992.
13. Zhuravlev, A.I., Bioantiokisliteli v regulatsii metabo-
lizma v norme i patologii (Bioantioxidants in Metabo-
lism Regulation under Normal and Pathological Condi-
tions), Moscow: Nauka, 1982.
35. Bassett, D.J.P., Bowen-Kelly, E., Brewster, E.L.,
Elbon, C.L., Reichenbaugh, S.S., Bunton, T., and
Kerr, J.S., Lung, 1988, vol. 166, pp. 335–369.
36. Bast, A. and Goris, R.J.A., Pharm. Weekbl. Sci., 1989,
14. Bast, A. and Haenen, G.R.M.M., Antioxidants in Ther-
apy and Preventive Medicine, New York: Plenum, 1990,
pp. 111–116.
15. Munday, R. and Winterboume, C.C., Biochem. Pharma-
col., 1989, vol. 38, pp. 4349–4352.
vol. 3, pp. 117–127.
37. Nakamura, H., Takada, S., Shimabuku, R., Matsuo, T., and
Obara, H., Kobe J. Med. Sci., 1987, vol. 33, pp. 53–63.
38. Kolosova, N.G., Lebedev, P.A., and Dikalova, A.E., Bull.
Exp. Biol. Med., 2004, vol. 137, pp. 249–251.
39. Shabalina, I.G., Shalbueva, N.I., Dikalova, A.E., Sinit-
sina, O.I., and Kolosova, N.G., Eksp. Klin. Farmakol.,
2001, vol. 64, pp. 34–36.
40. Prosenko, A.E., Markov, A.F., Khomchenko, A.S.,
Boiko, M.A., Terakh, E.I., and Kandalintseva, N.V.,
Neftekhimiya, 2006, vol. 46, pp. 471–475 [Pet. Chem.
(Engl. Transl.), vol. 46].
41. Prosenko, A.E., Dyubchenko, O.I., Terakh, E.I.,
Markov, A.F., Gorokh, E.A., and Boiko, M.A.,
Neftekhimiya, 2006, vol. 46, pp. 310–315 [Pet. Chem.
(Engl. Transl.), vol. 46].
42. Levin, D.E., Hollstein, M., Christman, E.A.,
Schwiers, E.A., and Ames, B.N., Proc. Natl. Acad.
Sci. USA, 1982, vol. 79, pp. 7445–7449.
43. De Flora, S., Bennicelli, C., Rovida, A., Scatolini, L.,
and Camoirano, A., Mutat. Res., 1994, vol. 307, pp. 157–
167.
16. Wengel, A., Enzymes–Tools and Targets, Basel: Karget,
1988, pp. 161–167.
17. Keniya, M.V., Lukash, A.I., and Gus’kov, E.P., Usp.
Sovrem. Biol., 1993, vol. 113, pp. 456–470.
18. Abramova, Zh.I. and Oksengendler, G.I., Chelovek i pro-
tivookislitel’nye veshchestva (Human andAntioxidants),
Leningrad: Nauka, 1985.
19. Beyer, R.E., Free Radical Biol. Med., 1990, vol. 8,
pp. 545–565.
20. Roginskii, V.A., Fenol’nye antioksidanty: reaktsionnaya
aktivnost' i effektivnost’ (Phenol Antioxidants: Reactiv-
ity and Efficiency), Moscow: Nauka, 1988.
21. Mickle, D.A.G., Li, R.K., Weisel, R.D., Tumiati, L.C.,
and Wu, T.W., J. Mol. Cell. Cardiol., 1990, vol. 22,
pp. 1297–1304.
22. Voskresenskii, O.N., Zhutaev, I.A., Bobyrev, V.N., and
Bezuglyi,Yu.V., Vopr. Med. Khim., 1982, vol. 1, pp. 14–27.
44. Grey, C.A. and Adlercreutz, P., Mutat. Res., 2003,
23. Sokolovskii, V.V., Vopr. Med. Khim., 1988, vol. 6, pp. 3–10.
vol. 527, pp. 27–36.
24. Sinitsyna, O., Krysanova, Zh., Ishchenko, A., Dikalova, A.,
Stolyarov, S., Kolosova, N.,Vasunina, E., and Nevinsky, G.,
J. Cell. Mol. Med., 2006, vol. 10, pp. 206–215.
45. Marczewska, J. and Koziorowska, J., Acta. Pol. Phar.,
2000, vol. 57, pp. 49–52.
46. Asad, N.R., Asad, L.M., Silva, A., Felzenswalb, I., and
Leitao,A.C., Acta. Biochim. Pol., 1998, vol. 45, pp. 677–
690.
25. Zenkov, N.K., Kandalintseva, N.V., Lankin, V.Z.,
Men’shchikova, E.B., and Prosenko, A.E., Fenol’nye
bioantioksidanty (Phenol Antioxidants), Novosibirsk:
Tipografiya SO RAMN, 2003.
26. Prosenko, A.E., Klepikova, S.Yu., Kandalintseva, N.V.,
Dyubchenko, O.I., Dushkin, M.I., Zenkov, N.K., and
Men’shchikova, E.B., Byull. SO RAMN, 2001, vol. 99,
pp. 114–119.
47. Eder, E., Favre, A., Stichtmann, C., and Deininger, C.,
Toxicol. Lett., 1989, vol. 48, pp. 225–234.
48. Imlay, J.A. and Linn, S., J. Bacteriol., 1987, vol. 169,
pp. 2967–2976.
49. Gomes, E.M., Souto, P.R.F., and Felzenszwalb, I.,
Mutat. Res., 1996, vol. 367, pp. 203–208.
27. Kandalintseva, N.V., Dyubchenko, O.I., Terakh, E.I.,
Prosenko, A.E., Shvarts, Ya.Sh., and Dushkin, M.I.,
Khim.-Farm. Zh., 2002, vol. 36, pp. 13–15.
50. Muller, J. and Janz, S., Environ. Mol. Mutagen., 1993,
vol. 22, pp. 157–163.
51. Nakayama, T., Hiramitsu, M., Osawa, T., and Kawak-
28. Bakhtina, I.A., Antip’eva, E.V., Prosenko, A.E.,
Strel’tsova, G.P., Dushkin, M.I., Zenkov, N.K.,
Men’shchikova, E.B., and Ragino, Yu.I., Byull. SO
RAMN, 2000, vol. 97–98, pp. 24–29.
ishi, S., Mutat. Res., 1993, vol. 303, pp. 29–34.
52. Duthie, S.J., Ma, A., Ros, M.A., and Collins, A.R., Can-
cer Research, 1996, vol. 56, pp. 1291–1295.
29. Asad, N.R. and Leitao, A.C., J. Bacteriol., 1991,
53. Cupples, C.G. and Miller, J.H., Proc. Natl. Acad. Sci.
USA, 1989, vol. 86, pp. 5345–5349.
vol. 173, pp. 2562–2568.
54. Miller, J.H. and Michael, M., Gene, 1996, vol. 179,
30. Hofnung, M. and Quillardet, P., Mutagenesis, 1986,
pp. 129–132.
vol. 1, pp. 319–330.
55. Bhatnagar, S.K. and Bessman, M.J., J. Biol. Chem.,
31. Maron, D.M. and Ames, B.N., Mutat. Res., 1983,
1988, vol. 263, pp. 8953–8957.
vol. 113, nos. 3–4, pp. 173–215.
56. Cabrera, M., Nghiem, Y., and Miller, J.H., J. Bacteriol.,
32. Issledovanie sinteticheskikh i prirodnykh antioksidantov
in vitro i in vivo // Sb. nauchn. statei (Study of Synthetic
1988, vol. 170, pp. 5405–5407.
and Natural Antioxidants In Vitro and In Vivo, Collec- 57. Nghiem, Y., Cabrera I., Cupples C.G., Miller J.H, Proc.
tion of Scientific Papers), Moscow: Nauka, 1992, p. 112. Natl. Acad. Sci. USA, 1988, vol. 85, pp. 2709–2713.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 4 2008

NEW PROMISING ANTIOXIDANTS
509
58. Michaels, M.L., Cruz, C., Grollman, A.P., and Miller, J.H., 60. Ehling, U.H., Machemer, J., Buselmaier, W., Dycka, J.,
Proc. Natl. Acad. Sci. USA, 1992, vol. 89, pp. 7022–7025.
Frohberg, H., Kratochvilova, J., Lang, R., Lorke, D.,
Muller, D., Peh, J., Rothrborn, G., Roll, R., Schulze-
Schencking, M., and Wiemann, H., Arch. Toxicol., 1978,
vol. 39, pp. 173–185.
59. Metodicheskie rekomendatsii Farmakologicheskogo
gosudarstvennogo komiteta RF “Otsenka mutagennykh
svoistv farmakologicheskikh sredstv” (Methodological
Recommendations of the Govermental Pharmacogolog-
ical Committee Evaluation of Mutagenic Properties of
Pharmaceuticals), Vedomosti farmakologicheskogo
komiteta, 1998, vol. 4, pp. 37–38.
61. Anderson, D., Bishop, J.B., Garner, R.C., Ostrosky-
Wegman, P., and Selby, P.B., Mutat Res, 1995, vol. 330,
pp. 115–181.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 4 2008