Synthesis and antioxidant activity of modified sterically hindered phenols

Article abstract of DOI:10.1134/S1070363216030166

Salicylaldehyde derivatives containing sterically hindered phenol fragments were synthesized. The compounds obtained are capable to offer protection against UV radiation.

Full text of DOI:10.1134/S1070363216030166

ISSN 1070-3632, Russian Journal of General Chemistry, 2016, Vol. 86, No. 3, pp. 602–606. © Pleiades Publishing, Ltd., 2016.
Original Russian Text © R.G. Tagasheva, S.V. Bukharov, G.N. Nugumanova, Yu.N. Oludina, M.A. Sazykina, I.S. Sazykin, E.Yu. Seliverstov, M.I. Khammami,
2016, published in Zhurnal Obshchei Khimii, 2016, Vol. 86, No. 3, pp. 461–465.
To the 100th Anniversary of A.N. Pudovik
Synthesis and Antioxidant Activity
of Modified Sterically Hindered Phenols
R. G. Tagashevaa, S. V. Bukharova, G. N. Nugumanovaa, Yu. N. Oludinaa,
M. A. Sazykinab, I. S. Sazykinb, E. Yu. Seliverstovb, and M. I. Khammamib
a Kazan National Research Technological University, ul. Karla Marksa 68, Kazan, Tatarstan, 420015 Russia
e-mail: tagashevar@mail.ru
b Southern Federal University, Ivanovskii Academy of Biology and Biotechnology, Rostov-on-Don, Russia
Received January 25, 2016
Abstract—Salicylaldehyde derivatives containing sterically hindered phenol fragments were synthesized. The
compounds obtained are capable to offer protection against UV radiation.
Keywords: sterically hindered phenols, acylhydrazone, thiosemicarbazone, UV radiation, protective activity
DOI: 10.1134/S1070363216030166
The diverse biological activity of hydrazones and
thiosemicarbazones is well known [1–3]. Hydrazones
have strong antioxidant properties; they can act as
peroxide radicals traps, non-radical hydroperoxide
destroyers, and can form metal complexes with
transition metal ions. Due to the low strength of the N–
H bond, these compounds may be more effective
peroxide radicals traps as compared with a typical
amine antioxidant diphenylamine. The NH-group of
hydrazone fragment provides the main contribution to
the antiradical activity of hydroxybenzaldehydes
hydrazones [4]. All this leads to a great interest in the
synthesis and study of hybrid structures based on
hydrazones, thiosemicarbazones, and phenol antioxi-
dants. Such compounds may show intramole-cular
synergism of the antioxidant action due to the high
activity of NH-group by reacting with peroxide
radicals and increased stability of the hindered phen-
oxyl radical (Scheme 1).
Here we report on the synthesis of sterically
hindered phenol derivatives of salicylaldehyde thios-
emicarbazone and acylhydrazone, as well as the study
of their antioxidant activity. Compounds 1–6 were
obtained with Scheme 2.
Thiosemicarbazones 1 and 5 were synthesized by
reacting alcoholic solutions of the corresponding
salicylaldehyde derivative with thiosemicarbazide
Scheme 1.
t-Bu
HO
t-Bu
t-Bu
.
.
NH__N=CH__B
HO
t-Bu
A
N__N=CH__B
+ ROO
+ ROOH
A
t-Bu
.
O
A
NH__N=CH__B
t-Bu
602

SYNTHESIS AND ANTIOXIDANT ACTIVITY
603
Scheme 2.
R1
R2
R3
OH
H2NNC(S)NH2 HCl
S
CH= NNCNH2
R1
1, 3, 5
R2
R3
OH
t-Bu
t-Bu
O
H2NNCCH2CH2
R1
OH
R2
R3
OH
CHO
t-Bu
OH
t-Bu
7, 8, 9
O
10
CH=NNCCH2CH2
2, 4, 6
t-Bu
R1 = R3 = H, R2 = OH (7, 1, 2); R1 = R3 = t-Bu, R2 = H (8, 3, 4); R1 = R3 =
OH , R2 = OH (9, 5, 6).
t-Bu
Scheme 3.
S
8
H2O
t-Bu
t-Bu
NH2NHCNH2
H2NNH2
.
_HOC(S)NH2
OH
HO
+
H2NNH2 H2O
2 8
t-Bu
NN=CH
t-Bu
CH=
11
hydrochloride in the presence of triethylamine
followed by addition of a catalytic amount of
trifluoroacetic acid.
The structure of obtained compounds was con-
firmed by NMR spectra. A double set of the signals of
1
some protons in the H NMR spectra of compounds 2,
4, and 6 indicate their existence as different geo-
metrical Z/E isomers.
Because of the low reactivity of the 2-hydroxy-3,5-
di-tert-butylbenzaldehyde 8, the complete conversion
in the reaction with thiosemicarbazide hydrochloride
was achieved only within more than 20 h.
Therefore the synthesis of thiosemicarbazone 3 in an
aqueous alcohol medium was complicated by
hydrolysis of thiosemicarbazide to give azine 11.
Structure of the latter was proved by its authentic
synthesis by reacting aldehyde 8 with hydrazine
hydrate (Scheme 3).
The assessment of antioxidant activity of
derivatives of hydrazones, thiosemicarbazones and
sterically hindered phenols was made by the example
of compounds 1–4, and 6. The ability to protect the
cells against oxidative stress (protective activity) of the
test substances was investigated in the concentration
range of 10–9–10–4 mol/L. The protective activity of
compounds 1–4 and 6 against UV radiation was
demonstrated by the example of bacterial lux-sensor
E. coli MG1655 (pKatG-lux).
Thiosemicarbazone 3 was obtained as a result of
the reaction of the aldehyde with a free thio-
semicarbazide 8 in the presence of trifluoroacetic acid.
As can be seen from the data given on the figure,
compounds 1–4, and 6 can effectively intercept the
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TAGASHEVA et al.
(1H, HC), 9.73 s (1H, OH), 11.15 s (1H, NH). Found,
%: С 45.31; Н 4.01; N 19.76. С8H9N3O2S. Calculated,
%: С 45.49; Н 4.29; N 19.89.
2
3
6
2,4-Dihydroxy-3,5-di-(3',5'-di-tert-butyl-4'-hyd-
roxyphenyl)benzaldehyde thiosemicarbazone (5)
was prepared similarly from 5 g (9 mmol) of 2,4-
dihydroxy-3,5-di-(3',5'-di-tert-butyl-4'-hydroxyphenyl)
benzaldehyde, 1.15 g (10 mmol) of thiosemicarbazide
hydrochloride, 1.4 mL (10 mmol) of triethylamine,
and 80 mL of ethanol; the reaction time was 4 h. Yield
4.7 g (80%), pale yellow powder, mp 190°C (decomp.).
1H NMR (CDCl3), δ, ppm: 1.39 s and 1.41 s (36H,
CMe3), 3.85 s and 3.99 s (4H, CH2-Ar), 5.07 s (1H,
OH1), 5.13 s (1H, OH2), 5.44 s (1H, OH3), 6.36 s and
6.48 s (2H, NH2), 6.94 s (1H, H4), 7.01 s and 7.10 s
(4H, ArH), 8.00 s (1H, HC=N), 9.67 s (1H, OH5), 9.84
s (1H, NH). Found, %: С 70.19; Н 8.82; N 6.15.
С38H53N3O4S. Calculated, %: С 70.44; Н 8.25; N 6.49.
4
5
1
Concentration, mol/L
Antioxidant activity of compounds 1–4 and 6 towards E.
coli MG1655 (pKatG-lux) biosensor when exposed to UV
rays (λ = 311 nm). The values of active substance
concentrations at which they reach the maximum activity
are given. (1) compound 1 (c = 1 × 10–7 mol/L), (2) com-
pound 2 (c = 1 × 10–8 mol/L), (3) compound 3 (c = 1 ×
10–5 mol/L), (4) compound 4 (c = 1 × 10–5 mol/L),
(5) compound 6 (c = 1 × 10–9 mol/L), (6) Trolox (c = 1 ×
10–4 mol/L).
3-(3,5-Di-tert-butyl-4-hydroxyphenyl)propanoic
acid 2,4-dihydroxybenzylidenehydrazide (2). A mix-
ture of 1.01 g (7.3 mmol) of 2,4-dihydroxy-benzal-
dehyde, 2.12 g (7.3 mmol) of 3-(3,5-di-tert-butyl-4-
hydroxyphenyl)propanoic acid and 20 mL of ethanol
was refluxed for 1.5 h. After cooling to room
temperature the precipitate was filtered off, washed
with ethanol, and dried in air to constant weight. Yield
hydrogen peroxide formed in intracellular and
intercellular environment when exposed to ultraviolet
rays. All investigated compounds have protective
properties, which is superior to activity of Trolox, a
water-soluble analog of natural antioxidant vitamin E.
Three of the compounds studied (1, 2, and 6) are active
at low concentrations (10–7–10–9 mol/L) and are of
interest as promising antioxidants.
1
2.6 g (87%), white powder, mp 240°C (decomp.). H
NMR (acetone-d6), δ, ppm: 1.43 s and 1.44 s (18H,
CMe3), 2.55 t and 2.92 t (4H, CH2CH2, 3JНН = 8.4 Hz),
5.80 s and 5.82 s (1H, OH), 6.40 br.s (1H, H3), 6.42
3
4
d.d and 6.46 d.d (1H, H5, JНН = 8.4, JНН = 2.4 Hz),
7.07 s and 7.12 s (2H, ArH), 7.13 d and 7.20 d (1H,
H6, 3JНН = 8.4 Hz), 8.16 s and 8.25 s (1H, CH=N), 8.79
s (1H, OH4), 10.07 s and 10.45 s (1H, OH2), 10.60 s
and 11.65 s (1H, NH). Found, %: С 70.02; Н 7.78; N
6.98. С24H32N2O4. Calculated, %: С 69.88; Н 7.82; N 6.79.
EXPERIMENTAL
1H NMR spectra were recorded on a Bruker
AVANCE-600 instrument using the residual proton
signals of the deuterated solvents as internal reference.
2,4-Dihydroxy-3,5-di-(3',5'-di-tert-butyl-4'-hydroxy-
phenyl)benzaldehyde was prepared as described in [5].
3,5-Di-tert-butyl-2-hydroxybenzaldehyde thiosemi-
carbazone (3) was prepared similarly from 0.2 g
(0.85 mmol) of 3,5-di-tert-butyl-2-hydroxybenzal-
dehyde, 0.077 g (0.85 mmol) of thiosemicarbazide,
2 drops of trifluoroacetic acid and 20 mL of ethanol;
reaction time was 2 h. Yield 0.23 g (88.5%), pale
2,4-Dihydroxybenzaldehyde thiosemicarbazone
(1). A solution of 1.5 g (11 mmol) of 2,4-dihydroxy-
benzaldehyde, 1.4 g (11 mmol) of thiosemicarbazide
hydrochloride, and 1.64 mL (12 mmol) of triethyl-
amine in 20 mL of ethanol was refluxed for 8 h. After
cooling to room temperature the precipitate was
filtered off, washed with ethanol, and dried in air to
constant weight. Yield 2.1 g (90%), pale yellow
1
yellow powder, mp 227–228°C. H NMR spectrum
(CDCl3), δ, ppm: 1.32 s (9Н, CMe3), 1.45 s (9 H,
CMe3), 6.62 s (2Н, NH2), 7.09 s (1H, ArH), 7.44 s
(1H, ArH), 8.17 s (1H, HC=N), 9.94 s (1H, OH), 10.24
s (1Н, NH). Found, %: С 62.61; Н 8.44; N 13.77.
С16H25N3OS. Calculated, %: С 62.50; Н 8.20; N 13.67.
Mass spectrum (MALDI), m/z 308: [M + H]+.
1
powder, mp 211–213°C. H NMR spectrum (DMSO-
3
4
d6), δ, ppm: 6.26 d.d (1H, H2, JНН = 8.5, JНН
=
2.05 Hz), 6.31 d (1H, H1, 4JНН = 2.05 Hz), 7.65 d (1H,
H3, 3JНН = 8.5 Hz), 7.72 s and 7.92 s (2H, NH2), 8.25 s
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 86 No. 3 2016

SYNTHESIS AND ANTIOXIDANT ACTIVITY
605
3-(3',5'-Di-tert-butyl-4'-hydroxyphenyl)propanoic
acid 3,5-di-tert-butyl-2-hydroxybenzylidenehydrazide
(4) was prepared similarly from 1.5 g (6.8 mmol) of
of hydrazine hydrate in 10 mL of methanol was stirred
at room temperature for 10 min. The precipitate was
filtered off, washed with methanol and dried. Yield
0.14 g (75%).
3,5-di-tert-butyl-2-hydroxybenzaldehyde, 1.98
g
(6.8 mmol) of 3-(3,5-di-tert-butyl-4-hydroxyphenyl)-
propanoic acid, 2 drops of acetic acid, and 30 mL of
ethanol; reaction time was 2 h. Yield 2.4 g (74%),
The biological activity of the test compounds was
investigated using fluorescent bacterial sensors E. coli
MG1655 (pXen7) and E. coli MG1655 (pKatG-lux). E.
coli MG1655 (pXen7) is constitutive biosensor used to
determine the substance toxicity [6]. The KatG
biosensor detects hydroperoxide formation in the cell
[6–8]. E. coli cells were grown on a Luria–Bertani (LB)
medium [9] supplemented with ampicillin (100 μg/mL).
1
white powder, mp 225–227°C. H NMR spectrum
(CDCl3), δ, ppm: 1.29 s, 1.32 c, 1.43 s and 1.45 s
(36H, CMe3), 2.59 t and 2.94–3.06 m (4H, CH2CH2),
5.09 s and 5.12 s (1H, OH), 6.95–6.99 m and 7.05–
7.10 m (1Н, Н2), 7.03 s and 7.13 s (2Н, Н3), 7.36–7.42
m (1Н, Н2), 7.97 s and 8.00 s (1Н, СН), 9.71 s and
9.94 s (1Н, ОН1), 10.61 s and 10.63 s (1Н, NH).
Found, %: С 75.93; Н 9.78; N 6.05. С32H48N2O3.
Calculated, %: С 75.55; Н 9.51; N 5.51.
To evaluate the antioxidant activity of the test
substances biosensor strains were exposed to ultra-
violet radiation with a wavelength of 311 nm as
described in [10]. A lamp PL-S 9W/01/2P (Phillips)
was used as the ultraviolet radiation source. The
radiation dose was 732 J = m–2 per min. Irradiation
was performed for 5 min. As a result of UV irradiation,
luminescence of aforementioned strains was induced,
which demonstrates that the formation of hydrogen
peroxide and organic peroxides happens in E. Coli
MG1655 (pKatG-lux) cells.
3-(3'',5''-Di-tert-butyl-4''-hydroxyphenyl)pro-
panoic acid 2,4-dihydroxy-3,5-di-(3',5'-di-tert-butyl-
4'-hydroxyphenyl)benzylidenehydrazide (6) was
prepared similarly from 2 g (3.5 mmol) of 2,4-di-
hydroxy-3,5-di-(3',5'-di-tert-butyl-4'-hydroxyphenyl)
benzaldehyde, 1.017 g (3.5 mmol) of 3-(3',5'-di-tert-
butyl-4'-hydroxyphenyl)propanoic acid, 2 drops of
acetic acid, and 30 mL of isopropyl alcohol; reaction
time was 6 h. Yield 2.1 g (70%), white powder, mp
To assess the protective activity of the test sub-
stances biosensor strains suspension was irradiated in
the absence and in the presence of the samples.
1
205°C (decomp.). H NMR spectrum (acetone-d6), δ,
ppm: 1.20 s, 1.23 s, 1.26 s and 1.28 s (54H, CMe3),
2.39 t and 2.68–2.82 m (4H, CH2CH2); 3.73 s, 3.74 s,
3.89 s and 3.90 s (4H, CH2-Ar); 5.54 s, 5.63 s, 5.65 s
and 5.66 s (3H, OH1), 6.60–7.15 m (6H, ArH), 7.51 s
and 7.58 s (1Н, ОН3), 7.96 s and 8.05 s (1H, CH), 9.87
s and 10.22 s (1H, OH2), 10.80 s and 11.69 s (1H,
NH). Found, %: С 76.95; Н 9.34; N 3.15. С54H76N2O6.
Calculated, %: С 76.38; Н 9.02; N 3.30.
Measuring the bioluminescence level was per-
formed for 2 h using a microplate thermostatic
luminometer LM-01T (Immunotech, Czech Republic).
To evaluate the impact of the factors on the
expression of the studied operons we calculated induc-
tion factor (Is).
Is = Le/Lc – 1.
(1)
3,5-Di-tert-butyl-2-hydroxybenzaldehyde bishydra-
zone (11). a. A mixture of 1.2 g (5 mmol) of 3,5-di-
tert-butyl-2-hydroxybenzaldehyde, 0.785 g (6 mmol)
of thiosemicarbazide hydrochloride, and 0.78 mL
(6 mmol) of triethylamine in 20 mL of ethanol was
refluxed for 8 h. The reaction mixture was cooled to
room temperature. After cooling to room temperature
the precipitate was filtered off, washed with ethanol,
and dried in air to constant weight. Yield 1.33 g (85%),
pale yellow powder, mp 209–210°C. 1H NMR
spectrum (CDCl3), δ, ppm: 1.35 s (18H, CMe3), 1.49 s
Here Lc is the luminescence intensity of the control
sample; Le is the luminescence intensity of the test
sample.
Statistically reliable excess of Le over Lk measured
by t-criterion [11] was considered as an indication of
the statistical significance of the induction effect.
Protective activity index was calculated by the formula (2).
A = (1 – Ia/Ip) × 100%.
(2)
Here, Ia is an induction factor of a bioluminescent
response of the investigated influence of the presence
of the protector; Ip is a factor of induction by the
studied influence.
4
(18H, CMe3), 7.18 d (2H, ArH, J = 2.4 Hz), 7.48 d
4
(2H, ArH, J = 2.4 Hz), 8.77 s (2H, HC=N), 11.90 s
(2H, OH).
b. A mixture of 0.2 g (0.85 mmol) of 3,5-di-tert-
butyl-2-hydroxybenzaldehyde and 0.02 g (0.4 mmol)
All experiments were repeated three times. Mean
values and variation indicators (the average margin of
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TAGASHEVA et al.
5. Kasymova, E.M., Burilov, A.R., Vagapova, L.I.,
Mukmeneva, N.A., Bukharov, S.V., Nugumanova, G.N.,
Krivolapov, D.B., Pudovik, M.A., Litvinov, I.A., and
Konovalov, A.I., Russ. Chem. Bull., 2006, vol. 55, no. 1,
p. 1171. DOI: 10.1007/s11172-006-0395-8.
error) of protecting effectiveness were calculated
according to three independent experiments. Statistical
processing was performed by standard biometric
formulas. The values of the confidence intervals were
calculated for p < 0.05 [12].
6. Zavilgelsky, G.B., Kotova, V.Yu., and Zavilgelsky, I.V.,
Mutat. Res., 2007, vol. 634, p. 172.
7. Lee, H.J. and Gu, M.B., Appl. Microbiol. Biotechnol.,
AKCNOWLEDGMENTS
2003, vol. 60, no. 5, p. 577.
This work was supported by the Southern Federal
University (grant no. 213.01-07-2014/12PChVG).
8. Lee, J.H., Youn, C.H., Kim, B.C., and Gu, M.B.,
Biosens. Bioelectron., 2007, vol. 22, no. 9–10, p. 2223.
9. Lushchak, V.I., Comp. Biochem. Physiol. (C), 2011,
REFERENCES
vol. 153, no. 2, p. 175.
1. Kitaev, Yu.P. and Buzykin B.N., Gidrazony
(Hydrazones), Moscow: Nauka, 1974.
2. Tomchin, A.B., Velezheva, V.S., and Shustov, E.B.,
Khim. Pharm. Zh., 1998, vol. 32, no. 2, p. 7.
3. Fedorova, O.V., Khim. Pharm. Zh., 1998, vol. 32, no. 2,
10. Oludina, Yu.N., Sazykina, M.A., Prazdnova, E.V.,
Sazykin, I.S., Khmelevtsova, L.E., Bukharov, S.V.,
Barsukova, T.A., Tagasheva, R.G., and Nugumanova, G.N.,
Pharm. Chem. J., 2015, vol. 48, no. 12, p. 787. DOI:
10.1007/s11094-015-1196-x.
11. Lakin, G.F., Biometriya (Biometry), Moscow: Vysshaya
p. 11.
Shkola, 1990.
4. Khizhan, E.I., Vinogradov, V.V., Morenko, V.V.,
Nikolaevskii, A.N., Khizhan, A.I., Zarechnaya, O.M.,
and Dmitruk, A.F., Russ. J. Gen. Chem., 2013, vol. 83,
no. 8, p. 1529. DOI: 10.1134/S1070363213080100.
12. Maniatis, T., Fritsch, E.F., and Sambrook, J., Molecular
Cloning: a Laboratory Manual, New York: Cold Spring
Harbor Laboratory, 1982.
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