Antioxidant and hepatoprotector activity of water-soluble 4-propylphenols containing hydrophilic groups in alkyl chains

Full text of DOI:10.1023/A:1019876403624

Pharmaceutical Chemistry Journal
Vol. 36, No. 4, 2002
ANTIOXIDANT AND HEPATOPROTECTOR ACTIVITY
OF WATER-SOLUBLE 4-PROPYLPHENOLS CONTAINING
HYDROPHILIC GROUPS IN ALKYL CHAINS
N. V. Kandalintseva,1 O. I. Dyubchenko,1 E. I. Terakh,1 A. E. Prosenko,1
Ya. Sh. Shvarts,2 and M. I. Dushkin2
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 36, No. 4, pp. 13 – 15, April, 2002.
Original article submitted November 12, 2001.
One of the main mechanisms in the pathogenesis of toxic
liver damage is destabilization of the membranes of
hepatocytes as a result of lipid peroxidation (LPO). In the
case of such liver disorders, a pronounced protective action
is offered by phenolic antioxidants such as a-tocopherol [1],
phenosane, Russia), and bis-(gamma-L-glutamyl)-L-cystei-
nyl-bis-glycine disodium salt (glutoxime, Russia).
EXPERIMENTAL CHEMICAL PART
potassium
phenosane,
synthetic
derivatives
of
Compounds I – VII were synthesized from the corre-
sponding 3-(4¢-hydroxyaryl)-1-halogenopropanes as de-
scribed previously [4, 5] and purified by double
recrystallization from ethanol. The main compound content
in the samples studied was 97 – 99%. The 1H NMR spectrum
of compound VIII was measured on a 500-MHz Bruker
spectrometer (Germany) using Si(CH3)4 as the external stan-
dard. The IR spectrum was recorded using a Vektor 22 Fou-
rier-transform spectrometer (Germany) using samples pellet-
ized with KBr (150 : 1). The melting points were determined
using a PTP device (Russia).
hydroxycynnamic acids, ionol, ubiquinone, and some other
natural compounds [2]. According to [3], some derivatives of
hydroxycynnamic acids are superior to a-tocopherol with re-
spect to hepatoprotector action. The high hepatoprotector ac-
tivity of hydroxycynnamic acid derivatives can be due to
their hydrophilicity, which accounts for the high rate of
transport and biological accessibility of these molecules.
Below we report on a comparative study of the antioxi-
dant activity and hepatoprotector properties of a series of wa-
ter-soluble 2,6-di-tert-butyl-4-propylphenol derivatives
(I – IV) and some analogous compounds (V – VIII) not con-
taining tert-butyl groups.
[3-(4¢-Hydroxyphenyl)propyl]diethylamine (VIII). To
a mixture of 4.15 ml (40 mmole) of diethylamine, 2.9 g
(20 mmole) of K2CO3, and 20 ml dioxane at 50°C was grad-
ually (during 0.5 h) added with stirring a solution of 2.15 g
(10 mmole) of 3-(4¢-hydroxyphenyl)-1-bromopropane in
20 ml of dioxane. The reaction mixture was kept for 5 h at
50°C, cooled, and extracted with benzene. The benzene ex-
tract was washed with a saturated aqueous NaCl solution,
dried over anhydrous Na2SO4, and saturated with dry hydro-
gen chloride. The precipitate was separated by filtration,
washed with benzene, dried, and recrystallized from water to
obtain 0.96 g (40%) of hydrochloride VIII; m.p.,
150 – 152°C; 1H NMR spectrum in D2O (d, ppm):
1.060 – 1.090 (t, 6H, CH2CH3), 1.808 (m, 2H, ArCH2CH2),
2.470 – 2.499 (t, 2H, ArCH2), 2.903 – 2.936 (t, 2H,
ArCH2CH2CH2N), 2.986 – 3.029 (q, 4H, NCH2CH3),
6.705 – 6.721 (d, 2H, Harom), 7.012 – 7.028 (d, 2H, Harom);
IR spectrum (n, cm – 1): 2958 (CH), 2666 (NH+);
C13H22ClNO.
X
HO
X
HO
V – VIII
I – IV
X = SO3Na (I, V), SSO3Na (II, VI), SC(NH2)2+Cl– (III, VII),
N(C2H5)2 × HCl (IV, VIII)
The reference substances were 2,6-di-tert-butyl-4-me-
thylphenol (ionol, Acros Organics, USA), b-(3,5-di-tert-bu-
tyl-4-hydroxyphenyl)propionate potassium salt (potassium
1
Novosibirsk State Pedagogical University, Novosibirsk, Russia.
Institute of Clinical Immunology, Siberian Division, Russian Academy of
2
Medical Sciences, Novosibirsk, Russia.
177
0091-150X/02/3604-0177$27.00 © 2002 Plenum Publishing Corporation

178
N. V. Kandalintseva et al.
[O2]/[RH] · 104
4
3
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
* p < 0.05, ** p < 0.01 with respect to control
1
2
4.0 30
20
3.5
20 mg/kg
40 mg/kg
10
*
*
b
0
3.0
0
0.2 0.4 0.6 0.8 1.0 1.2
–ln (1 – t/t)
*
*
*
2.5
2.0
1.5
1.0
0.5
0
*
*
*
*
*
*
*
**
*
**
**
20 40 60 80 100 120 140 160 180 200 220
Time, min
ÑÑl4
I
II III IV VI VII VII
Fig. 1. The kinetics of oxygen absorption in the course of
AIBN-initiated methyl oleate oxidation in chlorobenzene at 60°C in
the presence of (1 ) ionol (0.3 mM) and (2 ) isothiuronium chloride
III (0.5 mM); plots 3 and 4 in the inset represent anamorphoses of
curves 1 and 2, respectively.
Fig. 2. A histogram showing the effect of compounds I – VIII on
the ALT activity in the blood serum of mice with tetrachloromethane
hepatitis.
follows: methyl oleate, 1.48 M; AIBN, 12 mM; antioxidant,
0.3 – 0.6 mM.
Determining antiradical activity. The antiradical activ-
ity of compounds I – IV, ionol, and potassium phenosane was
characterized by the rate constant (k7) of their interaction
with hydroperoxide radicals of methyl oleate antiradical ac-
tivity and the inhibition coefficient ( f ) equal to the number
of chains terminated by a single antioxidant molecule. The k7
and f values were determined using a model reaction of the
AIBN-initiated methyl oleate oxidation in chlorobenzene at
60°C. Methyl oleate (Acros Organics, USA) was preliminary
purified from peroxides by double distillation in vacuum
(~ 170°C/2 – 3 Torr). The initiator AIBN (Acros Organics)
was purified by recrystallization from ethanol [6]. The work-
ing concentrations of components in a sample (20°C) were as
The volume of absorbed oxygen was determined by a
volumetric technique described in [6]. The oxygen pressure
in the system was 1 atm and the volume of oxidized sample
was 5 ml (20°C). The induction period (t) was determined
graphically as the point of intersection of two tangents to the
kinetic curve, the slopes of which amounted to 0.5 and 0.75
of the slope of a straight line corresponding to the
noninhibited oxidation reaction [6].
Figure 1 shows the kinetic curves of oxygen absorption
and their anamorphoses plotted as [O2]/[RH] versus
–ln(1 – t/t) for isothiuronium chloride III and ionol. Using
the slopes of anamorphoses, it was possible to determine the
k2/k7 ratio which, for the k2 value taken from [7], yielded
k7 = (2.6 ± 0.4) ´ 104 liter/(mole · sec). The coefficient f was
determined using the formula
TABLE 1. Characteristics of the Antiradical and General Antioxi-
dant Activity of the Synthesized Compounds
f ionol ×[ionol]×tAO
f AO
=
,
tionol ×[AO]
k7, 10 – 4
liter/(mole sec)
ID50 for LPO, mM
Compound
f
Cu2+
Fe2+
where fionol = 2 [7] (subscript AO refers to antioxidant).
I
23
15
36
15
2.3 ± 0.3
2.8 ± 0.5
2.7 ± 0.3
1.9 ± 0.2
…
1.1 ± 0.1
1.6 ± 0.1
1.2 ± 0.1
1.3 ± 0.1
…
II
EXPERIMENTAL BIOLOGICAL PART
III
14
13
IV
V
13
14
The general antioxidant activity of the synthesized com-
pounds was evaluated by determining their ability to inhibit
the formation of malonic dialdehyde (MDA) during the incu-
bation of low-density lipoproteins (LDLPs) with ambivalent
metal ions (Cu2+, Fe2+). The LDLP fraction
(1.019 – 1.063 g/ml) was isolated from the blood of donors
by the method of sequential ultracentrifugation in KBr solu-
tion density gradient (1050000g, 20 h; 105000g, 24 h) [8].
2800
400
470
550
3000
380
530
470
VI
VII
VIII
…
…
…
…
…
…
Potassium
phenosane
18.5
15
17
15
1.5 ± 0.1
2.6 ± 0.4
1.3 ± 0.1
Ionol
2.0

Antioxidant and Hepatoprotector Activity of Water-soluble 4-Propylphenols
179
PhO· + ROO· ® QP
The LDLPs were purified from residual KBr by chromatog-
raphy on a Sephadex G25 column. The protein content in
LDLPs was determined by the Lowry method. The isolated
LDLPs were oxidized at 37°C in the presence of CuSO4
(5 mM) or FeSO4 (25 mM) [9]. The antioxidant concentra-
tions were 1, 10, or 100 mM. Accumulation of the lipid
peroxidation (LPO) product (MDA) capable of interacting
with thiobarbituric acid was monitored by fluorescence spec-
troscopy (Hitachi Model P3000 spectrofluorimeter) [10]. The
antioxidant properties of the synthesized compounds were
evaluated by their effective concentrations producing a 50%
inhibition (ID50) of the MDA accumulation during a 30-min
incubation of LDLPs in the presence of metal ions.
Because of certain features of the spin density distribu-
tion and spatial hindrances with respect to ortho position, the
phenoxy radicals of 2,6-di-tert-butyl-4-alkylphenols attach
ROO× so as to form predominantly the para isomers of QP:
OOR
O
·O
R'
·
+ ROO
R'
The experimental toxic hepatitis in (C57Bl/6XCBA)F1
mice was induced by intraperitoneal injections of a 10%
tetrachloromethane solution in olive oil (0.2 ml per animal
weighing 25 g). The synthesized compounds were dissolved
in physiological solution and introduced intraperitoneally 2 h
before tetrachloromethane. The phenolic compounds were
used in the concentrations of 5 ´ 10 – 5 and 1 ´ 10 – 4 M/kg,
which corresponds to 20 and 40 mg/kg of glutoxime. The
degree of liver damage was evaluated by the
alanineaminotransferase (ALT) activity in the blood serum
[11] determined 24 h after tetrachloromethane introduction.
The ALT activity was estimated by an UV technique using a
Biocon reagent kit [12] and expressed in units of enzyme ac-
tivity per liter blood serum (U/liter). The experimental data
were statistically processed in terms of the classical Student’s
criterion.
Apparently, the large volume and considerable polarity
of the ionogenic fragment R¢ = CH2CH2CH2X hinder the at-
tack of the hydroperoxide radical of methyl oleate at the para
position of phenol ring. This leads to a decrease in the reac-
tion rate constant k7 and, hence, in the inhibition coefficient f.
The values of k7 for 2,6-di-tert-butyl-4-alkylphenols are
usually close to the rate constants for analogous compounds
not substituted at the ortho position [13]. However, com-
pounds V – VIII virtually do not inhibit the AIBN-initiated
oxidation of methyl oleate. This is probably explained by the
low stability of the phenoxy radicals formed by these com-
pounds.
Derivatives of the spatially-hindered phenols I – IV ex-
hibit a pronounced inhibition of the LPO of LDLPs, signifi-
cantly exceeding in this respect the ortho-unsubstituted
analogs V – VIII. Characterized by a lower antiradical activ-
ity than ionol, compounds II – IV are nevertheless compara-
ble with ionol in the ability to inhibit the LPO of LDLPs. The
difference between the antiradical and antioxidant activity of
these phenols can be related to the antioxidant properties of
ionogenic fragments – thiosulfonate, isothiuronium, and
alkylammonium. These groups probably account for the anti-
oxidant properties of the ortho-unsubstituted phenols
VI – VIII. Note that sulfonate V virtually does not affect the
oxidation of LDLPs.
RESULTS AND DISCUSSION
Experimental data on the antioxidant activity of the syn-
thesized compounds are presented in Table 1. As can be seen,
all the water-soluble 2,6-di-tert-butyl-4-alkylphenols (I – IV)
exhibit similar k7 values, close to the corresponding reaction
rate constant for ionol. This result is quite natural, since the
ionogenic fragments separated from the aromatic nucleus by
a hydrocarbon chain cannot significantly influence the reac-
tivity of the phenolic OH group or the stability of the
phenoxy radical. At the same time, compounds I – IV and
potassium phenosane are inferior to ionol with respect to the
inhibition coefficient f .
With respect to the ability of reducing ALT activity on
the toxic hepatitis model, the compounds studied can be ar-
ranged in the following order: alkylammonium
IV » isothiuronium III > thiosulfonate II > sulfonate I > po-
tassium phenosane » alkylammonium VIII > isothiuronium
VII > thiosulfonate VI > glutoxime (Fig. 2).
As is known [13], the antioxidant activity of phenolic
compounds is based on the reaction of PhOH with a
hydroperoxide radical:
Thus, the results of our investigation indicate that wa-
ter-soluble 2,6-di-(tert-butyl)-4-(n-propyl)phenol deriva-
tives, containing thiosulfonate, isothiuronium, and alkylam-
monium groups (compounds II – IV) as hydrophilic frag-
ments, can effectively inhibit oxidation of LDLPs in vitro
and produce a hepatoprotector effect in vivo. Therefore, these
substances are worthy of further preclinical investigation as
hepatoprotector drugs.
PhOH + ROO· ® PhO· + ROOH.
Under the conditions of initiated oxidation, the main
pathway of decomposition of the phenoxy radicals PhO× is
the formation of quinoid peroxides QP via the reaction

180
N. V. Kandalintseva et al.
5. N. V. Kandalintseva, O. I. Dyubchenko, A. E. Prosenko, et al.,
Khim.-Farm. Zh., 35(3), 22 – 25 (2001).
ACKNOWLEDGMENTS
6. V. F. Tsepalov, In Vivo and In Vitro Investigation of Synthetic
and Natural Antioxidants (A Coll. of Sci. Papers) [in Russian],
Nauka, Moscow (1992), pp. 16 – 26.
This study was supported by the Russian Foundation for
Basic Research, project Nos. 01-04-49306 and 02-04-07560.
7. G. D. Kadochnikova, in: Free-Radical Oxidation of Lipids: Ex-
periment and Clinics (A Coll. of Papers) [in Russian], Tyumen
(1997), pp. 5 – 21.
REFERENCES
8. Modern Methods in Biochemistry, V. N. Orekhovich (ed.), Mos-
cow (1977), pp. 533 – 537.
9. M. Dushkin, N. Zenkov, E. Menshikova, et al., Atherosclerosis,
114, 9 – 18 (1995).
10. K. Yagi, Biochem. Med., 15, 212 – 216 (1976).
11. D. N. Mayanskii, E. Wisse, and K. Decker, in: New Frontiers in
Hepatology (A Coll. of Papers) [in Russian], Novosibirsk
(1992).
12. H. Bergmeyer and A. Holder, Clin. Chem. Acta, 105, 147 – 152
(1980).
1. L. F. Vinogradova, T. S. Illarionova, E. V. Kharlitskaya, et al.,
Byull. Eksp. Biol. Med., 125(4), 417 – 419 (1998).
2. I. V. Sorokina, A. P. Krysin, T. B. Khlebnikova, et al., The Role
of Phenolic Antioxidants in Increasing Stability of Organic Sys-
tems with Respect to Free-Radical Oxidation [in Russian],
GPNTB SO RAN, Novosibirsk (1997), pp. 45 – 53.
3. S. M. Drogovoz, V. V. Slyshkov, and I. S. Sal’nikova, Eksp.
Klin. Farmakol., 56(5), 54 – 57 (1994).
4. A. E. Prosenko, S. Yu. Klepikova, N. V. Kandalintseva, et al.,
Byull. Sib. Otd. Ross. Akad. Med. Nauk, 99(1) (2001).
13. V. A. Roginskii, Phenolic Antioxidants: Reactivity and Efficacy
[in Russian], Nauka, Moscow (1988).