Synthesis and membrane protective activity of bis-sulfides derived from monoterpenoids and monosaccharides

Article abstract of DOI:10.1134/S1068162017030141

Hydroxyl- and chloroethyl derivatives of neomenthane- and isobornanethiol in yields of to 80% were synthesized. They served as the basis for the preparation of new bis-sulfides with diacetone-protected galacto- and fructopyranose fragments in yields up to 98%. The bis-sulfides synthesized were screened for membrane protective and antioxidant properties in a model cell system (in vitro) based on their ability to inhibit the H₂O₂-induced hemolysis of erythrocytes and retard the oxyhemoglobin oxidation.

Full text of DOI:10.1134/S1068162017030141

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2017, Vol. 43, No. 3, pp. 302–310. © Pleiades Publishing, Ltd., 2017.
Original Russian Text © S.V. Pestova, E.S. Izmest’ev, O.G. Shevchenko, S.A. Rubtsova, A.V. Kuchin, 2017, published in Bioorganicheskaya Khimiya, 2017, Vol. 43, No. 3, pp. 301–310.
Synthesis and Membrane Protective Activity of Bis-Sulfides Derived
from Monoterpenoids and Monosaccharides
S. V. Pestovaa, 1, E. S. Izmest’eva, O. G. Shevchenkob, S. A. Rubtsovaa, and A. V. Kuchina
aInstitute of Chemistry, Komi Science Centre, Ural Division, Russian Academy of Sciences, Syktyvkar, 167987 Russia
bInstitute of Biology, Komi Science Centre, Ural Division, Russian Academy of Sciences, Syktyvkar, 167000 Russia
Received June 1, 2016; in final form, June 20, 2016
Abstract⎯Hydroxyl- and chloroethyl derivatives of neomenthane- and isobornanethiol in yields of to 80% were
synthesized. They served as the basis for the preparation of new bis-sulfides with diacetone-protected galacto- and
fructopyranose fragments in yields up to 98%. The bis-sulfides synthesized were screened for membrane protective
and antioxidant properties in a model cell system (in vitro) based on their ability to inhibit the H2O2-induced
hemolysis of erythrocytes and retard the oxyhemoglobin oxidation.
Keywords: antioxidant, bis-sulfides, erythrocytes, membrane-protective activity, monosaccharide, oxidative hemolysis
DOI: 10.1134/S1068162017030141
INTRODUCTION
nontoxic or low-toxic fragments we first synthesized
bis-sulfides derived from monoterpenoids and
monosaccharides.
A wide application of sulfur-containing com-
pounds is based on their ability to stabilize polymeric
materials by inhibition of oxidative processes. A con-
siderable number of sulfur-containing inhibitors are
represented by bis-sulfides containing two sulfur atoms
separated by a hydrocarbon bridge [1]. The bis-sulfides
bearing glycoside fragments are widely used in the
organic synthesis of functionalities for the preparation of
dendrimers as artificial glycoproteins [2], photosensitiz-
ers [3], as well as in the synthesis of the agents with anti-
tuberculous [4] and antidiabetic [5] activities. Two sulfur
atoms, which can be involved in oxidation reactions, can
also provide antioxidant properties stipulated by the anti-
RESULTS AND DICUSSION
Previously, we obtained sulfides, sulfoxides [8, 9], and
disulfides [10] with various monoterpenoid and mono-
saccharide moieties. As a continuation of these studies,
we synthesized nonsymmetrical bis-sulfides (III) with
two sulfur atoms linked by an ethane bridge by the inter-
action of terpenyl sulfanyl ethyl chlorides (IА) or (IB)
with monosaccharide thiols (IIа) or (IIb) in different
combinations in the presence of a Cs2CO3/TBAI cata-
peroxide activity of sulfur-containing groups [6, 7]. An lytic system (Scheme 1). The yields of bis-sulfides were
essential factor supporting the use of bis-sulfides with maximal for neomenthane-bearing compounds (98%)
glycoside fragments in pharmacology is their low toxicity. and minimal in the case of isobornane- and diisopro-
Due to the necessity to develop methods of prepara- pylidenefructopyranose-derived compounds (63%)
tion of biologically active compounds bearing natural without regard to the monosaccharide component.
1" 2"
Cs2CO3, TBAI
S R
(IIIAa), (IIIAb), (IIIBa), (IIIBb)
+
R-SH
Terp S
Cl
Terp
S
C2H5OH
(IA, B)
(IIa, b)
7'
10'
9
7
1'
*
∗
5
10 O
6
O
7'
5' 3'
5'
5
3 O
O
O
7'
9'
Terp =
R =
O
3'
3
,
*
1
7
O
*
1'
8'
11
,
10
O
O
1
O
11
9
10'
A
B
b
a
Scheme 1.
1
Corresponding author: phone: +7 (821)224-1045; fax: +7 (821) 221-8477; e-mail: pestova-sv@chemi.komisc.ru.
Abbreviations: AAPH, 2,2-azo-bis(amidinopropane) dihydrochloride; TBAI, tetrabutylammonium iodide.
302

SYNTHESIS AND MEMBRANE PROTECTIVE ACTIVITY
303
1
(Scheme 2). Alcohols (VI) were synthesized by the
reaction of tosylates with 2-mercaptoethanol in the
presence of a base. The treatment of hydroxyethyl sul-
fides (VIА, В) with thionyl chloride in ether in the
presence of a catalytic quantity of pyridine resulted in
chlorides (IА, В). In their 13C NMR spectra an upfield
shift by ~17 ppm of the carbon resonance of the meth-
ylene group directly linked to the chlorine atom was
observed if compared with the resonances of similar
The H NMR spectra of bis-sulfides (III) con-
tained proton resonances characteristic for the mono-
saccharide fragments protected by isopropylidene
groups (а, b). If compared with starting chlorides (IА,
В) the resonances of bis-sulfides (III) C2'' protons
(3.60–3.69 ppm) shifted upfield (2.60–2.94 ppm),
whereas in 13C NMR spectra the resonance of C2''
atoms shifted upfield by ~10 ppm.
Terpenyl sulfanyl ethyl chlorides (IА) and (IB) were
obtained from L-menthol (IVА) and borneol (IVB)
via terpene tosylates (VА, В) and alcohols (VIА, В) atoms in alcohols (VIА, В).
HS
OH
SOCl2
Et2O
TsCl
Py
Cl
OH
Terp S
Terp S
Terp-OH
Terp-OTs
NaOH, DMF
(IVA, B)
(VA, B)
(IA, B)
(VIA, B)
TsCl
Py
R-OH
(VIIa)
R-OTs
(VIIIa)
LiAlH4
THF
R-SH
(IIa, b)
Terp, R =
R-SAc
(IXa, b)
*
I2, Ph3P
R-I
R-OH
(VIIb)
BzIm, PhMe
(VIIIb)
Scheme 2. * For terpene and monosaccharide fragments see Scheme 1.
Carbohydrate thiols (IIа) and (IIb) were synthe- the previously obtained data [10]. The presence of an
sized from isopropylidene-protected monosaccha- ethane bridge separating sulfur atoms resulted in an
rides (VIIа, b) via intermediate tosylate/iodide essential decrease in the hemolytic activity of the com-
(VIIIа)/(VIIIb) followed by the reduction of thioace- pounds. It is noteworthy that, in general, the bis-sul-
tates (IXа, b) obtained from them.
fides containing a neomenthane fragment (А) were
more toxic than similar compounds with an isobor-
nane moiety (B) (Fig. 1).
The primary evaluation of the biological activity of
the compounds under study was performed in vitro
with blood erythrocytes of laboratory animals. The
previously obtained sulfide (A-S-a) [8] and disulfide
(A-S-S-a) [10] bearing neomenthane (А) and diiso-
propylidenegalactopyranose (а) moiety were used as
comparatives:
The results of the study of membrane-protective
activity under the conditions of oxidative stress due to
the addition of hydrogen peroxide are shown in Figs. 2
and 3.
The analysis of the data in the Fig. 2 showed that
bis-sulfides with a diisopropylidenegalactopyranose
moiety (IIIAa) and (IIIBa) were somewhat more
active than the similar compounds containing a diiso-
propylidenefructose fragment (IIIAb) and (IIIBb),
although the difference was not always statistically sig-
nificant. The same trend was also found for the mem-
brane protective activity of the disulfides synthesized
previously and lacking a spacer between the sulfur
atoms [10].
S
S
S
O
O
O
O
O
O
O
O
O
O
A-S-S-a
A-S-a
The comparison of the compounds bearing both
neomenthane and diisopropylidenegalactopyranose
moieties demonstrated that the number of sulfur atoms
and the presence of an ethane bridge between them
impacted the membrane protective activity (Fig. 3). Bis-
sulfide (IIIAa) with a dimethylene spacer between the
sulfur atoms was the most active compound. Disulfide
(A-S-S-a) lacking this bridge was less active, although
The goal of the comparison was the evaluation of
the dependence of membrane-protective and antioxi-
dant properties on the number and position of sulfur
atoms.
Since the potential toxicity of the bis-sulfides and
the comparatives could considerably limit their use,
we first assessed their hemolytic activity.
Among the compounds tested, disulfide (A-S-S-a) the difference could be due to a considerably higher
demonstrated the highest toxicity (Fig. 1), which met hemolytic activity of the latter.
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304
PESTOVA et al.
10
0
5
15
20
25
Control
IIIAa
1
3
5
IIIBa
IIIAb
IIIBb
А-S-a
A-S-S-a
Hemolysis, %
Fig. 1. Hemolytic activities of the compounds at a concentration of 10 μM (ethanol solutions; 1, 3, and 5 h incubation).
Hemolysis, %
50
1
2
3
4
5
40
30
20
10
0
Control
IIIAa
IIIBa
IIIAb
IIIBb
Fig. 2. The effect of bis-sulfides (IIIAa), (IIIBa), (IIIAb), and (IIIBb) at a concentration of 10 μM on the degree of Н О -induced
2
2
erythrocyte hemolysis after 1–5 h incubation.
The next studies of antioxidant and membrane
According to the data in Fig. 4, all the four bis-sul-
fides (III) displayed a high membrane protective
activity relative to a peroxide radical without regard to
the structures of carbohydrate and terpene moieties.
protective properties of the compounds synthesized
were performed with ААРН as a source of radicals. It
is known that AAPH can form peroxide radicals upon
thermal degradation in an aqueous phase at physio-
logical temperatures. Unlike hydrogen peroxide, it
cannot penetrate cells and affect erythrocytes from the
outer membrane surface [11]. The primary target of
the Н2О2 action in erythrocytes is intracellular hemo-
globin, whereas in the case of AAPH the primary
effect is the free radical-induced damage of plasmatic
membrane.
At the same time, the number of sulfur atoms and
the spacer between them had a noticeable impact on
the biological activity of the compounds synthesized
(Fig. 5). The analysis of the dynamics of the hemolytic
process showed that bis-sulfide (IIIAa) containing
two sulfur atoms separated by a methylene bridge was
the most active, and compound (A-S-a) with one sul-
fur atom, the least active.
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SYNTHESIS AND MEMBRANE PROTECTIVE ACTIVITY
305
Hemolysis, %
50
1
2
3
4
5
40
30
20
10
0
Control
IIIAa
A-S-a
A-S-S-a
Fig. 3. The effect of (IIIAa), (A-S-a), and (A-S-S-a) at a concentration of 10 μM on the degree of Н О -induced erythrocyte
2
2
hemolysis after 1–5 h incubation.
The assessment of the cell death dynamics under hemolytic activity than structurally similar com-
conditions of acute oxidative stress allowed the con- pounds with an isobornyl group. The introduction of a
clusion that in general the compounds under study are dimethylene bridge between the sulfur atoms resulted
only active at early stages and have low activity in the in a decreased toxicity of the bis-sulfides if compared
final stage of the process.
with the corresponding disulfides lacking this frag-
ment. Under the conditions of oxidative stress induced
by a hydroxyl radical, bis-sulfides with a galactose moiety
were distinguished by a higher membrane protective
activity than the corresponding compounds with a fruc-
tose moiety. A similar dependency was also found upon
the analysis of the membrane protective activity of the
corresponding disulfides synthesized previously and
lacking a spacer between the sulfur atoms.
A low activity of the sulfide if compared with those
of disulfide and bis-sulfide can be explained by the
capacity of compounds with two sulfur atoms to dis-
play a higher antioxidant effect. At the same time, the
bis-sulfide, in which two sulfur atoms are separated by
a bridge, manifested a higher membrane protective
activity if compared with the disulfide, because the
effect of an electron acceptor group SO formed upon
the oxidation of one of the sulfide groups of com-
We showed that the number of sulfur atoms and the
pound (IIIAa) on the other sulfur atom was lower than presence of a spacer between them in the compounds
that in disulfide (А-S-S-a).
containing neomenthane and galactose fragments,
with all else being equal, had an impact on their mem-
brane protective and antioxidant activities. Bis-sulfide
(IIIAa) containing sulfur atoms separated by a
dimethylene spacer was the most active compound in
the model cell system of the acute oxidative stress
induced by either hydroxyl or peroxide radicals.
The same regularity was observed upon the analysis
of AOA evaluated by the ratio of the native (oxyhemo-
globin (oxyHb)) and oxidized hemoglobin forms
(methemoglobin (metHb) and ferryl hemoglobin (fer-
rylHb)) in erythrocyte hemolysates after induction of
the AAPH-induced oxidative stress (Fig. 6). Bis-sul-
fides (III) bearing two sulfur atoms separated by a
spacer were characterized by a higher AOA if com-
pared with a similar sulfide (A-S-a).
To summarize, we developed the methods of syn-
thesis of bis-sulfides containing terpene and mono-
saccharide fragments as well as terpene alcohols and
chlorides used for the preparation of these bis-sulfides.
The composition and structures of terpene thioetha-
nols, thioethyl chlorides, and bis-sulfides of various
EXPERIMENTAL
IR spectra were recorded on an IR-Fourier spec-
trometer in a thin film or KBr tablets. Melting points
1
were measured on a Gallencamp-Sanyo device. H
13
and C NMR spectra (δ, ppm; J, Hz) were recorded
on a Bruker Avance-300 spectrometer (300.17 MHz
for 1Н and 75.48 MHz for 13С) in CDCl3; chloroform
structures were confirmed by NMR and IR spectros- resonances as an internal standard. The complete
copy and element analysis.
1
assignment of H and 13C resonances was conducted
using dimeric homo-(1H–1H COSY, 1H–1H
The analysis of the results demonstrated the trend,
according to which the compounds with a neomen- NOESY) and heteronuclear (1H–13C HSQC, 1H–13C
thane fragment (А) were characterized by a higher HMBC) experiments. Optical rotation was measured
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 43 No. 3 2017

306
PESTOVA et al.
Hemolysis, %
100
90
80
70
60
50
40
30
20
10
0
1
2
3
4
5
Control
IIIAa
IIIBa
IIIAb
IIIBb
Fig. 4. The effect of bis-sulfides (IIIAa), (IIIBa), (IIIAb), and (IIIBb) at a concentration of 10 μM on the degree of ААРН-
induced erythrocyte hemolysis after 1–5 h incubation.
Hemolysis, %
100
1
2
3
4
5
90
80
70
60
50
40
30
20
10
0
Control
IIIAa
A-S-a
A-S-S-a
Fig. 5. The effect of (IIIAa), (A-S-a), and (A-S-S-a) at a concentration of 10 μM on the degree of ААРН-induced erythrocyte
hemolysis after 1–5 h incubation.
on an automatic digital P3002RS polarimeter (Kruss).
TLC was performed on Sorbfil plates eluting with a
CHCl3–Et2O mixture at various ratios; the com-
pounds were developed using phosphormolybdic acid
and KMnO4. Element analysis was carried out on an
automatic ЕА 1110 CHNS-O device. All the reactions
were performed in freshly distilled solvents. Column
chromatography was carried out on Alfa Aesar silica gel
(0.06–0.2 mm) in the same solvent systems as for TLC.
2-(((1S,2S,5R)-2-Isopropyl-5-methylcyclohexyl)-
thio)ethanol (VIА) and 2-(((1S,2S,4S)-1,7,7-trimeth-
ylbicyclo[2.2.1]hept-2-yl)thio)ethanol (VIB) were syn-
thesized by the reaction of 2-mercaptoethanol with p-
toluenesulfonates (V) in the presence of NaOH [13].
2-(((1S,2S,5R)-2-Isopropyl-5-methylcyclohexyl)-
thio)ethanol (VIА). The yield 78%. Viscous yellow liq-
20
uid,
+ 91.1 (с 1.30; CHCl3), Rf 0.41 (CHCl3–
[α]D
Et2O, 10 : 1). IR, ν, cm–1: 690 (C–S), 1047 (CН2OН),
1452, 2918, 3369 (OH). 1H NMR: 0.84–0.94 (m, 1Н,
L-menthol p-toluenesulfonate (VA) and borneol p-tolue-
nesulfonate (VB) were obtained as described in [12].
H4'e), 0.91 (d 3H, Me(7'), J 6.0), 0.93 (d, 3H,
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SYNTHESIS AND MEMBRANE PROTECTIVE ACTIVITY
307
2.5
2.0
1.5
1.0
0.5
0
metHb/oxyHb
ferrylHb/oxyHb
Control
IIIAa
IIIBa
IIIAb
IIIBb
A-S-a
A-S-S-a
Fig. 6. The effect of (IIIAa), (IIIBa), (IIIAb), (IIIBb), (A-S-a), and (A-S-S-a) at a concentration of 10 μM on the ratio of native
and oxidized hemoglobin forms in erythrocyte hemolysates under conditions of the oxidative stress in the presence of AAPH.
1
(CHCl3). IR, ν, cm–1: 700 (C–Cl), 1450, 2916. H
Me(10'), J 6.0), 0.97 (d, 3H, Me(9'), J 6.4), 1.08–1.37
(m, 3H, H2', H3'e, H6'e), 1.64–1.85 (m, 3H, Н8',
Н3'a, Н4'а), 1.87–2.06 (m, 2H, Н5', Н6'a), 2.37 (s,
1H, OH), 2.79 (t, 2H, H1'', J 5.9), 3.14–3.26 (m, 1H,
H1'), 3.73 (t, 2Н, Н2'', J 5.9). 13C NMR: 20.80 (С10'),
21.02 (С9'), 22.17 (С7'), 25.86 (С3'), 26.41 (C5'),
29.94 (C8'), 34.99 (С1''), 35.34 (С4'), 40.99 (С6'),
46.50 (С1'), 48.90 (С2'), 60.46 (С2''). Found, %: C,
66.35; H, 11.75; S, 14.83. C12H24OS. Calculated, %: C,
66.61; H, 11.18; S, 14.82.
NMR: 0.82–0.95 (m, 1Н, H4'e), 0.91 (d, 3H, Me(7'),
J 6.3), 0.93 (d, 3H, Me(10'), J 6.6), 0.97 (d, 3H,
Me(9'), J 6.6), 1.05–1.35 (m, 3H, H2', H3'e, H6'e),
1.58–1.81 (m, 3H, Н8', Н3'a, Н4'а), 1.87–2.05 (m,
2H, Н5', Н6'a), 2.80–2.96 (m, 2H, H1''), 3.15–3.30
(m, 1H, H1'), 3.60–3.69 (m, 2Н, Н2''). 13C NMR:
20.77 (С10'), 21.02 (С9'), 22.14 (С7'), 25.81 (С3'),
26.32 C(5'), 29.92 C(8'), 33.75 С(1''), 35.31 С(4'),
41.03 С(6'), 43.24 С(2''), 47.24 С(1'), 48.88 С(2').
Found, %: C, 61.42; H, 9.91; S, 13.76. C12H23SCl.
Calculated, %: C, 61.38; H, 9.87; S, 13.65.
2-(((1S,2S,4S)-1,7,7-Trimethylbicyclo[2.2.1]
hept-2-yl)thio)ethanol (VIB). The yield 75%. Viscous
20
((2-Chloroethyl)-((1S,2S,4S)-1,7,7-trimethylbicy-
clo[2.2.1]hept-2-yl)sulfide (IВ). The yield 80%. Vis-
yellow liquid,
+ 248.0 (с 0.30; CDCl3), Rf 0.29
[α]D
(CHCl3–Et2O, 10 : 1). IR, ν, cm–1: 677 (C–S), 1049
cous yellow liquid, 20 +21.1 (с 0.70; CDCl3), Rf 0.65
[α]D
(СН2ОН), 1456, 2951, 3385 (OH). 1H NMR: 0.86 (s,
3H, Me(9'), 1.00 (s, 3H, Me(8'), 1.03 (s, 3H, Me(10'),
1.13–1.25 (m, 2H, Н5'en, Н6'en)), 1.67–1.82 (m, 3H,
Н5'ex, Н6'ex, Н4'), 1.86–1.97 (m 2H, H3), 2.24 (s,
1H, OH), 2.60–2.69 (m, 1H, H2'), 2.72–2.82 (m, 2H,
H1''), 3.73 (t, 2Н, Н2'', J 5.9). 13C NMR: 14.01 (С10'),
20.17 (С8'), 20.44 (С9'), 27.33 (С5'), 38.00 (С6'),
38.52 (С3'), 41.23 (С1''), 45.90 (С4'), 47.32 (С1'),
49.46 (С7'), 54.39 (С2'), 60.32 (С2''). Found, %: C,
67.26; H, 10.39; S, 14.85. C12H22OS. Calculated, %:
C, 67.23; H, 10.34; S, 14.96.
1
(CHCl3). IR, ν, cm–1: 702 (C–Cl), 1453, 2920. H
NMR: 0.86 (s, 3H, Me(9')), 0.97 (s, 3H, Me(8')), 1.03
(s, 3H, Me(10')), 1.14–1.23 (m, 2H, Н5'en, Н6'en),
1.68–1.79 (m, 3H, Н5'ex), Н6'ex, Н4'), 1.88–1.98 (m,
2H, H3'), 2.62–2.70 (m, 1H, H2'), 2.84–2.94 (m, 2H,
H1''), 3.64 (t, 2Н, Н2'', J 8.1). 13C NMR: 14.04 С(10'),
20.15 С(8'), 20.41 С(9'), 27.34 С(5'), 36.90 С(6'),
38.52 С(3'), 41.37 С(1''), 43.17 С(2''), 45.89 С(4'),
47.32 С(1'), 49.57 С(7'), 55.45 С(2'). Found, %: C,
63.26; H, 8.65; S, 13.11. C12H21SСl. Calculated, %: C,
61.91; H, 9.09; S, 13.77.
(2-Chloroethyl)-((1S,2S,5R)-2-isopropyl-5-meth-
ylcyclohexyl)sulfide (IА)
and
(2-chloroethyl)-
1,2:3,4-Di-O-isopropylidene-6-О-(p-toluenesul-
fonyl)-α-D-galatopyranose (VIIIа) was synthesized as
((1S,2S,4S)-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl)-
sulfide (IВ) were obtained by the reaction of alcohols
with thionyl chloride used for the synthesis of chlo-
rides [14].
22
described in [15]. The yield 98%;
– 44.6 (с 0.30,
– 44.0 (с 0.28,
[α]D
22
CHCl ); ref. [15]: the yield 87%,
CHCl33).
[α]D
(2-Chloroethyl)-((1S,2S,5R)-2-isopropyl-5-meth-
ylcyclohexyl)sulfide (IА). The yield 78%. Viscous yel-
6-Deoxy-6-thioacetyl-1,2:3,4-di-O-isopropylidene-
α-D-galactopyranose (IXа) was obtained as described
[α]2D0
low liquid,
+19.0 (с 0.70; CDCl3), Rf 0.80
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PESTOVA et al.
22
96.64 C(1), 108.60 C(7), 109.28 C(10). Found, %: C,
in [16]. The yield 77%,
– 15.6 (с 0.30, CHCl3);
[α]D
62.35; H, 8.80; S, 13.72. C24H42S2O5. Calculated, %:
C, 60.72; H, 8.92; S, 13.51.
22
ref. [16]: yield 99%,
– 15.0 (с 1.80, CHCl3).
[α]D
1-Deoxy-1-iodo-2,3:4,5-di-O-isopropylidene-β-
1-[((1S,2S,4S)-1,7,7-Trimethylbicyclo[2.2.1]-
hept2-yl)sulfanyl]-2-(6-deoxy-1,2:3,4-di-O-isopro-
pylidene-α-D-galactopyranosyl-6-sulfanyl)ethane (IIIBa).
D-fructopyranose (VIIIb) was obtained as described in
22
[17]. The yield 64%,
– 47.6 (с 0.88, СНCl3).
1-Deoxy-1-thioacetyl-2,3:4,5-di-O-isopropylidene-
β-D-fructopyranose (IXb) was synthesized as
described in [18]. The yield 72%,
(CH ) CO); ref. [18]: the yield 76%, 22 –2.9 (с 1.00,
[α]D
[α]2D0
The yield 80%. Viscous yellow liquid,
–28.5
(с 0.50; CHCl3), Rf 0.51 (CHCl3–Et2O, 60 : 1). IR, ν,
cm–1: 646 (C–S), 1071 (С–О), 1169 (О–С–О), 2951.
1H NMR: 0.84 (s, 3H, Me(9')), 0.97 (s, 3H, Me(8')),
1.01 (s, 3H, Me(10')), 1.10–1.23 (m, 2H, Н5'en,
Н6'en), 1.36 (s, 3H, Me(8)), 1.38 (s, 3H, Me(12)),
1.48 (s, 3H, Me(11)), 1.57 (s, 3H, Me(9)), 1.66–1.78
(m, 3H, Н5'ex, Н6'ex, Н4'), 1.84–1.99 (m, 2H, H3'),
2.62–2.90 (m, 7H, Н2', Н2'', H1'', Н6), 3.84–3.94
(m, 1Н, Н5), 4.27–4,38 (m, 2Н, Н2, H3), 4.65 (dd,
1H, H4, J 7.8, 2.3), 5.55 (d, 1Н, Н1, J 5.0). 13C NMR:
14.01 С(10'), 20.20 С(8'), 20.42 С(9'), 24.47 С(12),
24.92 C(8), 26.00 C(11), 26.16C(9), 27.36 С(5'), 31.82
С(2''), 32.92 С(6), 34.76 С(1''), 38.53 С(6'), 41.19
С(3'), 45.90 С(4'), 47.33 С(1'), 49.48 С(7'), 54.93
С(2'), 68.02 C(5), 70.54 C(2), 70.90 С(4), 71.60 C(3),
96.64 C(1), 108.60 C(7), 109.28 C(10). Found, %: C,
61.27; H, 8.55; S, 13.54. C24H40S2O5. Calculated, %:
C, 60.98; H, 8.53; S, 13.57.
22
–3.1 (с 0.95,
[α]D
[α]D
3 2
СНCl3).
6-Deoxy-6-thio-1,2:3,4-di-O-isopropylidene-α-D-
galactopyranose (IIa) was obtained as described in
[19]. Viscous yellow liquid. The yield 91%, 22 – 70.0
[α]D
(c 0.47, CHCl3), ref. [19]: the yield 70%, – 88.7 (con-
centration not given, СНCl3).
1-Deoxy-1-thio-2,3:4,5-di-O-isopropylidene-β-
D-fructopyranose (IIb) was obtained as described in
[α]2D2
[20]. White powder. The yield 94%,
(c 0.40, CHCl3); ref. [20]: the yield 65%, not shown.
– 44.0
Monoterpene- and monosaccharide derived bis-sul-
fides. A carbohydrate-containing thiol (0.359 g,
1.3 mmol), cesium carbonate (0.424 g, 1.3 mmol), and
tetrabutylammonium iodide (0.480 g, 1.3 mmol) were
dissolved in EtOH (3 mL) under stirring in an argon
atmosphere. Terpenyl sulfanyl ethyl chlorides (IА) or
(IB) (1.0 mmol) were added in 5 min and the mixture
was refluxed for 6 h. The solvent was removed in vac-
uum and the product was isolated by column chroma-
tography on silica gel eluting with 60 : 1 CHCl3–Et2O.
1-((1S,2S,5R)-2-Isopropyl-5-methylcyclohexyl-
sulfanyl)-2-(1-deoxy-2,3:4,5-di-O-isopropylidene-β-
D-fructopyranosyl-1-sulfanyl)ethane (IIIAb). The
20
yield 98%. Viscous yellow liquid,
+10.6 (с 1.00;
[α]D
CHCl3), Rf 0.42 (CHCl3–Et2O, 60 : 1). IR, ν, cm–1:
1
640 (C–S), 1063 (C–O), 1167 (О–С–О), 2918. H
NMR: 0.89 (d, 3H, Me(7'), J 6.3), 0.92 (d, 3H,
Me(9'), J 6.3), 0.84–0.94 (m, 1H, H4'e), 0.96 (d, 3H,
Me(10'), J 6.6), 1.05–1.25 (d, 3H, H2', H3'e, H6'e),
1.37 (s, 3H, Me(12)), 1.48 (s, 3H, Me(8)), 1.50 (s, 3H,
Me(11)), 1.56 (s, 3H, Me(9)), 1.59–1.80 (m, 3H, H8',
H3'a, H4a'), 1.85–2.06 (m, 2H, Н5', H6'a), 2.68–
2.94 (m, 4H, Н2'', H1''), 2.84 (d, 1H, Н1А, J 13.8),
3.10 (d, 1H, Н1B, J 13.8), 3.14–3.20 (m, 1Н, H1'),
4.30 (d, 1Н, Н6А, J 13.0), 4.63 (d, 1Н, Н6B, J 12.6),
4.26 (d, 1H, H5, J 7.7), 4.34 (d, 1H, H3, J 2.5), 4.63
(dd, 1H, H4, J 8.0, 2.5). 13C NMR: 20.80 С(10'), 21.06
С(9'), 22.18 С(7'), 24.07 С(12), 25.49 С(10), 25.93
C(3'), 25.98 C(5'), 26.37 C(11), 26.63 C(9), 29.94
C(8'), 31.50 C(2''), 24.16 C(1''), 35.37 С(4'), 40.14
С(6'), 40.95 С(1), 46.94 С(1'), 48.94 С(2'), 61.68
С(6), 70.39 C(4), 70.89 С(5), 72.25 C(3), 103.64
C(2), 108.55 C(7), 109.06 C(10). Found, %: C, 63.10;
H, 8.77; S, 13.42. C24H42S2O5. Calculated, %: C,
60.72; H, 8.92; S, 13.51.
1-((1S,2S,5R)-2-Isopropyl-5-methylcyclohexyl-
sulfanyl)-2-(6-deoxy-1,2:3,4-di-O-isopropylidene-α-
D-galactopyranosyl-6-sulfanyl)ethane (IIIАа). The
20
yield 98%. Viscous yellow liquid,
– 8.0 (с 0.80;
[α]D
CHCl3), Rf 0.37 (CHCl3–Et2O, 60 : 1). IR, ν, cm–1:
646 (C–S), 999, 1071(C–O), 1169 (O–C–O), 1211,
1
2916. H NMR: 0.83–0.94 (m, 1Н, H4'e), 0.90 (d,
3H, Me(7'), J 6.6), 0.92 (d, 3H, Me(10'), J 6.6), 0.97
(d, 3H, Me(9'), J 6.6), 1.07–1.32 (m, 3H, H2', H6'e,
H3'e), 1.36 (с, 3H, Me(8)), 1.38 (s, 3H, Me(12)), 1.48
(s, 3H, Me(11)), 1.57 (s, 3H, Me(9)), 1.66–1.80 (m,
3H, Н4'а, Н8', Н3'a), 1.87–2.10 (m, 1H, Н5'), 1.88–
2.03 (m, 1H, Н6'a), 2.67–2.89 (m, 6H, H6, H1'',
H2''), 3.14–3.24 (m, 1H, H1'), 3.84–3.95 (m, 1H,
H5), 4.31–4.42 (m, 2Н, Н2, Н3), 4.65 (dd, 1H, H4, J
8.0, 2.2), 5.56 (d, 1Н, Н1, J 5.0). 13C NMR: 20.83
С(10'), 21.05 С(9'), 22.18 С(7'), 24.48 С(12), 24.92
С(8), 25.91 С(3'), 25.98 C(11), 25.99 C(5'), 26.36
C(9), 29.94 C(8'), 31.68 С(1''), 31.85 С(2''), 33.01
1-[((1S,2S,4S)-1,7,7-Trimethylbicyclo[2.2.1]hept-2-
С(6), 35.37 С(4'), 40.95 С(6'), 46.82 С(1'), 48.92 yl)sulfanyl]-2-(1-deoxy-2,3:4,5-di-O-isopropylidene-
С(2'), 68.00 С(5), 70.54 C(2), 70.91 С(4), 71.56 C(3), β-D-fructopyranosyl-1-sulfanyl)ethane (IIIBb). The
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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SYNTHESIS AND MEMBRANE PROTECTIVE ACTIVITY
309
20
forms (oxyHb, metHb, and ferrylHb) were calculated
based on the proper absorption coefficients [22]. Each
experiment was performed with five or six repeats. Sta-
tistical treatment and diagram plotting were carried
out with Microsoft Office Excel 2007 software.
yield 63%. Viscous yellow liquid,
–12.4 (с 0.34;
[α]D
CHCl3), Rf 0.36 (CHCl3–Et2O, 60 : 1). IR, ν, cm–1:
579 (C–S), 1063 (С–О), 1132, 1167 (О–С–О), 2941.
1H NMR: 0.84 (s, 3H, Me(8')), 0.96 (s, 3H, Me(9')),
1.01 (s, 3H, Me(10')), 1.10–1.22 (m, 2H, Н5'en,
Н6'en), 1.37 (s, 3H, Me(12)), 1.48 (s, 3H, Me(8)),
1.50 (s, 3H, Me(11)), 1.56 (s, 3H, Me(9)), 1.64–1.76
(m, 3H, Н5'ex, Н6'ex, Н4'), 1.83–1.94 (m, 2H, H3'),
2.61–2.91 (m, 6H, H2', H1'', H1А, H2''), 3.09 (d, 1H,
H1B, J 13.7), 3.79 (d, 1H, H6А, J 13.2), 3.94 (dd, 1H,
H6B, J 12.9, 1.4), 4.25 (d, 1Н, Н5 J 7.7), 4.33 (d, 1Н,
Н3, J 2.5), 4.63 (dd, 1H, H4, J 7.8, 2.6). 13C NMR:
14.00 (С10'), 20.19 С(8'), 20.41 С(9'), 24.05 С(12),
25.50 C(8), 25.97 C(11), 26.63 C(9), 26.36 С(5'),
34.10 С(2''), 34.53 С(1''), 38.51 С(6'), 39.98 C(1),
41.23 С(3'), 45.90 С(4'), 47.29 С(1'), 49.45 С(7'),
55.02 С(2'), 61.68 C(6), 70.38 С(4), 70.88 С(5), 72.21
(C3), 103.65 (C2), 108.54 C(7), 109.06 C(10). Found,
%: C, 61.78; H, 8.40; S, 13.28. C24H40O5S2. Calcu-
lated, %: C, 60.98; H, 8.53; S, 13.57.
For the evaluation of toxicity and antioxidant and
membrane protective activities of the compounds
under study we used 0.5% (v/v) suspension of mouse
erythrocytes in a phosphate buffer (PBS, рН 7.4).
Ethanol was used as a solvent. The in vitro toxicity of
the compounds under study was assessed by their
capacity to induce hemolysis. The ethanol solutions of
the compounds synthesized were added to an erythro-
cyte suspension and the mixture was incubated at 37°С
for 5 h in a temperature-controlled Biosan ES-20
shaker (Latvia). The control samples contained the
same volume of ethanol (0.1%). The membrane pro-
tective and antioxidant activities were evaluated by the
inhibition degree of the induced hemolysis, inhibition
of accumulation of secondary lipid peroxide products,
and oxyhemoglobin oxidation in erythrocytes. To this
end, the hemolysis was initiated 30 min after the addi-
tion of the tested solution into erythrocyte suspension
using 0.006% hydrogen peroxide or 5 mM AAPH. The
reaction mixture was incubated at 37°С for 5 h in a
temperature-controlled shaker under slow stirring.
ACKNOWLEDGMENTS
The authors are grateful to the researches of the
laboratory of physicochemical methods of the Insti-
tute of Chemistry, Komi Science Centre, Ural Divi-
sion, Russian Academy of Sciences, E. N. Zainullina,
S. P. Kuznetsov, and I. N. Alekseev for the registration
of NMR spectra and E.U. Ipatova, for IR spectra.
The work was supported by the Russian Founda-
tion for Basic Research, project no. 16-03-01064_а
and the Ural Division of Russian Academy of Sci-
ences, project no. 15-21-3-16.
The studies were performed using the equipment of
the Center of Collective Use “Chemistry” of the Insti-
tute of Chemistry, Komi Science Centre, Ural Divi-
sion, Russian Academy of Sciences. The studies of
biological activities were conducted in the Center of
Collective Use “Molecular Biology” of the Institute of
Biology, Komi Science Centre, Ural Division, Rus-
sian Academy of Sciences. The mice of the scientific
collection of experimental animals of Institute of Biol-
ogy of Komi Scientific Centre of Ural of the RAS were
used (http://www.ckp-rf.ru/usu/471933/).
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