Separation of racemic ortho-isobornylphenol into enantiomers and evaluation of their antioxidant activity

Article abstract of DOI:10.1134/S1068162011050049

Racemic ortho-isobornylphenol was separated into enantiomers through diastereomeric camphanates. The absolute configuration of chiral centers of the isolated products was determined by X-ray studies. Antioxidant activity and membrane-protective properties

Full text of DOI:10.1134/S1068162011050049

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2011, Vol. 37, No. 5, pp. 614–618. © Pleiades Publishing, Ltd., 2011.
Original Russian Text © E.V. Buravlev, I.Yu. Chukicheva, O.G. Shevchenko, K.Yu. Suponitsky, A.V. Kutchin, 2011, published in Bioorganicheskaya Khimiya, 2011, Vol. 37, No. 5,
pp. 685–689.
Separation of Racemic orthoꢀIsobornylphenol into Enantiomers
and Evaluation of Their Antioxidant Activity
, 1
E. V. Buravleva , I. Yu. Chukichevaa, O. G. Shevchenkob, K. Yu. Suponitskyc, and A. V. Kutchina
aInstitute of Chemistry, Komi Research Center, Ural Branch, Russian Academy of Sciences,
ul. Pervomaiskaya 48, Syktyvkar, GSPꢀ2, 167982 Russia
bInstitute of Biology, Komi Research Center, Ural Branch, Russian Academy of Sciences, Syktyvkar, 167000 Russia
cNesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences,
ul. Vavilova 28, Moscow, GSPꢀ1, 119991 Russia
Received February 9, 2011; in final form March 24, 2011
Abstract—Racemic orthoꢀisobornylphenol was separated into enantiomers through diastereomeric camphaꢀ
nates. The absolute configuration of chiral centers of the isolated products was determined by Xꢀray studies.
Antioxidant activity and membraneꢀprotective properties of the enantiomers were studied on the model of
H2O2ꢀinduced hemolysis of erythrocytes.
Keywords: orthoꢀisobornylphenol, enantiomers, diastereomers, antioxidant activity, membraneꢀprotective properties
DOI: 10.1134/S1068162011050049
1
INTRODUCTION
chloride. The mixture of camphanates (II) and (III)
was fractionated by the column chromatography on
silica gel. These compounds were hydrolyzed, and
The biological activity of chiral phenols (for
example, αꢀtocopherol [1], paraꢀnonylphenols [2],
isomers (+)ꢀ(
I) and (–)ꢀ(I) were obtained with >95%
and Δ8ꢀtetrahydrocannabinols [3]) is known to be
determined by the configuration of their chiral cenꢀ
ters. Isobornylphenols have a chiral alkyl fragment in
their structure and are analogues of natural phenol
antioxidants. In this connection, we pioneered the
purity. The diastereomeric purity of derivatives (II
)
1
and (III) was determined by H NMR spectroscopy.
The enantiomeric purity of the separated phenols was
evaluated by HPLC (scheme).
separation of racemic orthoꢀisobornylphenol (I) into
enantiomers and the evaluation of their antioxidant
properties.
The absolute configuration of compound (II) was
determined on the basis of configuration of the startꢀ
ing (
relative configuration, which was evaluated by Xꢀray
diffraction analysis (Fig. 1). The ( ,2 ,4 ) configꢀ
1S)ꢀcamphanic acid chloride and data on the
RESULTS AND DISCUSSION
1
R S S
uration was attributed to the chiral centers of the
isobornyl fragment of camphanate (II) and phenol
The separation method of racemic compounds
through their diastereomeric derivatives remains
important for modern organic chemistry. For examꢀ
ple, several racemic phenol compounds were sepaꢀ
rated via their camphanates [4, 5], camphorꢀ10ꢀsulꢀ
fonates [6, 7], and amides [8]. In this study, we have
(+)ꢀ(
ate (III) and phenol (–)ꢀ(
,2 ,4 ) configuration.
I
) that was prepared from it. Hence, camphanꢀ
I
) had the opposite
(1S R R
The structure of the terpene fragment of comꢀ
separated the racꢀ(
sponding diastereomeric esters (II) and (III), which
were prepared with the use of ( )ꢀcamphanic acid
I) racemate through the correꢀ
pound (II) in a crystal is analogous to that of the
aminomethyl derivatives of orthoꢀisobornylphenols
that we observed previously [9]. The value of the
C13–C12–C17–C22 torsion angle was –19.8(3) deg,
and the fragment of the ester group was almost perꢀ
pendicular to a plane of the aromatic ring
[85.32(11) deg]. Thus, the molecular structure of
compound (II) was mainly determined by intramoꢀ
lecular rather then intermolecular interactions.
1
S
Abbreviations: APꢀTBA, active products of 2ꢀthiobarbituric
acid; BHT, butylhydroxytoluene or 2,6ꢀdiꢀtertꢀbutylꢀ4ꢀmethꢀ
ylphenol (phenolic antioxidant); DMAP, 4ꢀdimethylaminopyriꢀ
dine; ferrylHb, ferrylhemoglobin; LPO, lipid peroxidation;
metHb, methemoglobin; oxyHb, oxyhemoglobin; PBS, phosꢀ
phate buffered saline.
1
Corresponding author: phone: (8212)21ꢀ99ꢀ16; fax: (8212)21ꢀ
84ꢀ77; eꢀmail: buravlevꢀev@chemi.komisc.ru.
614

SEPARATION OF RACEMIC orthoꢀISOBORNYLPHENOL INTO ENANTIOMERS
615
C(20)
C(24)
C(21)
C(19)
C(23)
C(22)
C(25)
C(18)
C(26)
C(17)
C(2)
C(12)
O(4)
C(13)
C(3)
C(10)
O(1)
O(2)
C(1)
C(5)
O(3)
C(11)
C(4)
C(6)
C(7)
C(14)
C(9)
C(16)
C(15)
C(8)
Fig. 1. General view of molecule (II) where the atoms are presented as thermal ellipsoids of atomic displacements with 50% probꢀ
ability.
(4S)
OR
(2S)
(1R)
4
O
6'
3
5
6
(
II) R = (1S)ꢀcamphanoyl
OH
1'
ii
9
8
5'
4'
11'
7
12
Cl
(+)ꢀ(
I
) R = H
9'
11
i
8O'
2
+
13
14
7'
1
(4R
)
3'
10'
16
10
OR
15
O
(2R)
racꢀ(I)
(1S)
(
(–)ꢀ( ) R = H
III) R = (1
S)ꢀcamphanoyl
ii
I
Scheme. The reaction conditions: (
i)
Triethylamine, DMAP, PhMe, reflux 5 h, chromatography on silica gel
;
(
ii) KOH/tetrahydrofuran, room temperature, 15 h.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 37
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2011

616
BURAVLEV et al.
rylHb was found. The antioxidant and membraneꢀ
protective activities of orthoꢀisobornylphenol and its
enantiomers in the concentration of 50 M were comꢀ
parable to those of BHT (the comparative substance)
(table). We found no differences in the biological
70
60
50
40
30
20
10
Control
BHT
μ
racꢀ(
I
)
activity of enantiomers (+)ꢀ(
these experimental conditions.
I) and (–)ꢀ(I) under
(+)ꢀ(
(–)ꢀ(
I
)
I
)
EXPERIMENTAL
The 1Н NMR and 13C NMR spectra (
, Hz) were recorded on a Bruker Avance II 300 specꢀ
δ, ppm;
J
trometer (Germany) in CDCl3 at a working frequency
of 300 and 75.5 MHz, respectively. Chloroform was
used as an internal standard. The IR spectra were
recorded on a Shimadzu IR Prestige 21 Fourier specꢀ
trometer (Japan) in tables with KBr. The specific rotaꢀ
tion was measured on a Kruss Optronic P3002RS
polarimeter (Germany). Melting points were deterꢀ
mined on a Koffler table. We previously synthesized
0
1
2
3
4
5
Incubation time, h
Fig. 2. Inhibition of the H O ꢀinduced hemolysis of erythroꢀ
2
2
cytes by racꢀ(I), (+)ꢀ(I), and (–)ꢀ(I) (50 µM) in comparison
with BHT. Every point is an average value of 2–4 repetitions.
the racemic orthoꢀisobornylphenol, racꢀ(
I) [14].
Chloride of ( )ꢀcamphanic acid, triethylamine, and
1
S
The antioxidant activity and membraneꢀprotective
4ꢀdimethylaminopyridine (Alfa Aesar, Great Britain
and SigmaꢀAldrich, United States) were used for the
syntheses of derivatives (II) and (III). The reactions
were monitored by TLC on Sorbfil plates. The subꢀ
stances were detected by the treatment of the plates
with the solution of Bromocresol Purple. Silica gel 60
properties were determined for the different forms of
orthoꢀisobornylphenol ( ). The method of the induced
I
erythrocyte hemolysis was a suitable model for studies
of these properties in vitro [10–12]. Both racemic
phenol racꢀ(
and (–)ꢀ( ) were shown to exhibit a pronounced
hemolytic effect in the concentration of 100 M and
I) and its enantiomeric phenols (+)ꢀ(I)
I
(70/230
matography.
Synthesis of derivatives (II) and (III). Phenol
racꢀ( (1.16 g, 5.0 mmol), chloride of ( )ꢀcamꢀ
μ, Alfa Aesar) was used for the column chroꢀ
μ
have no membraneꢀprotective properties under the
H2O2ꢀinduced oxidative stress. Earlier, hemolytic
activity was observed for high concentrations of new
hybrid spatially hindered phenols, ichfanes [13].
I)
1S
phanic acid (1.19 g, 5.5 mmol), triethylamine
(0.77 ml, 5.5 mmol), and DMAP (0.06 g, 0.5 mmol)
were refluxed in toluene (50 ml) upon stirring in the
argon atmosphere for 5 h. The reaction mixture was
cooled to room temperature, and the quaternary
ammonium salt was filtered off. The residue was evapoꢀ
rated and fractionated on a column with silica gel (PhH
was used as an eluent). Compounds (II) and (III) were
obtained with yields of 0.47 g (23%) and 0.64 g (31%)
Compounds racꢀ(
inhibited the Н2О2ꢀinduced hemolysis of erythrocytes
in the concentration of 50 M (Fig. 2). Analysis of the
I), (+)ꢀ(I), and (–)ꢀ(I) effectively
μ
content of the secondary products of LPO 4 h after the
incubation with the examined substances demonꢀ
strated the significant decrease in the APꢀTBA conꢀ
tent in the erythrocyte suspension.
An inhibiting effect of the tested compounds on the and diastereomeric purity of >98% and >95%, respecꢀ
H2O2ꢀinduced oxidation of oxyHb, metHb, and ferꢀ tively.
Parameters of H2O2ꢀinduced hemolysis of erythrocytes and their changes after the action of racꢀ(I), (+)ꢀ(I), and (–)ꢀ(I)
in the concentration of 50
µ
M in comparison with BHT
Parameters
Variation
metHb/oxyHb (5 h)
ferrylHb/oxyHb (5 h)
APꢀTBA, nmol/ml (4 h)
Control
BHT
0.91 0.09
0.305
0.32 0.01
0.238
2.95 0.10
1.93 0.07
1.87 0.08
1.73 0.06
1.81 0.12
racꢀ(I)
0.25 0.04
0.48 0.13
0.33 0.03
0.20 0.01
0.23 0.02
0.21 0.001
(+)ꢀ(
(–)ꢀ(
I
)
I)
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SEPARATION OF RACEMIC orthoꢀISOBORNYLPHENOL INTO ENANTIOMERS
617
2ꢀ((1
yl)phenyl(1
[2.2.1]heptaneꢀ1ꢀcarboxylate (II) was prepared as colꢀ flow rate of 1 ml/min at
orless crystals; mp. 127–128 С. f 0.28 (Sorbfil, benꢀ (+)ꢀ( ) and (–)ꢀ( ) were 17.3 and 19.5 min, respecꢀ
tively. The spectral characteristics of the enantiomers
R
,2
S
,4
S
)ꢀ1,7,7ꢀTrimethylbicyclo[2.2.1]heptꢀ2ꢀ by HPLC on a Chiralcel ODꢀH column eluted with
)ꢀ4,7,7ꢀtrimethylꢀ3ꢀoxoꢀ2ꢀoxabicyclo the mixture of hexane and isopropanol (99 : 1) at a
219 mn. Retention times of
S
,4
R
λ
°
R
I
I
zene); [α]2D2 –6.3 (
2953, 2878 (CH2, Me), 1792, 1748 (C=O), 1260
c
0.39, CHCl3); IR (KBr,
ν
, cm–1):
corresponded to those described in the literature for
racꢀ( [14].
2ꢀ((1 ,2
yl)phenol(+)ꢀ(I) was obtained as colorless crystals with
the yield of 0.16 g (95%); mp 97–98 ; the enantioꢀ
I)
1
(=C–O–C), 1094, 1053 (C–O–C); H NMR: 0.81
R
S,4S)ꢀ1,7,7ꢀTrimethylbicyclo[2.2.1]heptꢀ2ꢀ
(3 H, s, H10), 0.85 (3 H, s, H9), 0.90 (3 H, s, H8),
1.15 (6 H, s, H8', H9'), 1.17 (3 H, s, H10'), 1.24–1.46
(2 H, m, H3, H4), 1.59–1.70 (1 H, m, H5), 1.73–1.90
(3 H, m, 2H6 + H5'), 1.95–2.04 (1 H, m, H5'), 2.11–
2.26 (2 H, m, H3, H6'), 2.52–2.62 (1 H, m, H6'), 2.94
°
С
meric purity 98.5%; [α]2D3 +31.9 (
c
0.55, CHCl3)
.
2ꢀ((1 ,2 ,4 )ꢀ1,7,7ꢀTrimethylbicyclo[2.2.1]heptꢀ
S
R R
(1 H, t,
J
8.8, H2), 6.99–7.05, 7.17–7.25, and 7.45– 2ꢀyl)phenol(–)ꢀ(I) was obtained as colorless crystals
; the enanꢀ
7.50 (each of them 1 H, m, H13, H14, H15, H16); with the yield of 0.16 g (95%); mp 96–97
°С
13C NMR: 9.71 (C10'), 12.69 (C10), 16.76, 16.95
(C8', C9'), 20.61 (C9), 21.37 (C8), 27.36 (C5), 28.91,
31.06 (C5', C6'), 34.51 (C3), 40.19 (C6), 45.55 (C2),
46.16 (C4), 47.97 (C7), 50.09 (C1), 54.60, 54.90 (C4',
C7'), 90.67 (C1'), 121.72, 125.89, 126.42, 128.91
(C13, C14, C15, C16), 135.55, 149.69 (C11, C12),
166.32, 177.94 (C3', C8').
tiomeric purity 96.4%; [α]2D3 –32.1 (
c 0.72, CHCl3).
Xꢀray analysis of compound (II). Monocrystals of
compound (II) suitable for an Xꢀray experiment were
prepared by slow evaporation from a hexane solution.
The crystals of compound
(II), C26H34O4 were rhomꢀ
bic at 100 K. Their space group was
P
212121:
a
=
=
,
7.3272(10)
Å
,
,
b
Z
= 10.1382(13)
= 4,
Å
,
c
= 29.778(4) Å, V
Found, (%): C 76.06 and H 8.35 for C26H34O4
2212.0(5) Å3
M
= 410.53, dcalc = 1.233 g/cm3
(M
410.55). Calcd., (%): C 76.11 and H 8.30.
2ꢀ((1 ,2 ,4
2ꢀyl)phenyl(1 ,4
clo[2.2.1]heptaneꢀ1ꢀcarboxylate (III) was prepared as
colorless crystals; mp 123–124 С. Rf 0.25 (Sorbfil,
PhH); [α]2D2 –3.1 (
0.49, CHCl3); IR (KBr,
, cm–1):
μ
= 0.081 mm–1. Experimental intensities of 13873 reflecꢀ
S
R
S
R
)ꢀ1,7,7ꢀTrimethylbicyclo[2.2.1]heptꢀ
tions were measured on a SMART APEX2 difractoꢀ
meter ( (Moꢀ α) = 0.71073 Å, graphitic monochroꢀ
mator, ꢀscanning, < 29°). The starting massive of
R)ꢀ4,7,7ꢀtrimethylꢀ3ꢀoxoꢀ2ꢀoxabicyꢀ
λ
K
ω
2θ
°
measured intensities was processed using the SAINT
and SADABS programs from the APEX2 program
software [15]. After averaging the equivalent reflecꢀ
tions, 3332 independent reflections (Rint = 0.0656) was
obtained. They were used for deciphering and refineꢀ
ment. The structure was resolved by a direct method.
All the nonhydrogen atoms were localized in differenꢀ
tial syntheses of the electron density and refined by
F2hkl in anisotropic approximation. All the hydrogen
atoms were placed in geometrically calculated posiꢀ
tions and took into account during refinement in the
c
ν
2953, 2878 (CH2, Me), 1792, 1767 (C=O), 1261
(=C–O–C), 1105, 1047 (C–O–C); H NMR: 0.76
1
(3 H, s, H10), 0.85 (3 H, s, H9), 0.91 (3 H, s, H8), 1.12
and 1.17 (both 3 H, s, H8', H9'), 1.20 (3 H, s, H10'),
1.25–1.46 (2 H, m, H3, H4), 1.57–1.67 (1 H, m, H5),
1.75–1.88 (3 H, m, 2H6 + H5'), 1.97–2.07 (1 H, m,
H5'), 2.12ꢀ2.29 (2 H, m, H3, H6'), 2.56–2.65 (1 H, m,
H6'), 2.99 (1 H, t, J 8.8, H2), 6.99–7.05, 7.17–7.25,
and 7.45–7.51 (1 H, all m, H13, H14, H15, H16);
13C NMR: 9.69 (C10'), 12.81 (C10), 16.98, 17.01
(C8', C9'), 20.55 (C9), 21.45 (C8), 27.34 (C5), 29.10,
31.13 (C5', C6'), 34.47 (C3), 39.99 (C6), 45.54 (C2),
45.88 (C4), 48.00 (C7), 50.03 (C1), 54.51, 54.87 (C4',
C7'), 90.76 (C1'), 121.77, 125.94, 126.44, 129.04
(C13, C14, C15, C16), 135.49, 149.62 (C11, C12),
166.11, 177.74 (C3', C8').
“riding” model [
Uiso(H) = nUeq(C), where
n = 1.5 for
carbon atoms of methyl groups and
n
= 1.2 for the rest
of carbon atoms that are bound to the corresponding
hydrogen atoms]. The final values of invalidation facꢀ
tors were R1 = 0.0415 [it was calculated from Fhkl for
2913 reflections with I > 2σ(I)], wR2 = 0.1284 (it was
calculated from F2hkl for all the reflections), and
GOF = 1.059. All the calculations were performed
using the SHELXLꢀ97 program complex [16]. coordiꢀ
nates of the atoms and the temperature factors are
deponed in the Cambridge bank of structural data,
no. 827025.
Found, (%): C 76.17 and H 8.22 for C26H34O4
410.55). Calcd., (%): C 76.11 and H 8.30.
(M
Hydrolysis of the ester group of derivatives (II) and
(III). Compounds (II) or (III) (0.3 g, 7.3 mmol) were
dissolved in tetrahydrofuran (10 ml), 5 M aqueous
solution of KOH (3 ml) was added, and the reaction
mixture was stirred for 15 h at room temperature. The
organic layer was separated, washed with the saturated
Investigation of the antioxidant and membraneꢀproꢀ
tective properties. The 1% suspension of mouse erythꢀ
2
rocytes in PBS (pH 7.4) was used in the experiments.
solution of NaCl (2 ×10 ml), and dried over anhydrous
Na2SO4. The solvent was evaporated at a reduced presꢀ
sure. The residue was purified by column chromatogꢀ
raphy in the mixture of light petroleum and diethyl
ether (35 : 1). The enantiomeric purity was determined
2
The phosphateꢀsalt buffer was prepared by dissolution of one pelꢀ
let of PBS (SigmaꢀAldrich, United States) in 200 ml of deionized
water. The resulting solution contained 0.01 M phosphate buffer,
0.0027 M KCl, and 0.137 M NaCl (pH 7.4) at 25°C.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 37
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BURAVLEV et al.
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berg, V., Tetrahedron: Asymmetry, 2008, vol. 19,
pp. 216–222.
The solutions of compound (I) in ethanol were added
to the erythrocyte suspension and incubated for
10 min at 37 . The control samples contained no
°С
,
examined compounds. The alcohol solution of BHT
(antioxidant, 2,6ꢀdiꢀtertꢀbutylꢀ4ꢀmethylphenol) was
used as a comparative compound. The amount of ethꢀ
anol that was added to the erythrocyte suspension was
no higher than 0.1% of the total volume of the incubaꢀ
tion mixture. Hemolysis was initiated by the solution
of hydrogen peroxide. The reaction mixture was incuꢀ
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bated at slow stirring at 37°С. Aliquots were systematꢀ
ically taken and centrifuged at 3000 rpm for 5 min.
The degree of hemolysis was determined on a spectroꢀ
photometer at λ 541 nm according to the method [10].
8. Nikiforov, G.A., Nabiev, O.G., Chervin, I.I., and
Hemolysis was calculated from the optical density of
the supernatant (A) relative to the complete hemolysis
of the sample (B). The percentage of hemolysis was
calculated by the formula A/B × 100. The content of
the secondary products of LPO that reacted with
2ꢀthiobarbituric acid was determined on a spectroꢀ
photometer [17]. Accumulation of the products of the
hemoglobin oxidation was evaluated by analysis of the
absorption spectrum in the interval of wave lengths
from 500 to 710 nm. The content of various hemogloꢀ
bin forms (oxyHb, metHb, and ferrylHb) was calcuꢀ
lated subject to the corresponding extinction coeffiꢀ
cients [18].
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ACKNOWLEDGMENTS
This study was supported by the Russian Foundaꢀ
tion for Basic Research, project no. 10ꢀ03ꢀ01129ꢀa.
Russ. J. Org. Chem., 2008, vol. 44, pp. 62–66.
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