Synthesis and Antioxidant Properties of Hydroxycinnamic Acid Derivatives Containing Isobornyl Substituents

Article abstract of DOI:10.1007/s10600-019-02772-x

New hydroxycinnamic acid derivatives were synthesized by reacting malonic acid and 4-hydroxybenzaldehydes with isobornyl and/or alkyl substituents. The antiradical, antioxidant, and membrane-protective activities were assessed in various in vitro models.

Full text of DOI:10.1007/s10600-019-02772-x

DOI 10.1007/s10600-019-02772-x
Chemistry of Natural Compounds, Vol. 55, No. 4, July, 2019
SYNTHESIS AND ANTIOXIDANT PROPERTIES OF HYDROXYCINNAMIC
ACID DERIVATIVES CONTAINING ISOBORNYL SUBSTITUENTS
I. A. Dvornikova,1* E. V. Buravlev, I. V. Fedorova,
1
1
2
1
1
O. G. Shevchenko, I. Yu. Chukicheva, and A. V. Kuchin
New hydroxycinnamic acid derivatives were synthesized by reacting malonic acid and 4-hydroxybenzaldehydes
with isobornyl and/or alkyl substituents. The antiradical, antioxidant, and membrane-protective activities
were assessed in various in vitro models. The bulk of the substituent(s) in the o-position(s) relative to the
phenol hydroxyl had a substantial effect on the antioxidant properties of the tested acids.
Keywords: monoterpenoids, (E)-3-(4-hydroxyphenyl)acrylic acid, antioxidant activity, membrane-protective activity,
oxidative hemolysis.
Cinnamic acid derivatives are interesting because of the broad spectra of biological activity exhibited by representatives
of this compound class [1–4]. Derivatives of p-hydroxycinnamic acid are often used as promising starting compounds for
development of new multi-faceted conjugates because of their low toxicity and potent antioxidant (AO) properties [5]. Natural
monoterpenoids and their synthetic derivatives possessed various types of pharmacological activity [6]. Terpenophenols with
bicyclic bornyl and isobornyl moieties, i.e., synthetically available products derived from natural monoterpenoids [7], could
act as platforms for synthesizing cinnamic acid derivatives. Isobornylphenols and their derivatives were found to increase
brain blood flow without changing substantially systemic arterial pressure and to exhibitAO, hemorheological, and antiplatelet
properties [8, 9]. Herein, the synthesis of new 4-hydroxycinnamic acid derivatives with terpene isobornyl substituents on the
aromatic ring and results from tests of their AO properties in in vitro models are reported.
Cinnamic acid derivatives were synthesized by reacting the appropriate aldehyde 1–6 with malonic acid. The yields
of 7–12 were 78–91%. Analogs of p-hydroxycinnamic acid with substituents in the o-position to the phenol hydroxyl were
synthesized to study the AO activity (AOA) and included two isobornyls for 7 and 8 (meso-form and racemate, respectively);
isobornyl and t-butyl for 9, isobornyl and methyl for 10, and one isobornyl for 11. Acids 12 with two t-butyl groups and 13
with a propanoic acid fragment in the p-position relative to the OH group were synthesized to compare their biological
activities (Scheme 1).
The structures of newly synthesized compounds 7–11 and 13 were elucidated using spectral data. PMR spectra of
7
–11 showed resonances for double-bond protons at δ 6.23–6.29 and 7.48–7.57 ppm as doublets with H –H SSCCs of 16 Hz.
α β
This was consistent with the E-configuration of the C=C substituents. Carboxylic protons of 7–11 were observed as broad
singlets at δ 12.1 ppm. Phenol hydroxyl protons resonated as singlets at δ 8.11–8.67 ppm for 7–10 and δ 9.91 ppm for 11.
The lack of resonances for –CH=CH– protons and the appearance of broad triplets corresponding to methylene proton resonances
(
δ 2.61, 2.89 ppm) confirmed that the propanoic acid formed in 13.
The antiradical activity (ARA) of 7–13 was tested using diphenylpicrylhydrazyl (DPPH); AOA, on laboratory mouse
brain substrate. Their hemolytic activity was studied. AOA and membrane-protective (MP) properties were evaluated for
most compounds using an erythrocyte oxidative hemolysis model. The reference compounds were 2,6-diisobornyl-4-
methylphenol (14), an effective AO with various types of pharmacological effects [8, 9], and Trolox (6-hydroxy-2,5,7,8-
tetramethylchromane-2-carboxylic acid), a water-soluble vitamin E analog.
1
) Institute of Chemistry, Komi Scientific Center, Ural Branch, Russian Academy of Sciences, 48 Pervomaiskaya St.,
Syktyvkar, 167000 Russian Federation, e-mail: dvornikova-ia@chemi.komisc.ru; 2) Institute of Biology, Komi Scientific
Center, Ural Branch, Russian Academy of Sciences, 28 Kommunisticheskaya St., Syktyvkar, 167982 Russian Federation.
Translated from Khimiya Prirodnykh Soedinenii, No. 4, July–August, 2019, pp. 565–570. Original article submitted December
2
6, 2018.
658
0009-3130/19/5504-0658 ©2019 Springer Science+Business Media, LLC

TABLE 1. Comparative Assay of ARA and AOA (Test on Laboratory Mouse Brain Substrate) of Cinnamic Acid Derivatives
at Various Concentrations
ARA (% inhibition)
AOA (TBA-AP content, nM)*
Compound
1
00 μM
10 μM
100 μM
10 μM
1 μM
Trolox
94.5 ± 0.1
94.6 ± 0.1
94.6 ± 0.1
94.4 ± 0.1
94.7 ± 0.4
35.0 ± 1.0
94.0 ± 0.2
57.0 ± 1.2
83.5 ± 0.4
45.7 ± 0.9
29.2 ± 0.7
26.9 ± 0.5
25.6 ± 0.7
28.2 ± 0.8
12.3 ± 0.7
30.7 ± 0.6
10.9 ± 0.6
18.4 ± 1.0
3.4 ± 0.1
3.1 ± 0.2
2.6 ± 0.3
2.8 ± 0.2
2.6 ± 0.1
3.7 ± 0.2
3.4 ± 0.2
3.0 ± 0.2
4.5 ± 0.2
76.4 ± 0.7
3.4 ± 0.0
3.7 ± 0.2
4.6 ± 0.3
4.5 ± 0.3
55.8 ± 0.4
5.8 ± 0.4
4.5 ± 0.2
7.0 ± 0.1
103.9 ± 2.6
68.4 ± 0.6
90.8 ± 1.3
92.2 ± 0.5
77.4 ± 0.5
84.3 ± 0.8
105.5 ± 1.2
37.8 ± 0.3
70.5 ± 0.6
7
8
9
1
0
1
1
1
2
1
3
14
_
*
_____
TBA-AP concentrations in control (without compounds) and untreated samples (without oxidation initiation) were 88.0 ± 0.6
and 54.4 ± 0.1 nM, respectively.
OH
OH
OH
1
2
R
1
R
2
R
1
R
2
R
1
R
2
a
11
13
18
b
16
15
17
O
COOH
COOH
1
-6
7-12
13
8
9
7
1
1'
1
-5, 7-11, 13:R
1
=
1, 7, 13: R =
2
2, 8: R =
2
H
H
1
5
3
H
t
t
R
2
= Bu (3, 9); Me (4, 10), H (5, 11); R
= R
2
= Bu (6, 12)
a. CH (COOH) ; Py, CH (CH CH ) NH, PhH, 80°C; b. N H ⋅H O, EtOH, 78°C
2
2
2
2
2 2
2
4
2
Scheme 1*
Most of the new hydroxycinnamic acid derivatives were characterized by high ARA comparable with that of the
reference compounds (Table 1). The capability of the phenolic acids to reduce the stable radical DPPH depended on the
number of substituents in the o-positions. The least active compound 11 had one vacant o-position. A double bond also
affected the ARA. Compound 13 with a propanoic acid was less active than 7 with an acrylic acid. All tested derivatives were
far superior to Trolox and close to 14 in the ability to inhibit lipid peroxidation (LPO) in a substrate of brain tissue containing
high concentrations of natural lipids. Derivative 11 showed the lowest activity in this model (Table 1).
OH
HO
COOH
H
H
O
1
4
Trolox
Also, phenolic acids with two isobornyl substituents (7, 8, and 13) at a concentration of 10 μM had high hemolytic
activity (Fig. 1) that prevented further research of the AOA and MP properties of these compounds using blood erythrocytes as
the model. The cytotoxicity was minimized by reducing the concentration by an order of magnitude. This enabled further
studies of the compounds on living cells at a concentration of 1 μM. Earlier, high hemolytic activity for terpenophenols of
similar structures with free carboxylic acids were found by us to have high hemolytic activity [10].
_
*
2
_____
C atoms are numbered for convenience of interpreting the NMR spectra. Compounds 1, 7, and 13 were meso-stereoisomers;
–5 and 8–11, racemates (the structure of one enantiomer of which is shown in the scheme).
659

TABLE 2. Comparative Assay of MPA andAOA of Derivatives at Concentration 1 μM Using Laboratory Mouse Erythrocytes
MPA (% hemolysis)
3 h
AOA
Compound
metHb/oxyHb
ferrylHb/oxyHb
(
TBA-AP content, nM)
1
h
5 h
Control
18.3 ± 0.7
3.0 ± 0.2
2.7 ± 0.1
6.3 ± 0.7
11.8 ± 1.0
11.9 ± 1.9
8.6 ± 0.6
1.6 ± 0.1
35.9 ± 1.6
5.4 ± 0.8
4.1 ± 0.1
10.8 ± 0.6
24.1 ± 0.6
23.8 ± 0.6
15.6 ± 0.7
3.6 ± 0.1
41.6 ± 2.0
8.5 ± 0.8
5.4 ± 0.1
13.0 ± 0.5
31.9 ± 0.3
34.3 ± 0.4
19.0 ± 0.7
6.3 ± 0.1
1.32 ± 0.02
0.71 ± 0.06
0.59 ± 0.06
0.89 ± 0.02
1.03 ± 0.04
1.19 ± 0.04
1.0 ± 0.0
0.784 ± 0.053
0.520 ± 0.018
0.473 ± 0.015
0.595 ± 0.046
0.622 ± 0.020
0.764 ± 0.023
0.591 ± 0.032
0.527 ± 0.014
0.569 ± 0.016
0.472 ± 0.004
0.442 ± 0.003
0.441 ± 0.007
0.474 ± 0.005
0.480 ± 0.003
0.435 ± 0.008
0.439 ± 0.011
7
8
9
10
11
12
14
0.9 ± 0.0
9
7
0
0
5
0
0
3
1
0
0
C
7
8
9
10 11 12
- 1
13
-
10
μ
M
μM
Fig. 1. Comparative assay of hemolytic activity of cinnamic
acid derivatives 7–13 at concentrations 10 and 1 μM;
C is the control sample.
The MP activity (MPA) of the tested derivatives also depended on the number and nature of the substituents (Table 2).
The derivatives were placed in decreasing order of ability to protect cells from the effects of H O as 8 ≈ 14 > 7 > 9 > 12 > 10 > 11.
2
2
Thus, acids 8 and 7 with two isobornyl substituents possessed high MPA among the compounds synthesized by us with
having the maximum MPA. The MPA was shown by us earlier to depend on the number and structure of substituents in
8
the o-position relative to the phenol OH using prenylphenols and S-containing isobornylphenols as examples [11, 12].
The studies showed that all synthesized cinnamic acids except 11 reduced accumulation in erythrocytes of LPO
secondary products and inhibited oxidation of hemoglobin (Table 2). Compounds 7 and 8 had parameters on a par with
reference compound 14.
Thus, several 4-hydroxycinnamic acid derivatives with isobornyl substituents were synthesized and tested for AO
properties using in vitro models. The synthesized derivatives had high ARA that depended considerably on substituents in the
o-position relative to the phenol OH. The least active compound 11 had one vacant o-position. This same compound was
2
+
noticeably inferior to the other derivatives with respect to ability to inhibit Fe /ascorbate-induced LPO. The number and
volume of o-substituents affected strongly the activity of the derivatives in the erythrocyte oxidative hemolysis model. Cinnamic
acids 7 and 8 with two isobornyl moieties had the highest AOA and MPA; 11, the lowest.
EXPERIMENTAL
PMR and 13C NMR spectra were recorded in CDCl and DMSO-d on a Bruker Avance II 300 spectrometer (300 and
3
6
1
3
7
5 MHz, respectively). Resonances were assigned using C NMR spectra recorded in J-modulation mode, applying HSQC
in several instances. IR Diffuse reflectance spectra were recorded from KBr pellets on a Shimadzu IR Prestige 21 FT-IR
spectrometer. Melting points were determined on a Sanyo Gallenkamp MPD 350 apparatus and are uncorrected. Elemental
analyses used an automated Vario Micro cube analyzer in CHNS mode.
660

The course of reactions and purity of synthesized products were monitored by TLC on Sorbfil plates. Column
chromatography used silica gel (0.06–0.2 mm, Alfa Aesar). Malonic acid (pure grade) and previously synthesized aldehydes
1
–4 and 6 were used in the syntheses [13–16]. All solvents were dried and purified by standard methods.
The synthesized compounds were partially analyzed using equipment at the Khimiya Common Use Center (CUC),
IC, KSC, UB, RAS. Activities of compounds were investigated using equipment of the Molecular Biology CUC, IB, KSC,
UB, RAS. Animals from the scientific collection of experimental animals at the IB, KSC, UB, RAS were used in the work
(
http://www.ckp-rf.ru/usu/471933/).
4-Hydroxy-3-(1,7,7-trimethylbicyclo[2.2.1]heptan-exo-2-yl)benzaldehyde (5) was prepared by the literature method
[
17] with slight changes. A solution of 2-isobornylphenol (1.38 g, 6 mmol) [18] in MeOH (5.5 mL) was treated with a solution
of NaOH (3.6 g, 90 mmol) in H O (6 mL); heated to 60°C; treated dropwise with CHCl (9.6 mL, 120 mmol) over 20 min;
2
3
refluxed with stirring for 4 h; cooled in an ice bath; and treated dropwise with HCl solution (18%) to pH 6, H O (10 mL) to
2
dissolve the inorganic salt, and CHCl (20 mL). The organic layer was separated. The aqueous phase was extracted with
3
CHCl (2 × 15 mL). The combined organic extracts were dried over anhydrous Na SO and evaporated. The solid was
3
2
4
separated using column chromatography (eluent CHCl stabilized with 0.5% EtOH). The product was precipitated from a
3
–
1
mixture of C H –Et O. Yield 0.27 g (17%). Pale-pink powder, mp 152–154°C. C H O . IR spectrum (KBr, ν, cm ):
6
12
2
17 22 2
1
3
0
2
7
2
199, 3157 (ÎH), 2947, 2876, 1437, 1368 (CH , CH ), 1665 (Ñ=O). Í NMR spectrum (300 MHz, CDCl , δ, ppm, J/Hz):
3
2
3
.80 (3H, s, H-10), 0.85 (3H, s, H-8), 0.90 (3H, s, H-9), 1.28–1.56, 1.56–1.72, 1.78–1.98 (2H each, all m, Hà-3, 4, Í-5, 6),
.19–2.36 (1H, m Hb-3), 3.20 (1H, t, J = 8.7, H-2), 6.56 (1H, br.s, OH), 6.62 (1H, d, J = 8.2, H-13), 6.93 (1H, d, J = 8.1, H-14),
.90 (1H, s, H-16), 9.84 (1H, s, CHO). C NMR spectrum (75 MHz, CDCl , δ, ppm): 12.35 (CH , C-10), 20.33 (CH , C-9),
1
3
3
3
3
1.34 (CH , C-8), 27.46 (CH , C-5), 33.91 (CH , C-3), 39.85 (CH , C-6), 45.24, 45.54 (both CH, C-2, 4), 48.23 (C, C-7), 50.00
3
2
2
2
(
1
C, C-1), 115.56 (CH, C-13), 129.31, 131.13 (both C, C-11, 15), 129.85 (CH, C-14), 130.67 (CH, C-16), 161.12 (C, C-12),
91.81 (CH, CHO).
General Method for Synthesizing 7–12. A mixture of aldehyde 1–6 (1 mmol) and malonic acid (2.5 mmol) in C H
6
6
(
25 mL) was treated with Py (1 mL) and piperidine (0.1 mL), heated at 80°C for 6 h, cooled, treated with aqueous HCl (2 N),
and extracted with Et O. The organic extract was washed with H O, dried over Na SO , and evaporated at reduced pressure.
2
2
2
4
The product was precipitated from CHCl –petroleum ether.
3
(
E)-3-(4-Hydroxy-3,5-di-{1,7,7-trimethylbicyclo[2.2.1]heptan-exo-2-yl}phenyl)acrylic Acid (7)
meso-stereoisomer). Yield 78%, white powder, mp 240–242°Ñ, C H O . IR spectrum (KBr, ν, cm ): 3593, 2951, 1685,
625, 1598, 1458, 1442, 1273, 1176, 1145, 979, 860. Í NMR spectrum (300 MHz, DMSO-d , δ, ppm, J/Hz): 0.72, 0.79, 0.84
6
–
1
(
1
29 40 3
1
(
6H each, all s, CH -8, 8′, 9, 9′, 10, 10′), 1.30 (2H, m, H-5, 5′), 1.45–1.64 (6H, m, Hà-3, 3′, H-6, 6′), 1.80 (4H, m, H-4, 4′,
3
Ha-5, 5′), 2.28 (2Í, m, Hb-3, 3′), 3.26 (2Í, t, J = 8.6, H-2, 2′), 6.26 (1H, d, J = 15.9, H-18), 7.38 (2H, s, H-14, 16), 7.55 (1H,
1
3
d, J = 15.9, H-17), 8.11 (1H, s, OH), 12.09 (1Í, s, COOH). C NMR spectrum (75 MHz, DMSO-d , δ, ppm): 12.28 (CH ,
6
3
C-10, 10′), 19.86, 21.21 (all CH , C-8, 8′, 9, 9′), 27.04 (CH , C-5, 5′), 33.25 (CH , C-3, 3′), 38.52 (CH , C-6, 6′), 44.43, 44.92
3
2
2
2
(
both CH, C-2, 2′, 4, 4′), 47.66 (C, C-1, 1′), 49.62 (C, C-7, 7′), 114.79 (CH, C-18), 124.53 (C, C-15), 125.50 (CH, C-14, 16),
1
31.04 (C, C-11, 13), 145.36 (CH, C-17), 157.66 (C, C-12), 167.76 (ÑOOH).
(
E)-3-(4-Hydroxy-3,5-di-{1,7,7-trimethylbicyclo[2.2.1]heptan-exo-2-yl}phenyl)acrylicAcid (8) (racemate). Yield
–
1
8
1
6%, white powder, mp 249–251°Ñ, C H O . IR spectrum (KBr, ν, cm ): 3597, 3402, 2951, 1689, 1629, 1598, 1458, 1440,
384, 1265, 1172, 1143, 981, 858. Í NMR spectrum (300 MHz, DMSO-d , δ, ppm, J/Hz): 0.69 (6H, s, Í-10, 10′), 0.80, 0.85
29 40 3
1
6
(
6H each, s, H-8, 8′, 9, 9′), 1.30 (2H, m, H-5, 5′), 1.50–1.62 (6H, m, Hà-3, 3′, H-6, 6′), 1.80 (4H, m, H-4, 4′, 5, 5′), 2.17 (2Í,
m, Hb-3, 3′), 3.27 (2Í, t, J = 8.7, H-2, 2′), 6.23 (1H, d, J = 15.9, Í-18), 7.36 (2H, s, H-14, 16), 7.54 (1H, d, J = 15.9, Í-17),
1
3
8
2
2
1
.26 (1H, s, OH), 12.08 (1Í, s, COOH). C NMR spectrum (75 MHz, DMSO-d , δ, ppm): 12.30 (CH , C-10, 10′), 20.12,
6 3
1.19 (both CH , C-8, 8′, 9, 9′), 26.99 (CH , C-5, 5′), 33.81 (CH , C-3, 3′), 38.85 (CH , C-6, 6′), 44.44, 44.94 (both CH, C-2,
3
2
2
2
′, 4, 4′), 47.64 (C, C-1, 1′), 49.07 (C, C-7, 7′), 114.56 (CH, Ñ-18), 124.10 (C, C-15), 125.54 (CH, C-14, 16), 130.83 (C, C-11,
3), 145.43 (CH, Ñ-17), 157.21 (C, C-12), 167.75 (ÑOOH).
(
E)-3-(3-tert-Butyl-4-hydroxy-5-{1,7,7-trimethylbicyclo[2.2.1]heptan-exo-2-yl}phenyl)acrylic Acid (9). Yield
–
1
8
1
4%, white powder, mp 201–203°Ñ, C H O . IR spectrum (KBr, ν, cm ): 3585, 2954, 1683, 1624, 1597, 1429, 1274,
203, 1166, 981, 858. Í NMR spectrum (300 MHz, DMSO-d , δ, ppm, J/Hz): 0.69 (3H, s, Í-10), 0.78, 0.80 (3H each, s,
23 32 3
1
6
H-8, 9), 1.31 (1H, m, H-5), 1.38 (9H, s, C(CH ) ), 1.44–1.59 (2H, m, Hà-3, 6), 1.68–1.81 (3H, m, Hb-3, 6, H-4), 2.24 (1H, m,
3
3
Hb-3), 3.29 (1Í, t, H-2, overlapped by HOD resonance), 6.26 (1H, d, J = 15.9, Í-18), 7.30, 7.40 (1H each, s, H-14, 16), 7.57
(
(
1H, d, J = 15.9, H-17), 8.13 (1H, s, OH), 12.09 (1Í, s, COOH). 13C NMR spectrum (75 MHz, DMSO-d , δ, ppm): 11.97
6
CH , C-10), 19.98, 21.22 (CH , C-8, 9), 27.11 (CH , C-5), 29.54 (CH , C(CH ) ), 33.39 (CH , C-3), 34.42 (C, C(CH ) ),
3
3
2
3
3 3
2
3 3
661

3
8.34 (CH , C-6), 43.91 (CH, C-2), 44.97 (CH, C-4), 47.85 (C, C-1), 49.18 (C, C-7), 114.90 (CH, Ñ-18), 124.14, 126.03 (both
2
CH, C-14, 16), 124.71 (C, C-15), 131.56, 137.90 (both C, C-11, 13), 145.36 (CH, C-17), 156.99 (C, C-12), 167.84 (ÑOOH).
E)-3-(4-Hydroxy-3-methyl-5-{1,7,7-trimethylbicyclo[2.2.1]heptan-exo-2-yl}phenyl)acrylicAcid (10). Yield 87%,
(
–
1
pale-yellow powder, mp 215–217°Ñ, C H O . IR spectrum (KBr, ν, cm ): 3564, 2953, 1681, 1624, 1595, 1477, 1435,
382, 1276, 1188, 1174, 1147, 977, 854. Í NMR spectrum (300 MHz, DMSO-d , δ, ppm, J/Hz): 0.70 (3H, s, H-10), 0.80,
.85 (3H each, s, H-8, 9), 1.24–1.35 (1H, m, Hà-5), 1.44–1.59 (3H, m, Hà-3, 6), 1.68–1.81 (2H, m, H-4, Hb-5), 2.19 (3H, s,
2
0 26 3
1
1
0
6
ÑÍ -13), 2.13–2.25 (1H, m, Hb-3), 3.25 (1Í, t, J = 8.8, H-2), 6.25 (1H, d, J = 15.9, Í-18), 7.28, 7.34 (1H each, s, H-14, 16),
3
1
3
7
.48 (1H, d, J = 15.9, Í-17), 8.67 (1H, s, OH), 12.09 (1Í, s, COOH). C NMR spectrum (75 MHz, DMSO-d , δ, ppm): 12.11
6
(
CH , C-10), 16.80 (CH , ArCH ), 19.95, 21.18 (both CH , C-8, 9), 26.99 (CH , C-5), 33.38 (CH , C-3), 39.05 (CH , C-6),
3 3 3 3 2 2 2
4
1
4.46, 44.94 (both CH, C-2, 4), 47.48 (C, C-1), 49.18 (C, C-7), 114.82 (CH, C-18), 124.12, 124.61 (both C, C-13, 15), 126.26,
27.78 (both CH, C-14, 16), 130.58 (C, C-11), 144.87 (CH, C-17), 156.47 (C, C-12), 167.79 (ÑOOH).
(
E)-3-(4-Hydroxy-3-{1,7,7-trimethylbicyclo[2.2.1]heptan-exo-2-yl}phenyl)acrylic Acid (11). Yield 91%, beige
–
1
powder, mp 200–202°Ñ, C H O . IR spectrum (KBr, ν, cm ): 3587, 3394, 2951, 1678, 1624, 1597, 1502, 1431, 1265, 1220,
112, 983, 819, 570, 549. Í NMR spectrum (300 MHz, DMSO-d , δ, ppm, J/Hz): 0.71, 0.79, 0.86 (3H each, s, H-8, 9, 10),
1
9 24 3
1
1
6
1
.26–1.58 (4H, m, Ha-3, 5, H-6), 1.81 (2H, m, H-4, Hb-5), 2.24 (1Í, m, Hb-3), 3.17 (1Í, t, J = 8.8, H-2), 6.24 (1H, d, J = 16.0,
Í-18), 6.82, 7.34 (1Í each, d, J = 8.4, 8.4, H-13, 14), 7.47 (1H, s, Í-16), 7.51 (1H, d, J = 16.0, Í-17), 9.91 (1H, s, OH), 12.08
(
(
(
1Í, s, COOH). 13C NMR spectrum (75 MHz, DMSO-d , δ, ppm): 12.02 (CH , C-10), 19.88, 21.22 (both CH , C-8, 9), 27.01
6 3 3
CH , C-5), 32.99 (CH , C-3), 39.14 (CH , C-6), 44.24 (CH, C-2), 44.93 (CH, C-4), 47.51 (C, C-1), 49.26 (C, C-7), 114.60
2
2
2
CH, C-18), 115.03, 126.58, 128.44 (all CH, C-13, 14, 16), 124.54, 129.91 (both C, Ñ-11, 15), 144.89 (CH, C-17), 158.73 (C,
C-12), 167.85 (ÑOOH).
(
E)-3-(3,5-Di-tert-butyl-4-hydroxyphenyl)acrylicAcid (12). Yield 76%, mp 213–215°C. The spectral characteristics
agreed with those in the literature [19].
3-(4-Hydroxy-3,5-di-{1,7,7-trimethylbicyclo[2.2.1]heptan-exo-2-yl}phenyl)propionic acid (13) (meso-stereoisomer)
was prepared analogously to the literature method [20]. A solution of 7 (0.10 g, 0.23 mmol) in anhydrous EtOH (5 mL) was
treated with hydrazine hydrate (0.11 g, 2.29 mmol), stirred in air for 12 h, and evaporated. The solid was dissolved in EtOAc,
washed with H O, and dried over Na SO . The solvent was evaporated in vacuo. The solid was separated over a column
2
2
4
–
1
(
3
1
CHCl –MeOH eluent). Yield 0.05 g (50%), light-yellow powder, mp 185–187°Ñ, C H O . IR spectrum (KBr, ν, cm ):
3 29 44 3
1
603, 2951, 2875, 1710, 1456. Í NMR spectrum (300 MHz, CDCl , δ, ppm, J/Hz): 0.80, 0.84, 0.89 (6H each, s, H-8, 8′, 9, 9′,
0, 10′), 1.31–1.46 (4H, m, Ha-5, 5′, 6, 6′), 1.54–1.71 (4H, m, Ha-3, 3′, 6, 6′), 1.80–1.98 (4H, m, H-4, 4′, 5, 5′), 2.23 (2Í, m,
3
Hb-3, 3′), 2.61 (2Í, br.t, H-18), 2.89 (2Í, br.t, H-17), 2.98 (2Í, t, J = 8.5, H-2, 2′), 4.73 (1Í, br.s, OH), 6.98 (2H, s, H-14, 16).
1
3
C NMR spectrum (75 MHz, CDCl , δ, ppm): 12.54 (CH , C-10, 10′), 20.19, 21.46 (both CH , C-8, 8′, 9, 9′), 27.57 (CH ,
3
3
3
2
C-5, 5′), 30.81 (CH , Ñ-17), 34.02 (CH , C-3, 3′), 36.22 (CH , Ñ-18), 40.07 (CH , C-6, 6′), 45.51 (CH, C-4, 4′), 46.16 (CH,
2
2
2
2
C-2, 2′), 48.16 (C, C-1, 1′), 49.95 (C, C-7, 7′), 125.28 (CH, C-14, 16), 128.60 (C, C-11, 13), 130.25 (C, C-15), 152.54 (C,
C-12), 178.91 (ÑOOH).
AOA of the compounds was evaluated from their ability to inhibit LPO in substrate obtained from laboratory mouse
2+
brain (1 h after Fe /ascorbate-induced LPO) [21, 22]. Brain was homogenized (10%) in normal saline (pH 7.4) and centrifuged
for 10 min. Then, the supernatant (S1) containing H O, proteins, DNA, RNA, and lipids was collected. The tested compounds
2
were added to the supernatant as Me CO solutions (final concentrations 1, 10, and 100 μmol/L). Thirty minutes after initiating
2
LPO by adding freshly prepared FeCl and ascorbic acid [23], the samples were incubated for 1 h at 37°C with slow stirring.
2
The content of thiobarbituric-acid active products (TBA-AP) was determined on a Thermo Spectronic Genesys 20
5
–1
–1
spectrophotometer at λ 532 nm using an extinction coefficient of 1.56 × 10 L·mol ·cm [22, 24, 25].
ARA of the compounds was evaluated from their ability to react with DPPH [26]. The tested compounds were added
to DPPH in MeOH (final concentrations 10 and 100 μmol/L) and stirred for 30 min. The optical density of the solution was
measured at λ 517 nm on the spectrophotometer.
Hemolytic activity (cytotoxicity) and membrane-protective and antioxidant properties were evaluated using a
suspension of laboratory mouse blood erythrocytes in phosphate-buffered saline (pH 7.4).
Cytotoxicity was evaluated using an in vitro model of the ability to induce erythrocyte hemolysis. Compounds
dissolved in Me CO were added to erythrocyte suspension to final concentrations of 1 and 10 μM and incubated for 5 h at
2
3
7°C in a thermostatted shaker.
MPA and AOA were determined from the percent inhibition of induced hemolysis, reduced accumulation of LPO
secondary products, and oxidation of oxyhemoglobin in erythrocytes. MPA was evaluated from the percent inhibition of
662

H O -induced hemolysis of erythrocytes after incubation for 1, 3, and 5 h. For this, solutions of the tested compounds (final
2
2
concentration 1 μM) were added to erythrocyte suspension. Hemolysis was induced after 30 min by adding H O solution
2
2
(
0.006%). The reaction mixture was incubated in a thermostatted shaker at 37°C with slow stirring for 5 h. An aliquot was
taken from the incubation medium every 60 min and centrifuged for 5 min (1600 g). The extent of hemolysis was determined
from the hemoglobin content in the supernatant on the spectrophotometer at λ 524 nm [27]. The percent hemolysis was
calculated from the ratio to total hemolysis of the sample. AOA was evaluated from the ability of the compounds to stop
accumulation of TBA-AP in erythrocytes and from metHb/oxyHb and ferry1Hb/oxyHb ratios that characterized oxidation of
native hemoglobin and accumulation of oxidized species by the action of H O . The TBA-AP content was determined using
2
2
spectrophotometry as indicated above. Accumulation of hemoglobin oxidation products was evaluated by analyzing absorption
spectra in the range λ 540–640 nm. The oxyhemoglobin (oxyHb) and methemoglobin (metHb) contents were calculated
considering the corresponding extinction coefficients [28]. Each experiment was conducted 4–12 times. Statistical processing
used Microsoft Office Excel 2007 and 2010 software.
ACKNOWLEDGMENT
The research was financially supported by the RFBR in the framework of science Project No. 18-03-00950_a.
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