Synthesis, theoretical, and experimental study of radical scavenging activity of 3-pyridinol containing trans-resveratrol analogs

Article abstract of DOI:10.1007/s00044-018-2150-8

In the search of more powerful and versatile antioxidant agents for biomedical applications, a structural modification of the resveratrol scaffold was carried out. Six trans-resveratrol (TR) analogs with a trans-stilbazole framework were obtained. Quantum

Full text of DOI:10.1007/s00044-018-2150-8

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2150-8
ORIGINAL RESEARCH
Synthesis, theoretical, and experimental study of radical scavenging
activity of 3-pyridinol containing trans-resveratrol analogs
1
Olga I. Balakireva1 Irina V. Tarasova1 Alexey A. Burtasov1 Elena V. Semenova2
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Alexander V. Semenov
Pavel S. Petrov1 Olga V. Minaeva2 Nicolay A. Pyataev2
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Received: 24 December 2017 / Accepted: 30 January 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
In the search of more powerful and versatile antioxidant agents for biomedical applications, a structural modification of the
resveratrol scaffold was carried out. Six trans-resveratrol (TR) analogs with a trans-stilbazole framework were obtained.
Quantum calculations based on the density functional theory (DFT) were used to study the relationship between the structure
of synthesized compounds and antioxidant activity. Calculations have shown that derivatives 2b, 2c, and 2e should exhibit
higher antioxidant activity than TR, whereas 2a, 2d, and 3a are less effective antioxidants. The obtained data correlate well
with the results of the analysis carried out with diphenyl-1-picrylhydrazyl (DPPH). All compounds showed a lower
cytotoxicity compared to TR in the MTT test on mouse fibroblast cells L929.
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Keywords Resveratrol 2-Stilbazole 3-Pyridinol Antioxidant DFT
Introduction
digestive tract after oral administration. Thus the optimi-
zation of plant polyphenols bioavailability is the basis for
the full realization of their biomedical potential. The study
of the resveratrol antioxidant effect enhancement is impor-
tant due to the leading role in its physiological activity.
According to modern concepts, the antioxidant interaction
with active radicals can occur in several alternative
mechanisms (Scheme 1) (Mikulski et al. 2010).
The implementation of the first mechanism is determined
by the reducing activity of the antioxidant. For the HAT and
SPLET processes the stability of the resulting particles
(АrO• or АrO–) plays the key role. This stability is deter-
mined by the efficient electron density delocalization.
Thus, the most convenient descriptor for the antioxidant
activity evaluation is the adiabatic ionization potential (AIP)
for the first mechanism and the bond-dissociation enthalpy
(BDE) for the second process. Proton affinity (PA) and
electron transfer enthalpy (ETE) can be the descriptors for
the SPLET mechanism, as the estimation of the electron
transfer stage (Urbaniak et al. 2012). In addition, an
important descriptor allowing the formed radical АrO• sta-
bility evaluation is the spin density and electronic density of
HOMO delocalization.
Natural polyphenols are the subject of research due to the
wide range of biological effects associated with their anti-
oxidant activity. Resveratrol (1), the phytoalexin found in
grapes and some other plants, is one of the substances from
this class (Scalbert et al. 2005). Resveratrol has heart pro-
tecting (Kopp 1998) and anticancer (Baur and Sinclair
2006) activities, slows the aging process (Valenzano et al.
2006) and inhibits the neurodegenerative diseases devel-
opment (Karuppagounder et al. 2009). However, this wide
physiological activity potential of polyphenols, particularly
resveratrol, is limited by their low bioavailability. Resver-
atrol has poor water solubility and low absorption from the
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-018-2150-8) contains supplementary
material, which is available to authorized users.
* Alexander V. Semenov
salexan@mail.ru
1
Department of Organic Chemistry, Physics and Chemistry
It is known that reducing BDE, which can be achieved
by introducing donor substituents into the phenol system
(especially in para-position to the OH group), leads to the
antioxidant activity increasing. However, the significant
Institute, National Research Mordovia State University, 68
Bolshevistskaya Str., 430005 Saransk, Russia
2
Medicine Institute, National Research Mordovia State University,
68 Bolshevistskaya Str., 430005 Saransk, Russia

Medicinal Chemistry Research
water solubility and bioavailability by the quaternary
ammonium salts formation option. Second, it allows the
synthesis of analogs with greater antioxidant activity com-
pared to the natural prototype.
Materials and methods
Scheme 1 Alternative mechanisms of antioxidant action. a Electron
transfer from the antioxidant on the active radical lead to a cation
radicals and anion and the subsequent proton transfer from the cation
radical to the anion. This process named as single-electron transfer
followed by proton transfer (SET-PT). b Direct transfer of a hydrogen
atom from the antioxidant to the active radical. This process named as
hydrogen atom transfer (HAT). c The antioxidant deprotonation fol-
lowed by the transfer of the electron from the formed anion to the
active radical. Then the protonation of anion formed from the active
radical occurs. This process named as sequential proton loss electron
transfer (SPLET)
Experimental
Except where indicated, materials and reagents were used as
supplied by the suppliers without further purification. The
1H and 13C NMR spectra were recorded on a Jeol JNM-
ECX400 device (399.78 and 100.53 MHz, respectively) in
DMSO-d6. Chemical shifts were measured with reference to
1
the residual protons of the solvent (DMSO-d6, H, 2.49
ppm, 13C, 39.50 ppm). Optical density measurements were
carried out in the visible region at 517 nm on the “Shimadzu
UV2600 Vis” instrument, the thickness of the absorbing
layer was 10 mm. ESI-MS spectra were obtained on a
Thermo Scientific MSQ Plus mass spectrometer in positive
(negative) ion detection mode (source temperature 350 °C,
gas-nebulizer−nitrogen, gas flow 50 l/min, needle voltage
4.5 kV, voltage on cone 75 V, scanning in the range of
100–400 Da with a speed of 2 scans/s). The eluent is a
mixture of ACN and 0.1% formic acid in a ratio of 60:40, a
flow rate of 0.05 ml/min. Microwave syntheses were carried
out in the Monowave 300 reactor in special tightly sealed
ampoules made of borosilicate glass.
Fig. 1 Structures of resveratrol (1) and its analogs (2a–2e)
decreasing of BDEO-H leads to the decreasing of AIP (by
increasing the HOMO energy) to the values when the direct
reaction of the compound with molecular oxygen becomes
possible. It is difficult to prevent this interaction because it
will increase the prooxidant activity of the compounds or
even make them unstable in air.
The increasing of prooxidant properties is not a com-
pletely negative process. Thus studies of polyhydroxylated
resveratrol analogs showed a significant increase in their
cytotoxicity to cancer cells (HL-60 and Jurkat human leu-
kemia cells), associated with their prooxidative effects in
biological systems (Murias et al. 2005).
At the same time, the instability in the air is a crucial
parameter for the practical use. One of the solutions to this
problem is the transition from phenol substrates to 3-
hydroxy pyridine and pyrimidine derivatives. It was found
that this substitution increases the ionization potential of
more than 10 kcal/mole, while the O–H bond dissociation
energy O–H increasing only 1.1 kcal/mol (Pratt et al. 2001).
The presence of the π-deficient pyridine ring allowed to add
donor groups (including the most powerful dialkylamino
group) to the antioxidant structure. As the result, their
inhibitory activity increased significantly without increasing
the oxygen oxidation (Nara et al. 2008).
Synthesis
(E)-2-(4-Methoxystyryl)pyridine (3b)
A
mixture of picoline-2 (930 mg, 10 mmol), 4-
methoxybenzaldehyde (1.36 g, 10 mmol) and ZnCl2 (1 g,
7.35 mmol) was placed in the vial for the microwave reactor
and heated for 0.5 h at 200 °C. After cooling, acetone was
added to the reaction mixture, the insoluble precipitate was
filtered off, and the filtrate was evaporated. Then 10 mL of
water, 25 mL of concentrated ammonia solution were added
to the precipitate and boiled. After cooling, the mixture was
extracted with methylene chloride, the extract was dried
with anhydrous magnesium sulfate, and the solvent was
evaporated to yield 3b as a cream-pink solid (269 mg,
12%). The spectral data coincide with those described in the
literature (Mao et al. 2014).
In the present study we carried out the theoretical and
experimental research into resveratrol analogs containing 3-
pyridinol fragment (2a–2e) (Fig. 1).
This modification has been motivated by several pro-
blems. First, the presence of the pyridine ring increases the
(E)-4-(2-(Pyridin-2-yl)vinyl)phenol (3а)
A solution of stilbazole 3b (130 mg, 0.62 mmol) in CH2Cl2
(30 mL) was cooled to −10 °C with constant stirring, and
BBr3 (0.5 mL, 5.3 mmol) in CH2Cl2 (30 mL) was slowly

Medicinal Chemistry Research
added dropwise. At the end of the dropping, the mixture
was brought to a room temperature and stirred for 4 h under
an argon atmosphere. Then 20 mL of distilled water was
added dropwise to the reaction mixture. The aqueous layer
was separated, neutralized with a solution of ammonia to
pH 7, and extracted with ethyl acetate. The extract was
dried over anhydrous magnesium sulfate, and the solution
was evaporated to yield 3а as a yellow solid (100 mg, 83%);
mp 189–190 °C. The spectral data coincide with those
described in the literature (Choi et al. 2010).
(CH, C-4), 132.8 (CH, C-6), 135.0 (C, C-1′), 140.5 (CH, C-
b), 145.2 (C, C-2), 156.0 (C, C-5); MS (ESI) m/z calculated
for [C15H16NO+] = 226.12 (100.0%), 227.13 (16.2%)
found 226.07 (100.0%), 227.08 (17.0%).
(E)-5-methoxy-2-(4-methoxystyryl)-1-methylpyridin-1-ium
iodide (5b) Yellow solid (850 mg, 84%); mp 215–218 °С
1
(decomp.); H NMR (DMSO-d6, 400 MHz): δ 3.82 (3H, s,
OCH3), 3.98 (3H, s, OCH3), 4.34 (3H, s, NCH3), 7.04 (2H,
d, J = 8.7 Hz, H-3′, H-5′), 7.36 (1H, d, J = 16.0 Hz, H-a),
7.73–7.86 (3H, m, H-2′, H-6′, H-b), 8.15 (1H, dd, J = 9.2,
2.7 Hz, H-4), 8.40 (1H, d, J = 9.2 Hz, H-3), 8.71 (1H, d,
J = 2.7 Hz, H-6); 13C NMR (DMSO-d6, 100 MHz): 46.1
(CH3, NCH3), 55.4 (CH3, OCH3), 57.3 (CH3, OCH3), 114.5
(2CH, C-3′, C-5′), 114.5 (CH, C-a), 125.2 (CH, C-3), 127.8
(C, C-1′), 130.0 (2CH, C-2′, C-6′), 130.7 (CH, C-4), 132.2
1,2-Dimethyl-5-methoxypyridinium iodide (4)
Sodium (280 mg, 12.2 mmol) was dissolved in 4 mL of
absolute methanol in a vial for the microwave reactor, and
6-methylpyridin-3-ol (980 mg, 9 mmol) was added, heated
for 90 minutes at 100 °C. The mixture was cooled to room
temperature and methyl iodide (1.4 mL, 22.5 mmol) was
added, heated for 120 minutes at 100 °C. The solution was
evaporated, and the residue was recrystallized from ethanol
to yield 4 as a cream solid (1.76 g, 74%); mp 130–133 °С;
1H NMR (DMSO-d6, 400 MHz): δ 2.67 (3H, s, CH3), 3.95
(3H, s, OCH3), 4.19 (3H, s, NCH3), 7.92 (1H, d, J = 8.7 Hz,
H-3), 8.13 (1H, dd, J = 8.7, 2.7 Hz, H-4), 8.80 (1H, d, J =
2.7 Hz, H-6); 13C NMR (DMSO-d6, 100 MHz): δ 18.7
(CH3, CH3), 45.7 (CH3, NCH3), 57.2 (CH3, OCH3), 129.3
(CH, H-4), 130.9 (CH, H-3), 133.0 (CH, H-6), 147.9 (C, H-
2), 155.9 (C, H-5); MS (ESI) m/z calculated for [C8H12NO
+] = 138.09 (100.0%), 139.09 (8.7%) found 138.20
(100.0%), 139.20 (9.7%).
(CH, C-6), 140.5 (CH, C-b), 145.9 (C-2), 155.6 (C, C-5),
+
161.0 (C, C-4′); MS (ESI) m/z calculated for [C16H18NO2
]
= 256.13 (100.0%), 257.14 (17.3%) found 256.09
(100.0%), 257.12 (17.3%).
(E)-2-(3,4-dimethoxystyryl)-5-methoxy-1-methylpyridin-1-
ium iodide (5c) Yellow solid (870 mg, 80%); mp 206–208 °С
1
(decomp.); H NMR (DMSO-d6, 400 MHz): δ 3.82 (3H, s,
OCH3), 3.85 (3H, s, OCH3), 3.99 (3H, s, OCH3), 4.36 (3H, s,
NCH3), 7.05 (1H, d, J = 8.2 Hz, H-2′), 7.34–7.44 (3H, m, H-5′,
H-6′, H-a), 7.74 (1H, d, J = 16.0 Hz, H-b), 8.16 (1H, dd, J =
9.2, 2.7 Hz, H-4), 8.39 (1H, d, J = 9.2 Hz, H-3), 8.72 (1H, d,
J = 2.7 Hz, H-6); 13C NMR (DMSO-d6, 100 MHz): δ 46.2
(CH3, NCH3), 55.6 (CH3, OCH3), 55.8 (CH3, OCH3), 57.3
(CH3, OCH3), 110.6 (CH, C-5′), 111.7 (CH, C-2′), 114.5 (CH,
C-a), 122.9 (CH, C-6′), 125.2 (CH, C-3), 127.9 (C, C-1′), 130.7
(CH, C-4), 132.2 (CH, C-6), 140.9 (CH, C-b), 145.8 (C, C-2),
149.0 (C, C-3′), 151.0 (C, C-4′), 155.5 (C, C-5); MS (ESI) m/z
General procedure for the condensation of the salt (4) with
aromatic aldehydes
+
Salt 4 (700 mg, 2.64 mmol), the corresponding aldehyde
(2.64 mmol) and a few drops of piperidine in butan-1-ol
(10 mL) were placed in a round bottom flask equipped with
a reflux condenser. The reaction mixtures were boiled for
5–10 h. After the reaction was complete, the mixtures were
cooled; the precipitates were filtered off, washed with die-
thyl ether (with acetone in the case of compound 5a) to
yield the products of condensation.
calculated for [C17H20NO3 ] = 286.14 (100.0%), 287.15
(18.4%) found 286.12 (100.0%), 287.12 (17.2%).
(E)-2-(3,5-dimethoxystyryl)-5-methoxy-1-methylpyridin-1-
ium iodide (5d) Yellow solid (830 mg, 76%); mp
1
215–218 °С (decomp.); H NMR (DMSO-d6, 400 MHz): δ
3.81 (6H, s, 2OCH3), 4.00 (3H, s, OCH3), 4.37 (3H, s,
NCH3), 6.59 (1H, t, J = 2.1 Hz, H-4′), 6.97 (2H, d, J =
2.3 Hz, H-2′, H-6′), 7.51 (1H, d, J = 16.0 Hz, H-a), 7.70
(1H, d, J = 16.0 Hz, H-b), 8.20 (1H, dd, J = 9.2, 2.7 Hz, H-
4), 8.40 (1H, d, J = 9.2 Hz, H-3), 8.77 (1H, d, J = 2.7 Hz,
H-6); 13C NMR (DMSO-d6, 100 MHz): δ 46.3 (CH3,
NCH3), 55.4 (CH3, 2OCH3), 57.4 (CH3, OCH3), 102.1 (CH,
C-4′), 106.2 (2 CH, C-2′, C-6′), 117.6 (CH, C-a), 125.7
(CH, C-3), 130.6 (CH, C-4), 132.9 (CH, C-6), 136.9 (C, C-
1′), 140.6 (CH, C-b), 145.2 (C, C-2), 156.1 (C, C-5), 160.7
(2C, C-3′, C-5′); MS (ESI) m/z calculated for [C17H20NO3
+] = 286.14 (100.0%), 287.15 (18.4%) found 286.12
(100.0%), 287.12 (18.7%).
(E)-5-methoxy-1-methyl-2-styrylpyridin-1-ium iodide (5а)
1
Grey solid (490 mg, 53%); mp 213–215 °С (decomp.); H
NMR (DMSO-d6, 400 MHz): δ 4.01 (3H, s, OCH3), 4.38
(3H, s, NCH3), 7.42–7.50 (3H, m, H-3′, H-4′, H-5′), 7.52
(1H, d, J = 16.0 Hz, H-a), 7.77–7.84 (3H, m, H-2′, H-6′, H-
b), 8.20 (1H, dd, J = 9.6, 2.7Hz, H-4), 8.45 (1H, d, J =
9.6 Hz, H-3), 8.79 (1H, d, J = 2.7 Hz, H-6); 13C NMR
(DMSO-d6, 100 MHz): δ 46.2 (CH3, NCH3), 57.4 (CH3,
OCH3), 117.1 (CH, C-a), 125.7 (CH, C-3), 128.1 (2CH, C-
2′, C-6′), 128.9 (2CH, C-3′, C-5′), 130.1 (CH, C-4′), 130.6

Medicinal Chemistry Research
(E)-5-methoxy-1-methyl-2-(3,4,5-trimethoxystyryl)pyridin-1-
(DMSO-d6, 100 MHz): δ 115.5 (2CH, C-3′, C-5′), 122.2
(CH, C-3), 122.5 (CH, C-4), 124.7 (CH, C-a), 127.9 (2CH,
C-2′, C-6′), 127.9 (C, C-1′), 128.8 (CH, C-b), 137.7 (CH,
C-6), 146.9 (C, C-2), 152.3 (C, C-5), 157.3 (C, C-4′); MS
(ESI) m/z calculated for [C13H11NO2+H+] = 214.09
(100.0%), 215.09 (14.1%), found 214.14 (100.0%), 215.14
(14.7%); calculated for [C13H11NO2–H+] = 212.07
(100.0%), 217.08 (14.1%) found 212.08 (100.0%), 213.07
(13.8%).
ium iodide (5e) Yellow solid (815 mg, 70%); mp
1
201–203 °С (decomp.); Н NMR (DMSO-d6, 400 MHz): δ
3.72 (3H, s, OCH3), 3.86 (6H, s, 2OCH3), 4.00 (3H, s,
OCH3), 4.38 (3H, s, NCH3), 7.13 (2H, s, H-2′, H-6′), 7.45
(1H, d, J = 16.0 Hz, H-a), 7.73 (1H, d, J = 16 Hz, H-b),
8.20 (1H, dd, J = 9.2, 2.7 Hz, H-4), 8.38 (1H, d, J = 9.2 Hz,
H-3), 8.75 (1H, d, J = 2.7 Hz, H-6); 13С NMR (DMSO-d6,
100 MHz): δ 46.3 (CH3, NCH3), 56.2 (2 CH3, 2OCH3), 57.2
(CH3, OCH3), 61.9 (CH3, OCH3), 106.0 (2CH, C-2′, C-6′),
116.2 (CH, C-a), 125.4 (CH, C-3), 130.6 (CH, C-4), 130.8
(C, C-1′), 132.6 (CH, C-6), 139.6 (C, C-4′), 140.9 (CH, C-
b), 145.5 (C, C-2), 153.1 (2C, C-3′, C-5′); 155.9 (C, C-5);
MS (ESI) m/z calculated for [C18H22NO4+] = 316.15
(100.0%), 317.16 (19.5%) found 316.12 (100.0%), 317.13
(19.7%).
(E)-4-(2-(5-Hydroxypyridin-2-yl)vinyl)benzo-1,2-diol (2c)
1
Orange solid (94 mg, 53%); mp 174–175 °С (decomp.); H
NMR (DMSO-d6, 400 MHz): δ 6.72 (1H, d, J = 7.8 Hz, H-
5′), 6.79–6.91 (2H, m, H-6′, H-a), 6.97 (1H, d, J = 1.8 Hz,
H-2′), 7.12 (1H, dd, J = 8.2, 2.7 Hz, H-4), 7.22 (1H, d, J =
16.0 Hz, H-b), 7.34 (1H, d, J = 8.2 Hz, H-3), 8.10 (1H, d,
J = 2.7 Hz, H-6), 8.92 (1H, br s, OH–Ph), 9.01 (1H, br s,
OH–Ph), 9.92 (1H, br s, OH–Py); 13C NMR (DMSO-d6,
100 MHz): δ 113.3 (CH, C-2′), 115.7 (CH, C-5′), 118.8
(CH, C-6′), 122.2 (CH, C-3), 122.6 (CH, C-4), 124.5 (CH,
C-a), 128.4 (CH, C-b), 129.4 (C, C-1′), 137.4 (CH, C-6),
145.4 (C, C-3′), 145.7 (C, C-4′), 146.8 (C, C-2), 152.3 (C,
C-5); MS (ESI) m/z calculated for [C13H11NO3+H+] =
230.08 (100.0%), 231.09 (14.1%) found 230.08 (100.0%),
231.10 (14.2 %); calculated for [C13H11NO3–H+] = 228.07
(100.0%), 229.07 (14.1%) found 228.06 (100.0%), 229.08
(14.0%).
General procedure for the demethylation of salts (5а–e)
Salts 5a–e (0.78 mmol) and pyridinium chloride were added
to a round-bottomed flask equipped with a reflux condenser
at a rate of 670 mg (5.77 mmol) per CH3 group. The reac-
tion mixture was heated with stirring with a magnetic stirrer
at 200–210 °C for 3 h. At the end of the time, the reaction
mixture was poured onto the ice; the pH of the mixture was
adjusted to 7 with ammonia and then extracted with ethyl
acetate. The extracts were dried over anhydrous magnesium
sulfate, the solvent was evaporated. The residues were
washed with methylene chloride to yield the products.
(E)-5-(2-(5-Hydroxypyridin-2-yl)vinyl)benzo-1,3-diol (2d)
1
Grey solid (90 mg, 50%); mp 215–216 °С (decomp.); H
(E)-6-Styrylpyridin-3-ol (2а) Cream solid (80 mg, 52%);
mp 174–175 °С; H NMR (DMSO-d6, 400 MHz): δ 7.15
NMR (DMSO-d6, 400 MHz): δ 6.15 (1H, t, J = 2.1 Hz, H-
4′), 6.41 (2H, d, J = 1.8 Hz, H-2′, H-6′), 6.97 (1H, d, J =
16.0 Hz, H-a), 7.12 (1H, dd, J = 8.2, 2.7 Hz, H-4), 7.19
(1H, d, J = 16.0 Hz, H-b), 7.38 (1H, d, J = 8.2 Hz, H-3),
8.11 (1H, d, J = 2.7 Hz, H-6), 9.19 (2H, br s, 2OH-Ph), 9.93
(1H, br s, OH-Py); 13C NMR (DMSO-d6, 100 MHz): δ
102.4 (CH, C-4′), 104.7 (2 CH, C-2′, C-6′), 122.5 (CH, C-
3), 122.7 (CH, C-4), 127.3 (CH, C-a), 129.3 (CH, C-b),
137.7 (CH, C-6), 138.5 (C, C-1′), 146.3 (C, C-2), 152.7 (C,
C-5), 158.5 (2C, C-3′, C-5′); MS (ESI) m/z calculated for
[C13H11NO3+H+] = 230.08 (100.0%), 231.09 (14.1%)
found 230.07 (100.0%), 231.09 (14.7 %); calculated for
[C13H11NO3–H+] = 228.07 (100.0%), 229.07 (14.1%)
found 228.06 (100.0%), 229.07 (15.0%).
1
(1H, dd, J = 8.7, 2.7 Hz, H-4), 7.19 (1H, d, J = 16.5 Hz, H-
a), 7.25 (1H, t, J = 7.22 Hz, H-4′), 7.34–7.42 (4H, m, H-3′,
H-5′, H-3, H-b), 7.58 (2H, d, J = 7.3 Hz, H-2′, H-6′), 8.14
(1H, d, J = 2.7 Hz, H-6), 9.95 (1H, s, OH); 13С NMR
(DMSO-d6, 100 MHz): δ 122.4 (CH, H-3), 122.9 (CH, C-
4), 126.5 (2CH, C-2′, C-6′), 127.5 (CH, C-a), 127.9 (CH,
C-4′), 128.6 (2CH, C-3′, C-5′), 128.7 (CH, C-b), 136.8 (C,
C-1′), 137.9 (CH, C-6), 146.3 (C, C-2), 152.8 (C, C-5); MS
(ESI) m/z calculated for [C13H11NO+H+] = 198.09
(100.0%), 199.10 (14.2%), found 198.15 (100.0%), 199.15
(14.4%);
calculated
for
[C13H11NO–H+] = 196.08
(100.0%), 197.08 (14.2%) found 196.07 (100.0%), 197.07
(14.2%).
(E)-5-(2-(5-Hydroxypyridin-2-yl)vinyl)benzo-1,2,3-triol
(E)-6-(4-Hydroxystyryl)pyridin-3-ol (2b) Yellow solid
(105 mg, 63%); mp 210–211 °С (decomp.); 1H NMR
(DMSO-d6, 400 MHz): δ 6.76 (2H, d, J = 8.3 Hz, H-3′, H-
5′), 6.95 (1H, d, J = 16.0 Hz, H-a), 7.12 (1H, dd, J = 8.7,
2.7 Hz, H-4), 7.22–7.35 (2H, m, H-3, H-b), 7.40 (2H, d, J
= 8.7 Hz, H-2′, H-6′), 8.10 (1H, d, J = 2.7 Hz, H-6), 9.66
(1H, br s, OH-Ph), 9.92 (1H, br s, OH-Py); 13CNMR
(2e) Orange solid (100 mg, 52%); mp 130–133 °С
(decomp.); H NMR (DMSO-d6, 400 MHz): δ 6.50 (2H, s,
1
H-2′, H-6′), 6.79 (1H, d, J = 16.0 Hz, H-a), 6.95–7.14 (2H,
m, H-4, H-b), 7.33 (1H, d, J = 8.2 Hz, H-3), 8.08 (1H, d, J
= 2.7 Hz, H-6), 8.81 (3H, br s, 3OH-Ph), 9.70 (1H, br s,
OH-Py); 13C NMR (DMSO-d6, 100 MHz): δ 105.7 (2 CH,
C-2′, C-6′), 122.1 (CH, C-3), 122.4 (CH, C-4), 124.7 (CH,

Medicinal Chemistry Research
C-a), 127.4 (CH, C-b), 129.5 (C, C-4′), 133.5 (C, C-1′),
137.5 (CH, C-6), 146.1 (2C, C-3′, C-5′), 151.3 (C, C-2),
152.3 (C, C-5); MS (ESI) m/z calculated for [C13H11NO4
+H+] = 246.08 (100.0%), 247.08 (14.1%) found 246.08
(100.0%), 247.10 (15.5%); calculated for [C13H11NO4–H+]
= 244.06 (100.0%), 245.06 (14.1%) found 244.06
(100.0%), 245.06 (14.0%).
medium and 30 μl of the MTT solution of 5 mg/ml con-
centration was added. Colorimetric reaction was developed
after incubation with MTT (37 °C, 3 h) followed by the
addition of DMSO. The reaction was read spectro-
photometrically with a 492 nm filter and a background of
620 nm (microplate ELISA reader, EFOS 9305, Russia).
Incubations were tested in triplicate in 3 independent
experiments. Cytotoxicity was evaluated through the MTT-
derived colorimetric values. Those values were plotted to
generate dose–response curves.
DPPH radical scavenging activity
DPPH radical scavenging activity was assessed according to
the method of Shimada et al. (Shimada et al. 1992). To
determine the antiradical activity of the compounds
obtained and resveratrol, a series of solutions were prepared
by mixing an ethanol solution of DPPH in a final con-
centration of 45 μM and an ethanol solution of the corre-
sponding polyphenol at a final concentration of 0–1000 μM.
The solutions were held for 30 min in darkness at room
temperature. The optical density of the solutions was mea-
sured at 517 nm. TR was used as a positive control under
the same assay conditions. The negative control was with-
out any inhibitor. Lower absorbance at 517 nm represents
higher DPPH scavenging activity. The % DPPH radical
scavenging activity was calculated from the decrease in
absorbance at 517 nm in comparison with negative control.
Each series was repeated three times. DPPH radical’s con-
centration was calculated using the following equation:
DPPH scavenging effect (%) = (Ao–Ai)×100/Ao, where Ao
was the absorbance of the control and Ai was the absor-
bance in the presence of the sample.
All experiments were performed in three replicates. The
IC50 calculation was carried out in GraphPad Prizm 7.00.
Experimental data are expressed as mean values and stan-
dard errors and the significance of differences was analyzed
by ANOVA in Microsoft Excel 2010. Differences were
considered significant at p < 0.05. The IC50 based upon cell
growth in drug-free controls were estimated by curve fitting
using GraphPad Prizm 7.00.
Calculation
All computations were carried out by using the GAMESS
(US) package (Schmidt et al. 1993). The presented images
were generated by the Gabedit visualization program. Cal-
culations were performed for standard conditions at
298.15 K and 1.00 atm in the gas phase by DFT method
with hybrid functional B3LYP (Becke 1993). Geometry
optimization of the studied molecules was performed in
steps. First, the geometry of neutral molecules in their
ground states was optimized by a semi-empirical method
PM3 and then by using restricted B3LYP/6-31 G level of
theory. After that, the selection of the most favorable con-
formations was carried out. Then the obtained geometry
was reoptimized in 6–311 G(d,p) basis set. The calculations
for the radicals, anions, and cation radicals were performed
from the fully optimized structures of the neutral molecules
after H-atom or proton abstraction from the hydroxyl groups
or after electron abstraction. Since the radicals, anions, and
cation radicals are open-shell systems the unrestricted
B3LYP/6-311 G(d,p) level of theory for their full optimi-
zation was applied. In optimization of the structure of
radicals and cation radicals the spin contamination was
monitored. The values of the square of the total spin were
the range 0.76–0.78, so the contamination did not exceed
10%. Finally, to verify the stationarity of the energy states
obtained, vibrational spectra were calculated for all struc-
tures and their evaluation was performed for the absence of
negative frequencies. The calculated zero-point energies
(ZPE) values were scaled by a factor of 0.9805 (Scott and
Radom 1996) and used to correct the total energies.
Cells cultivation
Cells were kept in DMEM medium supplemented with 10%
fetal bovine serum (FBS), and confluent monolayers were
trypsinized, washed in DMEM, and applied in 96-well
microtiter plates (2 × 103 cells/well). Cells were incubated
in standard conditions during 48 h (Sanyo incubator, Japan;
37 °C, 5% CO2). Than new compounds and resveratrol
were added in final titrated 1:2 concentrations from 800 to
3.125 μM and were incubated with the cells in the same
conditions described above during 24 h. At same time
control vehicle (solvent used to dissolve the test com-
pounds) was added by using the same procedure, described
above, to the control cells.
MTT test procedure
MTT test was carried out by the procedure (Van de Loos-
drecht et al. 1994). Mouse fibroblast cells L929 (obtained
from the Collections tissue cultures of Ivanovsky Virology
Research Institute) were used. After all predetermined
incubation time, the medium was replaced with a fresh
The solvent (ethanol) effect was estimated on the main
descriptors of antioxidant activity since the results of the
following studies of antiradical activity in the DPPH test

Medicinal Chemistry Research
Scheme 2 General synthetic
route for synthesis of the
resveratrol analogs 2a–2e.
Reagents and conditions: (i) 1.
CH3ONa, CH3OH, MW. 2.
CH3I, MW; (ii) butanol,
piperidine; (iii) Py·HCl
strongly depend on the medium polarity. For this, a conductor
polarizable solvation model (CPCM) (Klamt and Schüürmann
1993) was used. In this model ethanol is assumed as a mac-
roscopic continuum characterized a dielectric constant ε =
24.85. The solvent effect was evaluated for the most stable
conformations of the molecules, their cation radicals, radicals,
and anions, fully optimized in the gas phase. SAR analysis
was carried out in Microsoft Excel 2010.
Fig. 2 Rotamers for compounds 2a–2e
stability of 4′-radical (comparing to 3- and 5-radical). To
assess the differences in this activity of OH groups in the
benzene and pyridine fragment along with stilbazole (2a)
(E)-4-(2-(pyridin-2-yl)vinyl)phenol (3a) was also synthe-
sized. First, (E)-2-(4-methoxystyryl)pyridine (3b) was syn-
thesized by the Knoevenagel condensation method
proposed in (Bialon 1902) with some modifications: repla-
cement of convection heating to microwave initiation,
which significantly decreased the reaction time and
increased the product yield. The product (3b) was deme-
thylated into (3a) by a standard procedure with BBr3.
Results and discussion
Chemistry
In this study five resveratrol analogs containing hydro-
xypyridine fragment (2a–2e) were synthesized. We have
proposed the efficient three-step synthesis for derivatives
(2a–2e) using more active N-methylpyridinium salt (4) as
methylene components on condensation step (Scheme 2).
The salt (4) was synthesized by methylation of 3-hydroxy-
5-methylpyridine under the microwave initiating. The con-
densation reaction of (4) with appropriate aromatic aldehydes
was carried out by boiling in butanol for 5–10 h in the pre-
sence of piperidine catalytic amount (Berdnikova et al. 2015)
afforded the yields of target N-methylstilbazoles (5a–5e)
ranged from 53 to 84%. All products are crystalline com-
pounds with E-configuration. The presence of Z-isomers in
Calculation
Since the free rotation around the single bond between the
pyridine and alkene fragments is possible, the nitrogen atom
can occupy two boundary positions relative to the double
bond protons (Fig. 2). According to the calculations from
two possible options, rotamer (b) with anti-configuration
possesses greater stability, which corresponds to the cal-
culation data (Castro et al. 2013) and X-Ray diffraction
analysis (Percino et al. 1997) for unsubstituted 2-stilbazole.
The distance between nitrogen atoms and hydrogen Hb in
all structures is in the range 2.477 to 2.504 Å, indicating a
certain contribution of weak hydrogen bond (Desiraju and
Steiner 2001) to the stabilization of the rotamer (b). Sub-
sequently, all calculations were performed for structures
with a skeleton rotamer (b). The most stable conformers of
2c and 2e also have intramolecular hydrogen bonds between
hydroxyl groups with lengths in the range 2.125 to 2.197 Å
(Fig. 3).
1
the reaction mixtures has not been detected by H NMR
spectra. The vicinal protons at C = C bond have doublet
signals with coupling constants about 16 Hz. Demethylation
of compounds (5a–5e) was carried out by heating to 200 °C
with freshly prepared anhydrous pyridinium chloride using
described protocol (Ruiz et al. 1997). Notably, this process
has been effective not only for N- but also for O-demethy-
lation. It allowed converting the compounds (5a–5e) into
target phenols (2a–2e) in a one-step process.
It is known that resveratrol hydroxyl group in the para-
position to the double bond has the highest activity
according to its lowest values of BDE and the highest

Medicinal Chemistry Research
Fig. 4 Formation of intramolecular hydrogen bonds for 4′-O-radical of
2e
electron density in the molecule allows selecting the regions
preferred for the radical attack. More active redox sites of
molecules have a high HOMO orbital density. The HOMO
distribution analysis (see Supplementary) shows a large
range of its delocalization, including both aromatic rings,
the double bond between them and hydroxyl groups in the
para positions to the double bonds. Thus the presence of a
number of active redox sites in the studied molecules was
suggested, first of all, 5′-OH- and/or 4-OH-groups. These
sites could be attacked by radicals in the real biological
systems. In contrast, OH groups in the meta-position should
be less sensitive to the interaction with free radicals. This
observation for trans-resveratrol is an excellent agreement
with the experimental data (Cao et al. 2003).
The spin density distribution in free radicals is a crucial
parameter for their stability, which is a fundamental factor
of antioxidant activity. The calculations show the spin
density delocalization throughout the molecule in all com-
pounds with the para-position of the radical center rela-
tively to ethylene units (see Supplementary). The cyclic
fragment directly containing the radical center and the vinyl
bond have the maximum localization. In the case of the
meta-position of the radical center relative to the central
alkene moiety, the spin density is delocalized almost
exclusively within the limits of benzene fragment.
Fig. 3 Formation of intramolecular hydrogen bonds for 2c and 2e
The energy values (see Supplementary) analysis showed
that all radicals are less stable than the neutral molecules
and the corresponding anions, so they can be more reactive
in biochemical reactions. The most important structural
parameters of the studied molecules and their radicals, such
as the C = C and O−H bonds lengths and orders, are given
in Supplementary.
It should be noted that the calculated parameters of the
central ethylene C = C bonds in the all studied molecules
have similar values. However, in the case of 5-O• and 4′-O•
radical forms C = C bonds are longer and have a reduced
order compared with the neutral molecules. An increasing
of length and a decreasing in the order of O−H bonds in the
case of an arrangement adjacent to the radical center were
noted, indicating the formation of a hydrogen bond between
these fragments. A shortening of O3′–H3′ and O5′–H5′
bonds in the case of 5-O• radicals was also indicated.
The calculated torsion angles values between two aro-
matic cycles and between cycles and O−H fragments indi-
cate the planar geometry of all studied structures, promoting
their participation in various stacking interactions. In addi-
tion, the planar conformation of free radicals and radical
cations should favor the delocalization of spin density and
the distribution of the unpaired electron, increasing the sta-
bility of radicals and, finally, antioxidant activity.
Thus, summarizing all given data the formation of
strongly resonance stabilized semiquinone structure could
be stated for the radicals formed from the hydroxyl located
at the para-positions relative to the ethylene moiety. The
presence of additional hydroxyl groups adjacent to the
radical center contributes to the delocalization and the
radical stabilization due to the formation of hydrogen bonds
(Fig. 4).
Based on the obtained data on total energies, the main
descriptors for the evaluation of antioxidant activity were
calculated (Table 1).
The energy, distribution of the electron density HOMO
in neutral molecules and delocalization of the spin density
in the radicals were estimated further. The HOMO energy is
an important parameter of the molecules reactivity related to
radicals. Molecules with a lower HOMO energy have lower
electron-donor ability. The HOMO distribution of the
It is known that electron-withdrawing groups (EWG)
stabilize the initial molecule and destabilize the corre-
sponding radical and cation radical, while electron-donating
groups (EDG) have the opposite effect. Thus, the replacing
of the benzene ring with pyridine should increase the AIP
and BDE of OH bond, but the addition of the hydroxyl

Medicinal Chemistry Research
Table 1 The B3LYP/6-311G(d,p) in the gas phase calculated values
Table 2 The B3LYP/6-311G(d,p) in the ethanol calculated values of
of the molecular descriptors of antioxidant activity [kcal/mol]
the molecular descriptors of antioxidant activity [kcal/mol]
Compound
AIP
BDE
PDE
PA
ETE
Compound
AIP
BDE
PDE
PA
ETE
3a
2a
2b
4′-OH 159.352 77.138 231.637 337.898 52.995
5-OH 160.656 76.423 229.618 336.138 54.040
5-OH 153.833 75.583 235.722 332.602 56.736
3a
2a
2b
4′-OH 107.634 85.533 23.460 55.380 93.130
5-OH 108.904 85.643 22.300 54.170 94.450
5-OH 103.014 83.993 26.540 54.860 92.110
4′-OH
76.269 236.409 334.205 55.819
4′-OH
84.343 26.890 56.480 90.840
2с
2d
2e
5-OH 152.180 69.517 231.367 337.308 45.964
2с
2d
2e
5-OH 101.974 83.953 27.540 54.880 92.050
3′-OH
4′-OH
72.244 234.094 330.297 55.702
68.087 229.937 325.334 56.508
3′-OH
4′-OH
82.363 25.950 52.950 92.390
77.733 21.320 50.570 90.140
5-OH 159.058 80.956 235.782 336.510 58.200
5-OH 109.264 90.693 26.990 53.640 100.030
3′-OH
5′-OH
80.623 235.449 340.821 53.556
81.031 235.858 340.562 54.224
3′-OH
5′-OH
90.823 27.120 59.080 94.720
90.573 26.870 59.160 94.390
5-OH 151.558 75.660 238.230 337.587 51.827
5-OH 101.774 84.053 27.840 54.350 92.680
3′-OH
4′-OH
5′-OH
73.162 235.732 336.184 50.733
60.991 223.560 327.255 47.490
73.003 235.573 336.904 49.853
3′-OH
4′-OH
5′-OH
83.503 27.290 53.130 93.350
73.063 16.850 47.850 88.190
82.473 26.260 53.400 92.050
TR
4′-OH 155.224 76.497 235.211 339.347 50.905
TR
4′-OH 104.864 84.773 25.470 56.660 91.090
Molecular descriptors are calculated by equations given further: AIP
= EArOH•+ – EArOH; BDE = HArO• + HH• – HArOH; PDE = HArO• + HH+
– HArOH•+; PA = HArO– + HH+ – HArOH; ETE = HArO• – HArO–
.
all structures studied is lower than in resveratrol due to the
effect of the electron-poor pyridine ring.
groups should decrease the energy of these processes. This
combined result is actually observed. According to data
from Table 1, AIP for isomeric stilbazoles 2a and 3a have
the highest values; while compounds (2b, 2c, 2e) have the
lower AIP compared with trans-resveratrol, i.e., have a high
ability for electronic transfer in the interacting with free
radicals in biological systems using the SET-PT mechan-
ism. The high AIP value for 2d, comparable to analog 2a
with the non-hydroxylated benzene ring, indicates a weak
effect of the meta located hydroxyl groups on the cation
radical stabilization, resulting in a low degree of π-deloca-
lization according to the spin density distribution in cation
radical 2d (see Supplementary).
The BDE values comparison for the pair of isomers 2a
and 3a shows that the O−H bond in the pyridine fragment
is slightly weaker than in the benzene ring and have the
strength similar to the O−H bond of trans-resveratrol,
determining its antioxidant properties. The addition of next
hydroxyls to the para position decreases the BDE, while the
meta-substituted derivative (2d) demonstrates the highest
bond strength in the series of compounds studied. The
presence of hydroxyl in the adjacent position significantly
decreases BDE due to the formation of intramolecular
hydrogen bonds.
The enthalpies of the second-stage reactions in the SET-
PT and SPLET mechanisms − PDE and ETE, respectively,
are also important for the overall energy evaluating.
An analysis of the data obtained shows the fine corre-
lation between the PDE and BDE changes, assuming the
similar effect of structural and electronic characteristics on
these parameters. The data obtained considering the solvent
effect are presented in Table 2.
The values of AIP, PDE, and PA are expected to be
significantly lower in the ethanol medium compared to the
gas phase. It is well known that charged particles are very
sensitive to the solvent polarity and their solvation reduces
the energy of the processes they participate. At the same
time, BDE values analysis (Tables 1 and 2) showed the
slight increase of these values in ethanol medium compared
to the gas phase. Thus, a more polar ethanol reduces the
ability of hydroxyl groups to homolytic dissociation.
Thus, from the thermodynamics viewpoint of the process
in the gas phase the HAT mechanism is predominant and in
the ethanol medium the process is preferably implemented by
the SPLET mechanism, which agrees well with the literature
data (Litwinienko and Ingold 2007). It should be noted that
the absolute values of the calculated descriptors in the gas
phase change upon transition to the ethanol medium, but the
relative order of antioxidant activity is generally preserved.
In contrast to previous descriptors, PA represents the
enthalpy of the first-stage reaction in the SPLET mechanism
and has the opposite dependence on the electronic effects of
the substituents, since EWG stabilizes ArO− and destabi-
lizes the parent structures and EDG have the opposite effect.
Indeed, PA for the most easily dissociating O−H bonds in
Radical scavenging activity
To study the radical scavenging activity the DPPH
scavenging assays were used (Blois 1958). It should be

Medicinal Chemistry Research
Table 3 The DPPH test IC50 values of the testing compounds and
resveratrol
some correlations between them. The data for the thermo-
dynamically more advantageous deprotonation process was
used to construct the correlation dependences for the com-
pounds with several hydroxyl groups. A correlation matrix
between the experimental values of pIC50 and calculated
descriptors in an ethanol medium was obtained (Table 4).
The Pearson agreement criterion was used for evaluating the
degree of linear dependence between two variables. Cor-
relation coefficients between variables are above 0.70 in
most cases. The low correlation between pIC50 and the PDE
value characterizing the second step of the SET-PT
mechanism is probably due to the fact that the first step is
the most important and determining for the mechanisms
with several steps from the thermodynamic point of view.
Next, we constructed correlation equations using a
multiple linear regression model corresponding to the
implementation of three main mechanisms of antioxidant
action. The relevance of the models was evaluated by three
criteria: the correlation coefficient (R), the value of the
Fisher coefficient (F) and the standard deviation (s):
Mechanism SET–PT:
Compound TR
3a
2a
2b
2c
2d
2e
IC50, µМ
93
521
1923 14.8
5.8
926
7.5
pIC50
1.968 2.716 3.283 1.170 0.763 2.966 0.875
Table 4 Correlation matrix for calculated antioxidant activity
descriptors and pIC50
BDE (O–H) PDE
PA
ETE
AIP
pIC50
BDE (O–H)
PDE
1
0.874
0.985
0.962
0.861
0.791
1
PA
0.912
1
ETE
0.759 0.901
0.505 0.795 0.913
0.398 0.702 0.883 0.986
1
AIP
1
pIC50
1
emphasized that there is a big difference between “anti-
radical” and “antioxidant” activity. The first one char-
acterizes only compounds ability to react with free radicals
(in a particular reaction), while the second shows the ability
to inhibit the oxidation process (involving many stages). In
addition, some complications in DPPH test may be caused
by partial ionization of the testing compounds, which
influences the rate of their reaction (Musialik and Litwi-
nienko 2005). However, DPPH test can be used as a simple
and rapid method of primary comparative evaluation of the
compounds antioxidant activity.
pIC50 ¼ 0:33ð 0:02ÞAIP À 0:04ð 0:02ÞPDE À 32:39ð 2:39Þ
n ¼ 6; R2 ¼ 0:98; s ¼ 0:17; F ¼ 107:96
Mechanism HAT:
pIC50 ¼ 0:14ð 0:05ÞBDE À 9:74ð 4:54Þ
n ¼ 6; R2 ¼ 0:62; s ¼ 0:77; F ¼ 6:66
Mechanism SPLET:
pIC50 ¼ À0:14ð 0:15ÞPA À 0:60ð 0:25ÞETE À 45:89ð 15:95Þ
n ¼ 6; R2 ¼ 0:82; s ¼ 0:60; F ¼ 7:10
The antioxidant concentration at which the 50% inhibi-
tion of DPPH occurs (IC50) was calculated from the graph
of the inhibition percentage versus the concentration of the
compound (see Supplementary), constructed from the
results of three independent measurements. The IC50 values
for the testing substances and the resveratrol as a reference
compound are shown in Table 3.
A lower IC50 value indicates a higher anti-radical activity
of the compound. Analysis of the Table 3 shows that
hydroxylstilbazoles (2b), (2c), and (2e), containing hydro-
xyl groups in the p-position to the double bond, both in the
pyridine and benzene fragments, have the highest activity in
the interaction with the DPPH radical. Hydroxystilbazoles
without OH groups in the p-position to the double bond, or
containing OH-groups only in one ring, show a low anti-
radical activity.
A quantitative estimation shows the good linear corre-
lation between the log IC50 and the descriptors describing
the SET-PT mechanism, satisfactory data were also
obtained for the SPLET mechanism. Thus, their realization
is most likely for the interaction of the testing substances
with DPPH, and the fine correlation between the calculated
and experimental values confirms the validity of the chosen
theoretical model and the calculation results. In the case of
the HAT mechanism, the low value of the correlation
coefficient indicates its low contribution to the overall
process under these experimental conditions. Indeed, the
HAT mechanism is the most likely process in the gas phase,
but a significant decrease in AIP values, and especially PA,
with a certain increase in BDE in the ethanol medium
makes the more favorable processes associated with the
electronic transfer.
SAR analysis
Cytotoxicity
Comparison of the calculated antioxidant activity descrip-
tors (in ethanol medium) for testing compounds with the
experimental data on the interaction with DPPH showed
MTT analysis (Mosmann 1983) is a sensitive, quantitative and
reliable colorimetric analysis of the viability, proliferation, and

Medicinal Chemistry Research
90
80
70
60
50
40
30
20
10
0
Table 5 The MTT test IC50 values of the testing compounds and
resveratrol
TR
3a
2a
TR
3a
2a
2b
2c 2d
2e
IC50, µM 19.16 24.65 205.20 531.00 –* 162.90 177.90
IC50 ðcompÞ
IC50ðTRÞ
–
1.3
10.4
27.7
–
8.5
9.3
*Not converged
1
10
100
1000
Compound 2c has the maximum cytotoxic activity in
the zone of average concentrations (400–50 μM), whereas
in smaller doses there is no difference with the control, but
in large doses this compound increases the number and/or
viability of the cells by 76%. Therefore, the untypical
shape of the survival curve of 2c caused the impossibility
of calculating the IC50 by the method of nonlinear
regression. The remaining data of half-maximal inhibitory
concentrations and confidence intervals are presented in
Table 5.
The studied compounds could be arranged as follows in
decreasing order of cytotoxicity: resveratrol > analog 3a >
2d > 2e > 2a > 2b. The paradoxical increasing of formazan
formation appearing at high concentrations (> 800 μM) of
test compounds (especially 2c, which has the lowest IC50
value in the DPPH test) is most likely due to the beginning
direct reduction of MTT by the test substances action. The
data requires clarification and it is possible to use other tests
and models of cell cultures.
C, μМ
120
100
80
60
40
20
0
TR
2b
2c
1
10
100
1000
C, μМ
140
120
100
80
TR
2d
2e
60
40
20
0
1
10
100
1000
Conclusion
C, μМ
Fig. 5 Comparison of MTT assay in L929 cells; data presented as
percentage of control
The results of DFT calculations and DPPH tests show that
the synthesized derivatives 2b, 2c, and 2e are more active
antiradical agents than resveratrol, whereas 3a, 2a, and 2d
have a lower ability to interact with free radicals in the
media under consideration. Thus, it was shown that the
antiradical activity of the obtained compounds strongly
depends on the number and position of the hydroxyl groups
in the stilbazole framework. At the same time, the medium
polarity affects the mechanism of the process essentially but
doesn’t affect the relative activity. MTT test data indicate
the lower cytotoxicity of the obtained compounds compared
to resveratrol, in contrast to which at concentrations <50 μM
they didn’t influence the cell growth of mouse fibroblasts
L929. Thus, the results obtained contribute to understanding
the pharmacological activity of the studied compounds
which can be used in food chemistry, medicine and phar-
macy as potential antioxidant agents.
activation of cells. Cytotoxicity of synthesized resveratrol
analogs was determined using the MTT method and mouse
fibroblast cells L929 (obtained from the Collections tissue
cultures of Ivanovsky Virology Research Institute).
The results of analysis of the MTT assay data are pre-
sented in Fig. 5 (and Table in Supplementary). It was shown
that most of the testing compounds reduced the viability of
L929 cells to varying degrees. The most significant cyto-
toxic effect, similar to resveratrol, was demonstrated by
analogs 2a and 3a, which reduced the number of fibroblasts
in comparison with the drug-free control over the whole
range of concentrations. Analogs 2b, 2c, and 2e were less
cytotoxic, they exerted a depressing effect on cell growth in
concentrations above 50 μM inclusively; at lower con-
centrations, these compounds did not affect cell growth.
Analog 2d was cytotoxic only at maximum concentrations:
from 800 to 200–100 μM, otherwise it indices did not differ
from the control values, but was significantly less toxic than
resveratrol
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.

Medicinal Chemistry Research
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