Synthesis of Pyridyl-4-Oxy-Substituted N-Hydroxy Amides of Cinnamic Acid as New Inhibitors of Histone Deacetylase Activity and Hepatitis C Virus Replication

Article abstract of DOI:10.1134/S1068162018040118

Three structural isomers of pyridyl-4-oxy-substituted N-hydroxy amides of cinnamic acid (PyOCHA) have been synthesized for the first time. The relationship between their antiviral activity against hepatitis C virus (HCV) and inhibition of histone deacetyl

Full text of DOI:10.1134/S1068162018040118

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2018, Vol. 44, No. 4, pp. 453–460. © Pleiades Publishing, Ltd., 2018.
Original Russian Text © M.V. Kozlov, A.Z. Malikova, K.A. Kamarova, K.A. Konduktorov, S.N. Kochetkov, 2018, published in Bioorganicheskaya Khimiya, 2018, Vol. 44, No. 4,
pp. 449–456.
Synthesis of Pyridyl-4-Oxy-Substituted N-Hydroxy Amides
of Cinnamic Acid as New Inhibitors of Histone Deacetylase Activity
and Hepatitis C Virus Replication
a, 1
a†
a
a
a
M. V. Kozlov , A. Z. Malikova , K. A. Kamarova , K. A. Konduktorov , and S. N. Kochetkov
a
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia
Received November 29, 2017; in final form, February 14, 2018
Abstract⎯ Three structural isomers of pyridyl-4-oxy-substituted N-hydroxy amides of cinnamic acid (PyO-
CHA) have been synthesized for the first time. The relationship between their antiviral activity against hepa-
titis С virus (HCV) and inhibition of histone deacetylases of different cellular localization has been studied.
Keywords: cinnamic hydroxamic acid, hepatitis C virus, histone deacetylases 1/2/6/8
DOI: 10.1134/S1068162018040118
INTRODUCTION
in the nucleus (HDAC1/2/3, class I), or in both the
cytoplasm and nucleus (HDAC8, class I), or only in the
cytoplasm (HDAC6/10, class IIВ and HDAC11, class
IV) [5]. At last, deacetylases HDAC4/5/7/9 of class
IIА, form the canonic active center and bind the acetyl-
ated lysine residues but have no deacetylation activity
because of the replacement of the tyrosine residue by the
histidine residue near the catalytic zinc ion [6].
Numerous studies have shown that HDACs in cell
cultures can affect the life cycle of pathogenic viruses
including influenza A virus and human immunodefi-
ciency virus [7, 8]. There is also feedback. For exam-
ple, hepatitis C virus (HCV) induces oxidative stress
and increases the activity of HDAC class I, which
inhibits expression of iron metabolism regulator hep-
cidin and, consequently, leads to excessive accumula-
tion of iron in the patient’s body [9].
The derivatives of N-hydroxy amide of cinnamic
acid (CHA) efficiently inhibit HDAC of classes I and
IIB [10–12]. The cinnamoyl CHA fragment ensures
the penetration of the hydroxamic residue into the
hydrophobic tunnel of the HDAC active center. At the
bottom of the tunnel, this residue forms the complex
bond with the catalytic zinc ion. Thus, the cinnamoyl
fragment is the linker of the optimal length between
the hydroxamic group and phenyl ring of CHA that
contains, as a rule, the additional aromatic substitu-
ent. The appropriate substituent dramatically
increases the binding strength of the inhibitor to
HDAC due to the additional interaction with the
enzyme active center.
The reversible posttranslational modification of
proteins plays a crucial role in regulating their biolog-
ical activity. In addition to phosphorylation, acetyla-
tion is the most common form of this modification.
Histone acetyltransferases (HAT) perform the transfer
of the acetyl group from acetyl coenzyme A to the
lysine residues in protein substrates whereas histone
deacetylases (HDAC) catalyze the reverse process.
Together, both groups of the enzymes support the
necessary level of acetylation of histones, transcription
factors, and other nuclear and cytoplasmic proteins.
The acetylation-deacetylation balance of proteins,
including enzymes, may not only determine their
binding to different targets but also influence their
enzymatic activity, cellular localization, and ubiqui-
tin-dependent proteasome degradation [1]. During
the cellular cycle, НАТ and HDAC are responsible for
epigenetic control of gene expression, regulate the growth
dynamics of tubulin microtubules and the actin cytoskel-
eton, and are involved in the aggresome formation [2].
Histone deacetylases, which are responsible for
deacetylation of the ε-N-acetyl-lysine residues form
the broad enzyme family consisting of four classes.
Histone deacetylases of classes I, II, and IV catalyze
the hydrolysis of the acetamide bond via coordination
of the carbonyl oxygen atom with the zinc ion in the
enzyme active center [3]. Histone deacetylases of class
III, also designated as SIRT (sirtuines), remove the
+
acetyl group using coenzyme NAD [4]. Zinc-depen-
dent histone deacetylases can be localized either only
Recently, it was shown that CHA suppresses the
reproduction of subgenomic and full-length replicons
of HCV in a cellular test system [13, 14]. The test sys-
tem is usually the human hepatoma cell line Huh7
1
Corresponding author: Phone: +7 (499) 135-05-90, fax:
7(499) 135-14-05, e-mail: kozlovmavi@gmail.com.
+
†
Deceased.
453

4
54
KOZLOV et al.
transfected by the specially designed viral RNA plus idine 1-oxide in DMF in the presence of Cs CO as a
2
3
strand, which encodes either all viral proteins (full- base. The temperature of the reaction varied in the
length replicon) or only nonstructural proteins range of 90–100°C for 1.5–3.5 h depending on the
responsible for the reproduction of viral RNA (subge- position of the hydroxyl group. After the solvent evap-
nomic replicon). In addition, the replicon contains the oration, the product was extracted from water by
sequence of the reporter protein (luciferase), the activ- n-BuOH and purified by silica gel chromatography.
ity of which is preserved after soft cell lysis and The yields of compounds (I-2), (I-3), and (I-4) were
strongly correlates with the viral RNA concentration 42, 51, and 42%, respectively.
[
15]. The testing of the CHA derivatives monomethyl-
Cinnamic acid esters (II-2,3,4) were synthesized
by the modified Wittig reaction for stabilized phos-
phorus ylides in aqueous media [17]. The refluxing of
benzaldehyde (I) and carboethoxymethylene triphen-
ylphosphorane in 1.2 M LiCl for 30 min resulted in the
mixture of E/Z isomeric olefins with multiple pre-
dominances of the E isomer. The products were iso-
lated by extraction with EtOAc, purified by silica gel
chromatography, and used further without separation
of the isomers. The yields of products (II-2), (II-3),
and (II-4) were 72, 76, and 85%, respectively.
ated at the N-hydroxyamide residue confirmed that it
is the CHA chelation ability that determines the effi-
ciency of HCV replication inhibition [14]. Moreover,
the profile of inhibitory activity of CHA para-CF -
3
derivative on a full panel of eleven human HDACs
pointed to HDAC6/8 as potential targets for the anti-
HCV action of this compound [16].
In this work, we synthesized three structural iso-
mers of pyridyl-4-oxy-substituted N-hydroxy amides
of cinnamic acid (PyO-CHA) and compared the
properties of each of them as inhibitors of both hepa-
titis C virus replication and activity of HDAC of
classes I and IIB.
The content of the E/Z isomers in the mixtures was
1
evaluated by H NMR spectroscopy by the relative
intensity of well-differentiated signals of C8 protons,
i.e., ~6.65 ppm (d, J ~ 16 Hz) and ~6.05 ppm (d, J ~
RESULTS AND DISCUSSION
1
2.5 Hz). The chemical shifts of the corresponding
Hydroxamic acids (IV-2,3,4) were prepared by the
four-step synthesis (Scheme). The pyridyl-4-oxy sub-
stituent for CHA derivatization was chosen due to its
ability to the donor-acceptor, hydrophobic, and stack-
ing interactions. Moreover, the bulk pyridine ring
could potentially discriminate the binding of isomeric
PyO-CHA in the active center of HDAC of different
classes.
proton of the E/Z isomers of cinnamic acid ethyl ester
are known from the literature, i.e., 6.44 ppm (Е iso-
mer, d, J 16.0 Hz) and 5.95 ppm (Z isomer, d, J
1
2.6 Hz) [18]. Interestingly, the values of the weight
ratio of the E/Z isomers were different in our experi-
ments, i.e., 10/1, 24/1, and 8/1 for (II-2), (II-3), and
(
II-4), respectively. The desired E isomeric products
were identified by the NMR spectra without the addi-
At the first stage of the synthesis, the correspond- tional separation of the isomers because of their prev-
ing hydroxybenzaldehyde was heated with 4-nitropyr- alence over Z isomers in the analyzed samples.
O
O
O
7
6
10
1
1
1
CHO
7
5
4
9 O
8
O
NHOH
6
3
1
CHO
5
4
i
ii
2
iii
iv
O
3 O
O
O
2
3'
OH
2
'
4'
5'
1'
N
O
N
O
N
N
(I-2, 3, 4)
(II-2, 3, 4)
(III-2, 3, 4)
(IV-2, 3, 4)
Scheme. Synthesis of cinnamic hydroxamic acid derivatives (IV-2, IV-3, and IV-4). (i) , 4-nitropyridine-1-oxide/
Cs CO /DMF/Δ; (ii), Ph P=CHCO Et/LiCl/H O/Δ; (iii),– PCl /CH Cl /Δ; (iv), NH OH/KOH/MeOH.
2
3
3
2
2
3
2
2
2
The pyridine-1-oxide group of cinnamic acid (III-2), (III-3), and (III-4) were obtained in quanti-
esters (II-2,3,4) was reduced by refluxing with PCl in tative yields.
3
CH Cl for 1 h. The reaction mixture was washed with
The final stage of the synthesis was the sequential
2
2
1
M Na CO . After evaporation of the organic solvent, addition of NH OH (15 equiv) in water and KOH
2
3
2
the mixture of E/Z isomeric products with the initial (2 equiv) in methanol to the methanol solution of cor-
weight ratio was used for the next stage. Compounds responding ethyl acrylate (III-2,3,4) at 0°C. Cooling
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 44
No. 4
2018

SYNTHESIS OF PYRIDYL-4-OXY-SUBSTITUTED
Table 1. Results of anti-HCV test of structural isomers of PyO-CHA
455
anti-HCV activity
Compound
isomer)
Inhibition
of histone deacetylases
(
EC , μM
CC , μM
SI
5
0
50
para-CF -СНА
HDAC6/8*
HDAC8
HDAC6
0.14*
0.45
0.15
0.16
25*
210
6.3
14
178*
470
42
3
IV-2 (ortho)
IV-3 (meta)
IV-4 (para)
HDAC1/2/6/8
88
*
Data from [13, 16].
was removed, and after 1.5 h, the mixture was neutral- 17-AAG did not so accelerate the NS3 degradation but
ized by the addition of NH OH ⋅ HCl (2 equiv), fol- slowed the Hsp90-dependent proteasomal degrada-
2
lowed by evaporation of 2/3 volume of the solvent. tion of NS5B viral polymerase [20, 21].
The mixture of E/Z isomeric hydroxamic acids was
extracted from water in EtOAc and the E isomer was
isolated by precipitation from methanol in a benzene-
EtOAc mixture. The yields of the individual isomers of
compounds (IV-2), (IV-3), and (IV-4) were 63, 59,
It is known from the literature that aryl- and het-
eroaryl-substituted CHAs inhibit the HDAC1/2/6/8
enzymes [10–12]. We have chosen these enzymes to
find out whether they can be responsible for the anti-
viral activity of PyO-CHA. The efficiency of the
HDAC1/2, HDAC6, and HDAC8 inhibition in the
41%, respectively.
The resulting compounds were tested for their Huh7 luc/neo cell culture when incubating with each
activity to inhibit the HCV replication in the Huh7 studied compound was evaluated by the accumulation
luc/neo cell culture, which supports the reproduction of the acetylated form of the corresponding protein
of full-length viral replicons (Table 1). Ortho-isomer substrate, i.e., H3 histone, α-tubulin, and protein
(
IV-2) significantly differs in its characteristics cohesin complex SMC3, respectively (Fig. 2a).
(
ЕС = 0.45 μM and СС = 210 μМ) from meta- and
50 50
It is important to note that compounds (IV-2) and
para-isomers (IV-3 and IV-4), which demonstrate (IV-3) exhibit the properties of selective inhibitors of
almost equal antiviral activity (ЕС = 0.15 and 0.16 μM) the HDAC8 and HDAC6 enzymes, respectively, in the
5
0
and twofold difference in cytotoxicity (СС = 6.3 and concentration range of 0–3 μM when the primary sup-
50
1
4 μM). The anti-HCV activity of all three structural pression of HCV virus replication is observed (Fig. 2a).
isomers of the resulting hydroxyamide PyO-CHA was Compound (IV-4) like most CHA para-derivatives
similar to that of one of the most active derivatives of [10], is a multipotent inhibitor of HDAC with pre-
cinnamic hydroxamic acid, para-CF -CHA [13, 16].
dominant inhibiting activity towards HDAC6. The
inhibition of HCV replication by compounds (IV-2)
and (IV-3) well correlates with the HDAC8 and HDAC6
inhibition (Fig. 2b). The role of these histone deacetyl-
ases as the targets for the anti-HCV action of CHA deriv-
atives was assumed earlier by the authors of [16].
3
The inhibition of HCV replication by the structural
isomers of PyO-CHA is accompanied by a synchro-
nous decrease in the concentration of two enzymes of
this virus, NS3 (helicase/proteinase) and NS5B
(
RNA-dependent RNA polymerase) (Fig. 1). The
suppression of HCV replication in the case of the
The results of the Western blot analysis also allow
inhibitor of heat shock protein Hsp90, 17-(allyl- one to make an important remark about the cytotoxic
amine)geldanamycin (17-AAG), leads to the inhibi- effect of the PyO-CHA derivatives. The СС50 values
tion of the NS5В degradation [19]. This effect was not for ortho-, meta-, and para-isomers (210, 6.3, and
observed in the case of PyO-CHA. Probably, in con- 14 μM, respectively) correlate well with the acetyla-
trast to what the authors believed, the presence of tion level of α-tubulin in cells after incubation with
(
1
IV-2)
(IV-3)
0.3
(IV-4)
0.3
Compound
Conc. (μM)
0
3
10
0
1
3
0
1
3
NS3
NS5B
α-Tub
Fig. 1. Degradation of viral NS3 and NS5B proteins. The Huh7-luc/neo cells were grown in the presence of PуO-CHA at the corre-
sponding concentration for 72 h, followed by the analysis of the content of the viral proteins and α-tubulin as the reference protein.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 44 No. 4 2018

4
56
KOZLOV et al.
(a)
(
IV-2)
1
(IV-3)
(IV-4)
Compound
Conc. (μM)
0
3
1
3
0
1
3
Ac-H3
H3
(
0.3
IV-2)
(IV-3)
(IV-4)
Compound
Conc. (μM)
0
1
3
0.3
1
3
0.3
1
3
Ac-α-Tub
α-Tub
(
IV-2)
1
(IV-3)
(IV-4)
Compound
0
0.3
3
10
0.3
1
3
0.3
1
3
Conc. (μM)
Ac-SMC3
SMC3
(b)
%
%
1
00
2.00
100
75
50
25
0
Compound (IV-2)
EC50 = 0.45 μM
Compound (IV-3)
EC50 = 0.15 μM
19
75
1.75
1.50
1.25
1.00
5
0
5
0
10
2
1
0
0.3
1.0
3.0
10.0
0
0.3
1.0
3.0
[PуO-CHA], μM
[PуO-CHA], μM
Inhibition (%) Ac-SMK3/SMC3 (norm)
Inhibition (%)
Ac-α-Tub/α-Tub (norm)
Fig. 2. Relationship between inhibition of HDAC and suppression of HCV replication. (a) The Huh7-luc/neo cells were grown
in the presence of PуO-CHA at the corresponding concentration for 24 h, followed by the analysis of the content of the acetylated
forms of substrate proteins. The content of the proteins in Western blots was evaluated by densitometry. (b) The values of HDAC
inhibition were calculated as the Ac-substrate/substrate ratio in the presence of the inhibitor normalized to the same ratio without
the inhibitor (right axis). Inhibition of the replicon is expressed as a percentage (left axis).
each compound (Fig. 2a). Probably, the cytotoxicity of the HDAC enzymes of different classes by the result-
level of the PyO-CHA isomers is largely determined ing compounds. The found correlation between inhibi-
by the efficacy of the HDAC6 inhibition.
tion of both HCV replication and intracellular HDAC6
and HDAC8 activity is of undoubted interest for further
study of the antiviral action of CHA derivatives.
Thus, for the first time, we describe in this work the
synthesis of the isomeric pyridyl-4-oxy-derivatives of
CHA and show their antiviral activity in the system of
the full-length HCV replicon. The introduction of the
pyridyl-4-oxy-substituent into different positions of
the phenyl ring of cinnamic acid N-hydroxyamide
EXPERIMENTAL
We used 2-hydroxybenzaldehyde, cesium carbon-
makes it possible to affect the selectivity of inhibition ate (Acros Organics); lithium chloride (Merck);
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 44
No. 4
2018

SYNTHESIS OF PYRIDYL-4-OXY-SUBSTITUTED
457
3
-hydroxybenzaldehyde,
-nitropyridine-1-oxide, carbethoxymethylene triph- and Н4'). C NMR spectrum (CDCl ): 190.34 (С7),
4-hydroxybenzaldehyde, 7.20 (2Н, d, J 8.6, H3 and Н5), 6.97 (2Н, d, J 7.7, H2'
13
4
3
enylphosphorane (Ph P=CHCO Et), phosphorus (III)
3
2
159.20 (С4), 155.68 (С3'), 141.02 (С1' and С5'),
chloride, and 50% aqueous hydroxylamine (Sigma- 133.73 (С1), 132.30 (С2 and С6), 119.97 (С3 and С5),
Aldrich). The column chromatography was carried out 115.97 (С2' and С4').
on silica gel (Kieselgel, 0.060–0.200 mm, Acros Organ-
Compounds (II-2), (II-3), and (II-4) were synthe-
ics) using the elution systems mentioned in the text. TLC
sized by the unified method. Carbomethoxyethylene
was performed on the plates of Kieselgel 60 F254
triphenylphosphorane (2.09 g, 6 mmol) and LiCl
(Merck) in a CHCl –EtOH (10 : 1) mixture.
3
(
1.27 g, 30 mmol) were added to the solution of corre-
NMR spectra (δ, ppm; J, Hz) were recorded on an sponding pyridine-1-oxide (I-2, I-3, or I-4) (1.07 g,
AMX III-400 spectrometer (Bruker, Germany) using 5 mmol) in water (25 mL). The reaction mixture was
1
4
00 MHz for H NMR (internal standard: Me Si; sol- refluxed under stirring for 30 min, followed by wash-
4
1
3
ing with EtOAc (2 × 25 mL). The combined organic
vents: CDCl or DMSO-d ) and 100.6 MHz for С
3
6
extracts were washed with 5 M NaCl (10 mL) and
NMR with suppression of carbon-proton interaction
solvents: CDCl or DMSO-d ).
dried over Na SO . The solvent was evaporated in vac-
(
2
4
3
6
uum, and the residue was dissolved in MeCN
4
-(2-Formylphenoxy)pyridine-1-oxide (I-2). 4-Nitro-
(
10 mL). The product was isolated by silica gel chro-
pyridine-1-oxide (2.10 g, 15 mmol) and Cs CO
2
3
matography using a CHCl –EtOH mixture (10 : 1) as
3
(
4.52 g, 15 mmol) were added to the solution of sali-
an eluent. The selected fractions were evaporated, the
wet residue was washed with Et O (2 × 10 mL), and
cylic aldehyde (1.83 g, 15 mmol) in DMF (15 mL).
The reaction mixture was stirred at 90°С for 2 h, fol-
lowed by evaporation. The residue was dissolved in
water (20 mL) and washed with n-BuOH (2 × 20 mL
and 10 mL). The combined organic extracts were
evaporated to the final volume of 25 mL. The product
was isolated by silica gel chromatography using an
2
the residue was filtered and air dried. The yields of the
products were 72, 76, and 85%; the weight ratios for
E/Z isomers were 10 : 1, 24 : 1, and 8 : 1 for compounds
(
II-2), (II-3), and (II-4), respectively.
(E)-4-(2-(3-Ethoxy-3-oxyprop-1-enyl)phenoxy)pyri-
1
i-PrOH-EtOH mixture (1 : 1) as an eluent. R 0.27. dine-1-oxide (II-2), R 0.45. Н NMR spectrum
f
f
The chosen fractions were evaporated; the oil residue (DMSO-d ): 8.16 (2Н, d, J 7.7, Н1' and H5'), 7.97
6
was washed with EtOAc (15 mL); the precipitate was (1Н, d, J 7.7, H6), 7.70 (1Н, d, J 16.1, H7), 7.51 (1Н, t, J
filtered, washed with n-hexane (2 × 5 mL), and air- 7.6, H4), 7.33 (1Н, t, J 7.5, H5), 7.17 (1Н, d, J 8.1,
dried. The resulting pyridine-1-oxide (I-2) (1.35 g, Н3), 7.03 (2Н, d, J 7.1, H2' and Н4'), 6.69 (1Н, d, J
4
2%) was further used without additional purification. 16.1, H8), 4.16 (2Н, q, J 7.0, H10), 1.22 (3Н, t, J 7.0,
1
13
Н NMR spectrum (CDCl ): 10.22 (1 H, s, Н7), 8.28 H11). C NMR spectrum (DMSO-d ): 165.89 (С9),
3
6
(
2 H d, J 7.6, Н1' and H5'), 7.95 (1 H, dd, J 7.7, J 1.6, 153.76 (С2 or С3'), 152.81 (С2 or С3'), 140.17 (С1' and
1
2
H6), 7.66 (1 H, td, J 8.2, J 1.7, Н4), 7.40 (1 Н, t, J С5'), 137.01 (С7), 132.34 (С4), 128.87 (С6), 125.86
1
2
7
.6, H5), 7.10 (1 Н, d, J 8.2, H3), 6.95 (2 Н, d, J 7.6, (С1), 125.76 (С5), 120.47 (С3 or С8), 120.32 (С3 or
13
С8), 115.40 (С2' and С4'), 60.13 (С10), 14.04 (С11)
H2' and Н4'). C NMR spectrum (CDCl ): 187.91
3
(С7), 157.46 (С2 or С3'), 155.43 (С2 or С3'), 141.04
С1' and С5'), 136.32 (С4), 130.73 (С1), 127.98 (С6),
(E)-4-(3-(3-Ethoxy-3-oxyprop-1-enyl)phenoxy)-
(
1
1
pyridine-1-oxide (II-3), R 0.56. Н NMR spectrum
f
26.56 (С5), 120.98 (С3), 115.08 (С2' and С4').
(
DMSO-d ): 8.15 (2Н, d, J 7.6, Н1' and H5'), 7.65
6
-(3-Formylphenoxy)pyridine-1-oxide (I-3). The (1Н, d, J 16.1, H7), 7.62–7.57 (2Н, m, H2 and Н4),
4
reaction was carried out at 100°С for 1.5 h. The yield of 7.49 (1Н, t, J 7.9, H5), 7.21 (1Н, dd, J 8.1, J 1.5, H6),
1
2
pyridine-1-oxide (I-3) was 1.65 g (51%). R 0.31
f
7.04 (2Н, d, J 7.6, H2' and Н4'), 6.69 (1Н, d, J 16.1,
1
(
i-PrOH). Н NMR spectrum (CDCl ): 9.99 (1H, s, H8), 4.18 (2Н, q, J 7.1, H10), 1.25 (3Н, t, J 7.1, H11).
3
1
3
Н7), 8.30 (2H d, J 7.6, Н1' and H5'), 7.77 (1Н, d, J
C NMR spectrum (DMSO-d ): 165.97 (С9), 155.02
6
7
.6, H6), 7.62 (1Н, t, J 7.9, H5), 7.58 (1Н, dd, J 2.2, (С3), 153.78 (С3'), 143.17 (С7), 140.06 (С1' and С5'),
1
J 1.5, H2), 7.35 (1Н, ddd, J 8.1, J 2.5, J 0.9, H4), 136.48 (С1), 130.81 (С5), 125.09 (С6), 121.46 (С4),
2
1
2
3
13
1
19.55 (С2 or С8), 119.10 (С2 or С8), 115.69 (С2' and
6
.94 (2Н, d, J 7.6, H2' and Н4'). C NMR spectrum
С4'), 60.04 (С10), 14.10 (С11).
(
CDCl ): 190.65 (С7), 157.71 (С3 or С3'), 154.69 (С3 or
3
С3'), 141.12 (С1' and С5'), 138.75 (С1), 131.47 (С5), 127.77
(E)-4-(4-(3-Ethoxy-3-oxyprop-1-enyl)phenoxy)-
(С4), 126.19 (С6), 120.02 (С2), 115.04 (С2' and С4').
1
pyridine-1-oxide (II-4), R 0.50. Н NMR spectrum
f
4
-(4-Formylphenoxy)pyridine-1-oxide (I-4). The (DMSO-d ): 8.17 (2Н, d, J 7.6, Н1' and H5'), 7.81
6
reaction was carried out at 100°С for 3.5 h. i-PrOH (2Н, d, J 8.7, H2 and Н6), 7.66 (1Н, d, J 16.0, H7),
was used as an eluent for chromatography. The yield of 7.18 (2Н, d, J 8.7, H3 and Н5), 7.08 (2Н, d, J 7.6, H2'
1
pyridine-1-oxide (I-4) was 1.35 g (42%). R 0.35. Н and Н4'), 6.60 (1Н, d, J 16.0, H8), 4.19 (2Н, q, J 7.1,
f
13
NMR spectrum (CDCl ): 9.97 (1Н, s, H7), 8.26 (2H H10), 1.25 (3Н, t, J 7.1, H11). C NMR spectrum
3
d, J 7.7, Н1' and H5'), 7.94 (2Н, d, J 8.7, H2 and Н6), (DMSO-d ): 166.13 (С9), 156.33 (С4), 153.18 (С3'),
6
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 44 No. 4 2018

4
58
KOZLOV et al.
1
43.22 (С7), 140.13 (С1' and С5'), 130.76 (С1), 130.49 cooled to 0°С. The cooling was removed, and after 1.5
(С2 and С6), 119.62 (С3 and С5), 117.86 (С8), 116.18 h of incubation, NH OH ⋅ HCl (0.49 g, 7 mmol) was
2
(С2' and С4'), 59.95 (С10), 14.13 (С11).
added to the reaction mixture, which was stirred for
another 10 min. The solvent (~20 mL) was evaporated
in vacuum. The residue was dissolved in water (30 mL)
and washed with EtOAc (2 × 30 mL). The combined
Compounds (III-2), (III-3), and (III-4) were syn-
thesized by the unified method. Corresponding pyri-
dine-1-oxide (II-2, II-3, or II-4) (1.00 g, 3.51 mmol)
was dissolved in CH Cl (35 mL), followed by the
organic extracts were dried over Na SO , and the sol-
2
4
2
2
vent was evaporated in vacuum. The residue was dis-
solved in a minimal volume of methanol, followed by
the addition of a 5-fold volume of a benzene-EtOAc
mixture (1 : 2). After standing overnight at 4°С, the
precipitate was filtered, washed with a benzene-
EtOAc mixture (1 : 1) (5 mL), and air dried. The yields
of the target products were 63, 59 and 41% of the orig-
inal content, respectively. The isomeric purity of the
addition of PСl (0.96 g, 7 mmol). The reaction mix-
3
ture was refluxed for 1 h and washed then with 1 M
Na CO (35 mL). The water extract was additionally
2
3
washed with CH Cl (2 × 25 mL). The combined
2
2
organic extracts were dried over Na SO , and the sol-
2
4
vent was evaporated in vacuum. The products were
obtained in quantitative yields with the initial weight
ratios of E/Z isomers
1
compounds was confirmed by Н NMR spectra as
(
Е)-Ethyl 3-(2-(pyridine-4-yloxy)phenyl)acrylate
described in [18].
1
(
III-2). R 0.60. Н NMR spectrum (DMSO-d ): 8.48
f
6
(
Е)-N-Hydroxy-3-(2-(pyridine-4-yloxy)phenyl)-
(
2Н, d, J 6.2, Н1' and H5'), 7.99 (1Н, d, J 7.8, H6),
.64 (1Н, d, J 16.1, H7), 7.54 (1Н, t, J 7.7, H4), 7.37
1Н, t, J 7.5, H5), 7.19 (1Н, d, J 8.1, Н3), 6.91 (2Н, d,
1
acrylamide (IV-2), R 0.29. Н NMR spectrum
f
7
(
DMSO-d ): 10.93 (1Н, s, CONHOH), 9.06 (1Н, s,
(
6
CONHOH), 8.47 (2Н, d, J 6.1, Н1' and H5'), 7.76
(1Н, d, J 7.6, H6), 7.54–7.33 (3Н, m, H4, Н5 and
Н7), 7.18 (1Н, d, J 8.0, H3), 6.89 (2Н, d, J 6.1, H2'
J 6.2, H2' and Н4'), 6.68 (1Н, d, J 16.1, H8), 4.13 (2Н,
13
q, J 7.1, H10), 1.20 (3Н, t, J 7.1, H11). C NMR spec-
trum (DMSO-d ): 165.83 (С9), 163.72 (С3'), 151.94
6
13
and Н4'), 6.60 (1Н, d, J 15.9, H8). C NMR spectrum
DMSO-d ): 163.91 (С3'), 162.20 (С9), 151.42 (С2,
(
С2), 151.64 (С1' and С5'), 136.95 (С7), 132.35 (С4),
(
1
28.86 (С6), 126.46 (С1), 126.18 (С5), 121.63 (С3 or
6
С1' and С5'), 131.25 (С4 or С7), 131.11 (С4 or С7),
28.22 (С6), 127.54 (С1), 126.32 (С5), 121.87 (С3 or
С8), 120.33 (С3 or С8), 111.78 (С2' and С4'), 60.10
(
1
С10), 14.00 (С11).
Е)-Ethyl 3-(3-(pyridine-4-yloxy)phenyl)acrylate
III-2). (III-3), R 0.70. Н NMR spectrum (DMSO-
С8), 121.80 (С3 or С8), 111.71 (С2' and С4').
(
1
(Е)-N-Hydroxy-3-(3-(pyridine-4-yloxy)phenyl)-
(
f
1
acrylamide (IV-3), R 0.17. Н NMR spectrum
d ): 8.47 (2Н, d, J 6.2, Н1' and H5'), 7.70–7.62 (2Н,
f
6
(
DMSO-d ): 10.74 (1Н, s, CONHOH), 9.08 (1Н, s,
m, H4 and Н7), 7.60 (1Н, s, H2), 7.51 (1Н, t, J 7.9,
6
H5), 7.22 (1Н, dd, J 8.0, J 1.9, H6), 6.93 (2Н, d, J CONHOH), 8.47 (2Н, d, J 5.7, Н1' and H5'), 7.61–
1
2
7
.43 (3Н, m, H4, Н5 and Н7), 7.37 (1Н, s, H2), 7.17
6
.2, H2' and Н4'), 6.69 (1Н, d, J 16.1, H8), 4.18 (2Н,
13
(1Н, d, J 6.5, H6), 6.94 (2Н, d, J 5.7, H2' and Н4'),
q, J 7.1, H10), 1.24 (3Н, t, J 7.1, H11). C NMR spec-
13
6
.51 (1Н, d, J 15.8, H8). C NMR spectrum (DMSO-
trum (DMSO-d ): 165.95 (С9), 163.74 (С3'), 154.02
6
d ): 163.74 (С3'), 162.38 (С9), 154.08 (С3), 151.50
(
С3), 151.44 (С1' and С5'), 143.14 (С7), 136.53 (С1),
6
(
С1' and С5'), 137.35 (С1), 137.18 (С7), 130.89 (С5),
24.52 (С6), 121.37 (С4), 120.49 (С2), 119.14 (С8),
12.14 (С2' and С4').
1
30.82 (С5), 125.43 (С6), 122.34 (С4), 120.16 (С2 or
1
С8), 119.54 (С2 or С8), 112.08 (С2' and С4'), 60.03
1
(С10), 14.09 (С11).
(
Е)-Ethyl 3-(4-(pyridine-4-yloxy)phenyl)acrylate
(Е)-N-Hydroxy-3-(4-(pyridine-4-yloxy)phenyl)-
1
1
(
III-4), R 0.67. Н NMR spectrum (DMSO-d ): 8.49 acrylamide (IV-4), R 0.25. Н NMR spectrum
f
6
f
(
2Н, dd, J 4.7, J 1.5, Н1' and H5'), 7.83 (2Н, d, J 8.6, (DMSO-d ): 10.76 (1Н, s, CONHOH), 9.07 (1Н, s,
1 2
6
H2 and Н6), 7.68 (1Н, d, J 16.1, H7), 7.20 (2Н, d, J CONHOH), 8.48 (2Н, d, J 6.0, Н1' and H5'), 7.67
8
.6, H3 and Н5), 6.98 (2Н, dd, J 4.7, J 1.5, H2' and (2Н, d, J 8.3, H2 and Н6), 7.49 (1Н, d, J 15.8, H7),
1
2
7
.19 (2Н, d, J 8.5, H3 and Н5), 6.96 (2Н, d, J 6.0, H2'
Н4'), 6.61 (1Н, d, J 16.0, H8), 4.19 (2Н, q, J 7.1, H10),
13
13
and Н4'), 6.45 (1Н, d, J 15.8, H8). C NMR spec-
trum (DMSO-d ): 163.48 (С3'), 162.63 (С9), 154.50
1
.26 (3Н, t, J 7.1, H11). C NMR spectrum (DMSO-
d ): 166.11 (С9), 163.30 (С3'), 155.38 (С4), 151.53
6
6
(
С4), 151.53 (С1' and С5'), 137.24 (С7), 132.01 (С1),
29.51 (С2 and С6), 120.78 (С3 and С5), 119.06 (С8),
12.36 (С2' and С4').
(
С1' and С5'), 143.22 (С7), 131.14 (С1), 130.49 (С2
1
and С6), 120.59 (С3 and С5), 118.03 (С8), 112.52 (С2'
1
and С4'), 59.96 (С10), 14.12 (С11).
Compounds (IV-2), (IV-3), and (IV-4) were syn-
Cell cultures. The Huh7 cell line was cultured in the
thesized by the unified method. 50% Aqueous DMEM + F12 (2 : 1) medium containing 10% FBS
NH OH (3.12 mL, 52.5 mmol) and 2 M KOH in (Hy Clone), 2 mM L-glutamine, penicillin (100
2
methanol (3.50 mL, 7 mmol) were successively added U/mL), and streptomycin (100 /mL) at 37°C and 5%
under stirring to the solution of corresponding ethyl CO . The cells were reseeded every three days in the
2
acrylate (III-2, III-3, or III-4) (0.94 g, 3.5 mmol) quantitative ratio of 1 : 3 or 1 : 5. The culture of the
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 44
No. 4
2018

SYNTHESIS OF PYRIDYL-4-OXY-SUBSTITUTED
459
Huh7-luc/neo line was grown under the same condi- H3(K9/K14) 9677S, CST); in dilution 1 : 10000 to α-
tions in the presence of additional 330 μg G418/mL.
tubulin (T5168, Sigma); in dilution 1 : 2000 to histone
H3 (Histone H3 9715S, CST); in dilution 1 : 500 to the
acetylated form of SMC3 (MABE1073, EMD Milli-
pore) and 1 : 2000 to SMC3 (5996S, CST). The mix-
tures were incubated overnight at 4°С, followed by
washing with PBST. The conjugate of horseradish per-
oxidase with secondary specific antibodies (anti-
mouse and anti-rabbit, Santa Cruz) were added in
dilution 1 : 10000, and the mixtures were incubated for
Antiviral activity. The Huh7-luc/neo cell culture
was reseeded into 48-well plate in the medium without
antibiotic G418. After 48 h (30–40% monolayer), the
studied compounds at different concentrations were
added to the culture medium. After the incubation at
3
7°C in 5% СО for three days (monolayer in the con-
2
trol probe was 100%), the medium was removed. The
cells were washed with phosphate buffered saline
5
0 min at room temperature. The membranes were
(
PBS) and lysed, followed by the evaluation of the
washed with PBST, and the signal was visualized using
the ECL kit (Pierce-Thermo scientific) and High-per-
formance ECL film (GE Healthcare).
luciferase activity by the Luciferase Assay System kit
(
Promega) according to the manufacturer’s protocol.
Chemiluminescence was measured on a Thermo
luminometer (Labsystems).
Cytotoxicity. The Huh7 cell culture was reseeded
into 96-well plate. After 24 h (30–40% monolayer),
the studied compounds at different concentrations
were added to the culture medium. After the incuba-
ACKNOWLEDGMENTS
The work was supported by the grants from the
Program of Fundamental Research of the State Acad-
emies of Sciences for 2013-2020 (no. 01201363818),
Russian Scientific Foundation (no. 14-50-00060),
and Presidium of RAS (“Fundamental research for
the development of biomedical technology” for 2014-
tion at 37°C in 5% СО for three days (monolayer in
2
the control probe was 100%), the cell viability was
evaluated using the MTT kit (Sigma-Aldrich) accord-
ing to the manufacturer’s protocol.
Sample preparation for Western blot analysis. The
cell cultures were reseeded in a 6-well plate. After 24 h
2
017)
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Translated by A. Levina
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 44
No. 4
2018