Structural isomers of cinnamic hydroxamic acids block HCV replication via different mechanisms

Article abstract of DOI:10.1016/j.ejmech.2019.111723

A set of ortho-, meta- and para-substituted cinnamic hydroxamic acids (CHAs) was synthesized. In each series of structural isomers, a phenyl substituent was linked to an aromatic ring of the parent cinnamic acid via a linker of one to four atoms in length

Full text of DOI:10.1016/j.ejmech.2019.111723

European Journal of Medicinal Chemistry 183 (2019) 111723
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Structural isomers of cinnamic hydroxamic acids block HCV
replication via different mechanisms
Maxim V. Kozlov*, Konstantin A. Konduktorov, Alsu Z. Malikova 1, Kamila A. Kamarova,
Anastasia S. Shcherbakova, Pavel N. Solyev, Sergey N. Kochetkov
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilova 32, Moscow, 119991, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
A set of ortho-, meta- and para-substituted cinnamic hydroxamic acids (CHAs) was synthesized. In each
series of structural isomers, a phenyl substituent was linked to an aromatic ring of the parent cinnamic
acid via a linker of one to four atoms in length. Using a cell test system with the full-length replicon of
hepatitis C virus (HCV), we established a relationship between the suppression of HCV replicon propa-
gation and the inhibition of class I/IIb histone deacetylases (HDACs). Anti-HCV activity correlated with
the inhibition of HDAC8 in the case of ortho-CHAs, while in the case of meta-CHAs it correlated with the
inhibition of HDAC1/2/3 and HDAC6. The antiviral activity of para-CHAs was many times stronger than
that of meta-CHAs with about the same efficiency of HDAC1/2/3/6 inhibition, which indicated the ex-
istence of an additional cell target that does not belong to the studied group of HDACs.
© 2019 Elsevier Masson SAS. All rights reserved.
Received 25 August 2019
Accepted 19 September 2019
Available online xxx
Keywords:
Cinnamic hydroxamic acids
Anti-HCV activity
Cell viability
Inhibition of histone deacetylases
1. Introduction
transcription factor FoxO1 [4,5].
HDACs form an extensive family of enzymes which includes
The success achieved in the therapy of chronic hepatitis C (CHC)
due to the creation of effective direct acting antivirals (DAAs) pro-
vides an opportunity to search for new approaches to the treatment
of CHC, including the development of antiviral host targeting
agents (HTAs) [1]. The combined application of DAAs and HTAs
appears promising e hypothetically, it can lead to a sustained
virologic response with a short duration of the therapy, thereby
reducing the risk of emergence of DAA-resistant strains of hepatitis
C virus (HCV) [2].
Histone deacetylases (HDACs) are of interest as anti-HCV host
targets due to a close interweaving of HCV propagation and activ-
ities of HDACs. Very recently, Hamdane et al. have convincingly
demonstrated a major epigenetic reprogramming in the hepato-
cytes of patients with CHC, which correlates with specific genome-
wide changes in the acetylated form of histone H3 (H3K27ac) [3].
Earlier, Chen et al. have shown that both gluconeogenic activity and
HDAC9 expression levels are increased in liver tissues from HCV-
infected patients, and the key role in abnormal glucose homeo-
stasis is played by HDAC3/9-dependent deacetylation of the
zinc-dependent HDACs classes I, II, and IV that hydrolyze the ε-N-
acetyllysine residues of protein substrates, coordinating the oxygen
atom of the acetyl group with zinc ion. HDACs class III, also referred
to as SIRTs (sirtuins), use the coenzyme NADþ for deacetylation [6].
As for zinc-dependent HDACs, they can be located either in the
nucleus (HDAC1/2/3 class I), or in the nucleus and cytoplasm
(HDAC8 class I and HDAC4/5/7/9 class IIa), or in the cytoplasm
(HDAC6/10 class IIb and HDAC11 class IV). It is likely that HDACs
class IIa do not have an independent deacetylation activity, but
after entering the nucleus, they interact with HDAC3 comprising a
part of regulatory transcription complexes and play the role of
signal transducers [7,8].
Epigenetic changes induced by HCV may be caused by the
activation of HDACs, which is promoted by oxidative stress [9]. The
observed increase in the activity of nuclear HDACs led to a decrease
in the level of H3K9ac and suppression of expression of a negative
regulator of iron absorption, hepcidin. It is worth noting that the
inhibition of HDACs with a small molecule compound, trichostatin
A, caused the simultaneous accumulation of H3K9ac, activation of
hepcidin expression, and suppression of HCV replication [10].
Subsequent use of the acetyl-histone H3 chromatin immunopre-
cipitation assay allowed to identify two more genes e osteopontin
(OPN) and apolipoprotein-A1 (Apo-A1), the expression of which,
* Corresponding author.
E-mail address: kozlovmv@eimb.ru (M.V. Kozlov).
Deceased.
1
https://doi.org/10.1016/j.ejmech.2019.111723
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

2
M.V. Kozlov et al. / European Journal of Medicinal Chemistry 183 (2019) 111723
similar to the expression of the hepcidin gene (LEAP-1), is closely
interconnected with HCV infection and HDAC inhibition [11,12].
HDAC6 has a strong nuclear export signal that results in the
cytoplasmic retention of HDAC6, which excludes its participation in
histone deacetylation [13]; nonetheless, a selective inhibition of
HDAC6 leads to suppression of HCV replication [14]. Deacetylating
activity towards numerous proteins, enzymes and transcription
factors as well as ubiquitin-binding activity and interaction with a
molecular motor, dynein, make HDAC6 involved in the most
important cellular processes (cell division, oxidative stress, auto-
phagy etc) that can both contribute to viral infection and counteract
it [15e17]. Unfortunately, the inherent polyfunctionality of HDAC6
makes it difficult to identify the mechanisms of its influence on
HCV replication.
The activity of both nuclear and cytoplasmic HDACs is sup-
pressed in the presence of a variety of hydroxamic inhibitors,
including derivatives of cinnamic hydroxamic acid (CHAs) [18]. In
the structure of CHAs, the phenyl residue of cinnamic acid (linker) is
most often bound to a hydrophobic aromatic or heterocyclic sub-
stituent (cap) via a connection unit, the length of which usually does
not exceed four atoms. The introduction of a suitable substituent
dramatically increases affinity of HDAC inhibitors due to an addi-
tional interaction with the active site of the enzyme. While para and
meta derivatives of CHA are multipotent inhibitors of histone
deacetylases, ortho derivatives often exhibit selectivity for HDAC8
[19].
The SAR studies of CHAs as inhibitors of HCV replication
demonstrated that the introduction of small substituents (Me,
MeO, CF3, etc.) into the phenyl ring or exocyclic cinnamic acid
residue significantly affected the potency of anti-HCV action of
CHAs [20,21]. Predictably, the introduction of more bulky pyridyl-
4-oxy-substituent into different positions of the phenyl ring of
CHAs not only changed the potency of HCV suppression, but also
affected the selectivity of HDAC inhibition [22]. In this work, we
synthesized a set of structural ortho, meta and para isomers of
phenyl derivatives of CHA with connection units of various lengths.
Using a test system with the full-length HCV replicon, we evaluated
the potency of inhibition of HCV replication in parallel with the
acetylation of HDACs protein substrates for each series of com-
pounds. Our data clearly indicate a correlation between the inhi-
bition of HDACs and anti-HCV action of ortho- and meta-CHAs,
while the antiviral activity for para-CHAs was most likely deter-
mined by a different cell target.
synthesis, isomeric hydroxybenzaldehydes were alkylated with
phenethyl bromide or 2-phenoxyethyl bromide in DMF in the
presence of K2CO3 as a base. The length of connection unit between
the phenyl rings of the substituent and the cinnamic acid residue
varied from 1 to 4 atoms; as for ortho-, meta- and para-metoxy
derivatives of CHA, they were used in the biological assays as
reference compounds that do not possess a cap structure.
2.2. Antiviral activity and cytotoxicity
All synthesized ortho-, meta- and para-CHAs were tested as
antiviral agents in the cell test system with the full-length HCV
replicon that additionally encodes the luciferase gene. The strength
of the chemiluminescent signal obtained in the described assay was
proportional to the amount of viral RNA [23]. In parallel, the effect
on cell viability was determined for all compounds using the MTT
test. The results of testing are presented in detail in Table 1 and for
greater clarity are reproduced on a three-dimensional histogram
(Fig. 1A).
As can be seen from Fig. 1A: (i) para-PhO/BnO/PHEO-CHAs
blocked HCV replication many times more strongly than ortho and
meta analogues; (ii) ortho-PhO/BnO/PHEO/PEGO-CHAs were
significantly less cytotoxic than meta and para analogues; (iii) in
comparison with MeO derivatives, phenyl ortho and para, but not
meta derivatives of CHA demonstrated an increased selectivity of
antiviral action; (iv) differences in the antiviral properties of meta-
and para-, but not ortho-PEGO-CHAs, were diminished due to the
connection unit length of 4 atoms.
Based on the results of testing of the entire set of compounds, a
scatter plot (Fig. 1B) of cytotoxicity versus antiviral activity
expressed as pCC50 and pEC50, respectively, was constructed; a
significant moderate positive correlation was found between them
(r ¼ 0.520, P < 0.05). However, it is easy to see that a selection of five
meta-CHAs, together with two methoxy ortho and para derivatives,
formed a separate group of compounds (‘5 mþ2 M0), the antiviral
activity of which statistically correlated with cytotoxic effect
significantly stronger and more reliably (rm ¼ 0.950, Р < 0.002). As
expected, on the scatter plot of pEС50 versus lg(TI), an indicator of
the selectivity of the antiviral effect, the values of lg(TI) for com-
pounds of the ‘5 mþ2 M’ group were minimal and were located in
the narrow range of values e 0.31 (Fig. 1C). At the same time,
phenyl ortho and para derivatives of CHA demonstrated the values
of lg(TI) ꢀ 2, which gives the evidence to suggest that the mecha-
nism of HCV replication suppression is not associated with the
cytotoxic effect of these compounds.
2. Results and discussion
2.1. Chemistry
2.3. Inhibition of HDACs
A set of 15 compounds was synthesized according to the com-
mon synthetic procedures shown in Scheme 1. At the final stage,
hydroxamic acids were prepared by means of activation of the
corresponding cinnamic acids by carbonyldiimidazole in DMF with
subsequent hydroxyaminolysis, as described earlier [21]. At the
previous stage, commercially not available cinnamic acids (CAs)
were synthesized by the Knoevenagel condensation of corre-
sponding benzaldehydes with malonic acid. And at the first stage of
2.3.1. Inhibition of HDAC1/2/3
Our interest in the relationship between suppression of HCV
replication by CHAs and their inhibition of HDACs class I was
generated by convincing data on the involvement of these enzymes
in the development of HCV infection [3,9e12]. However, it is
obvious that deregulation of gene expression in the presence of
CHAs may also be the cause of the cytotoxic effect of these com-
pounds. An analysis of the scatter plots of inhibition of HDACs class
Scheme 1. Reagents and conditions: (i) RBr, K2CO3, DMF, 70e90 ꢁC, 1e2 h; (ii) HO2CCH2CO2H, (CH2)5NH, Py, 120 ꢁC, 3 h; (iii) CDI, DMF, 2 h followed by NH2OH$HCl overnight.

M.V. Kozlov et al. / European Journal of Medicinal Chemistry 183 (2019) 111723
3
Table 1
Anti-HCV activity and cytotoxicity of tested CHAsa.
Rb/Lb
Structure and denotation of ReCHAs EC50
(m
M)c/CC50
(m
M)c/TI (¼ CC50/EC50
)
MetO/1
meta-MeO-CHA
ortho-MeO-CHA
para-MeO-CHA
0.57 0.14/17 3.4/30
1.1 0.17/30 7.5/27
0.56 0.040/15 4.5/27
PhO/1
BnO/2
meta-PhO-CHA
0.40 0.071/20 6.9/50
para-PhO-CHA
0.054 0.0043/20 6.7/370
ortho-PhO-CHA
0.40 0.10/66 9.4/165
meta-BnO-CHA
0.18 0.066/6.6 1.7/37
ortho-BnO-CHA
0.25 0.068/31 6.6/120
para-BnO-CHA
0.042 0.0083/9.9 2.7/240
PHEO/3
PEGO/4
para-PHEO-CHA
0.13 0.032/16 2.8/120
ortho-PHEO-CHA
0.32 0.065/83 2.5/260
meta-PHEO-CHA
0.30 0.090/12 1.6/41
para-PEGO-CHA
0.063 0.017/5.7 1.6/91
meta-PEGO-CHA
0.11 0.033/6.0 1.2/55
ortho-PEGO-CHA
0.37 0.022/45 15/120
a
Cells were treated with different concentrations of the compounds for 72 h, replicon inhibition and cell viability were measured as described in the Experimental Section;
bR is the substituent abbreviation and L is the length of connection unit as number of atoms; cEC50 and CC50 are presented as the mean value and standard deviation of at least
four and six independent experiments, correspondingly.
I versus antiviral activity (pEC50) as well as cytotoxicity (pCC50
)
the ‘5 mþ2 M’ group (rm ¼ 0.910, P < 0.005) only slightly differed
from the indices in the entire set of compounds. Thus, the inhibition
of HDAC1/2/3 by CHAs was responsible for their cytotoxic effect
with a high degree of reliability, while the antiviral activity was
statistically associated with the inhibition of HDAC1/2/3 only in the
‘5 mþ2 M’ group of compounds.
allows to answer an important question: in the presence of which
CHAs the inhibition of class I HDACs predominantly suppresses
replicon propagation, and for which it mainly reduces viability of
the cells.
The efficiency of inhibition of HDAC1/2/3 was initially assessed
using the Western blot analysis for the content of the acetylated
form of histone H3 (H3K9/14ac) in the cell lysates. The quantitative
indicator of inhibition of HDAC1/2/3 was the ratio of the H3K9/14ac
content to the content of unmodified histone H3 normalized to the
same ratio in the absence of the inhibitor (Ac-H3/H3; Fig. 2A). To
construct the corresponding scatter plots, we chose the Ac-H3/H3
2.3.2. Inhibition of HDAC6
The participation of HDAC6 in the HCV life cycle remains
obscure but detectable. Earlier, using two selective HDAC6 in-
hibitors as an example, we showed the concurrence of the curves of
replicon inhibition and accumulation of the acetylated form of
a-
values obtained at a CHA concentration of 3 mM, at which the
level of H3 acetylation varied in the widest possible range (Fig. 2В,
C).
tubulin ( -TubK40ac), for which HDAC6 is the main deacetylating
a
enzyme in the cells of the test system [14,22]. Then, having at our
disposal a set of 15 structurally related CHAs, we planned to reliably
study the statistical relationships between HDAC6 inhibition and
antiviral activity as well as the cytotoxic effect of these compounds.
The statistical relationship between the accumulation of H3K9/
14ac and the values of рЕС50 for the entire set of CHAs was mod-
erate and not significant (r ¼ 0.495, P < 0.1). However, for the
compounds of the ‘5 mþ2 M’ group, the correlation was very strong
and highly significant (rm ¼ 0.928, P < 0.005). Such correlation in-
dicators are usually typical for a causal relationship between
events; in any case, they reliably guarantee the possibility of its
existence. Unexpectedly, a strong and highly significant correlation
(r ¼ 0.741, P < 0.002) between the values of H3K9/14ac and pCC50
was observed for the entire set of compounds, which, in addition to
other evidence, corroborated the correctness of the results of
Western blot analysis and cell viability testing selected for the
scatter plot. It is important to note that the correlation indices in
The results of Western blot analysis for the content of
K40ac in cell lysates are presented in Fig. 3A. A quantitative indi-
cator of the inhibition of HDAC6 was the ratio of the content of
TubK40ac to the content of unmodified -Tub normalized to the
a-Tub-
a
-
a
same ratio in the absence of the inhibitor (Ac-Tub/Tub; Fig. 3A). To
construct the correlation dependences (Fig. 3B and C), we chose the
values of Ac-Tub/Tub that were obtained at a CHA concentration of
3
m
M, at which the acetylation level of
possible range, as well as in the case of the histone H3 acetylation.
Interestingly, a noticeable increase in the content of -TubK40ac
during cell incubation with ortho derivatives of CHA was observed
a-Tub varied in the widest
a

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M.V. Kozlov et al. / European Journal of Medicinal Chemistry 183 (2019) 111723
Fig. 1. (A) Antiviral activity (EC50) and cytotoxicity (CC50) of CHAs tested in the cell system with full-length HCV replicon. (B) Scatter plot of cytotoxicity (pCC50) versus antiviral
activity (pEC50); r is the correlation coefficient calculated for the full set of CHAs, and rm is the correlation coefficient calculated for the ‘5 mþ2 M’ group of compounds (meta-MeO/
PhO/BnO/PHEO/PEGO-CHAs plus ortho- and para-MeO-CHAs); P is the significance of the corresponding correlation. (C) Scatter plot of antiviral activity (pEC50) versus selectivity
(lg(TI)).
only in the case of two compounds e ortho-MeO-CHA and ortho-
BnO-СНА.
selective HDAC8 inhibitor, demonstrated a positive correlation with
the accumulation of the acetylated form of the protein of the
cohesin complex SMC3 (SMC3K105/106ac) [22], which is a bona
fide protein substrate of HDAC8 [24]. The results of Western blot
analysis for the content of SMC3K105/106ac after the incubation of
cells in the presence of phenyl ortho, meta and para derivatives of
СНА are presented in Fig. 4A. As expected, which was nevertheless
surprising, the inhibition of HDAC8 with CHAs even at high con-
centrations took place only in the case of ortho derivatives, with one
A moderate and insignificant correlation (r ¼ 0.509, P < 0.1)
between the accumulation of a-TubK40ac and pEC50 for the entire
set of CHAs became strong and increased in significance
(rm ¼ 0.838, P < 0.05) for the ‘5 mþ2 M’ group of compounds. At the
same time, a strong and highly significant correlation (r ¼ 0.865,
P < 0.001) between a-TubK40ac and pCC50 did not fundamentally
change (rm ¼ 0.919, P < 0.005) upon the transition from the entire
set of compounds to the ‘5 mþ2 M0 group. Thus, the results of the
SAR study of CHAs as HCV replication blockers and inhibitors of
HDAC6 and HDAC1/2/3 almost completely repeated each other. As a
result, we could confidently accept the inhibition of these subtypes
of HDACs as the main source of the cytotoxic effect of the studied
CHAs, as well as the anti-HCV activity for the ‘5 mþ2 M’ group of
compounds.
exception of m-PEGO-CHA at 10 mM concentration.
To compare the curves of HCV replication suppression and
HDAC8 inhibition, the accumulation of SMC3K105/106ac in cells
after their treatment with ortho-CHAs was additionally measured
at concentrations of 0.1, 0.3 and 1
mM (Fig. 4A). As shown in Fig. 4B,
the WB signal was noticeable, starting from 0.1
mM concentration of
all ortho-CHAs, and then increased in parallel with the suppression
of HCV replication, and only in the case of o-BnO-CHA, the replicon
inhibition was significantly outpacing the accumulation of
SMC3K105/106ac. Interestingly, in contrast to other ortho de-
rivatives, o-BnO-CHA inhibited both HDAC1/2/3 and HDAC6;
perhaps, it was the polypotent effect of this compound that caused
the observed ‘gap effect’.
Using all data obtained (24 pairs of values), we built a scatter
plot of logarithm of the normalized ratio of Ac-SMC3/SMC3 versus
replicon inhibition and calculated the corresponding correlation
2.3.3. Inhibition of HDAC8
The lack of correlation between the suppression of HCV repli-
cation and the inhibition of HDAC1/2/3/6 for phenyl ortho de-
rivatives of CHA led us to view HDAC8 as another possible antiviral
target, since it is ortho isomers that have an increased selectivity for
HDAC8 [19]. Currently, the relationship of HDAC8 with HCV repli-
con propagation remains in question. In our hands, the inhibition of
HCV replication with pyridyl-4-oxy ortho derivative of CHA, a new

M.V. Kozlov et al. / European Journal of Medicinal Chemistry 183 (2019) 111723
5
Fig. 2. (A) Western blot analysis for the content of the acetylated forms of histone H3 (H3K9/14ac) in Huh7-luc/neo cells treated with CHAs for 24 h. (B and С) Scatter plots of the
values of pEC50 and pCC50 versus the relative content of H3K9/14ac in the presence of 3 M CHAs; r is the correlation coefficient calculated for the entire set of CHAs and rm is the
correlation coefficient, calculated for the ‘5 mþ2 M’ group of compounds; P is the significance of the corresponding correlation.
m
coefficient, the value of which with very high reliability (r ¼ 0.832,
Р < 0.001) corroborated the existence of a strong statistical rela-
tionship between inhibition of HDAC8 and anti-HCV activity of
ortho-CHAs (Fig. 4C).
suppression of HCV replication. However, in order to demonstrate
the EC50 values at the level of 40e60 nM concentrations, para-CHAs
should probably inhibit a more susceptible antiviral target X, which
does not belong to the studied group of HDACs. This assumption
explains rather well why phenyl meta derivatives of CHA as well as
methoxy derivatives of CHA showed similar patterns of anti-HCV
action, forming a separate group of compounds ‘5 mþ2 M’
(Figs.1e3). Indeed, since the phenyl meta derivatives of CHA did not
have the required molecular geometry, while the methoxy de-
rivatives of CHA did not contain an aromatic substituent at all, all of
these compounds were unable to inhibit either HDAC8 targeted by
ortho-CHAs or hypothetical target X for para-CHAs, but exclusively
HDAC1/2/3/6.
2.4. Independent antiviral effect of para-CHAs on HDACs
All the results obtained in this work indicated that the antiviral
activity of phenyl para derivatives of CHA can not be interpreted as
a result of the inhibition of HDACs class I/IIb. This can be visually
demonstrated using the graphs of the concentration dependence of
HCV replication suppression and accumulation of H3K9/14ac and
a
-TubK40ac for each of the para-CHAs (Fig. 5). It can be seen that in
the presence of 0.3 M of any of the compounds, 80e90% inhibition
m
of the replicon was already taking place, while the level of acety-
lation of protein substrates was only beginning to grow, and neither
the rate of this growth nor the maximum achievable values
3. Conclusion
Using a set of structural isomers of CHA carrying a phenyl sub-
stituent in the aromatic ring of a cinnamic acid residue, we were
able to establish statistical relationships between inhibition of
HDACs class I/IIb and suppression of HCV replicon propagation.
correlated with the corresponding values of ЕС50
Most likely, the inhibition of HDAC1/2/3/6 with para-CHAs upon
reaching the concentration of 0.3 M and higher contributes to the
.
m

6
M.V. Kozlov et al. / European Journal of Medicinal Chemistry 183 (2019) 111723
Fig. 3. (A) Western blot analysis for the content of the acetylated form of
values of pEC50 and pCC50 versus the relative content of -TubK40ac in the presence of 3
correlation coefficient, calculated for the ‘5 mþ2 M’ group of compounds; P is the significance of the corresponding correlation.
a-tubulin (
a
-TubK40ac) in Huh7-luc/neo cells treated with CHAs for 24 h. (B and С) Scatter plots of the
a
m
M CHA; r is the correlation coefficient calculated for the entire set of CHAs and rm is the
Anti-HCV activity correlated strongly and significantly with the
inhibition of HDAC8 in the case of ortho-CHAs, while in the case of
meta-CHAs it correlated in a similar manner with the inhibition of
HDAC1/2/3 and HDAC6. The antiviral activity of para-CHAs was
many times stronger than that of meta-CHAs, despite about the
same efficiency of HDAC1/2/3/6 inhibition. Based on this observa-
tion, we suggested the existence of an additional antiviral target,
the identification of which is of unquestionable interest. Finally, the
results of the SAR study will be useful for the design of new de-
rivatives of cinnamic hydroxamic acid e host targeting agents that
suppress HCV replication.
Daltonics micrOTOF-Q II hybrid quadrupole time-of-flight mass
spectrometer using electrospray ionization (ESI); measurements
were done in positive ion mode. Cinnamic acids formed stable
complexes of association ions with potassium impurity ions [25],
which were observed as an overlaid sharp peak on the base peak
chromatogram. TLC was performed on the plates of Kieselgel 60
F254 (Merck) and the column chromatography was carried out on
silica gel (Kieselgel, 0.035e0.070 mm, Acros Organics) using the
elution systems mentioned in the text.
4.1.2. Synthesis of ortho-, meta- and para-MeO-CHA
4. Experimental section
4.1. Chemistry
(iii) To a solution of the corresponding acid (0.71 g, 4.0 mmol) in
DMF (4 ml), CDI (0.65 g, 4.0 mmol) was added. After 2 h incu-
bation, NH2OH$HCl (0.56 g, 8.0 mmol) was added, the mixture
was stirred for 15 min and kept overnight (~18 h). Vessel con-
tents were mixed with o-xylol (8 ml) and evaporated. The res-
idue was dissolved in EtOAc (25 ml) and washed with 4%
NaHCO3 (25 ml) followed by H2O (25 ml), dried over Na2SO4 and
evaporated. The product was isolated by silica gel chromatog-
raphy using a CHCl3eEtOHeAcOH mixture (100 : 10: 1) as an
eluent. The selected fractions were evaporated, the crude
4.1.1. General
NMR spectra (d, ppm; J, Hz) were registered on a AMX III-400
spectrometer (Bruker, Germany) at 400 MHz for 1H NMR (internal
standard: Me4Si; solvent: DMSO‑d6) and 100.6 MHz for 13C NMR
with suppression of carbon-proton interaction (solvent: DMSO‑d6).
High-resolution mass spectra (HRMS) were registered on a Bruker

M.V. Kozlov et al. / European Journal of Medicinal Chemistry 183 (2019) 111723
7
Fig. 4. (A) Western blot analysis for the content of the acetylated forms of SMC3 (SMC3K105/106ac) in Huh7-luc/neo cells treated with CHAs for 24 h. (B) The relationship between
inhibition of HDAC8 and HCV replication blocking with CHAs; inhibition of the replicon for 72 h is expressed as a percentage (left axis); the values of HDAC8 inhibition were
calculated as the Ac-SMC3/SMC3 normalized ratio (right axis). (С) Scatter plot of logarithm of the relative content of SMC3K105/106ac (in the presence of 0, 0.1, 0.3, 1, 3 and 10
ortho-PhO/BnO/PHEO/PEGO-CHAs for 24 h) versus replicon inhibition (%); r and P are the correlation coefficient and its significance.
mM of
Fig. 5. The relationship between the inhibition of HDAC1/2/3/6 and HCV replication blocking with para-CHAs; the replicon inhibition for 72 h is expressed as a percentage (left
axis); the values of HDAC1/2/3 and HDAC6 inhibition for 24 h were calculated as the Ac-H3/H3 and Ac-Tub/Tub normalized ratios (right axis).
1H), 3.86 (s, 3H). 13C NMR (75 MHz, DMSO‑d6)
d 163.66, 157.99,
product was washed with a small volume of non-polar solvent
(benzene, 1,2-DCE or CHCl3), the obtained precipitate was
filtered and air dried. The yields of o-, m- and p-MeO-CHA were
19, 25 and 40%, respectively.
133.84, 131.28, 128.32, 123.68, 121.16, 119.91, 112.17, 56.02. ESI-
HRMS m/z: Calcd for
C
10H11NO3 [MþH]þ 194.0812, found
194.0816; calcd [MþNa]þ 216.0631, found 216.0638.
(E)-N-Hydroxy-3-(3-methoxyphenyl)acrylamide
(meta-MeO-
CHA): 1H NMR (400 MHz, DMSO)
d 10.78 (s, 1H), 9.10 (bs, 1H), 7.42
(E)-N-Hydroxy-3-(2-methoxyphenyl)acrylamide
(ortho-MeO-
CHA): 1H NMR (400 MHz, DMSO)
d
10.69 (s, 1H), 8.95 (s, 1H), 7.68
(d, J ¼ 15.8 Hz, 1H), 7.31 (t, J ¼ 7.9 Hz, 1H), 7.17e7.06 (m, 2H), 6.94 (d,
J ¼ 6.6 Hz, 1H), 6.50 (d, J ¼ 15.8 Hz, 1H), 3.78 (s, 3H). 13C NMR
(d, J ¼ 15.8 Hz, 1H), 7.50 (d, J ¼ 7.9 Hz, 1H), 7.36 (t, J ¼ 7.8 Hz, 1H),
7.07 (d, J ¼ 8.1 Hz, 1H), 6.98 (t, J ¼ 7.5 Hz, 1H), 6.50 (d, J ¼ 16.0 Hz,
(101 MHz, DMSO)
d 162.66, 159.56, 138.16, 136.25, 129.89, 119.71,

8
M.V. Kozlov et al. / European Journal of Medicinal Chemistry 183 (2019) 111723
119.49, 115.16, 112.63, 55.08. ESI-HRMS m/z: Calcd for C10H11NO3
[MþH]þ 194.0812, found 194.0808; calcd [2 M þ H]þ 387.1551,
found 387.1550.
119.41, 118.06. ESI-HRMS (m/z): Calcd for
C
15H12O3 [MþH]þ
241.0859, found 241.0861; calcd [M þ NH4]þ 258.1125, found
258.1128; calculated [MþNa]þ 263.0679, found 263.0683; calcd
[6 M þ K]2 þ 740.2212, found 740.2166; calcd [6 M þ K]þ
1479.4350, found 1479.4278.
(E)-N-Hydroxy-3-(4-methoxyphenyl)acrylamide
(para-MeO-
CHA): 1H NMR (400 MHz, DMSO)
d
9.78 (bs, 2H), 7.48 (d, J ¼ 8.3 Hz,
2H), 7.33 (d, J ¼ 15.8 Hz, 1H), 6.95 (d, J ¼ 8.6 Hz, 2H), 6.31 (d,
(E)-3-(3-Phenoxyphenyl)acrylic acid (meta-PhO-CA): 1H NMR
J ¼ 15.9 Hz, 1H), 3.78 (s, 3H). 13C NMR (75 MHz, DMSO‑d6)
d
163.60,
(400 MHz, DMSO)
d
12.41 (bs, 1H), 7.57 (d, J ¼ 16.0 Hz, 1H),
160.64, 137.78, 129.40, 128.08, 117.5, 114.80, 55.70. ESI-HRMS m/z:
Calcd for C10H11NO3 [MþH]þ 194.0812, found 194.0813; calcd
[2 M þ H]þ 387.1551, found 387.1558.
7.49e7.34 (m, 5H), 7.15 (t, J ¼ 7.4 Hz, 1H), 7.06e6.96 (m, 3H), 6.51 (d,
J ¼ 16.0 Hz, 1H). 13C NMR (101 MHz, DMSO)
d 167.30, 157.01, 156.40,
143.05, 136.29, 130.42, 130.01, 123.54, 123.26, 120.14, 120.10, 118.60,
118.13. ESI-HRMS (m/z): Calcd for C15H12O3 [MþH]þ 241.0859,
found 241.0864; calcd [5 M þ K]2þ 620.1818, found 620.1772; calcd
[6 M þ K]2þ 740.2212, found 740.2179; calcd [4 M þ K]þ 999.2777,
found 999.2716. calcd [5 M þ K]þ 1239.3564, found 1239.3512.
(E)-3-(4-Phenoxyphenyl)acrylic acid (para-PhO-CA): 1H NMR
4.1.3. Synthesis of ortho-, meta- and para-PhO-CHA
(iii) To a solution of the corresponding acid (0.72 g, 3.0 mmol) in
DMF (3 ml), CDI (0.50 g, 3.0 mmol) was added. After 2 h incu-
bation, NH2OH$HCl (0.21 g, 3.0 mmol) was added, the mixture
was stirred for 15 min and kept overnight (~18 h). The solution
was poured into H2O (25 ml) and washed with EtOAc (25 ml);
the organic layer was washed with H2O (20 ml), dried over
Na2SO4 and evaporated. The o- and m-PhOCHA or p-PhOCHA
were isolated by silica gel chromatography using EtOAc or a
CHCl3eEtOHeAcOH mixture (100 : 10: 1) as eluents, corre-
spondingly. The selected fractions were evaporated, the crude
product was washed with a small volume of non-polar solvent
(1,2-DCE or CCl4), the obtained precipitate was filtered and air
dried. The yields of o-, m- and p-PhO-CHA were 20, 28 and 21%,
respectively.
(400 MHz, DMSO)
d
12.30 (bs, 1H), 7.70 (d, J ¼ 8.7 Hz, 2H), 7.58 (d,
J ¼ 16.0 Hz,1H), 7.42 (t, J ¼ 8.0 Hz, 2H), 7.19 (t, J ¼ 7.4 Hz,1H), 7.07 (d,
J ¼ 7.7 Hz, 2H), 6.99 (d, J ¼ 8.7 Hz, 2H), 6.44 (d, J ¼ 16.0 Hz, 1H). 13
C
NMR (101 MHz, DMSO‑d6)
d 167.56, 158.58, 155.67, 143.12, 130.12,
129.23, 124.11, 119.32, 118.17, 117.96. ESI-HRMS (m/z): Calcd for
C
15H12O3 [MþH]þ 241.0859, found 241.0864; calcd [5 M þ K]2þ
620.1818, found 620.1779; calcd [6 M þ K]2þ 740.2212, found
740.2181; calcd [4 M þ K]þ 999.2777, found 999.2722.
4.1.4. Synthesis of ortho-, meta- and para-BnO-CHA
(iii) To a solution of the corresponding acid (0.51 g, 2.0 mmol) in
DMF (2 ml), CDI (0.33 g, 2.0 mmol) was added. After 2 h incu-
bation, NH2OH$HCl (0.16 g, 2.3 mmol) was added, the mixture
was stirred for 15 min and kept overnight (~18 h). The solution
was poured into H2O (20 ml) and the product was isolated from
oily residue by silica gel chromatography using a CHCl3eEtOH
mixture (100 : 10) as an eluent. The selected fractions were
evaporated, the crude product was washed with a small volume
of non-polar or low polar solvents (1,2-DCE, MeCN or EtOH), the
obtained precipitate was filtered and air dried. The yields of o-,
m- and p-BnO-CHA were 26, 22 and 17%, respectively.
(E)-N-Hydroxy-3-(2-phenoxyphenyl)acrylamide
(ortho-PhO-
CHA): 1H NMR (400 MHz, DMSO‑d6)
d
10.80 (s, 1H), 9.04 (s, 1H),
7.75e7.57 (m, 2H), 7.38 (bs, 3H), 7.21 (bs, 1H), 7.13 (bs, 1H),
6.99e6.95 (m, 2H), 6.91 (d, J ¼ 7.2 Hz, 2H), 6.57 (d, J ¼ 15.5 Hz, 1H).
13C NMR (101 MHz, DMSO)
d 162.64, 156.81, 154.47, 132.28, 130.97,
130.10, 128.07, 126.37, 124.30, 123.40, 120.74, 119.55, 118.02. ESI-
HRMS (m/z): Calcd for
C
15H13NO3 [MþH]þ 256.0968, found
256.0968; calcd [MþNa]þ 278.0788, found 278.0788.
(E)-N-Hydroxy-3-(3-phenoxyphenyl)acrylamide
(meta-PhO-
d 10.71 (bs, 1H), 9.04 (bs, 1H),
CHA): 1H NMR (400 MHz, DMSO)
7.46e7.36 (m, 4H), 7.33 (d, J ¼ 7.3 Hz, 1H), 7.23e7.11 (m, 2H), 7.02
(E)-3-(2-(Benzyloxy)phenyl)-N-hydroxyacrylamide (ortho-BnO-
(dd, J ¼ 15.5, 7.7 Hz, 3H), 6.43 (d, J ¼ 15.8 Hz, 1H). 13C NMR
CHA): 1H NMR (400 MHz, DMSO)
d 10.72 (bs, 1H), 9.00 (bs, 1H), 7.77
(101 MHz, DMSO‑d6)
d
162.39, 157.11, 156.33, 137.50, 136.86, 130.50,
(d, J ¼ 15.9 Hz,1H), 7.53 (d, J ¼ 7.2 Hz,1H), 7.49e7.28 (m, 6H), 7.14 (d,
130.05, 123.62, 122.73, 119.99, 119.44, 118.71, 116.88. ESI-HRMS (m/
z): Calcd for C15H13NO3 [MþH]þ 256.0968, found 256.0969; calcd
[MþNa]þ 278.0788, found 278.0788.
J ¼ 8.2 Hz, 1H), 6.98 (t, J ¼ 7.3 Hz, 1H), 6.50 (d, J ¼ 15.9 Hz, 1H), 5.21
(s, 2H). 13C NMR (101 MHz, DMSO)
d 163.04, 156.31, 136.85, 133.01,
130.67, 128.48, 127.85, 127.46, 123.67, 120.92, 119.42, 113.12, 69.55.
ESI-HRMS (m/z): Calcd for C16H15NO3 [MþH]þ 270.1125, found
270.1125; calcd [MþNa]þ 292.0944, found 292.0948.
(E)-N-Hydroxy-3-(4-phenoxyphenyl)acrylamide
(para-PhO-
CHA): 1H NMR (400 MHz, DMSO)
d 10.70 (bs, 1H), 8.99 b(s, 1H), 7.57
(d, J ¼ 8.2 Hz, 2H), 7.49e7.38 (m, 3H), 7.18 (t, J ¼ 7.4 Hz, 1H), 7.06 (d,
(E)-3-(3-(Benzyloxy)phenyl)-N-hydroxyacrylamide (meta-BnO-
J ¼ 7.9 Hz, 2H), 7.00 (d, J ¼ 8.6 Hz, 2H), 6.38 (d, J ¼ 15.8 Hz, 1H). 13
C
CHA): 1H NMR (400 MHz, DMSO‑d6)
d 9.92 (bs, 1H), 7.51e7.36 (m,
NMR (101 MHz, DMSO)
d
162.79, 157.82, 155.87, 137.49, 130.07,
5H), 7.36e7.28 (m, 2H), 7.21 (s, 1H), 7.14 (d, J ¼ 7.5 Hz, 1H), 7.02 (d,
129.90, 129.25, 123.92, 119.11, 118.38, 117.95. ESI-HRMS (m/z): Calcd
J ¼ 8.1 Hz, 1H), 6.48 (d, J ¼ 15.8 Hz, 1H), 5.14 (s, 2H). 13C NMR
for C15H13NO3 [MþH]þ 256.0968, found 256.0973.
(101 MHz, DMSO‑d6) d 162.52, 158.63, 137.89, 136.88, 136.30, 129.91,
(ii) Preparation of ortho-, meta- and para-PhO-CA. To the
mixture of the corresponding aldehyde (2.52 g, 12.7 mmol) and
malonic acid (1.99 g, 19.1 mmol), dry pyridine (6.5 ml) and piperi-
dine (0.2 ml) were successively added. The mixture was stirred at
120 ꢁC for 3 h, cooled to 10 ꢁC and acidified with HCl (~14 ml) at 1:1
ratio with cooling and stirring. After 30 min, the precipitate was
filtered, washed 3 times with H2O (20 ml) and air dried. The yields
of o-, m- and p-PhO-CA were 92, 91 and 96%, respectively.
(E)-3-(2-Phenoxyphenyl)acrylic acid (ortho-PhO-CA): 1H NMR
128.37, 127.79, 127.66, 119.89, 119.55, 115.92, 113.58, 69.18. ESI-
HRMS (m/z): Calcd for
C
16H15NO3 [MþH]þ 270.1125, found
270.1125; calcd [MþNa]þ 292.0944, found 292.0944.
(E)-3-(4-(Benzyloxy)phenyl)-N-hydroxyacrylamide
(para-BnO-
CHA): 1H NMR (400 MHz, DMSO)
d 10.64 (bs, 1H), 8.95 (bs, 1H), 7.50
(d, J ¼ 8.2 Hz, 2H), 7.47e7.30 (m, 6H), 7.04 (d, J ¼ 8.6 Hz, 2H), 6.32 (d,
J ¼ 15.7 Hz, 1H), 5.14 (s, 2H). 13C NMR (101 MHz, DMSO)
d 163.07,
159.31, 137.89, 136.73, 128.95128.36, 127.80, 127.61, 116.61, 115.15,
69.25. ESI-HRMS (m/z): Calcd for C16H15NO3 [MþH]þ 270.1125;
found 270.1128; calcd [MþNa]þ 292.0944, found 292.0948.
(ii) Preparation of ortho-, meta- and para-BnO-CA was per-
formed exactly as described for PhO-CA in section 4.1.3. The yields
of the corresponding structural isomers were 87, 96 and 94%.
(E)-3-(2-(Benzyloxy)phenyl)acrylic acid (ortho-BnO-CA): 1H NMR
(400 MHz, DMSO)
d
12.44 (s, 1H), 7.89 (dd, J ¼ 7.8, 1.2 Hz, 1H), 7.79
(d, J ¼ 16.2 Hz, 1H), 7.46e7.35 (m, 3H), 7.21 (t, J ¼ 7.5 Hz, 1H), 7.15 (t,
J ¼ 7.4 Hz, 1H), 6.98 (d, J ¼ 7.8 Hz, 2H), 6.92 (d, J ¼ 8.1 Hz, 1H), 6.60
(d, J ¼ 16.1 Hz, 1H). 13C NMR (101 MHz, DMSO)
d 167.48, 156.73,
154.72, 137.50, 131.86, 130.16, 128.57, 125.62, 124.26, 123.54, 120.61,

M.V. Kozlov et al. / European Journal of Medicinal Chemistry 183 (2019) 111723
9
(400 MHz, DMSO)
d
12.27 (bs, 1H), 7.89 (d, J ¼ 16.2 Hz, 1H), 7.69 (dd,
DMSO‑d6) d 163.20, 159.25, 138.73, 136.78, 130.45, 129.43, 128.78,
J ¼ 7.7, 1.4 Hz, 1H), 7.51e7.31 (m, 6H), 7.17 (d, J ¼ 8.4 Hz, 1H), 6.99 (t,
126.76, 120.41, 119.94, 116.31, 113.54, 68.67, 35.41. ESI-HRMS (m/z):
Calcd for C17H17NO3 [MþH]þ 284.1281, found 284.1280; calcd
[MþK]þ 322.0840, found 322.0843.
J ¼ 7.5 Hz, 1H), 6.53 (d, J ¼ 16.2 Hz, 1H), 5.21 (s, 2H). 13C NMR
(101 MHz, DMSO)
d 167.74, 156.71, 138.59, 136.69, 131.56, 128.47,
127.92,127.57,122.83,120.92,119.38,113.06, 69.71. ESI-HRMS (m/z):
(E)-N-Hydroxy-3-(4-phenethoxyphenyl)acrylamide (para-PHEO-
Calcd for
C
16H14O3 [MþH]þ 255.1016, found 255.1016; calcd
CHA): 1H NMR (400 MHz, DMSO)
d 10.64 (bs, 1H), 8.96 (bs, 1H), 7.48
[M þ NH4]þ 272.1281, found 272.1281; calcd [MþNa]þ 277.0835,
found 277.0833; calcd [2 M þ Na]þ 531.1778, found 531.1781; calcd
[6 M þ K]2þ 782.2681, found 782.2624; calcd [7 M þ K]2þ 909.3152,
found 909.3092; calcd [5 M þ K]þ 1309.4346, found 1309.4241;
calcd [6 M þ K]þ 1563.5289, found 1563.5204.
(d, J ¼ 8.3 Hz, 2H), 7.40 (d, J ¼ 15.8 Hz, 1H), 7.35e7.27 (m, 4H),
7.25e7.19 (m, 1H), 6.96 (d, J ¼ 8.6 Hz, 2H), 6.32 (d, J ¼ 15.8 Hz, 1H),
4.22 (t, J ¼ 6.8 Hz, 2H), 3.03 (t, J ¼ 6.8 Hz, 2H). 13C NMR (101 MHz,
DMSO‑d6)
d 163.14, 159.43, 138.19, 137.97, 128.99, 128.89, 128.25,
127.40, 126.24, 116.52, 114.85, 68.20, 34.79. ESI-HRMS (m/z): Calcd
for C17H17NO3 [MþH]þ 284.1281, found 284. 1292; calcd [MþNa]þ
306.1101, found 306.1115.
(ii) Preparation of ortho-, meta- and para-PHEO-CA was per-
formed exactly as described for PhO-CA in section 4.1.3, except that
after cooling and stirring for 30 min the obtained conglomeration
of oily crystals was carefully crushed up and the resulting solids
were filtered, washed 3 times with H2O (30 ml) and air dried. The
yields of the corresponding structural isomers were 98, 84 and 88%.
(E)-3-(2-Phenethoxyphenyl)acrylic acid (ortho-PHEO-CA): 1H
(E)-3-(3-(Benzyloxy)phenyl)acrylic acid (meta-BnO-CA): 1H NMR
(400 MHz, DMSO)
d
12.37 (s, 1H), 7.56 (d, J ¼ 16.0 Hz, 1H), 7.47 (d,
J ¼ 7.3 Hz, 2H), 7.44e7.29 (m, 5H), 7.25 (d, J ¼ 7.6 Hz, 1H), 7.06 (dd,
J ¼ 8.1, 1.7 Hz, 1H), 6.55 (d, J ¼ 16.0 Hz, 1H), 5.15 (s, 2H). 13C NMR
(101 MHz, DMSO‑d6) d 167.45,158.64,143.72,136.89,135.63,129.88,
128.35, 127.77, 127.67, 120.95, 119.56, 116.98, 113.76, 69.26. ESI-
HRMS (m/z): Calcd for
C
16H14O3 [MþH]þ 255.1016, found
255.1018; calcd [MþNa]þ 277.0835, found 277.0833; calcd
[6 M þ K]2þ 782.2681, found 782.2629; calcd [7 M þ K]2þ 909.3152,
found 909.3104; calcd [8 M þ K]2þ 1036.3624, found 1036.3564;
calcd [5 M þ K]þ 1309.4346, found 1309.4249.
NMR (400 MHz, DMSO)
d
12.26 (bs, 1H), 7.84 (d, J ¼ 16.2 Hz, 1H),
7.64 (d, J ¼ 7.6 Hz, 1H), 7.41e7.26 (m, 5H), 7.21 (t, J ¼ 7.1 Hz, 1H), 7.07
(d, J ¼ 8.2 Hz, 1H), 6.96 (t, J ¼ 7.4 Hz, 1H), 6.50 (d, J ¼ 16.2 Hz, 1H),
4.25 (t, J ¼ 6.6 Hz, 2H), 3.08 (t, J ¼ 6.6 Hz, 2H). 13C NMR (101 MHz,
(E)-3-(4-(Benzyloxy)phenyl)acrylic acid (para-BnO-CA): 1H NMR
(400 MHz, DMSO‑d6)
d
12.19 (s, 1H), 7.62 (d, J ¼ 8.7 Hz, 2H), 7.55 (d,
J ¼ 16.0 Hz, 1H), 7.45 (d, J ¼ 7.3 Hz, 2H), 7.39 (t, J ¼ 7.3 Hz, 2H), 7.33
DMSO) d 167.79, 156.87, 138.76, 138.25, 131.61, 129.00, 128.48,
(dd, J ¼ 8.4, 5.9 Hz, 1H), 7.04 (d, J ¼ 8.7 Hz, 2H), 6.38 (d, J ¼ 16.0 Hz,
128.24, 126.24, 122.58, 120.67, 119.31, 112.47, 68.77, 34.95. ESI-
1H), 5.15 (s, 2H). 13C NMR (101 MHz, DMSO‑d6)
d
167.72, 159.96,
HRMS (m/z): Calcd for
C
17H16O3 [MþH]þ 269.1172, found
143.59, 136.69, 129.84, 128.39, 127.84, 127.64, 127.01, 116.62, 115.15,
69.31. ESI-HRMS (m/z): Calcd for C16H14O3 [MþH]þ 255.1016, found
255.1016; calcd [MþNa]þ 277.0835, found 277.0833; calcd
[2 M þ Na]þ 531.1778, found 531.1780; calcd [6 M þ K]2þ 782.2681,
found 782.2632; calcd [7 M þ K]2þ 909.3152, found 909.3067; calcd
[4 M þ K]þ 1055.3403, found 1055.3341; calcd [5 M þ K]þ
1309.4346, found 1309.4210.
269.1175; calcd [M þ NH4]þ 286.1438, found 286.1441; calcd
[MþNa]þ 291.0992, found 291.0995; calcd [2 M þ Na]þ 559.2091,
found 559.2102.
(E)-3-(3-Phenethoxyphenyl)acrylic acid (meta-PHEO-CA): 1H
NMR (400 MHz, DMSO)
d
12.39 (s, 1H), 7.55 (d, J ¼ 16.0 Hz, 1H),
7.39e7.17 (m, 9H), 6.97 (dd, J ¼ 8.0, 1.7 Hz, 1H), 6.55 (d, J ¼ 16.0 Hz,
1H), 4.23 (t, J ¼ 6.9 Hz, 2H), 3.04 (t, J ¼ 6.9 Hz, 2H). 13C NMR
(101 MHz, DMSO)
d 167.57, 158.78, 143.86, 138.30, 135.70, 129.91,
4.1.5. Synthesis of ortho-, meta- and para-PHEO-CHA
128.96, 128.30, 126.27, 120.82, 119.57, 116.80, 113.42, 68.21, 34.93.
ESI-HRMS (m/z): Calcd for C17H16O3 [MþH]þ 269.1172, found
269.1178; calcd [M þ NH4]þ 286.1438, found 286.1447; calcd
[MþNa]þ 291.0992, found 291.0995; calcd [2 M þ Na]þ 559.2091,
found 559.2108; calcd [6 M þ K]2þ 824.3151, found 824.3120; calcd
[7 M þ K]2þ 958.3700, found 958.3665; calcd [8 M þ K]2þ
1092.4250, found 1092.4213.
(iii) To a solution of the corresponding acid (0.80 g, 3.0 mmol) in
DMF (3 ml), CDI (0.50 g, 3.0 mmol) was added. After 2 h incu-
bation, NH2OH$HCl (0.21 g, 3.0 mmol) was added, the mixture
was stirred for 15 min and kept overnight (~18 h). The solution
was diluted with 10% KHCO3 (25 ml) and washed with a mixture
of EtOAc (30 ml) and MeOH (8 ml). Organic layer was filtered,
washed with brine (15 ml), dried over Na2SO4 and evaporated.
The product was isolated by silica gel chromatography using a
CHCl3eEtOH mixture (10 : 1) as an eluent. The selected fractions
were evaporated, the crude product was washed with a small
volume of non-polar solvent (toluene or CCl4), the obtained
precipitate was filtered and air dried. The yields of o-, m- and p-
PHEO-CHA were 22, 25 and 20%, respectively.
(E)-3-(4-Phenethoxyphenyl)acrylic acid (para-PHEO-CA): 1H
NMR (400 MHz, DMSO)
d
12.19 (s, 1H), 7.60 (d, J ¼ 8.6 Hz, 2H), 7.54
(d, J ¼ 16.0 Hz, 1H), 7.39e7.26 (m, 4H), 7.22 (dt, J ¼ 8.1, 4.1 Hz, 1H),
6.96 (d, J ¼ 8.6 Hz, 2H), 6.36 (d, J ¼ 16.0 Hz, 1H), 4.23 (t, J ¼ 6.8 Hz,
2H), 3.03 (t, J ¼ 6.8 Hz, 2H). 13C NMR (101 MHz, DMSO)
d 167.75,
160.07, 143.64, 138.15, 129.87, 128.89, 128.26, 126.82, 126.25, 116.49,
114.83, 68.23, 34.78. ESI-HRMS (m/z): Calcd for C17H16O3 [MþH]þ
269.1172, found 269.1175; calcd [MþNa]þ 291.0992, found
291.1000; calcd [2 M þ Na]þ 559.2091, found 559.2105; calcd
[6 M þ K]2þ 824.3151, found 824.3110; calcd [7 M þ K]2þ 958.3700,
found 958.3663; calcd [4 M þ Na]þ 1111.4029, found 1111.3984;
calcd [5 M þ Na]þ 1379.5129, found 1379.5067.
(E)-N-Hydroxy-3-(2-phenethoxyphenyl)acrylamide (ortho-PHEO-
CHA): 1H NMR (400 MHz, DMSO)
d 10.73 (bs, 1H), 9.00 (bs, 1H), 7.71
(d, J ¼ 15.9 Hz,1H), 7.49 (d, J ¼ 7.3 Hz, 1H), 7.40e7.27 (m, 5H), 7.21 (t,
J ¼ 7.1 Hz, 1H), 7.06 (d, J ¼ 8.3 Hz, 1H), 6.95 (t, J ¼ 7.4 Hz, 1H), 6.52 (d,
J ¼ 15.9 Hz, 1H), 4.24 (t, J ¼ 6.8 Hz, 2H), 3.11 (t, J ¼ 6.7 Hz, 2H). 13C
(i) Preparation of ortho-PHEO-BA. Phenethyl bromide (3.70 g,
20 mmol), K2CO3 (3.04 g, 22 mmol) and TEBAC (0.50 g, 2.2 mmol)
were added to the solution of salicylic aldehyde (2.68 g, 22 mmol)
in DMF (20 mL). The reaction mixture was stirred at 90ꢁС for 1 h,
followed by supplementing of the additional portion of phenethyl
bromide (1.85 g, 10 mmol) and K2CO3 (3.04 g, 22 mmol) into the
vessel, after which heating and stirring continued for 1 h 40 min.
After cooling, the vessel contents were filtered, salts were washed 2
times on a filter with o-xylol (10 ml) and combined filtrates were
evaporated. The residue was dissolved in DCM (50 ml) and washed
2 times with 1 M KOH (40 ml), followed by H2O (40 ml, 2 times) and
NMR (101 MHz, DMSO)
d 163.18, 156.66, 138.28, 133.49, 130.71,
129.03, 128.26, 127.94, 126.25, 123.32, 120.66, 119.46, 112.47, 68.77,
34.97. ESI-HRMS (m/z): Calcd for C17H17NO3 [MþH]þ 284.1281,
found 284.1281; calcd [MþNa]þ 306.1101, found 306.1101.
(E)-N-Hydroxy-3-(3-phenethoxyphenyl)acrylamide (meta-PHEO-
CHA): 1H NMR (300 MHz, DMSO)
d 10.71 (s,1H), 9.04 (s,1H), 7.44 (d,
J ¼ 15.8 Hz, 1H), 7.38e7.27 (m, 5H), 7.26e7.19 (m, 1H), 7.13 (d,
J ¼ 6.3 Hz, 2H), 6.95 (d, J ¼ 8.3 Hz, 1H), 6.48 (d, J ¼ 15.8 Hz, 1H), 4.22
(t, J ¼ 6.8 Hz, 2H), 3.05 (t, J ¼ 6.8 Hz, 2H). 13C NMR (75 MHz,

10
M.V. Kozlov et al. / European Journal of Medicinal Chemistry 183 (2019) 111723
brine (40 ml); the organic layer was dried over Na2SO4 and evap-
orated. A spontaneous crystallization of the product took place
within 10 min, after which the crude product was resuspended in
hexane (20 ml), filtered and air dried. The yield was 47%.
322.1051.
(E)-N-Hydroxy-3-(4-(2-phenoxyethoxy)phenyl)acrylamide (para-
PEGO-CHA): 1H NMR (400 MHz, DMSO‑d6)
9.96 (bs, 2H), 7.51 (d,
d
J ¼ 8.2 Hz, 2H), 7.42 (d, J ¼ 15.8 Hz, 1H), 7.30 (t, J ¼ 7.9 Hz, 2H),
2-Phenethoxybenzaldehyde
(ortho-PHEO-BA):
1H
NMR
7.04e6.92 (m, 5H), 6.33 (d, J ¼ 15.8 Hz,1H), 4.33 (dd, J ¼ 13.3, 5.0 Hz,
(400 MHz, DMSO) 10.29 (s, 1H), 7.74e7.54 (m, 2H), 7.44e7.27 (m,
d
4H). 13C NMR (101 MHz, DMSO‑d6)
d 163.06, 159.34, 158.25, 137.88,
4H), 7.27e7.17 (m, 2H), 7.04 (t, J ¼ 7.5 Hz, 1H), 4.34 (t, J ¼ 6.6 Hz, 2H),
129.49, 129.03, 127.64, 120.74, 116.70, 114.91, 114.47, 66.49, 66.11.
ESI-HRMS (m/z): Calcd for C17H17NO4 [MþH]þ 300.1230, found
300.1236; calcd [MþNa]þ 322.1050, found 322.1055.
3.10 (t, J ¼ 6.5 Hz, 2H). 13C NMR (101 MHz, DMSO)
d 189.02, 160.81,
138.23, 136.36,128.96,128.26,127.43,126.29,124.24,120.64,113.58,
69.01, 34.78. ESI-HRMS (m/z): Calcd for C15H14O2 [MþH]þ 227.1067,
found 227.1068; calcd [MþNa]þ 249.0886, found 249.0893.
(ii) Preparation of ortho-, meta- and para-PEGO-CA was per-
formed exactly as described for PHEO-CA in section 4.1.5. The
yields of the corresponding structural isomers were 97, 94 and
93%.
(i) Preparation of meta- and para-PHEO-BA. Phenethyl bromide
(1.85 g, 10 mmol), K2CO3 (1.52 g, 11 mmol) were added to the so-
lution of the corresponding aldehyde (1.22 g, 10 mmol) in DMF
(10 mL). The reaction mixture was stirred at 90ꢁС for 2 h. After
cooling, the vessel contents were filtered, salts were washed on a
filter with o-xylol (2 ꢂ 5 ml) and combined filtrates were evapo-
rated. The residue was dissolved in DCM (25 ml) and washed 2
times with 1 M KOH (40 ml), followed by H2O (40 ml, 2 times) and
brine (40 ml); the organic layer was dried over Na2SO4 and evap-
orated. The product was isolated by silica gel chromatography using
a n-hexaneeEtOAc mixture (6 : 1) as an eluent. The yields of m- and
p-PHEO-BA in the form of oily liquid were 33 and 31%, respectively.
3-Phenethoxybenzaldehyde (meta-PHEO-BA): 1H NMR (400 MHz,
(E)-3-(2-(2-Phenoxyethoxy)phenyl)acrylic acid (ortho-PEGO-CA):
1H NMR (400 MHz, DMSO‑d6)
d
12.29 (s, 1H), 7.87 (d, J ¼ 16.2 Hz,
1H), 7.69 (d, J ¼ 7.7 Hz, 1H), 7.40 (t, J ¼ 7.8 Hz, 1H), 7.30 (t, J ¼ 8.0 Hz,
2H), 7.15 (d, J ¼ 8.3 Hz, 1H), 7.09e6.97 (m, 3H), 6.95 (t, J ¼ 7.3 Hz,
1H), 6.57 (d, J ¼ 16.2 Hz, 1H), 4.52e4.22 (m, 4H). 13C NMR (101 MHz,
DMSO)
d 167.89, 158.32, 156.88, 138.77, 131.69, 129.53, 128.71,
122.93, 121.13, 120.84, 119.54, 114.67, 113.07, 67.12, 66.19. ESI-HRMS
(m/z): Calcd for C17H16O4 [MþH]þ 285.1121, found 285.1125; calcd
[M þ NH4]þ 302.1387, found 302.1392; calcd [MþNa]þ 307.0941,
found 307.0944; calcd [2 M þ NH4]þ 586.2435, found 586.2450;
calcd [2 M þ Na]þ 591.1989, found 591.2004.
DMSO‑d6)
d 9.96 (s, 1H), 7.53e7.47 (m, 2H), 7.45e7.41 (m, 1H),
7.36e7.20 (m, 6H), 4.27 (t, J ¼ 6.8 Hz, 2H), 3.06 (t, J ¼ 6.8 Hz, 2H). 13
C
NMR (101 MHz, DMSO)
d
192.92, 158.91, 138.18, 137.63, 130.35,
(E)-3-(3-(2-Phenoxyethoxy)phenyl)acrylic acid (meta-PEGO-CA):
128.93, 128.29, 126.29, 122.16, 121.27, 113.95, 68.41, 34.78. ESI-
HRMS (m/z): not determined.
1H NMR (400 MHz, DMSO)
d
12.36 (bs, 1H), 7.57 (d, J ¼ 16.0 Hz, 1H),
7.37e7.23 (m, 5H), 7.06e6.90 (m, 4H), 6.57 (d, J ¼ 16.0 Hz, 1H),
4.41e4.26 (m, 4H). 13C NMR (101 MHz, DMSO)
167.51, 158.65,
4-Phenethoxybenzaldehyde (para-PHEO-BA): 1H NMR (400 MHz,
d
DMSO)
d
9.86 (s, 1H), 7.85 (d, J ¼ 8.8 Hz, 2H), 7.38e7.27 (m, 4H),
158.26, 143.75, 135.71, 129.93, 129.48, 120.98, 120.71, 119.63, 116.74,
114.45, 113.47, 66.42, 66.14. ESI-HRMS (m/z): Calcd for C17H16O4
[MþH]þ 285.1121, found 285.1124; calcd [M þ NH4]þ 302.1387,
found 302.1392; calcd [MþNa]þ 307.0941, found 307.0945; calcd
[2 M þ NH4]þ 586.2435, found 586.2451; calcd [2 M þ Na]þ
591.1989, found 591.2001; calcd [5 M þ K]2þ 730.2474, found
730.2431; calcd [6 M þ K]2þ 872.2998, found 872.2943; calcd
[7 M þ K]2þ 1014.3522, found 1014.346813; calcd [8 M þ K]2þ
1156.4047, found 1156.4006.
7.26e7.19 (m, 1H), 7.12 (d, J ¼ 8.7 Hz, 2H), 4.30 (t, J ¼ 6.9 Hz, 2H),
3.06 (t, J ¼ 6.8 Hz, 2H). 13C NMR (101 MHz, DMSO)
d 191.24, 163.40,
138.01, 131.80, 129.64, 128.96, 128.33, 126.36, 114.95, 68.61, 34.71.
ESI-HRMS (m/z): not determined.
4.1.6. Synthesis of ortho-, meta- and para-PEGO-CHA
(iii) To a solution of the corresponding acid (0.85 g, 3.0 mmol) in
DMF (3 ml), CDI (0.50 g, 3.0 mmol) was added. After 2 h incu-
bation, NH2OH$HCl (0.21 g, 3.0 mmol) was added, the mixture
was stirred for 15 min and kept overnight (~18 h). The solution
was poured into H2O (9 ml), and after 1 h the precipitate was
filtered and air dried. The product was purified by recrystalli-
zation. Recrystallizing solvents were EtOH for o- and p-PEGO-
CHA and EtOAc for m-PEGO-CHA. The yields of o-, m- and p-
PEGO-CHA were 17, 35 and 17%, respectively.
(E)-3-(4-(2-Phenoxyethoxy)phenyl)acrylic acid (para-PEGO-CA):
1H NMR (400 MHz, DMSO‑d6)
d
12.19 (s, 1H), 7.64 (d, J ¼ 8.7 Hz, 2H),
7.56 (d, J ¼ 16.0 Hz, 1H), 7.36e7.24 (m, 2H), 7.02 (d, J ¼ 8.8 Hz, 2H),
6.98 (d, J ¼ 8.1 Hz, 2H), 6.94 (d, J ¼ 7.3 Hz, 1H), 6.39 (d, J ¼ 16.0 Hz,
1H), 4.39e4.28 (m, 4H). 13C NMR (101 MHz, DMSO)
d 167.74, 159.98,
158.24, 143.60, 129.90, 129.48, 127.04, 120.75, 116.65, 114.87, 114.47,
66.53, 66.08. ESI-HRMS (m/z): Calcd for C17H16O4 [MþH]þ 285.1121,
found 285.1129; calcd [M þ NH4]þ 302.1387, found 302.1393; calcd
[MþNa]þ 307.0941, found 307.0951; calcd [2 M þ Na]þ 591.1989,
found 591.2008.
(E)-N-Hydroxy-3-(2-(2-phenoxyethoxy)phenyl)acrylamide
(ortho-PEGO-CHA): 1H NMR (300 MHz, DMSO)
d
10.02 (bs, 2H), 7.75
(i) Preparation of ortho-PEGO-BA. 2-phenoxyethyl bromide
(4.02 g, 20 mmol), K2CO3 (3.04 g, 22 mmol) and TEBAC (0.50 g,
2.2 mmol) were added to the solution of salicylic aldehyde (2.68 g,
22 mmol) in DMF (20 mL). The reaction mixture was stirred at 90ꢁС
for 45 min. After cooling to room temperature, the vessel contents
were filtered, salts were washed on a filter with o-xylol (2 ꢂ 10 ml)
and combined filtrates were evaporated. The residue was sus-
pended in MeOH (10 ml); after incubation at 10ꢁС for 45 min, the
precipitate was filtered, washed 2 times on a filter with MeOH
(10 ml) and air dried. The yield of o-PEGO-BA was 48%.
(d, J ¼ 15.9 Hz,1H), 7.54 (d, J ¼ 7.3 Hz,1H), 7.44e7.26 (m, 3H), 7.15 (d,
J ¼ 8.1 Hz, 1H), 7.09e6.92 (m, 4H), 6.53 (d, J ¼ 16.0 Hz, 1H), 4.40 (s,
4H). 13C NMR (75 MHz, DMSO)
d 163.56, 158.79, 157.03, 133.62,
131.25, 130.00, 128.19, 124.16, 121.58, 121.28, 120.06, 115.15, 113.53,
67.57, 66.65. ESI-HRMS (m/z): Calcd for
C
17H17NO4 [MþH]þ
300.1230, found 300.1231; calcd [MþNa]þ 322.1050, found
322.1049.
(E)-N-Hydroxy-3-(3-(2-phenoxyethoxy)phenyl)acrylamide (meta-
PEGO-CHA): 1H NMR (400 MHz, DMSO‑d6)
d 10.57 (bs, 1H), 9.21 (bs,
1H), 7.44 (d, J ¼ 15.7 Hz, 1H), 7.37e7.24 (m, 3H), 7.16 (d, J ¼ 8.3 Hz,
2-(2-Phenoxyethoxy)benzaldehyde (ortho-PEGO-BA): 1H NMR
2H), 7.06e6.86 (m, 4H), 6.48 (d, J ¼ 15.8 Hz, 1H), 4.62e4.13 (m, 4H).
(400 MHz, DMSO)
d 10.34 (s, 1H), 7.74e7.61 (m, 2H), 7.37e7.23 (m,
13C NMR (101 MHz, DMSO‑d6)
d
162.62, 158.67, 158.27, 138.15,
3H), 7.09 (t, J ¼ 7.5 Hz, 1H), 7.03e6.90 (m, 3H), 4.48 (dd, J ¼ 5.4,
136.35, 130.00, 129.51, 120.75, 119.93, 119.53, 115.75, 114.48, 113.39,
3.3 Hz, 2H), 4.39 (dd, J ¼ 5.5, 3.2 Hz, 2H). 13C NMR (101 MHz,
66.38, 66.15. ESI-HRMS (m/z): Calcd for
C
17H17NO4 [MþH]þ
DMSO‑d6) d 189.15, 160.84, 158.36, 136.40, 129.52, 127.51, 124.51,
300.1230, found 300.1234; calcd [MþNa]þ 322.1050, found
121.05, 120.85, 114.63, 114.04, 67.63, 66.11. ESI-HRMS (m/z): Calcd

M.V. Kozlov et al. / European Journal of Medicinal Chemistry 183 (2019) 111723
11
for C15H14O3 [MþH]þ 243.1016, found 243.1019; calcd [MþNa]þ
265.0835, found 265.0841.
10 min) at 4 ꢁC, the supernatant was neutralized with 1.5 M Tris-
HCl, pH 8.8 (one-tenth of the volume), followed by the addition
of a 5x Laemmly buffer (one-fifth of the volume).
(i) Preparation of meta- and para-PEGO-BA. 2-Phenoxyethyl
bromide (4.02 g, 20 mmol) and K2CO3 (2.76 g, 20 mmol) were
added to the solution of the corresponding hydroxybenzaldehyde
(2.44 g, 20 mmol) in DMF (20 mL). The reaction mixture was stirred
during its heating from 70ꢁС to 90ꢁС within 1.5 h and incubated at
90ꢁС for another 30 min. After cooling to room temperature, the
vessel contents were poured into H2O (60 ml) and the suspension
was stirred at 10ꢁС for 45 min; the precipitate was filtered, air dried
and washed 2 times on a filter with n-hexane (5 ml) and air dried.
The yields of m- and p-PEGO-BA were 79 and 76%, respectively.
3-(2-Phenoxyethoxy)benzaldehyde (meta-PEGO-BA): 1H NMR
4.2.4. Western blot
The proteins from the cellular lysates were separated by elec-
trophoresis in 10% PAAG containing 0.1% SDS and then transferred
to a nitrocellulose membrane by electroblotting. The membrane
was treated with 5% low-fat dry milk (Bio-Rad, USA) in PBS that
contained 0.05% Tween-20 (PBST) for 60 min at room temperature.
The primary antibodies in dilution 1 : 10,000 were added to
a-
tubulin (T5168, Sigma, USA); in dilution 1 : 2000 to histone H3
(9715S, CST) and SMC3 (5996S, CST); in dilution 1 : 1000 to the
acetylated form of a-tubulin (anti-acetyl lysine antibody, ab80178,
(400 MHz, DMSO‑d6)
d 9.99 (s, 1H), 7.59e7.46 (m, 3H), 7.38e7.25
(m, 3H), 7.02e6.89 (m, 3H), 4.40 (dt, J ¼ 5.2, 3.1 Hz, 2H), 4.34 (dt,
Abcam, UK) and the acetylated form of histone H3 (anti-acetyl
histone H3(K9/K14), 9677S, CST); in dilution 1 : 500 to the acety-
lated form of SMC3K105/106ac (anti-acetyl SMC3 antibody (K105/
106), MABE1073, EMD Millipore, USA). The mixtures were incu-
bated overnight at 4 ꢁC, followed by washing with PBST. The con-
jugate of horseradish peroxidase with secondary specific antibodies
(anti-mouse and anti-rabbit, Santa Cruz, USA) were added in dilu-
tion 1 : 10,000, and the mixtures were incubated for 50 min at room
temperature. The membranes were washed with PBST, and the
signal was visualized using the ECL kit (Pierce-Thermo Scientific,
USA) and High-performance ECL film (GE Healthcare, USA).
J ¼ 4.5, 3.2 Hz, 2H). 13C NMR (101 MHz, DMSO)
d 192.84, 158.85,
158.23, 137.64, 130.37, 129.49, 122.50, 121.41, 120.75, 114.47, 113.82,
66.69, 66.08. ESI-HRMS (m/z): not determined.
4-(2-Phenoxyethoxy)benzaldehyde (para-PEGO-BA): 1H NMR
(400 MHz, DMSO‑d6)
d
9.88 (s, 1H), 7.88 (d, J ¼ 8.6 Hz, 2H), 7.30 (t,
J ¼ 7.9 Hz, 2H), 7.18 (d, J ¼ 8.7 Hz, 2H), 7.01e6.91 (m, 3H), 4.44 (dd,
J ¼ 5.5, 3.2 Hz, 2H), 4.34 (dd, J ¼ 5.4, 3.2 Hz, 2H). 13C NMR (101 MHz,
DMSO‑d6)
d 191.17, 163.23, 158.16, 131.72, 129.76, 129.45, 120.75,
114.94, 114.44, 66.84, 65.94. ESI-HRMS (m/z): Calcd for C15H14O3
[MþH]þ 243.1016, found 243.1020.
4.2. Biological materials and methods
4.2.5. Statistical analysis
Data on the luciferase activity and cell viability were presented
as the mean value and standard deviation of at least four and six
independent experiments, correspondingly. The extent of correla-
tion between two variables was quantified by calculating the
Pearson's product moment correlation coefficient (r), while its
significance (P) was evaluated by the t-test.
4.2.1. Antiviral activity
The Huh7-luc/neo cell culture was reseeded into a 48-well plate
in the medium without antibiotic G418. After 48 h (30e40%
monolayer), the studied compounds at different concentrations
were added to the culture medium. After the incubation at 37 ꢁC in
5% CO2 for three days (monolayer in the control probe was 100%),
the medium was removed. The cells were washed with phosphate
buffered saline (PBS) and lysed, followed by the evaluation of the
luciferase activity by the Luciferase Assay System kit (Promega,
USA) according to the manufacturer's protocol. Chemiluminescence
was measured on a Thermo luminometer (Labsystems, USA).
Acknowledgments
The work was supported by the Russian Foundation for Basic
Research (Grant # 17-04-00175) and by the Program of Funda-
mental Research of the State Academies of Sciences (Grant #
01201363818).
4.2.2. Cell viability
The Huh7 cell culture was reseeded into a 96-well plate. After
24 h (30e40% monolayer), the studied compounds at different
concentrations were added to the culture medium. After the in-
cubation at 37 ꢁC in 5% CO2 for three days (monolayer in the control
probe was 100%), the cell viability was evaluated using the MTT kit
(Sigma-Aldrich, USA) according to the manufacturer's protocol.
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