Synthesis of (Z)-N-hydroxy-3-methoxy-3-phenylacrylamide as new selective inhibitor of hepatitis C virus replication

Article abstract of DOI:10.1134/S1068162016010076

According to recently published results, cinnamic hydroxamic acid (CHA) inhibits replication of hepatitis C virus (HCV). We synthesized a structural analogue of CHA, i.e., (Z)-N-hydroxy-3-methoxy3-phenylacrylamide, which inhibited HCV replication five tim

Full text of DOI:10.1134/S1068162016010076

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2016, Vol. 42, No. 2, pp. 191–197. © Pleiades Publishing, Ltd., 2016.
Original Russian Text © M.V. Kozlov, A.A. Kleymenova, K.A. Konduktorov, A.Z. Malikova, K.A. Kamarova, R.A. Novikov, S.N. Kochetkov, 2016, published in Bioorganicheskaya
Khimiya, 2016, Vol. 42, No. 2, pp. 214–220.
Synthesis of (Z)ꢀNꢀHydroxyꢀ3ꢀMethoxyꢀ3ꢀPhenylacrylamide
as New Selective Inhibitor of Hepatitis C Virus Replication
M. V. Kozlov1, A. A. Kleymenova, K. A. Konduktorov, A. Z. Malikova,
K. A. Kamarova, R. A. Novikov, and S. N. Kochetkov
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences,
ul. Vavilova 32, Moscow, 119991 Russia
Received July 1, 2015; in final form, August 7, 2015
Abstract—According to recently published results, cinnamic hydroxamic acid (CHA) inhibits replication
of hepatitis C virus (HCV). We synthesized a structural analogue of CHA, i.e., (
Z)ꢀNꢀhydroxyꢀ3ꢀmethoxyꢀ
3ꢀphenylacrylamide, which inhibited HCV replication five times more selectively than CHA. It was found
that both compounds did not inhibit deacetylation of Acꢀ
of which is important for virus replication.
αꢀtubulin with histone deacetylase 6, the activity
Keywords: cinnamic hydroxamic acid, hepatitis C virus, histone deacetylase 6, Acꢀαꢀtubulin
DOI: 10.1134/S1068162016010076
1
INTRODUCTION
culture of human hepatocytes [4–10]. Among studied
compounds, two classes of biologically active hydroxꢀ
amates can be distinguished, i.e., inhibitors of matrix
metalloproteinases [4, 5, 9] and histone deacetylases
[6, 8]. Probably, the antiꢀHCV activity of benzoic, cinꢀ
namic, and pyridine hydroxamic acids described in
the literature is also associated with inhibition of
enzymes of these two classes; however, the mechanism
of the inhibition of the HCV replication is still unclear
[7, 9, 10].
Currently, therapy of hepatitis C is based on direct
action drugs, which efficiently inhibit viral proteins
and enzymes. A widely used drug Harvoni (Gilead
Sciences, Inc., United States) is a mixture of blocker
of the NS5A protein and the inhibitor of RNAꢀ
dependent RNA and NS5B polymerase of the virus.
The sustained antiviral response of this drug is
observed in 90% of patients [1–3]. The relatively
short duration of the treatment (12 to 24 weeks)
reduces the occurrence of crossꢀresistant forms of the
virus in the patient’s body. However, taking into
account the number of HCVꢀinfected patients (about
200 million people), the number of patients immune
to Harvoni may reach 20 million people. Therefore,
the search for new therapeutic targets remains releꢀ
vant, especially as regards proteins of the host cell,
which play an important role in intracellular virus
reproduction.
In 2013, we found that, in contrast to benzohyꢀ
droxamic acid (BHA, SI =14 and ЕC50 = 12 µM),
its orthoꢀmethoxyꢀderivative demonstrated a 4ꢀfold
increase in the selectivity while maintaining the
activity (oꢀMeOꢀBHA, SI = 53 and ЕC50 = 12 µM)
[7]. Taking into account both these data and the
high antiꢀHCV activity of cinnamic hydrozamic
acid (CHA, ЕC50 = 0.86 µM [9]), we decided to
According to literature data, hydroxamic acids can
selectively inhibit the propagation of HCV replicon in
synthesize the 3ꢀmethoxy analogue of CHA (3ꢀ
MeOꢀCHA), which combined the structural eleꢀ
Abbreviations: BHA, benzohydroxamic acid; CHA, cinnamic
hydroxamic acid; HDAC6, histone deacetylase 6; HCV, hepatiꢀ
tis C virus.
ments of оꢀМеОꢀВHА and CHA and to study the
antiviral characteristics of this compound (see forꢀ
mulas).
1
Corresponding author: phone: +7 (499) 135ꢀ0590; fax: +7 (499)
135ꢀ1405; eꢀmail: kozlovmv@hotmail.com.
191

192
KOZLOV et al.
OMe
OMe
CONHOH
1
2'
5'
3
CONHOH
CONHOH
CONHOH
3'
4'
1'
6'
2
BHA
EC50 = 15 μМ
SI = 14
CHA
EC50 = 0.86 μМ
SI = 45
3ꢀMeOꢀCHA
oꢀMeOꢀBHA
EC50 = 12 μМ
SI = 53
Formulas
.
Molecular structure of 3ꢀMeOꢀCHA containing
ꢀMeOꢀBHA and CHA.
structural elements of
o
RESULTS AND DISCUSSION
The synthesis of cinnamic hydroxamic acid (CHA)
and its monomethyl derivatives ( )–(III) is carried out
by activation of the acid component with carbonyl
diimidazole in dimethylformamide, followed by the
(Scheme 1). Imidazole is formed during the activation
of the initial acid while synthesizing hydroxamic acid
and acts then as a base that deprotonates hydrochloride.
After evaporation of the solvent, the products were
extracted by ethyl acetate from water and were purified
I
condensation with hydroxylamine component by silica gel chromatography. The yields were 20–30%.
Comp. R1 R2 R3
R1
R1
O
R1
O
CHA H
H
Me H
H
H
O
i
ii
(
(
(
I
II
III
)
NR2OR3
Ph
OH
Ph
Im
Ph
)
H
H
Me H
Me
)
H
•
Scheme 1. Synthesis of CHA and compounds
Im, imidazole residue
(I)–(III). (i), CDI/DMF; (ii), NHR OR3 HCl;
2
.
To synthesize the 3ꢀmethoxy analogue of CHA trimetyl orthoformate mixture in the presence of cataꢀ
VII), we used commercially available ethyl benzoyꢀ lytic amount of TsOH gave dimethylketal (IV), which
lacetate as the starting compound (Scheme 2). The was evaporated and isolated by extraction with nꢀhexꢀ
(
boiling of this
βꢀketoester for 50 min in a methanolꢀ ane in a yield of 92%.
MeO
Ph
MeO
O
OMe
O
O
i
MeO OMe
CO2Et
ii
iii
+
CO2Et
CO2Et
Ph NHOH Ph
(VI) 3ꢀMeOꢀCHA (VII)
hydroxyꢀ3ꢀmethoxyꢀ3ꢀphenylacrylamides VI) and (VII).
; (iii), NH OH HCl/KOH/MeOH.
NHOH
Ph
Ph
(IV)
Scheme 2. Synthesis of
(V)
(E)ꢀ and (
Z
)
ꢀNꢀ
(
•
(i
), HC(OCH ) /TsOH/MeOH/
3 3
Δ
; (ii),
Δ
2
Examples of thermal enolization of ketals to correꢀ sponds to the content of isomers in the mixture (23
and 77%).
sponding vinyl esters are known from the literature
At the final stage of the synthesis, a cooled methaꢀ
nol solution of NH2OH (2 equiv.) and KOH (1 equiv.)
was added to the reaction mixture containing comꢀ
pound (V), and the mixture was incubated at room
temperature for 18 h. The solution was neutralized,
the solvent was evaporated, and an aqueous base
(pH ~8) was added to the residue, followed by washing
with hexane. The products were extracted with methꢀ
ylene chloride. Hydroxamic acids (VI) and (VII) were
separated from each other and from an unidentified
[11]. Similarly, pyrolysis of compound (IV) led to the
mixture of (
E
/
Z
)ꢀisomers of ethylꢀ3ꢀmethoxyꢀphenyꢀ
lacrylate (V) (Scheme 2). The reaction proceeds at
190°C for 15 min with a quantitative yield. The PMR
spectrum in Fig. 1 showed that the proton signals of
two isomers were grouped in the regions of the proton
chemical shifts of the aromatic ring, exocyclic double
C–C bond, the methyl residue of enol ester, and ethyl
ester residue. The ratio of the intensity of two signals
inside each group is constant (about 1/3.4) and correꢀ side product by silica gel chromatography.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
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SYNTHESIS OF (
Z
)ꢀN
ꢀHYDROXYꢀ3ꢀMETHOXYꢀ3ꢀPHENYLACRYLAMIDE
193
E (q)
3.94
I (t)
1.07
OCH3
D (q) G (s)
4.12 3.78
A (m)
7.43
B (s) C (s)
5.65 5.31
H (t)
1.23
CO2Et
(V)
F (s)
3.79
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0
ppm
1
Fig. 1. H NMR spectrum of ethyl 3ꢀmethoxyꢀ3ꢀphenylacrylate (V) (E/Z)ꢀisomer mixture. Multiplet A, phenyl residue protons;
singlets B/C, alkene protons at C2; quadruplets D/E and triplets H/I, protons of the ester ethyl groups; and singlets F/G, protons
of enol ether methyl groups.
PMR spectra of compounds (VI) and (VII) after ence in culture media and methods of the cytotoxicity
chromatographic purification are presented in Fig. 2. evaluation which were used in our work and in [9].
The signal of C2 proton in compound (VI) (5.23 ppm)
The data in the table show that methylation of the
hydroxamic residue dramatically reduces the antiviral
is shifted upfield by 0.28 ppm compared with that in
compound (VII) (5.51 ppm). A similar difference in
chemical shifts (0.34 ppm) is observed between the
main (5.31) and minor (5.65 ppm) signals of C2 proꢀ
activity and selectivity of
(EC50 = 75 M and SI = 3.3) and makes
compound (III) completely inactive (EC50 > 300
N
ꢀmethylated compound (II
ꢀmethylated
M).
)
μ
О
μ
tons in the PMR spectrum of isomers (V) (Fig. 1).
These results imply that the binding strength between the
inhibitor and the target protein is determined by the
chelating ability of the hydroxamic residue.
Most likely, the spatial proximity of the phenyl ring
to the hydroxamic acid residue in (E)ꢀisomer (VI)
Thus, hydroxamate (VI) is the product of the converꢀ
sion of the ester, the content of which is 77% in the
mixture. This means that the yields of compounds (VI
)
and (VII) are 10 and 18%, respectively.
The absolute configuration of each product was
determined by 2D 1Hꢀ1H NOESY NMR spectrosꢀ
copy. The spectrum of compound (VI) contains crossꢀ
pecks at 5.23–3.69 ppm (data not shown), which indiꢀ
cates that the C2 protons and protons of 3ꢀMeO group
are located on one side of the double bond. This is posꢀ
sterically hinders the chelation of the metal ion in the
active center of the target protein. This is confirmed by
a 30ꢀfold decrease in the antiviral activity of this comꢀ
pound in comparison with the activity of (
VII) (EC50 = 180 and 5.7 M, respectively). At last,
the methoxy group at position 3 of compound (VII
Z)ꢀisomer
(
μ
)
sible only for the (Е)ꢀisomer (Fig. 2). On the other
increases the selectivity of its action by a factor of almost
five compared to CHA, but unfortunately, this group
decreases simultaneously the antiviral efficiency by a facꢀ
hand, the spectrum of compound (VII) contains
crossꢀpeaks at 5.51–7.54 ppm (data not shown),
which indicates the interaction of the C2 proton with
protons of the aromatic ring (H2' and H6'), which can
tor of three (EC50 = 5.7 and 2.0 μM, respectively). This
result shows that the structural analogies between benꢀ
zoic and cinnamic hydroxamic acids as HCV inhibitors
are unjustified and cannot be used in the future.
occur only in the (Z)ꢀisomer (Fig. 2).
At the next stage, we examined all prepared comꢀ
pounds as inhibitors of the HCV replication. CHA and
Histone deacetylase 6 (HDAC6) is known to be
compound (
activity (ЕC50 = 2.0 and 2.3
cytotoxicity (CC50 = 20 M for both compounds) lates the acetylated AcꢀLys40 residue of
(table). These values are in satisfactory agreement Аcꢀ ꢀtubulin) in microtubes [13]. The evaluation of
with published data (ЕC50 = 0.86 and 3.2 M, respecꢀ the level of Аcꢀ ꢀtubulin by westernꢀblotting makes it
I
) demonstrate close values of antiviral localized in mammalian cells mostly in cytoplasm,
M, respectively) and where it binds to the tubulin cytoskeleton and deacetyꢀ
ꢀtubulin
μ
μ
α
(
α
μ
α
tively) despite the authors use of hepatocytes of the possible to monitor the cytoplasmic activity of
Huhꢀ7/HCV1bꢀRluc line [9], which maintain the HDAC6. In recent papers, we conformed the direct
reproduction of the subgenomic HCV replicon. Howꢀ correlation between the inhibition of the HDAC6
ever, the cytotoxicity values significantly differ from activity and the inhibition of the HCV virus replicaꢀ
the published data (CC50 = 39 and 59
μM, respecꢀ tion by paraꢀderivatives of benzhydroxamic acid [8,
tively). A possible reason for this discrepancy is the differꢀ 10]. CHA is known to selectively inhibit the HDAC6
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
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KOZLOV et al.
B (s)
8.58
C (ddt)
7.35
D (s)
5.23
E (s)
3.69
A (s)
10.22
OCH3
O
NHOH
(VI)
10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5
ppm
B (m)
7.44
D (s)
8.79
E (s)
5.51
F (s)
3.76
C (s)
10.32
A (m)
7.54
O
OCH3
NHOH
(
VII)
10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5
ppm
1
Fig. 2. H NMR spectra of (
E
)ꢀ and (
Z
)ꢀNꢀhydroxyꢀ3ꢀmethoxyꢀ3ꢀphenylacrylamide (VI) and (VII).
activity (IC50 = 22 nM) [12]. However, the incubation of known now for kinase p38 MAPK [14], cyclooxygenꢀ
the Huh7ꢀluc/neo cell line in the presence of CHA or ase COXꢀ2 [15], and kinase PDKꢀ1 [16].
compound (VII) at concentrations up to 10 or 100
respectively, is not accompanied by any noticeable
accumulation of Аcꢀ ꢀtubulin (data not shown).
μM,
As a result of this work, we have obtained new data
showing that the antiꢀHCV activity of CHA is due to
metal ion chelation in the active center of the target
protein and this chelation is possible only when the
α
It is hard to imagine that when penetrating into
cells, CHA can no longer bind to the active center of
HDAC6 at concentrations, which is 500 times higher
than the IC50 value. The result can most likely be
explained by a phenomenon of substrate selectivity of
this inhibitor. The fact is that the real biological subꢀ
strate of HDAC6 is Аcꢀαꢀtubulin, i.e., the protein
with a molecular weight of about 50 kDa but not the
peptide used for testing in vitro [12]. The formation of
phenyl and hydroxamic groups are located in trans
ꢀ
position relative to the double C–C bond of the exoꢀ
cyclic residue. We have synthesized a new HCV inhibꢀ
itor,
(Z)ꢀNꢀhydroxyꢀ3ꢀmethoxyꢀ3ꢀphenylacrylaꢀ
mide, and have found that the 3ꢀmethoxy substituent
increases the selectivity of the antiviral effect of the
obtained compound by a factor of five as compared to
CHA. The fact that Аcꢀαꢀtubulin is not accumulated
during incubation of cells with Huh7ꢀluc/neo in the
presence of CHA gives reason to believe that this
hydroxamate possesses substrate selectivity relative to
HDAC6.
the Аcꢀ
for the ability of CHA to compete for the catalytic
center with ꢀacetylated Lys40 residue. At the same
αꢀtubulin–HDAC6 complex can be crucial
εꢀN
time, CHA may retain its ability to inhibit deacetylaꢀ
tion of other protein substrates of HDAC6 and,
thereby, to block the HCV replication [8, 10]. If the
assumed substrate selectivity of CHA as the HDAC6
inhibitor will be confirmed, this will expand the list of
EXPERIMENTAL
We used 1,1'ꢀcarbonyl diimidazole (CDI), hydroxꢀ
examples of substrateꢀselective inhibition, which is ylamine hydrochloride,
Nꢀmethyl hydroxylamine
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
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SYNTHESIS OF (
Z
)ꢀN
ꢀHYDROXYꢀ3ꢀMETHOXYꢀ3ꢀPHENYLACRYLAMIDE
195
Results of antiꢀHCV test of structural analogues of CHA
Compound Structure
EC50,
μM
CC50,
μM
SI
CONHOH
CHA
2.0/0.86*
2.3/3.2*
20/39*
20/59*
10/45*
Ph
Ph
Me
(I)
8.7/18*
CONHOH
CON(Me)OH
CONHOMe
(II)
75
250
3.3
Ph
(III
)
>300
>1000
–
Ph
OMe
(
VI
)
180
5.7
700
280
3.9
49
Ph
CONHOH
OMe
3ꢀMeOꢀCHA (VII
)
CONHOH
Ph
*Data published in [9].
hydrochloride, methoxylamine hydrochloride, benꢀ
zoyl acid ethyl ester, trimethyl orthoformate, toluene
Cinnamic acid
(А). 1H NMR spectrum: 10.73 (1H, s, NH), 9.00 (1H, s,
OH), 7.55 (2H, d, 6.9 Hz, H2' and H6'), 7.46 (1H, d,
15.9 Hz, H3), 7.43–7.34 (4H, m, H3, H3', H4' and
H5'), 6.47 (1H, d,
15.8 Hz, H2). 13C NMR spectrum:
162.68 (C1), 138.28 (C3), 134.78 (C1'), 129.38 (C'4),
128.87 (C'3 and C'5), 127.41 (C'2 and C'6), 119.08
(C2).
Nꢀhydroxylamide (CHA), Rf 0.29
J
sulfonic acid monohydrate (TsOH
⋅ H2О), triethyꢀ
J
lamine (NEt3), ꢀdiisopropylethylamine (DIEA),
N
J
and dimethylformamide (DMF) (SigmaꢀAldrich,
United States). TLC was performed on Kieselgel 60
F254 plates (Merck, Germany) in systems CHCl3–
EtOH, 10 : 1 (A); CHCl3–EtOH, 20 : 1 (B); PhCH3–
MeCN, 20 : 1 (C).
(
Е Nꢀhydroxyꢀ3ꢀphenylꢀ2ꢀbutenamide (I), Rf 0.37
)ꢀ
(А). 1H NMR spectrum: 11.25–8.29 (2H, m,
NHOH), 7.48 (2H, d, 7.4, H2' and H6'), 7.44–7.33
NMR spectra (δ, ppm; J, Hz) were recorded on a
J
AMX IIIꢀ400 spectrometer (Bruker, Germany) with
400 MHz for 1H NMR (internal standard, Me4Si; solꢀ
vent, DMSOꢀd6) and 100.6 MHz for 13C NMR (solꢀ
vent DMSOꢀd6, suppression of carbon–proton interꢀ
action).
(3H, m, H3', H4' and H5'), 6.04 (1H, s, H2), 2.49
(3H, s, CH3). 13C NMR: 163.89 (C1), 148.08 (C3),
142.03 (C1'), 128.48 (C'3 and C'5), 128.43 (C'4),
125.78 (C'2 and C'6), 117.22 (C2), 16.66 (CH3)
Cinnamic acid ꢀhydroxyꢀ ꢀmethylamide
0.38 (B). 1H NMR spectrum: 10.06 (1H, s, OH), 7.64
(2H, d, 6.8, H2' and H6'), 7.50 (1H, d, 15.9, H3),
.
N
N
(II), Rf
CHA and compounds (I)–(III) were synthesized by
the same method. The starting acid (4 mmol) was disꢀ
solved in DMF (4 mL), followed by the addition of
1,1'ꢀcarbonyldiimidazole (0.65 g, 4 mmol). After
incubation for 2 h, the corresponding hydroxylamine
hydrochloride derivative (8 mmol) was added, and the
reaction mixture was stirred until its complete dissoluꢀ
tion. After incubation for 18 h, the solvent was evapoꢀ
rated, water (15 mL) was added to the residue, folꢀ
J
J
7.46–7.34 (3H, m, H3', H4' and H5'), 7.23 (1H, d,
J
15.9 Hz, H2), 3.22 (3H, s, NCН3). 13C NMR: 165.56
(C1), 140.74 (C3), 134.93 (C1'), 129.57 (C'4), 128.84
(C'3 and C'5), 127.76 (C'2 and C'6), 117.28 (C2),
36.04 (NCH3)
.
Cinnamic acid
0.38 (B). H NMR spectrum: 11.27 (1H, s, NH),
7.64–7.46 (3H, m, H2', H6' and H3), 7.46–7.35 (3H,
N
ꢀhydroxyꢀNꢀmethylamide (III), Rf
1
lowed by washing with EtOAc (2 × 15 mL). Organic
fractions were combined, dried over Na2SO4, and
evaporated. The products were isolated in yields of
20–30% by silica gel chromatography using a chloroꢀ
formꢀethanol mixture as eluent.
m, H3', H4' and H5'), 6.42 (1H, d, J 15.9, H2), 3.67
(3H, s, ОCН3). 13C NMR: 165.62 (C1), 139.51 (C3),
134.56 (C1'), 129.65 (C'4), 128.87 (C'3 and C'5),
127.56 (C'2 and C'6), 118.51 (C2), 63.31 (OCH3).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
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KOZLOV et al.
1
Ethylꢀ3,3ꢀdimethoxyꢀ3ꢀphenylpropionate
Trimethyl orthoformate (20 mL) and TsOH
(500 mg) were added to the solution of ethyl benzoyꢀ
lacetate (5.76 g, 30 mmol) in methanol (25 mL), and
the mixture was boiled for 50 min. The reaction mixꢀ
ture was cooled to room temperature, followed by the
addition of NEt3 (0.27 mL). After evaporation of the
(IV). Rf 0.23 (B), H NMR spectrum: 10.32 (1H, s, OH),
⋅
Н2О
8.79 (1H, s, NH), 7.55–7.53 (2H, m, H2' and H6'
Phe), 7.46–7.41 (3H, m, H3', H4' and H5' Phe), 5.51
(1H, s, H2), 3.76 (3H, s, ОCН3). 13C NMR: 162.52
(C1 or C3), 161.82 (C1 or C3), 134.88 (C1'), 129.69
(C'4), 128.55 (C'3 and C'5), 126.58 (C'2 and C'6),
101.14 (C2), 59.71 (ОСН3)
.
solvent, the residue was washed with nꢀhexane (50 and
Cell cultures. Cells of the Huh7 line were grown in
a DMEM + F12 (2 : 1) medium containing 10% fetal
bovine serum (Hy Clone), 2 mM Lꢀglutamine, peniꢀ
25 mL). The combined organic extracts were succesꢀ
sively washed with 2% NaHCO3 (50 mL) and water
(50 mL) and dried over Na2SO4. The solvent was
evaporated, and dimethylketal (IV) was obtained in a cillin (100 U/mL), and streptomycin (0.1 mg/mL) at
yield of 6.57 g (92%) and was used without additional
purification.
f 0.58 (C). 1H NMR: 7.42–7.27 (5H, m,
H2'ꢀH6'), 3.78 (2H, q, 7.1, OCН2CН3), 3.13 (6Н, s,
37°C and 5% СО2. Cells were passaged by 1 : 3 or 1 : 5
R
diluting every three days. Cells of the Huh7ꢀluc/neo
lines were grown under the same conditions in the
presence of additional antibiotic geneticin (G418,
0.33 mg/mL).
J
ОCН3), 2.94 (2Н, s, CН2CO2Et), 0.88 (3H, t,
J 7.1,
OCН2CН3). 13C NMR: 167.68 (C1), 139.54 (C1'),
127.74 (C4'), 127.65 (C'3 and C'5), 126.62 (C'2 and
C'6), 100.98 (C3), 59.36 (OCH2CH3), 48.36 (OCH3),
Antiviral activity. The Huh7ꢀluc/neo cells were
passaged into 48ꢀwell plate (without G418). After
incubation for 24 h (40–50% confluence), the studied
compounds at different concentrations were added to
42.81 (C2), 13.57 (OCH2CH3)
)ꢀEthylꢀ3ꢀmethoxyꢀ3ꢀphenylacrylate (V) was
synthesized by boiling ethylꢀ3,3ꢀdimethoxyꢀ3ꢀpheꢀ
nylpropionate (IV) (6.11 g, 25.7 mmol) at 190 C for
15 min. A mixture of isomers (V) (5.29 g, 100%)
was obtained in a 3.4 : 1 ratio and was used for subseꢀ
quent reaction.
f 0.43 (C). 1H NMR specꢀ
.
(E/Z
the cell culture. After three days of incubation at 37°C
°
and 5% СО2 (monolayer in the control probe was
100%), the medium was removed, cells were washed
with PBS and lysed. Luciferase activity of the referꢀ
ence protein was evaluated using the Luciferase Assay
System (Promega) according to manufacturer’s proꢀ
tocol. Chemiluminescence was measured by a
Thermo luminometer (Labsystems).
E/Z
E
ꢀisomer: R
trum (fragment): 5.31 (1H, s, H2), 3.94 (2H, q,
CН2CН3), 3.78 (3Н, s, ОCН3), 1.07 (3H, t,
J
J
7. 1,
7.1,
1
CН2CН3).
Z
ꢀisomer
:
R
f 0.43 (C). H NMR spectrum
(fragment): 5.65 (1H, s, H2), 4.12 (2H, q,
CН2CН3), 3.79 (3Н, s, ОCН3), 1.23 (3H, t,
CН2CН3).
J
J
7.1
7.1,
,
Cytotoxicity. The Huh7ꢀluc/neo cells were pasꢀ
saged into a 96ꢀwell plate. After incubation for 24 h
(40–50 % confluence), the studied compounds at difꢀ
ferent concentrations were added to the cell culture.
(E
/
Z
)ꢀ
N
ꢀHydroxyꢀ3ꢀmethoxyꢀ3ꢀphenylacrylaꢀ
HCl (2.82 g, 39.6 mmol)
mide (VI and VII). NH2OH
⋅
in MeOH (15 mL) and KOH (3.36 g, 60 mmol) in
MeOH (9 mL) were dissolved under heating to 50°C.
The second solution was added to the first one under
stirring, and the mixture was immediately cooled to
After three days of incubation at 37°C and 5% СО2
(monolayer in the control probe was 100%), the cell
viability was evaluated by the MTT test according to
manufacturer’s protocol (SigmaꢀAldrich).
0°
C
. After 10 min, the precipitated KCl was separated
by filtration, and the mixture of ꢀizomers ( ) was
E
/
Z
V
Westernꢀblot. Proteins from cell lysates were sepaꢀ
rated by electrophoresis in 10% PAAG containing
0.1% SDS and were transferred on nitrocellulose
membrane by the electromigration method. The
membrane was treated with 5% nonfat dry milk (Bioꢀ
Rad) in PBST (phosphate saline buffer, pH 7.4, conꢀ
taining 0.1% Tweenꢀ20) for 60 min at room temperaꢀ
ture. Primary antibodies to acetylated lysine (ab80178,
Abcam) at 1 : 1000 dilution and antibodies to tubulin
(Sigma) at 1 : 10000 dilution were added to memꢀ
added to the resultant solution. After incubation for
6 h, another portion of KOH (1.10 g, 19.6 mmol) was
added to the reaction mixture, and the reaction was
left to proceed overnight, followed by the addition of
AcOH (2 mL) and evaporation. The residue was disꢀ
solved in Н2О (35 mL) and after the addition of 5 M
KOH (~1 mL to pH 8), it was washed with
nꢀhexane
(30 mL) and CН2Cl2 (2 35 mL). Fractions containing
×
methylene chloride were combined, dried over
Na2SO4 and evaporated. Isomers of hydroxamic acids
were separated by silica gel chromatography using a
branes, which were incubated at 4°С overnight, folꢀ
lowed by washing with PBST. The conjugate of horseꢀ
radish peroxidase with specific secondary antibodies
(antimouse, antirabbit, Santa Cruz) was added at
1 : 10000 dilution and incubated for 50 min at room
temperature. Membranes were washed with PBST,
and the signal was visualized using the ECL kit
(PierceꢀThermo Scientific) and a High performance
CH2Cl2–MeOH mixture (20 : 1). Eꢀisomer (VI) was
obtained in a yield of 0.60 g (10%). Rf 0.14 (B),
1H NMR spectrum: 10.22 (1H, s, OH), 8.58 (1H, s,
NH), 7.47–7.24 (5H, m, H2'ꢀH6' Phe), 5.23 (1H, s,
2H), 3.69 (3H, s, OCH3). 13C NMR: 163.68 (C1 and
C3), 135.34 (C1'), 128.84 (C'4), 128.58 (C'3 and C'5),
127.24 (C'2 and C'6), 92.86 (C2), 55.68 (ОCН3)
.
Z
ꢀisomer (VII) was obtained in a yield of 0.31 g (18%). ECL film (GE Healthcare).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
No. 2
2016

SYNTHESIS OF (
Z
)ꢀN
ꢀHYDROXYꢀ3ꢀMETHOXYꢀ3ꢀPHENYLACRYLAMIDE
197
8. Kozlov, M.V., Kleymenova, A.A., Konduktorov, K.A.,
Malikova, A.Z., and Kochetkov, S.N., Biochemistry
(Moscow), 2014, vol. 79, pp. 637–642.
ACKNOWLEDGMENTS
The work was supported by a grant of the Russian
Scientific Foundation no. 14ꢀ50ꢀ00060.
9. Ai, T., Xu, Y., Qiu, L., Geraghty, R.J., and Chen, L.,
J. Med. Chem., 2015, vol. 58, pp. 785–800.
10. Kozlov, M.V., Kleymenova, A.A., Romanova, L.I.,
Konduktorov, K.A., Kamarova, K.A., Smirnova, O.A.,
Prassolov, V.S., and Kochetkov, S.N., Bioorg. Med.
Chem. Lett., 2015, vol. 25, pp. 2382–2385.
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Translated by A. Levina
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
No. 2
2016