Synthesis and biological activity of hydrazones of o- and p-hydroxybenzoic acids. Spatial structure of 5-Bromo-2-hydroxybenzylidene-4-hydroxybenzohydrazide

Article abstract of DOI:10.1134/S1070363217100097

A series of hydrazones based on hydrazides of o- and p-hydroxybenzoic acids have been prepared. N-(5-Bromo-2-hydroxybenzylidene)-4-hydroxybenzohydrazide has been studied by X-ray diffraction analysis; its molecule forms hydrogen bond with a solvating ethanol molecule. Biological activity of the synthesized hydrazones towards cathepsin Е and(or) elastase of human neutrophils has been determined.

Full text of DOI:10.1134/S1070363217100097

ISSN 1070-3632, Russian Journal of General Chemistry, 2017, Vol. 87, No. 10, pp. 2299–2306. © Pleiades Publishing, Ltd., 2017.
Original Russian Text © O.A. Nurkenov, Zh.B. Satpaeva, I.A. Schepetkin, A.I. Khlebnikov, K.M. Turdybekov, T.M. Seilkhanov, S.D. Fazylov, 2017, published
in Zhurnal Obshchei Khimii, 2017, Vol. 87, No. 10, pp. 1639–1646.
Synthesis and Biological Activity of Hydrazones
of o- and p-Hydroxybenzoic Acids. Spatial Structure
of 5-Bromo-2-hydroxybenzylidene-4-hydroxybenzohydrazide
O. A. Nurkenova*, Zh. B. Satpaevaa, I. A. Schepetkinb,c, A. I. Khlebnikovd,e,
K. M. Turdybekovf, T. M. Seilkhanovg, and S. D. Fazylova
a Institute of Organic Synthesis and Coal Chemistry of Kazakhstan,
ul. Alikhanova 1, Karaganda, 100008 Kazakhstan
*e-mail: nurkenov_oral@mail.ru
b Montana State University, Bozeman, USA
c Russian-Speaking Academic Science Association (RASA) Center,
National Research Tomsk Polytechnic University, Tomsk, Russia
d National Research Tomsk Polytechnic University, Tomsk, Russia
e Polzunov Altai State Technical University, Barnaul, Russia
f Buketov Karaganda State University, Karaganda, Kazakhstan
g Ualikhanov Kokshetau State University, Kokshetau, Kazakhstan
Received May 15, 2017
Abstract—A series of hydrazones based on hydrazides of o- and p-hydroxybenzoic acids have been prepared.
N-(5-Bromo-2-hydroxybenzylidene)-4-hydroxybenzohydrazide has been studied by X-ray diffraction analysis;
its molecule forms hydrogen bond with a solvating ethanol molecule. Biological activity of the synthesized
hydrazones towards cathepsin Е and(or) elastase of human neutrophils has been determined.
Keywords: hydrazone, hydroxybenzoic acid hydrazide, cathepsin Е, neutrophil elastase, molecular docking
DOI: 10.1134/S1070363217100097
The interest to hydrazones has recently emerged
[1, 2] due to a broad range of their biological activity:
1 and 2 with a series of aromatic aldehydes (Scheme 1).
The condensation reaction was performed by heating
(60–70°С) equimolar amounts of the aldehyde and the
hydrazides in ethanol medium during 3–5 h. The target
compounds were obtained with 70–90% yield.
antidepressant,
antiinflammatory,
antimalarial,
antimicrobial, antiviral, and antitumor [3, 4]. For
example, ftivazide, saluzide, and related drugs are used
in the tuberculosis treatment [5, 6]. Inhibitors of many
enzymes like cyclooxygenase, monoaminoxidase, epoxide
hydrolase, and various cathepsins have been prepared
based on hydrazones [7–11]. Proteolytic enzymes
cathepsin Е (EC 3.4.23.34) and elastase of neutrophil
(EC 3.4.21.37) play important part in pathogenesis of
autoimmune diseases and tumors growth [12–14]. In
view of the above, this study aimed to synthesize the
derivatives of N-arylidene hydrazones and investigate
their antiinflammatory activity in the cellular system
in vitro as well as the inhibiting activity with respect to
proteolytic enzymes cathepsin Е and elastase of neutrophil.
The products 3–16 were readily crystallizable white
substances, readily soluble in many organic solvents.
The IR spectra of N-arylidene hydrazones of o- and
p-hydroxybenzoic acids 3–16 did not contain bands of
the NH2 group vibrations. The bands of the NH group
stretching vibrations were observed at 3285–3355 cm–1.
The hydrazone С=О stretching vibrations were found
at 1675–1690 cm–1. A group of characteristic bands at
1600–1440 cm–1 was assigned to the stretching vibra-
tions of the aromatic ring.
1
The downfield part of the Н NMR spectrum of
2-hydroxy-N-(2-hydroxybenzylidene)benzohydrazide 6
contained the signals of methine protons of the
Hydrazones 3–16 were synthesized via the conden-
sation of hydrazides of o- and p-hydroxybenzoic acids
2299

2300
NURKENOV et al.
Fig. 1. COSY 1H–1H correlations in compound 6.
Fig. 2. HMQC 1H–13C correlations in compound 6.
aromatic systems: 6.74–6.90 (Н4, Н6, Н15, Н17), 7.26
(Н5), 7.47 (Н3), 7.67 (Н18), and 7.79 (Н16) ppm. The
most downfield signals (10.14, 11.40, and 11.89 ppm)
with integral intensity 1Н were assigned to the protons
of hydroxy and amino groups.
The 13C NMR spectrum of compound 6 contained
the signals of the СН groups of two benzene rings at
115–132 ppm: 115.67 (С15), 116.93 (С6), 119.22 (С17),
119.81 (С4), 128.63 (С18), 130.18 (С3), 130.27 (С16),
and 131.66 (С5) ppm. Quaternary atoms of the aromatic
systems were assigned to the signals at 115.50 (С2),
123.72 (С13), 157.98 (С14), and 161.46 (С1) ppm. The
signal at 148.18 ppm corresponded to the sp2-hyb-
ridized С12 atom. The downfield signal at 166.48 ppm
was assigned to the carbonyl atom С8.
7
OH
6
5
19
1
HO
O
8
C
4
15
17
14
2
The H–H and C–H spin-spin coupling in compound
6 via one or several bonds were observed by means of
two-dimensional NMR spectroscopy COSY and
HMQC (Figs. 1 and 2).
10
12
11
3
13
18
16
NHN=CH
6
Scheme 1.
O
7
7R2
R2
R1
R1
R
C
H
O
O
8
C
12
C
10 11
NHN=CH__R3
NHNH2
3_16
1, 2
19HO
15
17
15
14
18
R3 =
1
2
_
1
2
_
(1, 3, 2, 10),
F
16 (1, 4, 2, 11),
Br
R = OH, R = H (1, 3 9); R = H, R = OH (2, 10 16);
18
19
19
OH
15
18
15
14
15
OH
16
17
14
14
(1, 5, 2, 12),
OCH3
(1, 6, 2, 13),
(1, 9, 2, 16).
(1, 7, 2, 14),
17
18
17
18
17
18
N (1, 8, 2, 15),
OH
15
14
OCH2CH3
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 87 No. 10 2017

SYNTHESIS AND BIOLOGICAL ACTIVITY OF HYDRAZONES
To elucidate the spatial structure of the derivatives
2301
of N-arylidenehydrazones of p-hydroxybenzoic acid
we performed the X-ray diffraction study of a solvate
of N-(5-bromo-2-hydroxybenzylidene)-4-hydroxybenzo-
hydrazide 11 with ethanol (Fig. 3). The obtained data
revealed that the bond lengths and bond angles in
compound 11 were close to the standard ones [15]. The
molecule of compound 11 was practically planar, but
the phenyl cycles were slightly twisted with respect to
each other (the dihedral angle was 5.9°). In the crystal
each molecule of compound 11 formed the N1–Н···О4
(x, y, z) hydrogen bond [N···O 2.956(5), Н···О 2.22(4) Å,
N–Н···О 164(2)°] with an ethanol molecule. More-
over, the O1–H···O2 intermolecular hydrogen bond
was formed [O···O 2.243(4), Н···О 1.89(5) Å, angle
O–Н···О 174(5)°], leading to the organization of mole-
cules of compound 11 in chains along the a0c diagonal
in parallel with the plane formed by that diagonal and
the b axis.
Fig. 3. General view of a molecule of ethanol solvate of
N-(5-bromo-2-hydroxybenzylidene)-4-hydroxybenzohyd-
razide 11.
phenyl)methylidene]benzohydrazide 22 (IC50 14.4 µM).
Hence, the introduction of chlorine or bromine atoms
in the structure of hydrazones can be considered a
promising approach to prepare new inhibitors of
cathepsin Е.
Compounds 3–16 were tested for antiinflammatory
activity in vitro in a culture of human monocytic cells
(MonoMac-6 cell line) reflected by the suppression of
the lipopolysaccharide-induced production of cyto-
kines interleukin-6 and tumor necrosis factor in the
MonoMac-6 cells. The cells were treated with a solution
of the tested compound in DMSO (final concentration
of DMSO in the culture medium equaled 1%) during
30 min, and then bacterial lipopolysaccharide was
added (0.5 µg/mL). The level of cytokines was eva-
luated by means of enzyme-linked immunosorbent assay
after 24 h incubation of the MonoMac-6 cells (37°С,
5% CO2). The obtained data revealed that none of the
studied hydrazones suppressed the lipopolysaccharide-
induced production of cytokines interleukin-6 and
tumor necrosis factor in the MonoMac-6 cells.
The highest inhibiting activity with respect to the
neutrophils elastase was observed for compound 25
(IC50 21.1 µM). Molecular docking of the active
compounds in the structure of the neutrophils elastase
[17] revealed that benzoyl groups of the active
compounds 4 and 25 were located in the region of the
central pocket of the active site of the elastase,
similarly to the position of the co-crystallized peptide
inhibitor of the elastase, but did not penetrate deeply in
the pocket. The benzene rings adjacent to the imine
fragment of the hydrazones were located in the wide
part of the hydrophobic pocket, near the surface of the
protein. Notably, the benzoyl fragments of molecules 4
and 25 formed three hydrogen bonds with Ser214 and
Val216, the OH group acting as the donor and acceptor
of the hydrogen bonds; this evidently facilitated the
attaching of the molecules in the active center of the
elastase favorable for the inhibition. This is
exemplified in Scheme 2 by the location of the
hydrogen bonds and hydrophobic interactions for
hydrazone 25.
Various hydrazones exhibited the inhibiting activity
towards certain proteases including cathepsins [7, 10,
16]. Therefore, we evaluated the effect of the prepared
compounds 3–16 on the enzymatic activity of cathepsin
E and neutrophil elastase. Since some of the tested
compounds inhibited the enzymatic activity (see the
table), to better reveal the structure–activity rela-
tionship we also studied the commercial analogs of the
prepared hydrazones, 17–25 (Vitas-M Laboratory
Ltd.). It was found that the compounds 4, 17–19, 21–
24 containing bromine (4, 17, 21, 22), chlorine (18),
three hydroxyls (19, 23), or four hydroxyls (24) in the
structure exhibited the activity towards cathepsin Е
(IC50 < 50 µM.). The highest activity was observed in
the case of 5-bromo-2-hydroxy-N'-[(1E)-(2-hydroxy-
The positioning of the inactive hydrazones 3 and 6
in the active site of the neutrophils elastase were
noticeably different from that of the active inhibitors.
The benzene rings of molecules of compounds 3 and 6
penetrated deeply in the central hydrophobic pocket of
the enzyme.
In summary, the condensation of hydrazides of o-
and p-hydroxybenzoic acids with different aromatic
aldehydes afforded a series of hydrazones, some of
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 87 No. 10 2017

2302
NURKENOV et al.
Inhibiting activity of the hydrazone towards cathepsin Е and elastase of human neutrophils
R2
OH
R3
R4
R5
R6
NH
R1
N
N
O
N
O
R7
R4
3_7, 9_14, 16_25
8 (o-OH), 15 (p-OH)
Cathepsin Е Elastase
Comp.
no.
R1
R2
R3
R5
R6
R7
IC50, µMa
3
4
H
H
OH
OH
OH
OH
OH
–
H
H
H
H
H
H
F
H
–
49.9
–
–
33.3
–
H
H
H
H
H
H
H
H
OH
H
H
Br
H
H
H
5
OCH3
H
6
OH
H
–
–
7
OH
54.9
97.8
8
–
9
H
H
H
H
H
H
H
OH
H
OCH2CH3 OH
H
–
–
78.8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
OH
OH
OH
OH
OH
–
H
H
H
H
H
H
H
H
H
H
H
F
H
–
–
–
–
–
OH
H
H
OH
H
60.8
–
OCH3
H
OH
H
H
89.7
66.5
OH
H
–
H
OH
H
H
H
OCH2CH3 OH
H
94.2
34.2
21.9
36.7
59.0
20.1
14.4
32.3
33.5
79.2
64.0
–
H
OH
OH
OH
OH
OH
OH
H
H
H
H
Br
Cl
H
H
H
OH
OH
OH
OH
OH
OH
OH
H
H
H
80.8
–
H
H
OH
H
H
H
H
OH
OH
H
H
–
H
H
H
Br
H
60.4
–
Br
OH
OH
H
H
H
H
H
H
OH
H
–
H
OH
OH
H
OH
42.3
21.1
H
H
N(CH2CH2CH3)2
H
a (–) the enzyme activity is not suppressed at the substrate concentration <100 µM.
them were found promising for the synthesis of new
inhibitors of cathepsin Е.
tively) in DMSO-d6. Chemical shifts were measured
relative to residual protons or carbon atoms of the solvent.
The reaction course and the purity of the products were
monitored by means of thin-layer chromatography on
EXPERIMENTAL
1Н and 13С spectra were recorded using a JNN-ECA
Silufol
UV-254
plates
(isopropanol–benzene–
Jeol 400 spectrometer (399.78 and 100.53 MHz, respec-
ammonia, 10 : 5 : 2, developing in iodine vapor).
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 87 No. 10 2017

SYNTHESIS AND BIOLOGICAL ACTIVITY OF HYDRAZONES
2303
Scheme 2.
was added to a stirred solution of 0.76 g (0.005 mol) of
o-hydroxybenzoic acid hydrazide in 10 mL of 2-propanol.
The mixture was stirred during 30 min at 60–70°С.
The reaction completeness was monitored by means of
TLC. After cooling the mixture to ambient the fine
crystalline precipitate was filtered off and recrystal-
lized. Yield 1.08 g (83.8%), mp 250–251°С (1,4-
R
N
Val216
O
O
H
HN
N
R
Phe215
Phe192
O
H
1
dioxane–hexane, 1 : 2). Н NMR spectrum, δ, ppm:
2
6.23 d (1Н, СН1Ar, JНН = 7.5 Hz), 6.62 t (1Н, СН2
J
,
Ar
НН = 8.4 Hz), 6.45 t (1Н, СН3Ar, 2JНН = 8.5 Hz), 6.77 t
O
R
(1Н, СН4Ar, 2JНН = 7.3 Hz), 7.74 d (2Н, СН15,17Ar, 2JНН
=
8.6 Hz), 7.89 d (2Н, СН14,18Ar, JНН = 8.5 Hz), 8.57 s
2
NH
R
Ser214
Tyr224
Val216
Gly218
N
(1Н, N=CH), 10.45 s (1Н, ОН), 11.85 s (1Н, NH–N).
Hydrazides 4–16 were prepared similarly.
Arg217A
N-(5-Bromo-2-hydroxybenzylidene)-2-hydroxy-
benzohydrazide (4). Yield 1.35 g (80%), mp 295°С.
1Н NMR spectrum, δ, ppm: 6.90 d (1Н, СН15Ar, 2JНН
=
X-ray diffraction study of compound 11. The
intensities of 6311 reflections (3293 independent ones,
Rint 0.0516) were measured using an Xcalibur Ruby
diffractometer (Oxford Diffraction) (CuKα, graphite
monochromator, φ,θ-scanning, 4.03 ≤ θ ≤ 76.13°) at
293 K. The crystals were monoclinic, C14H11N2O3Br·
C2H5OH, а 18.233(4), b 17.857(4), c 13.191(3) Å, β
130.31(3)°, V 3275(1) Å3, Z 8, space group C2/с, dcalc
1.546 g/cm3, μ 3.618 mm–1. The dataset was processed
(including the accounting for absorption) using SAINT
and SADABS software.
8.8 Hz), 6.95 t (1Н, СН2Ar, JНН = 7.4 Hz), 7.02 d (1Н,
СН1Ar, JНН = 8.0 Hz), 7.32 d (1Н, СН4Ar, JНН
=
7.6 Hz), 7.42 t (1Н, СН3Ar, JНН = 8.4 Hz), 7.42 d (1Н,
СН16Ar, JНН = 8.7 Hz), 7.76 s (1Н, СНAr), 8.57 s (1Н,
N=CH), 10.32 s (1Н, ОН7), 11.42 s (1Н, ОН), 11.98 s
(1Н, NH–N).
2-Hydroxy-N-(4-methoxybenzylidene)benzohyd-
razide (5). Yield 1.12 g (83%), mp 215–218°С (2-pro-
panol). 1Н NMR spectrum, δ, ppm: 3.76 s (3Н, ОСН3),
6.26 d (1Н, СН1Ar, JНН = 7.5 Hz), 6.48 t (1Н, СН3
,
Ar
J
НН = 8.5 Hz), 6.65 t (1Н, СН2Ar, JНН = 8.4 Hz), 6.80 d
Structure of compound 11 was solved via the direct
method. Positions of the non-hydrogen atoms were
refined in the anisotropic approximation by full-matrix
least-squares method. The hydrogen atoms at N1, О1,
O3', and O4 were found from differential synthesis, and
their positions were refined in the isotropic
approximation. Other hydrogen atoms were put in the
geometry defined positions and refined in the isotropic
approximation with fixed position and thermal
parameters using the rider model. The structure was
solved and refined using SHELXS-97 and SHELXL-
97 software packages [18, 19]. 1750 independent
reflections with I ≥ 2σ(I) were used in the refinement,
225 parameters were refined. The final divergence
(1Н, СН4Ar, JНН = 7.1 Hz), 6.84 d (2Н, СН15,17Ar, 3JНН
=
3
8.7 Hz), 7.60 d (2Н, СН14,18Ar, JНН = 8.7 Hz), 8.35 s
(1Н, N=СН), 10.01 s (1Н, ОН), 11.49 s (1Н, NН). 13С
NMR spectrum, δС, ppm: 55.81 (ОСН3), 114.84
(СН2,6Ar), 115.51 (СН15,17Ar), 119.81 (С4Ar), 129.06
(СН14,18Ar), 130.11 (СН3,5Ar), 147.36 (N=СН), 161.21
(С1Ar, С16Ar), 163.21 (С=О).
2-Hydroxy-N-(2-hydroxybenzylidene)benzohyd-
razide (6). Yield 1.15 g (90%), mp 273–275°С
(EtOH). 1Н NMR spectrum, δ, ppm: 6.84–6.90 m (4Н,
3
СН4,6,15,17Ar), 7.26 t (1Н, СН5Ar, JНН = 7.3 Hz), 7.47 d
(1Н, СН3Ar, 3JНН = 8.7 Hz), 7.67 d (1Н, СН18Ar, 3JНН
=
7.8 Hz), 7.79 t (1Н, СН16Ar, 3JНН = 8.2 Hz), 8.56 s (1Н,
N=СН), 10.14 s (1Н, ОН19), 11.40 s (1Н, ОН7), 11.89
s (1Н, NH). 13С NMR spectrum, δС, ppm: 115.12
(С2Ar), 115.67 (СН15Ar), 116.93 (СН6Ar), 119.22
(С17Ar), 119.81 (СН4Ar), 123.72 (С13Ar), 128.12
(СН18Ar), 130.27 (СН16Ar, СН3Ar), 131.66 (СН5Ar),
148.18 (N=СН), 157.98 (С14Ar), (С1Ar), 166.48 (С=О).
factors were as follows: R1 0.0501, R2 0.0938 [over
W
reflections with I ≥ 2σ(I)], R1 0.1085, WR2 0.1190 (over
all reflections), GooF 0.969. Peaks of residual
electronic density: Δρ 0.258 and –0.430 е/Å3. Crystallo-
graphic data were deposited at the Cambridge
Crystallographic Data Centre (CCDC 1546889).
2-Hydroxy-N-(4-hydroxybenzylidene)hydrazide
(7). Yield 1.06 g (83%), mp 277–278°С (EtOH). Н
N-(4-Fluorobenzylidene)-2-hydroxybenzohyd-
razide (3). p-Fluorobenzaldehyde (0.63 g, 0.0051 mol)
1
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 87 No. 10 2017

2304
NURKENOV et al.
NMR spectrum, δ, ppm: 6.83 d (2Н, СН15,17Ar, 2JНН
=
=
3JНН = 8.7 Hz), 7.61 d (2Н, СН14,18Ar, JНН = 8.7 Hz),
3
2
3
3
8.2 Hz), 6.92 d.d (2Н, СН4,6Ar, JНН = 16.0, JНН
7.77 d (2Н, СН3,5
,
JНН = 7.8 Hz), 8.34 s (1Н,
Ar
7.6 Hz), 7.38 t (1Н, СН5Ar, 2JНН = 7.8 Hz), 7.54 d (2Н,
СН14,18Ar, 2JНН = 8.2 Hz), 7.86 d (1Н, СН3Ar, 2JНН = 7.8 Hz),
8.33 s (N=СН), 9.92 br.s (1Н, ОН19), 11.66 s (1Н,
NH), 11.96 br.s (1Н, ОН7). 13С NMR spectrum, δC,
ppm: 116.20 (С2Ar), 116.29 (СН15,17Ar), 117.85
(СН6Ar), 119.38 (СН4Ar), 125.60 (С13Ar), 128.81
(СН3Ar), 129.62 (СН14,18Ar), 134.25 (СН5Ar), 149.78
(N=СН), 159.85 (С16Ar), 160.21 (С1Ar), 165.25 (С=О).
N=СН), 10.03 s (1Н, ОН), 11.46 s (1Н, NН). 13С
NMR spectrum, δC, ppm: 55.80 (ОСН3), 114.85
(СН2,6Ar), 115.51 (СН15,17Ar), 124.61 (С4Ar), 127.64
(С13Ar), 129.05 (СН14,18Ar), 130.11 (СН3,5Ar), 147.34
(N=СН), 161.21 (С1Ar, С16Ar), 163.19 (С=О).
4-Hydroxy-N-(2-hydroxybenzylidene)benzohyd-
razide (13). Yield 0.85 g (67%), mp 260°С (EtOH).
1Н NMR spectrum, δ, ppm: 6.85 d (2Н, СН2,6Ar, JНН
=
2-Hydroxy-N-(pyridin-4-ylmethylidene)benzohyd-
razide (8). Yield 1.09 g (91%), mp 238–239°С
(EtOH). 1Н NMR spectrum, δ, ppm: 6.95 m (2Н, СН4,6Ar),
8.7 Hz), 6.90 d (2Н, СН15,17Ar, JНН = 8.8 Hz), 7.74 d
(2Н, СН3,5Ar, JНН = 7.8 Hz), 7.54 d (2Н, СН14,18
,
Ar
2JНН = 8.2 Hz), 8.28 s (1Н, N=CH), 9.98 br.s (2Н,
7.41 t (1Н, СН5Ar, JHH = 4.9 Hz), 7.63 d (2Н, СН14,18
,
ОН7,19), 11.40 s (1Н, NH).
Ar
J
HH = 3.9 Hz), 7.84 d (1Н, СН3Ar), 8.42 s (1Н, N=CH),
4-Hydroxy-N-(4-hydroxybenzylidene)benzohyd-
razide (14). Yield 0.94 g (73.8%), mp 265°С (EtOH).
1Н NMR spectrum, δ, ppm: 6.80 m (4Н, СН2,6,15,17Ar),
7.50 d (2Н, СН14,18Ar, JНН = 6.9 Hz), 7.74 d (2Н,
СН5,3Ar, JНН = 8.2 Hz), 8.28 s (1Н, N=CH), 9.95 br.s
(2Н, ОН7,19), 11.40 s (1Н, NH). 13С NMR spectrum,
δC, ppm: 115.49 (СН2,6Ar), 116.21 (СН15,17Ar), 124.63
(С4Ar), 126.03 (С13Ar), 129.21 (СН14,18Ar), 130.07
(СН5,3Ar), 147.74 (N=CH), 159.75 (С16Ar), 161.04 (С1Ar),
163.07 (С=О).
8.62 d (2Н, СН15,17Ar), 11.96 br.s (2Н, NH, ОН). 13С
NMR spectrum, δC, ppm: 116.83 (С2Ar), 117.77
(СН6Ar), 119.61 (С4Ar), 121.59 (СН14,18Ar), 129.46
(СН3Ar), 134.47 (СН5Ar), 141.89 (С13Ar), 146.63 (N=CH),
150.81 (СН15,17Ar), 159.17 (С1Ar), 165.34 (С=О).
N-(3-Ethoxy-4-hydroxybenzylidene)-2-hydroxy-
benzohydrazide (9). Yield 0.9 g (60.6%), mp 196–
1
198°С (С6Н6). Н NMR spectrum, δ, ppm: 1.33 t (3Н,
3
СН3, JHH = 6.9 Hz), 4.01–4.06 m (2Н, ОСН2), 6.84 d
3
(1Н, СН3Ar, JHH = 8.2 Hz), 6.94 d.d (2Н, СН18,20
,
Ar
3
3JHH = 7.6, 13.5 Hz), 7.07 d (1Н, СН4Ar, JHH
=
4-Hydroxy-N-(pyridin-4-ylmethylidene)benzohyd-
razide (15). Yield 1.07 g (89%), mp 261–263°С (EtOH).
8.2 Hz), 7.28 s (1Н, СН6Ar), 7.34–7.41 m (1Н, СН19Ar),
7.88 d.d (1Н, СН21Ar, 3JHH = 7.8, 13.7 Hz), 8.30 s (1Н,
N=СH).
1Н NMR spectrum, δ, ppm: 6.84 d (2Н, СН2,6Ar, JНН
=
8.9 Hz), 7.60 d (2Н, СН14,18Ar, JНН = 6.0 Hz), 7.79 d
(2Н, СН3,5Ar, JНН = 6.9 Hz), 8.37 s (N=CH), 8.60 d
(2Н, СН15,17Ar, JНН = 6.0 Hz), 10.12 br.s (1Н, NH),
11.83 s (1Н, ОН). 13С NMR spectrum, δC, ppm:
115.62 (СН2,6Ar), 121.41 (СН14,18Ar), 124.07 (С4Ar),
130.48 (СН3,5Ar), 142.26 (С13Ar), 144.83 (N=CH),
150.75 (СН15,17Ar), 161.45 (С1Ar).
N-(4-Fluorobenzylidene)-4-hydroxybenzohyd-
razide (10). Yield 0.87 g (67.5%), mp 183–185°С
1
(propanol-2). Н NMR spectrum, δ, ppm: 6.81 d (2Н,
2
СН2,6Ar, J = 8.7 Hz), 7.78 t (2Н, СН3,5Ar, J = 8.7 Hz),
2
7.74 d (2Н, СН15,17Ar, J = 8.6 Hz), 7.89 d (2Н,
2
СН14,18Ar, J = 8.5 Hz), 8.58 s (1Н, N=CH), 10.50 s
N-(3-Ethoxy-4-hydroxybenzylidene)-4-hydroxy-
benzohydrazide (16). Yield 1.37 g (91.9%), mp 240–
242°С (EtOH). 1Н NMR spectrum, δ, ppm: 1.02 t (3Н,
(1Н, ОН), 11.83 s (1Н, NHN).
N-(5-Bromo-2-hydroxybenzylidene)-4-hydroxy-
benzohydrazide (11). Yield 0.85 g (51.1%), mp 293–
3
3
СНСН3, JHH = 6.9 Hz), 1.31 t (3Н, ОСН2СН3, JHH
=
295°С (EtOH). 1Н NMR spectrum, δ, ppm: 6.88 d (2Н,
6.9 Hz), 3.33–3.43 m (1Н, СНСН3), 3.93–4.04 m (2Н,
2
СН2,6Ar, JНН = 8.7 Hz), 6.90 d (1Н, СН15Ar, JНН
=
ОСН2СН3), 6.80 d (2Н, СН18,20Ar, 3JHH = 8.2 Hz), 6.82
8.8 Hz), 7.42 d (1Н, СН16Ar, JНН2 = 8.7 Hz), 7.76 s (1Н,
d (1Н, СН3Ar, 3JHH = 3.7 Hz), 7.03 d (1Н, СН4Ar, 3JHH
7.8 Hz), 7.24 s (1Н, СН6Ar), 7.77 d (2Н, СН17,21Ar, 3JHH
=
=
СН18Ar), 7.83 d (2Н, СН3,5 JНН = 8.5 Hz), 8.57 s
Ar,
(1Н, N=CH), 10.15 s (1Н, ОН7), 11.42 s (1Н, ОН),
11.98 s (1Н, NH–N).
8.7 Hz), 8.27 s (1Н, N=CH). 13С NMR spectrum, δC,
ppm: 15.24 (ОСН2СН3), 19.06 (СНСН3), 56.58
(СНСН3), 64.43 (ОСН2СН3), 110.87 (СН6Ar), 115.49
(СН18,20Ar), 116.07 (СН4Ar), 122.42 (СН3Ar), 124.64
(С5Ar), 126.47 (С16Ar), 130.07 (СН17,21Ar), 147.70
(N=CH), 149.60 (С2Ar), 161.06 (С1Ar), 163.15 (С19Ar).
4-Hydroxy-N-(4-methoxybenzylidene)benzohyd-
razide (12). Yield 1.21 g (90%), mp 220°С (EtOH).
1Н NMR spectrum, δ, ppm: 3.76 s (3Н, ОСН3), 6.83 d
3
(2Н, СН15,17Ar, JНН = 8.7 Hz), 6.97 d (2Н, СН2,6
,
Ar
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 87 No. 10 2017

SYNTHESIS AND BIOLOGICAL ACTIVITY OF HYDRAZONES
Activity with respect to elastase was estimated as
ACKNOWLEDGMENTS
2305
the ability of the compound to inhibit hydrolysis of
synthetic fluoro-genic substrate N-methylsuccinyl-Ala-
Ala-Pro-Val-7-amino-4-methylcoumarin (Calbiochem)
by elastase of human neutrophils (Calbiochem). The
formation of the fluorescent product was detected
under excitation at 355 nm from the intensity of
emission at 460 nm using a Fluoroskan Ascent FL
instrument (Thermo Scientific) [20]. Activity with
respect to cathepsin E was estimated as the ability of
the compound to inhibit hydrolysis of synthetic
fluorogenic substrate of cathepsin E (1756.0 Da, Enzo
Life Sciences, Inc.). The formation of the fluorescent
product was detected under excitation at 325 nm from
the intensity of emission at 410 nm using the same
instrument.
This study was financially supported by the
Ministry of Education and Science of Russian
Federation (project no. 4.6660.2017/8.9).
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