Design and Bioevaluation of Novel Hydrazide-Hydrazones Derived from 4-Acetyl-N-Substituted Benzenesulfonamide

Article abstract of DOI:10.1134/S1068162020050052

Abstract: In this research, a series of hydrazine-hydrazone derivatives (Ia–g), (IIa–h) were synthesized to discover new antioxidant and anticholinesterase agents. The structures of synthesized compounds were characterized by spectroscopic data using UV, IR, ¹H, ¹³C NMR, mass spectroscopy, and elemental analysis. The bio-evaluation of the synthesized compounds (Ia–g), (IIa–h) were evaluated according to in vitro activity assays. The results of β-carotene/linoleic acid assay showed that among the synthesized compounds, the (Ib), (Ie), (IIb–IIe), and (IIh) compound exhibited higher activity for the lipid peroxidation inhibitory activity. In the DPPH free scavenging activity and the cation radical scavenging activity in ABTS^?+ activity, compound (IIb) was found to be more active. In the CUPRAC reduced power assay, the A_0.5 values of all synthesized compounds were better than α-TOC. In AChE assay, compound (IIb) exhibited the most activity with IC₅₀ = 11.12 ± 0.74 μM, while the compounds (Ib–g) and (IIb–h), exhibited excellent activity than the positive standard galantamine (IC₅₀ = 46.06 ± 0.10 μM) in the BChE assay.

Full text of DOI:10.1134/S1068162020050052

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2020, Vol. 46, No. 5, pp. 702–714. © Pleiades Publishing, Ltd., 2020.
Design and Bioevaluation of Novel Hydrazide-Hydrazones Derived
from 4-Acetyl-N-Substituted Benzenesulfonamide
E. Bozkurta, Y. Sıcakb, E. E. Oruç-Emrea, 1, A. Karaküçük Iyidoğana, and M. Öztürkc
aDepartment of Chemistry, Faculty of Arts and Sciences, Gaziantep University, Gaziantep, 27410 Turkey
bDepartment of Medicinal and Aromatic Plants, Köyceğiz Vocational School, Muğla Sıtkı Koçman University,
Muğla, 48800 Turkey
cDepartment of Chemistry, Faculty of Sciences, Muğla Sıtkı Koçman University, Muğla, 48800 Turkey
Received January 1, 2020; revised March 18, 2020; accepted March 22, 2020
Abstract—In this research, a series of hydrazine-hydrazone derivatives (Ia–g), (IIa–h) were synthesized to
discover new antioxidant and anticholinesterase agents. The structures of synthesized compounds were char-
acterized by spectroscopic data using UV, IR, 1H, 13C NMR, mass spectroscopy, and elemental analysis. The
bio-evaluation of the synthesized compounds (Ia–g), (IIa–h) were evaluated according to in vitro activity
assays. The results of β-carotene/linoleic acid assay showed that among the synthesized compounds, the (Ib),
(Ie), (IIb–IIe), and (IIh) compound exhibited higher activity for the lipid peroxidation inhibitory activity. In
the DPPH free scavenging activity and the cation radical scavenging activity in ABTS•+ activity, compound
(IIb) was found to be more active. In the CUPRAC reduced power assay, the A0.5 values of all synthesized
compounds were better than α-TOC. In AChE assay, compound (IIb) exhibited the most activity with IC50
=
11.12 0.74 μM, while the compounds (Ib–g) and (IIb–h), exhibited excellent activity than the positive stan-
dard galantamine (IC50 = 46.06 0.10 μM) in the BChE assay.
Keywords: sulfonamide, hydrazone, antioxidant activity, anticholinesterase inhibitory activity, Lipinski’s
rules
DOI: 10.1134/S1068162020050052
INTRODUCTION
niazid (antimicrobial), nifuroxazide (intestinal anti-
septic), nitrofurantoin, and nifuratel (urine antisep-
tic), iproniazid and isocarboxazid (antidepressants)
[11]. In the literature, it has been reported that hydra-
zide-hydrazone derivatives exhibit antioxidant and
anticholinesterase activity [12–15].
In protecting the organism’s health status, the bal-
ance between the antioxidant systems of the body with
free radicals is essential [16]. Extreme reactive oxygen
species (ROS) production eventuate oxidative stress
that may be the reason for fatal damage to living cell
structures [17]. Oxidative stress has also related some
of the common diseases such as cancer, neurodegen-
erative, cardiovascular, inflammatory, and autoim-
mune. It is associated with the pathology of Alzhei-
mer’s disease, as well [18].
Alzheimer’s disease is degeneration of the central
nervous system, also is known as the most common
form of dementia. As a progressive neurologic disor-
der, it is characterized mainly by premature senile
mental deterioration that results in behavioral abnor-
malities in the patient [19]. The acetylcholinesterase
(AChE) inhibitory compounds were used to treat Alz-
heimer’s disease due to the deficiency of the insuffi-
Sulfonamides (sulfa drugs) are significant bioactive
synthetic medicines. Over the last decades, many sulfa
drugs have been developed and began using in the
treatment. [1]. The brinzolamide, which is a carbonic
anhydrase inhibitor, has been used to reduce intraoc-
ular pressure in patients with open-angle glaucoma or
ocular hypertension [2]. An anti-glaucoma agent, dor-
zolamide, is used to decrease the production of aque-
ous humor [3]. Another sulfonamide drug sultiame
having anticonvulsant activity is reported for the cure
of West syndrome and epilepsy [4]. Besides, studies
have shown that sulfonamides exhibit antioxidant and
anticholinesterase activities [5–9].
Another essential organic compound class, hydra-
zones (–CONHN=CH–) are formed by the reaction
of hydrazine and ketones/aldehydes [10]. Hydrazones
can be used as intermediates to synthesize coupling
products using the active hydrogen of the azomethine
group. Besides, they are very effective organic com-
pounds and used as drugs in the treatment such as iso-
1 Corresponding author: phone: + (903) 423-17-29-98; fax:
+ (903) 423-60-10-32; e-mail: oruc@gantep.edu.tr.
702

DESIGN AND BIOEVALUATION
703
cient amount of acetylcholine (ACh). However, these absorption bands accompanied by the C=N absorp-
drugs have undesired side effects. Therefore, the tion band between the 1591–1607 cm–1 region that
was good evidence for the presence of an azomethine
linkage. An additional strong band in the 1637–
1671 cm–1 region, attributed to a carbonyl stretching,
confirmed the hydrazone feature of all the compounds
[21–24]. The asymmetric and symmetric stretching
bands of the SO2NH group of the synthesized mole-
improvement of novel effective antioxidants and
AChE inhibitory compounds having fewer side effects
are desired. According to some literature, using anti-
oxidants may decrease the progression of Alzheimer’s
disease and reduce neuronal degeneration [20].
In our body, which is under oxidative stress, oxygen
is divided into two atoms that do not have a paired
electron. These atoms that travel alone and have miss-
ing electrons are called free radicals. However, elec-
trons like to go around in pairs. That’s why free radi-
cals in our bodies travel our entire body to find another
electron. During this circulation, cells, proteins, and
DNA are damaged. Free radicals can cause many dis-
eases. It can lead to diseases such as diabetes, Alzhei-
mer’s, and Parkinson’s disease, vascular occlusion. In
recent years, free radicals formed in our body have
become a severe health problem with the increase of
oxidation-related diseases. It is an advantage for an
antioxidant compound if it inhibits or minimizes sev-
eral diseases related to oxidative stress. Thus, the novel
hydrazide-hydrazones, combined with the sulfon-
amide group to increase the pharmacological activi-
ties, were designed herein. These compounds (Ia–g),
(IIa–h), derived from 4-acetyl-N-substituted benzene-
sulfonamide, were synthesized and evaluated for their
in vitro antioxidant and anticholinesterase activities.
cules were in the range of 1335–1347 cm–1 and 1144–
1166 cm–1, respectively [25–28]. The absorption
bands associated with substituents connected to the
hydrazide-hydrazone skeleton appeared in the
expected regions [29].
1
In the H NMR spectra of the hydrazide-hydra-
zone derivatives, the proton of –CONHN= group res-
onated as a singlet at 10.49–11.19 ppm. The disappear-
ance of the proton peaks of the free amino group is evi-
dence of hydrazide-hydrazone synthesis. These data
were compatible with the literature records [30–33].
Moreover, resonating of the proton of –SO2NH group
as a singlet at the range of 10.32–10.92 ppm was
another proof of the hydrazide-hydrazone group for-
mation. In the structure of compounds i and ii, the
protons of –CH3 had resonance at 2.59 ppm as the
electron density, after the formation of imine
(‒N=C–) resonated at 2.28–2.38 ppm. In the struc-
ture of compounds (Ia–h), the protons in ortho and
meta positions relative to the sulfonamide group in the
A ring resonated in the range of 7.03–7.24 ppm. In the
structure of compounds (IIa–h), however, they dis-
played a peak at 7.83 ppm due to the electron-with-
drawing property of the carboxylic acid. Chemical
shifts of the protons belonging to the rings C and D of
the compounds (If), (IIf) were observed in the range
of 7.56–8.00 ppm, and the amide proton was observed
in the range of 10.80–10.81 ppm [34]. The protons of
the C and D rings of the compounds (Ig), (IIg) reso-
nated in the range of 7.29–7.44 ppm, and the tertiary
hydroxyl proton was detected in the range of 7.23–
7.24 ppm [35, 36].
RESULTS AND DISCUSSION
In this work, the new hydrazide-hydrazones (Ia–
g), (IIa–h) derivated from 4-acetyl-N-substituted
benzenesulfonamide were synthesized. In the first
step, the substituted benzoylhydrazine derivatives
were prepared by the treatment of substituted benzoyl-
chloride with hydrazine hydrate. In the second step,
sulfonamide compounds were synthesized via the
treatment of 4-acetylbenzenesulfonylchloride with
substituted aromatic amines. In the last step, substi-
tuted hydrazide-hydrazone derivatives were obtained
by interacting substituted benzoylhydrazine with sul-
fonamide compounds in methanolic solution. The
synthetic route followed for the preparation of the tar-
get molecules was shown outlined in Scheme 1.
In this research, the ultraviolet and visible (UV-
Vis) spectra of all hydrazide-hydrazones derivatives
were carried out (dissolved) in DMSO. In the UV-Vis
spectra of hydrazone groups (Ia–g), (IIa–h), the K
bands caused by transitions of n → σ* and π → π* of nitro-
gen in –C=N– group were detected at 274–285 nm.
Also, characteristic R bands originating from the n →
π* transition, containing the carbonyl moiety of the
hydrazide group (C=O), were at 295–315 nm.
In the 13C NMR spectrum of the compound i and
ii, it was found that the –CH3 carbon in the acetyl
group resonated at 27.44–27.45 ppm, the aromatic
carbons resonated in the range 118.86–143.58 ppm,
the carbonyl carbons in the acetyl group were detected
at 197.63–197.68 ppm, carbonyl carbon of COOH in
the structure of compound ii was at 167.15 ppm, and
the values herein were consistent with the literature
data [27]. The resonance of the imine bond (–C=N)
carbon in the range of 152.72–154.94 ppm was com-
patible with the literature data [37]. The methyl
(‒CH3) carbon of hydrazones was a resonance in the
range of 13.80–15.47 ppm, and the aromatic carbons
were resonance in the range of 115.58–133.10 ppm,
the carboxylic acid carbonyl carbon was a resonance in
the range of 167.18–167.74 ppm, the carbonyl (‒C=O)
carbon bound to the hydrazone group was found to have
The IR spectra of hydrazide-hydrazones (Ia–g),
(IIa–h) provided the 3137–3354 cm–1 absorption as
the weak NH band. Both weak NH and C–H bending resonance in the range of 163.23–169.37 ppm.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 46 No. 5 2020

704
BOZKURT et al.
O
R
+
R
Cl
O
HO
O
S
O
O
NaOH
R1
NH2
+
Cl
C CH3
O
NH2NH2 · H2O
O
S
O
H
O
C CH3
R1
N
NH2
O
R
N
H
iii
i = R1: -H
ii = R1: -COOH
HF
C8
HG
HD
HE
HC
HA
O
O
HH
C1
H
H
R1
N S
C N N C R
O C
CH3
C3
C7
C10
C9
6
C12
C11
C5
C4
C2
HB
C14
C15
HJ
C14
C13
C15
C14
C13
C15
C16
C14
C13
C15
C16
C16 C17
C13
C16
C15
N
F
Cl
C14
HI
HI
HJ
HI
HJ
HI
HJ
C13
OHH
a
b
c
d
I
R
HK
C14
C15
C14
C15
C19
C18
C17
C20
C21
HN
C14
C15
C16
HJ
O
C13
C16
C13
C16
C13
H
N
NO2
Br
HK
HL
HI
HJ
HI
HJ
HL
HM
HI
HJ
e
f
h
g
O
O
N S
H O
C N N C R
CH3
H
(Ia−g) series
O
O
HOOC
N S
C N N C R
H O
CH3
H
(IIa−h) series
Scheme 1. Synthetic pathway of substituted hydrazide-hydrazones (Ia–g), (IIa–h).
The mass spectra of the synthesized compounds (I, IIg) bearing the nonsubstituted phenyl ring in the
II, Ia–g, IIa–h) were run using ESI (+) and (–) tech-
nique. According to the results, the compounds (Ib,
Ic, Id, Ig, IIa–d, IIf, and IIh) exhibited [M+1] molec-
ular ion peaks while compounds (II, Ie, IIe) [M–1],
and the compounds (I, Ia) [M+2], and the compounds
structure, the ions formed as a result of the fragmenta-
tion of the phenyl ring and the cleavage ions of the
phenyl ring were detected. Compounds (IIa–h) bear-
ing acid groups have been generally found to form ions
resulting from the cleavage of the α-cleavage relative to
(If and IIg) [M–2]. The compounds (I, Ia–g, IIa, IIf, the acid carbonyl, –COOH (m/z 45). Formed ions
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DESIGN AND BIOEVALUATION
705
with cleavage of the pyridine ring (m/z 78) in com- the DPPH, which resulted in the decrease of absorp-
pounds (Ib) and (IIb) and the cleavage products of this tion at 517 nm [39–42, 44]. The lower absorbance at
ring were detected. In general, when the mass spectra 517 nm indicates higher DPPH free radical scavenging
of all synthesized compounds were examined, it, activity. In the DPPH free scavenging activity, com-
unlike the molecular ion, has been found that the deg- pounds (IIb) (IC50 = 26.77 0.31 μM) demonstrated the
radation proceeds from 8 points. In Scheme 2, frag-
mentation points on the skeletal structure of the syn-
thesized compounds were indicated.
best activity. Moreover, all series (Ia–g), (IIa–h) exhibited
more activity than BHT (IC50 = 53.80 0.92 μM).
ABTS (2,2′-azino-di-[3-ethylbenzthiazoline sul-
fonate) cation radical scavenging activity method is
based on the neutralizes of ABTS cation radical by an
antioxidant. The ABTS cation radical is produced by
the oxidation of ABTS molecule with strong oxidizing
agents, such as K2S2O7 in water. The produced green-
ish radical absorbs at 734 nm. The advantage of this
radical is the solubility in inorganic and organic sol-
vents. Therefore, using this assay, both hydrophilic
and hydrophobic compounds can be tested [45].
Another advantage of the ABTS assay is that the bulky
compounds can approach this molecule to transfer
electrons easily when compared with the DPPH mol-
ecule. In this assay, lower absorbance exhibits the
higher ABTS cation radical scavenging activity [39–
O
8
7
6
2 H
N
1
5
4
3
O
S
N
R
N
C
CH3
H
O
Scheme 2. Fragmentation points
on the skeletal structure of the synthesized compounds.
Biological Activity
Antioxidant activity evaluation. The synthesized
compounds (Ia–g, IIa–h) were screened for their anti-
oxidant activity using four different assays (Table 1).
The α-tocopherol and BHT (butylated hydroxyl tolu-
ene) were used as positive standards to compare the
activity. In general, among the synthesized molecules
(Ia–g), (IIa–h) series, the antioxidant activity of
compounds (IIa–h) series were founded to be active
than compounds (Ia–g) derivatives. β-carotene-lin-
oleic acid assay is based on the discoloration of the yel-
lowish color of a β-carotene having an absorption
band at 470 nm. The singlet oxygen oxidizes the dou-
ble bonds of linoleic acid added to the oxygenated
media, which resulted in the lipid peroxyl radicals (L•
or LOO•). The produced radicals attach the β-caro-
tene to degrade it. The antioxidant in the media neu-
tralizes the radicals or stops the radicalic degradation
radicals by transferring Hydrogen radical or scavenge
the singlet oxygen, which accelerates the radicalic deg-
radation [38–42]. Therefore, the higher absorbance at
470 nm indicates the higher antioxidant activity.
According to the β-carotene/linoleic acid assay results,
the compounds (IIb) (IC50 = 10.21 0.12 μM), (Ib)
42, 46]. In ABTS assay, compound (IIb) (IC50
4.13 0.54 μM) was the most active compound
among the tested compounds (Ia–g), (IIa–h). Com-
=
pounds (Ib) (IC50 = 7.08 0.45 μM), (IIe) (IC50
=
7.45 0.50 μM), (Ie) (IC50 = 9.69 0.28 μM), (IIc)
(IC50 = 10.88 0.63 μM), (Ic) (IC50 = 11.85 0.18 μM),
(IId) (IC50 = 13.51
0.49 μM), and (Id) (IC50 =
14.41 1.13 μM) indicated good ABTS•+ cation radi-
cal scavenging activity.
The CUPRAC (Cupric Reducing Antioxidant
Capacity) assay tests the electron giving power of the
antioxidant. The antioxidant reduces the Cupric to
Cuprous. The reduction potential is monitored by
using the neocuproine ligand. The neocuproine ligand
and cuprous can occur complex molecule which
absorbs at 450 nm. Therefore, the amount of reduc-
tion of Cupric to cuprous indicates the power of anti-
oxidants. Accordingly, the higher absorbance at
450 nm suggests the higher Cupric reducing antioxi-
dant capacity [40–42, 47, 48]. In the CUPRAC assay,
compound (IIb) (A0.5 = 8.88 0.01 μM) possessed the
highest activity. The cupric reducing the antioxidant
capacities of synthesized compounds (Ia–g), (IIa–h)
were better than α-TOC (A0.5 = 40.41 0.03 μM).
In this study, according to the antioxidant activity
assay results of the synthesized compounds, they
exhibit better antioxidant activity than other com-
pounds with the potential of forming free radicals by
weakening the NH bond in the hydrazone group of the
compounds (Ib) and (IIb) containing the heteroatom
in the ring and compound (Ic), (Id), (IIc) and (IId)
containing the halogen group bound to the ring.
(IC50 = 12.83
0.19 μM), (IIc) (IC50 = 14.75
0.48 μM), (IId)
0.44 μM), (IIe) (IC50 = 16.20
(IC50 = 18.83 0.19 μM), (Ie) (IC50 = 20.16 0.35 μM),
and (IIh) (IC50 = 20.16 0.37 μM) exhibited higher
lipid peroxidation inhibitory activity among the syn-
thesized compounds. The compounds (IIb), (Ib),
(IIc), (IIe), (IId), (Ie), and (IIh) exhibiting high lipid
peroxidation inhibitory activity sadi that they have the
abilities of radical hydrogen transfer.
DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate)
free radical scavenging activity method is based on the
neutralizes of DPPH radical by an antioxidant. The
DPPH is a stable free radical absorbing at 517 nm
wavelength. When the DPPH radical get an electron
from an antioxidant, the absorption at 517 nm
decreases [43]. This free radical is stable at room tem-
Acetyl- and butyryl-cholinesterase inhibitory activi-
perature. Therefore, it can be said that the antioxidant, ties evaluation. The only known hypothesis to treat
if approaches DPPH molecule, transfers electrons to Alzheimer’s disease is to inhibit the acetylcholinester-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 46 No. 5 2020

706
BOZKURT et al.
Table 1. Antioxidant activity results of synthesized compoundsa
DPPH• assay IC50, ABTS•+ assay IC50,
Sample
code
β-Carotene/linoleic acid assay IC50, μM
CUPRAC A0.50, μM
μM
μM
(I)
(II)
(Ia)
(Ib)
(Ic)
(Id)
(Ie)
(If)
(Ig)
(IIa)
(IIb)
(IIc)
(IId)
(IIe)
(IIf)
(IIg)
(IIh)
44.18 0.42
39.56 0.90
35.47 0.54
12.83 0.19
22.77 0.33
27.14 0.60
20.16 0.35
33.82 0.66
30.49 0.76
29.14 0.88
10.21 0.12
14.75 0.44
18.36 0.61
16.20 0.48
25.88 0.68
22.84 0.52
20.16 0.37
58.19 0.54
54.93 0.71
52.06 0.18
30.36 0.85
33.99 1.14
36.76 0.53
32.01 0.17
49.40 0.44
44.26 0.68
50.65 0.94
26.77 0.31
33.24 0.55
35.73 0.26
30.29 0.75
45.74 0.59
43.52 0.36
39.44 0.58
53.80 0.92
40.11 0.22
36.77 0.34
34.17 0.03
7.08 0.45
11.85 0.18
14.41 1.13
9.69 0.28
29.16 0.40
30.49 0.07
33.41 0.89
4.13 0.54
10.88 0.63
13.51 0.49
7.45 0.50
25.37 1.08
29.70 0.44
17.30 0.67
2.81 0.43
42.08 0.00
38.45 0.01
33.66 0.03
8.88 0.00
18.77 0.04
21.64 0.01
16.47 0.02
28.30 0.02
24.11 0.00
20.35 0.01
8.40 0.01
9.73 0.01
10.04 0.00
14.26 0.00
16.27 0.00
13.90 0.04
13.15 0.03
3.84 0.00
BHTb
α-TOCb
2.44
4.63
0.07
0.11
12.16 0.16
4.93 0.39
40.41 0.03
a
b
Values expressed are means SD of three parallel measurements. p < 0.05, significantly different with student’s t-test. Reference
compounds.
ase (AChE) and butyrylcholinesterase (BChE), which respectively, which were lower than 25 μM. The said
are the chief enzymes of Alzheimer’s disease. There- compounds they were exhibited better activity among
fore, acetylcholinesterase inhibitory drugs are used for synthesized compounds (Ia–g), (IIa–h). In the BChE
the treatment of patients. The inhibition of both assay, however, the IC50 of all compounds (Ia–g),
enzymes lessens the symptoms of Alzheimer’s disease
by increasing the communication between nerve end-
ings and the activities in cholinergic pathways in the
brain [49]. Accordingly, the anticholinesterase activity
method is based on the inhibition of AChE and BChE
enzymes. In a control test, where there is no testing
compound, the enzyme hydrolyzes the acetylthiocho-
line or butyrylthiocholine to give thiocholine. The lat-
ter reacts with the DTNB to give a yellow color which
can be measured at 412 nm. When any inhibitory com-
pound added to the medium to be tested for its activity,
the compound inhibits the enzymes; thus, thiocholine
occurs a lesser amount, which resulted in lower absor-
bances. Therefore, the lower absorbance indicates
higher activity. In other words, lower absorbance indi-
cates higher acetylcholinesterase and butyrylcholines-
terase inhibitory activity of the compound.
(IIa–h) except compounds (Ia) and (IIa), were lower
than galantamine used as a drug in mild Alzheimer
patients. The compounds (IIb), (Ib), (IIe), (IIc),
(IId), (Ie), (Ic), (Id), (IIh), (IIg), (Ig), (IIf), and (If)
from synthesis series exhibited excellent activity with
IC50 values of 14.26 0.68, 17.25 0.37, 20.74 0.62,
21.88 0.82, 26.65 0.49, 26.65 0.54, 31.19 0.84,
35.20 0.68, 36.35 0.40, 40.72 0.43, 43.34 0.60,
and 45.51 0.61 μM, respectively. The galantamine
possessed 46.06 0.10 μM IC50 value in the same con-
ditions. As a result, the compounds (IIb) and (Ib) the
AChE and BChE against show better activity at low
absorbance by inhibiting both enzymes better than
other compounds.
Druglikeness Properties
The in vitro anticholinesterase activity of synthe-
sized compounds (Ia–g), (IIa–h) against AChE and
BChE were given in Table 2. In AChE inhibitory
assay, the IC50 values of compounds (IIb), (Ib), (IIe),
According to Lipinski’s rules of five, our results
showed that the molecular weights of compounds
except for (If), (IIf–g) were not greater than 500 at the
range of 393.47–499.59 Da. The logP values of all
(IIc), and (IId) were 11.12
0.74, 15.28
0.33, hydrazones were smaller than 5 at the range of 2.49–
18.23 0.64, 20.42 0.71, and 23.19
0.56 μM, 4.67. The number of groups that accepted hydrogen
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DESIGN AND BIOEVALUATION
707
Table 2. Acetyl-, and butyryl-cholinesterase inhibitory
pounds. According to the antioxidant activity results,
these two compounds may have a significant impact
on the prevention of radical-induced oxidative stress.
In anticholinesterase activity, compounds (IIb), (Ib),
(IIe), (IIc), (IId), (Ic), and (Id) showed considerable
activity against both AChE and BChE enzymes. Nota-
bly, the BChE activity of the compounds containing
the heteroatom in the ring, the halogen, and nitro
groups in the phenyl ring were was found to be more
excellent from the galantamine used as standard.
According to the data in Table 3, suggested that com-
pounds (IIb) and (Ib) from novel synthesized hydra-
zide-hydrazone derivatives with tertiary amine groups
can be evaluated as both AChE and BChE enzyme
inhibitors, which may have promising features for the
treatment of Alzheimer’s disease.
activities of synthesized compoundsa
Anticholinesterase inhibitory activity
Sample
AChE assay IC50,
μM
BChE assay IC50,
μM
(I)
(II)
(Ia)
(Ib)
(Ic)
(Id)
(Ie)
(If)
(Ig)
(IIa)
(IIb)
(IIc)
(IId)
(IIe)
(IIf)
(IIg)
(IIh)
49.30 0.67
47.79 0.44
45.05 0.26
15.28 0.33
28.09 0.33
33.70 0.56
25.07 0.85
41.23 0.82
38.04 0.55
44.52 0.28
11.12 0.74
20.42 0.71
23.19 0.56
18.23 0.64
40.14 0.40
36.18 0.83
30.49 0.50
4.50 0.09
54.47 0.06
51.73 0.19
48.76 0.49
17.25 0.37
31.19 0.84
35.20 0.68
26.65 0.54
45.51 0.61
40.72 0.43
48.36 0.79
14.26 0.68
21.88 0.82
26.65 0.49
20.74 0.62
43.34 0.60
40.52 0.78
36.35 0.40
46.06 0.10
EXPERIMENTAL
All chemicals were purchased from Sigma-Aldrich
(St. Louis, MO, USA), Alfa Aesar, Fluka, and Merck.
The reactions and the purities of the compounds were
monitored by thin-layer chromatography (TLC) on
silica gel 60 F254 aluminum sheets purchased from
Merck (Darmstadt, Germany). Melting points were
recorded by open capillaries on the Stuart melting
point SMP30 apparatus and are uncorrected. C, H, N,
S percent of the compounds were detected by Thermo
Scientific Flash 2000 (Finnegan MAT, USA) elemen-
tal analyzer. UV spectra, PG Instruments brand T80 +
UV/Vis Spectrometer spectrometry was in the range
of 190 to 1100 nm wavelengths. FTIR spectra were
recorded on Perkin Elmer Frontier spectrometer by
attenuated total reflectance (ATR) apparatus
(Waltham, Massachusetts, USA). Mass spectra were
recorded on Ab-SciEx 3200 Q-Trap MSMS detector
with an electrospray ionization probe (Framingham,
MA, USA). NMR spectra were recorded on Brucker
Avance-400 MHz spectrometer (Billerica, MA, USA)
by using DMSO-d6 as a solvent and TMS as an inter-
nal standard. Bioactivity measurements were carried
out on a 96-well microplate reader, SpectraMax
340PC384, Molecular Devices (USA), at the Depart-
ment of Chemistry, Mugla Sıtkı Kocman University.
Galantamineb
a
Values expressed are means
SD of three parallel measure-
b
ments. p < 0.05, significantly different with student’s t-test.
Reference compounds.
atoms (n-ON) was less than 10 except compounds
(IIe) and (IIf), and the number of groups that donated
hydrogen atoms (n-OHNH) was less than 5, which
were within the Lipinski’s rules. All data for the calcu-
lation of absorption (%ABS), according to Zhao et al.
[50] and TPSA values were shown in Table 3.
CONCLUSIONS
Novel hydrazide-hydrazones (Ia–g), (IIa–h) deri-
vatized from 4-acetyl-N-substituted benzenesulfon-
amide were synthesized. The structures of synthesized
Synthesis of 4-acetyl-N-phenyl benzenesulfonamide
compounds were confirmed by spectroscopy methods (i or I). To a solution of 4-acetylbenzenesulfonyl chlo-
1
as UV-Vis, IR, H NMR, 13C NMR, mass spectros-
copy, and elemental analysis (C, H, N, S). Antioxi-
dant and anticholinesterase activities of the synthe-
sized compounds (Ia–g), (IIa–h) were reported for
the first time in this study. The (Ia–g) and (IIa–h)
hydrazone series in both activity assays were found to
be more active than the starting materials. Fifteen dif-
ferent hydrazone derivatives tested for their antioxi-
dant potency by in vitro antioxidant and anticholines-
terase activities. Compound (IIb) and (Ib), bearing
heteroatom on the aromatic ring, were found to be the
ride (1 mmol) in acetone, 5 g of silicagel was added.
2 mmol of aniline was added dropwise to the stirred
mixture at room temperature. After the reaction has
been completed (as indicated by TLC), methanol was
added and the silica gel was removed by filtration. The
methanol solution was taken in a beaker and then puri-
fied water was added dropwise to crystallize the prod-
uct by precipitation. The crystalline product was
washed with water and filtered and dried [51]. This
compound was previously synthesized by Gioiello
[52]. Yield: 83%; orange solid, mp 99–101°C; IR
most potent antioxidant agents among all tested com- (ν, cm–1): 3273 (N–H); 3092, 3042 (aromatic C–H);
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 46 No. 5 2020

708
BOZKURT et al.
1
Table 3. Druglikenees properties of hydrazones (Ia–g,
IIa–h)*
2962, 2885 (aliphatic C–H); 1687 (C=O); H NMR
(400 MHz) (DMSO-d6/TMS) δ (ppm): 2.59 (s, 3H,
HB); 7.04 (t, 1H, J1 = 6.8 Hz, J2 = 6.8 Hz, HH); 7.10 (d,
2H, J = 8.4 Hz, HF); 7.24 (t, 2H, J1 = 8.0 Hz, J2 =
8.0 Hz, HG); 7.88 (d, 2H, J = 8.4 Hz, HD); 8.08 (d,
Compound LogP TPSA
MW
87.63 393.47
2.49 100.52 394.46
nOH nOHNH
(Ia)
3.78
6
7
2
2
2
2
2
3
2
3
3
3
3
3
3
4
3
4
(Ib)
2H, J = 8.4 Hz, HC); 10.47 (s, 1H, HE); 13C NMR
(100 MHz) (DMSO-d6/TMS) δ (ppm): 27.44 (C2);
120.74, 124.87, 127.50, 129.48, 129.72 (C4, C5, C8, C9,
C10); 137.75, 140.20, 143.58 (C3, C6, C7); 197.68 (C1);
(Ic)
(Id)
3.94
4.46
87.63 411.46
87.63 427.91
6
6
9
8
(Ie)
(If)
3.74 133.46 438.46
4.67 116.73 512.59
MS (m/z) (%): 277 [M+2]; UV-Vis (DMSO, λmax
,
nm): 296, 280; Anal. calc. for C14H13NO3S: C, 61.07;
H, 4.76; N, 5.09; S, 11.65%; found: C, 61.09; H, 4.85;
N, 5.00; S, 11.09%.
(Ih)
(Ig)
4.59
87.63 472.36
6
7
8
9
4.06 107.86 499.59
3.69 124.93 437.48
2.40 137.82 438.46
3.85 124.93 455.47
4.37 124.93 471.92
3.65 170.75 482.47
4.58 154.03 556.60
4.50 124.93 516.37
3.97 145.16 543.60
(IIa)
(IIb)
(IIc)
(IId)
(IIe)
(IIf)
(IIh)
(IIg)
Synthesis of 4-(4-acetylphenylsulfonamido)benzoic
acid (ii or II). 7.3 mmol of 4-aminobenzoic acid was
added to 10 mL of purified water and the pH of the
reaction mixture was maintained to 9 with aqueous
NaHCO3 solution. 7.3 mmol of 4-acetylbenzenesulfo-
nyl chloride was then added in small portions to the
reaction mixture and the reaction was stirred at room
temperature. After the reaction has been completed (as
indicated by TLC), the pH of the reaction mixture was
changed to 2 with 1M HCl. The resulting precipitate
was filtered off with water and dried. The crude prod-
uct was purified by crystallization from methanol [53],
this compounds was previously synthesized by Deng
and Mani [27]. Yield: 84%; cream solid, mp 251–
8
8
11
10
8
9
* These parameters were determined with Molinspiration calcula-
tion software and Molsoft software.
acetic acid was added to the reaction mixture. The
253°C; IR (ν, cm–1): 3259 (N–H); 3110 (aromatic C– mixture was refluxed on a water bath for 2 h. After
1
cooling the mixture, the precipitate was filtered, dried,
and recrystallized from ethanol [55].
H); 2836 (aliphatic C–H); 1677 (C=O); H NMR
(400 MHz) (DMSO-d6/TMS) δ (ppm): 2.60 (s, 3H,
HB); 7.22 (d, 2H, J = 8.4 Hz, HF); 7.82 (d, 2H, J = 8.8
Hz, HG); 7.95 (d, 2H, J = 8.4 Hz, HD); 8.11 (d, 2H,
J = 8.4 Hz, HC); 10.99 (s, 1H, HE); 12.81 (s, 1H, HH);
4-(1-(2-Benzoylhydrazinylidene)ethyl)-N-phenyl-
benzenesulfonamide (Ia). Yield: 47%; white solid, mp
222–225°C; IR (ν, cm–1): 3378, 3137 (N–H); 3024
(aromatic C–H); 2953, 2882 (aliphatic C–H); 1687
(C=O); 1600 (C=N); 1H NMR (400 MHz) (DMSO-
d6/TMS) δ (ppm): 2.36 (s, 3H, HB); 7.04 (t, 1H, J1 =
8.0 Hz, J2 = 6.8 Hz, HH); 7.10 (d, 2H, J = 7.2 Hz, HF);
7.24 (t, 2H, J1 = 7.6 Hz, J2 = 8.0 Hz, HG); 7.52 (t, 2H,
J1 = 6.8 Hz, J2 = 6.8 Hz, HJ); 7.59 (d, 1H, J = 6.4 Hz,
HK); 7.80–7.97 (m, 6H, HC, HD, HI); 10.33 (s, 1H,
13C NMR (100 MHz) (DMSO-d6/TMS) δ (ppm):
27.45 (C2); 118.86, 126.42, 127.54, 129.64, 131.28 (C4,
C5, C8, C9, C10); 140.42, 142.02, 143.24 (C3, C6, C7);
167.15 (C11); 197.68 (C1); MS (m/z): 317.7 [M–1]; UV-
Vis (DMSO, λmax, nm): 293, 275; Anal. calc. for
C15H13NO5S: C, 56.42; H, 4.10; N, 4.39; S, 10.04%;
found: C, 55.96; H, 3.87; N, 4.36; S, 9.91%.
HE); 10.89 (s, 1H, HA); 13C NMR (100 MHz)
(DMSO-d6/TMS) δ (ppm): 14.86 (C2); 120.72,
Synthesis of the substituted hydrazide derivatives
(iii). Firstly 4-substituted aroyl chloride (10 mmol)
was reacted with phenol (10 mmol in 100 mL of 10% 124.69, 127.28, 127.47, 128.72, 129.65, 132.07, 134.37
sodium hydroxide solution) to form 4-substituted (C4, C5, C8, C9, C10, C14, C15, C16); 137.40, 138.02,
phenyl benzoate. The crude product was washed with
water and recrystallized from ethanol. Then the 4-sub-
stituted phenyl benzoate (5 mmol) was reacted with
hydrazine hydrate (10 mmol) in methanol. The mix-
ture was refluxed and monitored by TLC. The crude
product was washed with water and recrystallized from
ethanol [54].
Synthesis of substituted hydrazide-hydrazone deriv-
atives (Ia–g), (IIa–h). To a solution of 1 mmol hydra-
zide derivatives (iii) in 10 mL, methanol was added a
solution of 1 mmol acetylbenzenesulfonamide deriva-
tives (i or ii) in 10 mL methanol. A few drops of glacial
140.20, 142.59 (C3, C6, C7, C13); 147.7 (C1), 163.2
(C12); MS (m/z) (%): 394.7 [M+2]; UV-Vis (DMSO,
λ
max, nm): 305, 279; Anal. calc. for C21H19N3O3S: C
64.10; H 4.87; N 10.68; S 8.15%; found: C 63.42; H
4.72; N 10.77; S 8.23%.
N-Phenyl-4-[1-[2-(pyridine-4-carbonyl)hydraz-
inylidene]ethyl]benzenesulfonamide (Ib). Yield 46%;
white solid, mp 232–235°C; IR (ν, cm–1): 3284 (N–
H); 3067 (aromatic C–H); 2870, 2815 (aliphatic C–
1
H); 1671 (C=O); 1598 (C=N); H NMR (400 MHz)
(DMSO-d6/TMS) δ (ppm): 2.38 (s, 3H, HB); 7.04–
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DESIGN AND BIOEVALUATION
709
7.24 (m, 5H, HF, HG, HH); 7.81–7.82 (m, 4H, HD, (aromatic C–H); 2962, 2891 (aliphatic C–H); 1665
(C=O); 1600 (C=N); 1521 (NO2 asymmetric stretch-
ing band); 1341 (SO2 asymmetric stretching band);
1158 (SO2 symmetric stretching band); H NMR
HI); 8.00 (d, 2H, J = 7.6 Hz, HC), 8.78 (d, 2H, J = 7.6
Hz, HJ); 10.36 (s, 1H, HE); 11.13 (s, 1H, HA); 13C
NMR (100 MHz) (DMSO-d6/TMS) δ (ppm): 15.17
(C2); 120.75, 122.41, 124.72, 127.31, 127.69, 129.67,
150.59 (C4, C5, C8, C9, C14, C15); 133.58 (C10); 137.99
(C7); 140.49 (C3); 141.41 (C13); 142.31 (C6); 154.94
(C1); 163.28 (C12); MS (m/z) (%): 395.1 [M+2]; UV-
Vis (DMSO, λmax, nm): 305, 279; Anal. calc. for
C20H18N4O3S: C 60.90; H 4.60; N 14.20; S 8.13%;
found: C 60.30; H 4.46; N 14.37; S 8.27%.
4-(1-(2-(4-Fluorobenzoyl)hydrazono)ethyl)-N-
phenylbenzenesulfonamide (Ic). Yield 56%; cream
solid, mp 225–227°C; IR (ν, cm–1): 3317 (N–H);
3073 (aromatic C–H); 2944, 2876, 2811 (aliphatic C–
H); 1666 (C=O); 1603 (C=N); 1H NMR (400 MHz)
(DMSO-d6/TMS) δ (ppm): 2.36 (s, 3H, HB); 7.03 (t,
1H, J1 = 7.6 Hz, J2 = 7.2 Hz, HH); 7.11 (d, 2H, J = 7.2 Hz,
HF); 7.24 (t, 2H, J1 = 7.6 Hz, J2 = 7.2 Hz, HG); 7.35 (t,
2H, J1 = 8.8 Hz, J2 = 8.8 Hz, HJ); 7.80 (d, 2H, J = 8.0 Hz,
HC); 7.96 (m, 4H, HD, HI); 10.33 (s, 1H, HE); 10.91 (s,
1
(400 MHz) (DMSO-d6/TMS) δ (ppm): 2.39 (s, 3H,
HB); 7.04 (t, 1H, J1 = 6.8 Hz, J2 = 6.8 Hz, HH); 7.11 (d,
2H, J = 8.0 Hz, HF); 7.24 (t, 2H, J1 = 7.6 Hz, J2 =
8.0 Hz, HG); 7.82–8.13 (m, 6H, HC, HD and HI); 8.35
(d, 2H, J = 6.8 Hz, HJ); 10.34 (s, 1H, HE); 11.19 (s,
1H, HA); 13C NMR (100 MHz) (DMSO-d6/TMS) δ
(ppm): 15.19 (C2); 120.73, 123.90, 124.71, 127.31,
129.66, 130.04, 130.98 (C4, C5, C8, C9, C14, C15); 135.11,
137.97, 140.08, 140.44, 142.32 (C3, C6, C7, C10, C13);
151.38 (C16); 154.83 (C1); 163.23 (C12); MS (m/z) (%):
436.6 [M–1]; UV-Vis (DMSO, λmax, nm): 300; Anal.
calc. for C21H18N4O5S: C, 57.53; H, 4.14; N, 12.78; S,
7.31%; found: C 56.99; H, 4.01; N, 12.79; S, 7.42%.
N-(4-(2-(1-(4-(N-Phenylsulfamoyl)phenyl)-ethyliden)-
hydrazinocarbonyl)phenyl)benzamide (If). Yield 77%;
white solid, mp 259–261°C; IR (ν, cm–1): 3345,
3280 (N–H); 3029 (aromatic C–H); 2918, 2846 (ali-
1H, HA); 13C NMR (100 MHz) (DMSO-d6/TMS) δ phatic C–H); 1654 (C=O); 1591 (C=N); 1339
(SO2 asymmetric stretching band); 1161 (SO2 sym-
(ppm): 14.78 (C2); 115.67, 120.71, 124.73, 127.40,
129.67, 131.29 (C4, C5, C8, C9, C14, C15); 130.75, metric stretching band); 1H NMR (400 MHz)
(DMSO-d6/TMS) δ (ppm): 2.38 (s, 3H, HB); 7.04 (t,
1H, J1 = 7.2 Hz, J2 = 7.6 Hz, HH); 7.11 (d, 2H, J =
8.4 Hz, HF); 7.24 (t, 2H, J1 = 8.4 Hz, J2 = 7.2 Hz, HG);
7.56 (t, 2H, J1 = 6.8 Hz, J2 = 7.6 Hz, HM); 7.63 (t, 1H,
J1 = 7.6, J2 = 7.2 Hz, HN); 7.80 (d, 2H, HC); 7.93–8.00
(m, 8H, HD, HI, HJ and HL); 10.33 (s, 1H, HE); 10.51
135.82, 137.97, 140.21, 142.53 (C3, C6, C7, C10, C13);
153.53 (C1); 164.27 (C16), 166.35 (C12); MS (m/z)
(%): 411.9 [M+1]; UV-Vis (DMSO, λmax, nm): 305,
282; Anal. calc. for C21H18FN3O3S: C 61.30; H 4.41; N
10.21; S 7.79%; found: C 60.25; H 4.25; N 10.12; S
7.65%.
(s, 1H, HK); 10.80 (s, 1H, HA); 13C NMR (100 MHz)
(DMSO-d6/TMS) δ (ppm): 14.73 (C2); 119.83,
120.72, 121.35, 124.70, 127.29, 127.45, 128.06, 128.78,
129.66 (C4, C5, C19, C8, C9, C14, C21, C20, C15); 132.31,
134.56, 135.11, 136.60, 138.03, 140.14, 140.71 (C3, C6,
C7, C10, C13, C16, C18); 142.67 (C1); 164.22 (C12);
4-(1-(2-(4-Chlorobenzoyl)hydrazono)ethyl)-N-
phenylbenzenslfonamide (Id). Yield 47%; white solid,
mp 210–213°C; IR (ν, cm–1): 3313, 3156 (N–H);
3078, 3029 (aromatic C–H); 2944, 2968, 2898 (ali-
phatic C–H); 1637 (C=O); 1598 (C=N); 1347 (SO2
asymmetric stretching band); 1156 (SO2 symmetric
165.84 (C17); MS (m/z) (%): 510.7 [M–2]; UV-Vis
(DMSO, λmax, nm): 315, 278; Anal. calc. for
C28H24N4O4S: C, 65.61; H, 4.72; N, 10.93; S, 6.26%;
found: C, 64.76; H, 4.59; N, 11.04; S, 6.10%.
1
stretching band); H NMR (400 MHz) (DMSO-
d6/TMS) δ (ppm): 2.36 (s, 3H, HB); 7.03 (t, 1H, J1 =
7.2 Hz, J2 = 7.2 Hz, HH); 7.11 (d, 2H, J = 8.0 Hz, HF);
7.24 (t, 2H, J1 = 7.6 Hz, J2 = 8.0 Hz, HG); 7.59 (d, 2H,
J = 8.0 Hz, HJ); 7.80 (d, 2H, J = 7.6 Hz, HC); 7.92–
7.98 (m, 4H, HD and HI); 10.34 (s, 1H, HE); 10.96 (s,
1H, HA); 13C NMR (100 MHz) (DMSO-d6/TMS) δ
(ppm): 14.97 (C2); 120.71, 124.69, 127.40, 128.00,
4-(1-(2-(2-Hydroxy-2,2-diphenylacetyl)hydrazono)-
ethyl)-N-phenylbenzensulfon-amide (Ig). Yield 85%;
orange solid, mp 228–231°C; IR (ν, cm–1): 3268
(N‒H); 3082, 3020 (aromatic C-H); 2972 (aliphatic
129.66, 133.10 (C4, C5, C8, C9, C14, C15); 130.55, C–H); 1650 (C=O); 1600 (C=N); 1342 (SO2 asym-
136.95, 138.01, 139.04, 140.36, 142.47 (C3, C6, C7, C10, metric stretching band); 1166 (SO2 symmetric stretch-
ing band); 1H NMR (400 MHz) (DMSO-d6/TMS) δ
C13, C16); 149.63 (C1); 162.27 (C12); MS (m/z) (%):
429 [M+2]; UV-Vis (DMSO, λmax, nm): 305, 274; (ppm): 2.38 (s, 3H, HB); 7.04 (t, 1H, J1 = 7.6 Hz, J2 =
Anal. calc. for C21H18ClN3O3S: C, 58.94; H, 4.24; N, 7.6 Hz, HH); 7.09 (d, 2H, J = 8.0 Hz, HF); 7.23 (t, 2H,
9.82; S, 7.49%; found: C, 58.24; H, 4.11; N, 9.88; S, J1 = 7.2 Hz, J2 = 7.2 Hz, HG); 7.29–7.38 (m, 10H, HL,
7.22%.
HL', HK, HK', HJ and HJ'); 7.80 (d, 2H, J = 8.0 Hz,
HC); 7.94 (d, 2H, J = 8.0 Hz, HD); 10.32 (s, 1H, HE);
10.49 (s, 1H, HA); 13C NMR (100 MHz) (DMSO-
d6/TMS) δ (ppm): 13.83 (C2); 81.16 (C13); 120.83,
4-(1-(2-(4-Nitrobenzoyl)hydrazono)ethyl)-N-phen-
ylbenzensulfonamide (Ie). Yield 70%; yellow solid,
mp 239–241°C; IR (ν, cm–1): 3311 (N–H); 3083
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BOZKURT et al.
124.75, 127.30, 127.53, 127.85, 127.99, 128.23, 129,64 (ppm): 2.36 (s, 3H, HB); 7.22 (d, 2H, J = 8.4 Hz, HF);
(C4, C5, C8, C9, C15, C16, C17); 135.83, 137.97, 140.32,
142.40, 143.87 (C3, C6, C7, C10, C14); 152.85 (C1);
7.34 (t, 2H, J1 = 8.8 Hz, J2 = 8.8 Hz, HJ); 7.82 (d, 2H,
J = 8.4 Hz, HG); 7.87 (d, 2H, J = 8.4 Hz, HC); 7.94–
169.36 (C12); MS (m/z) (%): 500.6 [M+1]; UV-Vis 7.96 (m, 4H, HD and HI); 10.84 (s, 2H, HA and HE);
12.73 (s, 1H, HH); 13C NMR (100 MHz) (DMSO-
d6/TMS) δ (ppm): 14.78 (C2); 115.58, 118.75, 118.88,
126.23, 127.33, 127.65, 129.64, 131.24 (C4, C5, C8, C9,
C10, C14, C15); 130.90, 139.91, 142.39, 142.86 (C3, C6,
C7, C13); 145.52 (C1); 164.17 (C16); 166.50 (C12); 167.18
(C11); MS (m/z) (%): 457.1 [M+2]; UV-Vis (DMSO,
(DMSO, λmax, nm): 295; Anal. calc. for C28H25N3O4S:
C, 67.32; H, 5.04; N, 8.41; S, 6.42%; found: C, 66.36;
H, 5.00; N, 8.48; S, 6.12%.
4-(4-(1-(2-Benzoylhydrazono)ethyl)phenylsulfon-
amido)benzoic acid (IIa). Yield 48%; white solid, mp
244–246°C; IR (ν, cm–1): 3327 (N–H); 3042 (aro-
matic C–H); 2931, 2864 (aliphatic C–H); 1688, 1652
(C=O); 1606 (C=N); 1335 (SO2 asymmetric stretch-
λ
max, nm): 305, 279; Anal. calc. for C22H18FN3O5S: C,
58.02; H, 3.98; N, 9.23; S, 7.04%; found: C, 57.81; H,
3.91; N, 8.51; S, 6.92%.
1
ing band); 1158 (SO2 symmetric stretching band); H
NMR (400 MHz) (DMSO-d6/TMS) δ (ppm): 2.36 (s,
3H, HB); 7.21 (d, 2H, J = 8.4 Hz, HF); 7.52 (t, 2H,
J1 = 7.2 Hz, J2 = 8.0 Hz, HJ); 7.59 (t, 1H, J1 = 7.6 Hz,
J2 = 7.6 Hz, HK); 7.81 (d, 2H, J = 8.4 Hz, HG); 7.88–
8.11 (m, 6H, HC, HD and HI); 10.90 (s, 2H, HA and
HE); 12.77 (s, 1H, HH); 13C NMR (100 MHz)
(DMSO-d6/TMS) δ (ppm): 14.75 (C2); 118.76, 127.31,
127.64, 128.71, 129.6, 131.23, 132.05 (C4, C5, C8, C9,
C14, C15, C16); 126.23, 134.40, 139.90, 142.31, 142.91
(C3, C6, C7, C10, C13); 146.78 (C1); 1623.44 (C12);
167.18 (C11); MS (m/z) (%): 438.0 [M+1]; UV-Vis
(DMSO, λmax, nm): 305, 279; Anal. calc. for
C22H19N3O5S: C, 60.40; H, 4.38; N, 9.61; S, 7.33%;
found: C, 60.15; H, 4.37; N, 9.18; S, 7.39%.
4-(4-(1-(2-(4-Chlorobenzoyl)hydrazono)ethyl)phe-
nylsulfonamido)benzoic acid (IId). Yield 45%; white
solid, mp 276–278°C; IR (ν, cm–1): 3260 (N–H);
3080 (aromatic C–H); 2953, 2829 (aliphatic C–H);
1674, 1663 (C=O); 1607 (C=N); 1336 (SO2 asymmet-
ric stretching band); 1160 (SO2 symmetric stretching
1
band); H NMR (400 MHz) (DMSO-d6/TMS) δ
(ppm): 2.36 (s, 3H, HB); 7.22 (d, 2H, J = 8.4 Hz, HF);
7.59 (d, 2H, J = 8.4 Hz, HJ); 7.81 (d, 2H, J = 8.8 Hz,
HG); 7.88–8.09 (m, 6H, HC, HD and HI); 10.92 (s,
2H, HA and HE); 12.78 (s, 1H, HH); 13C NMR
(100 MHz) (DMSO-d6/TMS) δ (ppm): 15.47 (C2);
119.31, 126.77, 127.90, 128.23, 129.35, 131.07 (C4, C5,
C8, C9, C10, C14, C15); 131.79, 140.60, 142.81, 143.34
(C3, C6, C7, C13); 146.09 (C1); 167.74 (C11); MS (m/z)
4-(4-(1-(2-Isonicotinoylhydrazono)ethyl)phenylsul-
fonamido)benzoic acid (IIb). Yield 41%; white solid,
mp 288–290°C; IR (ν, cm–1): 3364 (N–H); 3075
(aromatic C–H); 2966 (aliphatic C–H); 1687, 1656
(C=O); 1606 (C=N); 1338 (SO2 asymmetric stretch-
(%): 473.0 [M+1]; UV-Vis (DMSO, λmax, nm): 305,
284; Anal. calc. for C22H18ClN3O5S: C, 55.99; H,
3.84; N, 8.90; S, 6.79%; found: C, 55.43; H, 3.71; N,
8.87; S, 6.87%.
1
ing band); 1161 (SO2 symmetric stretching band); H
4-(4-(1-(2-(4-Nitrobenzoyl)hydrazono)ethyl)phe-
nylsulfonamido)benzoic acid (IIe). Yield 49%; yellow
solid, mp 281–283°C; IR (ν, cm–1): 3325 (N–H);
3108, 3047 (aromatic C–H); 2935, 2869 (aliphatic C–
H); 1682, 1665 (C=O); 1603 (C=N); 1336 (SO2 asym-
metric stretching band); 1157 (SO2 symmetric stretch-
NMR (400 MHz) (DMSO-d6/TMS) δ (ppm): 2.38 (s,
3H, HB); 7.22 (d, 2H, J = 7.6 Hz, HF); 7.81–7.83 (m,
4H, HG and HC); 7.89 (d, 2H, J = 8.0 Hz, HD); 8.03
(d, 2H, J = 8.0 Hz, HI); 8.78 (d, 2H, J = 8.0 Hz, HJ);
10.90 (s, 1H, HE); 11.14 (s, 1H, HA); 12.79 (s, 1H,
HH); 13C NMR (100 MHz) (DMSO-d6/TMS) δ
(ppm): 15.11 (C2); 118.79, 122.43, 123.39, 126.24,
127.35, 127.87, 131.25, (C4, C5, C8, C9, C10, C14);
140.20, 141.42, 142.62, 142.94 (C3, C6, C7, C13); 147.68
(C1); 150.57 (C15); 163.37 (C12); 167.20 (C11); MS
ing band); 1H NMR (400 MHz) (DMSO-d6/TMS) δ
(ppm): 2.38 (s, 3H, HB); 7.22 (d, 2H, J = 7.6 Hz, HF);
7.81 (d, 2H, J = 7.6 Hz, HG); 7.89–8.13 (m, 6H, HC,
HD and HI); 8.35 (d, 2H, J = 8.4 Hz, HJ); 10.87 (s, 1H,
HE); 11.19 (s, 1H, HA); 13C NMR (100 MHz)
(DMSO-d6/TMS) δ (ppm): 15.15 (C2); 118.78,
123.89, 126.23, 127.35, 127.53, 127.82, 129.63, 130.05,
131.23 (C4, C5, C8, C9, C10, C14, C15); 140.20, 142.29,
142.62 (C3, C6, C7); 149.66 (C16); 154.66 (C1); 163.26
(m/z) (%): 439.0 [M+1]; UV-Vis (DMSO, λmax, nm):
305, 279; Anal. calc. for C21H18N4O5S: C, 57.53; H,
4.14; N, 12.78; S, 7.31%; found: C, 56.85; H, 3.94; N,
12.71; S, 7.25%.
(C12); 167.17 (C11); MS (m/z) (%): 480.6 [M–1]; UV-
Vis (DMSO, λmax, nm): 354, 319, 285; Anal. calc. for
C22H18N4O7S: C, 54.77; H, 3.76; N, 11.61; S, 6.65%;
found: C, 54.12; H, 3.81; N, 11.27; S, 6.55%.
4-(4-(1-(2-(4-Fluorobenzoyl)hydrazono)ethyl)phe-
nylsulfonamido)benzoic acid (IIc). Yield 69%; white
solid, mp 239–241°C; IR (ν, cm–1): 3264 (N–H);
3073 (aromatic C–H); 2824 (aliphatic C–H); 1675,
1657 (C=O); 1604 (C=N); 1336 (SO2 asymmetric
stretching band); 1160 (SO2 symmetric stretching
band); 1H NMR (400 MHz) (DMSO-d6/TMS) δ
4-(4-(1-(2-(4-Benzamidobenzoyl)hydrazono)-
ethyl)phenylsulfonamido)benzoic acid (IIf). Yield 78%;
white solid, mp 291–293°C; IR (ν, cm–1): 3354,
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DESIGN AND BIOEVALUATION
711
516.6 [M+1]; UV-Vis (DMSO, λmax, nm): 305, 279;
Anal. calc. for C22H18BrN3O5S: C, 51.17; H, 3.51; N,
8.14; S, 6.21%; found: C, 50.63; H, 3.39; N, 8.20; S,
3308 (N–H); 3029 (aromatic C–H); 2918 (aliphatic
C–H); 1680, 1656 (C=O); 1607 (C=N); 1347 (SO2
asymmetric stretching band); 1163 (SO2 symmetric
1
stretching band); H NMR (400 MHz) (DMSO- 6.12%.
d6/TMS) δ (ppm): 2.38 (s, 3H, HB); 7.22 (d, 2H, J =
8.8 Hz, HF); 7.56 (t, 2H, J1 = 7.6 Hz, J2 = 7.2 Hz, HM);
7.63 (t, 1H, J1 = 7.2 Hz, J2 = 7.2 Hz, HN); 7.82 (d, 2H,
Biological Activity
Determination of antioxidant activity. The antioxi-
dant activity was measured using four complementary
assays. The β-carotene-linoleic acid assay differs from
the others in that the antioxidant gives to media the
hydrogen radical. In this assay, the antioxidant com-
pound also scavenges the singlet oxygen and also
transfers electrons to stop the radicalic degradation.
Therefore, β-carotene-linoleic acid assay, lipid perox-
J = 8.4 Hz, HJ); 7.88 (d, 2H, J = 8.0 Hz, HG); 7.93–
8.00 (m, 8H, HC, HD, HI and HL); 10.52 (s, 1H, HE);
10.81 (s, 1H, HA); 13C NMR (100 MHz) (DMSO-
d6/TMS) δ (ppm): 14.73 (C2); 118.74, 119.82, 120.72,
126.21, 127.33, 127.61, 128.24, 128.92, 131.25 (C4, C5,
C8, C9, C10, C14, C15, C19, C20); 132.31, 133.14, 135.10,
135.17, 139.95, 142.32, 142.97 (C3, C6, C7, C13, C16,
C18, C21); 145.99 (C1); 164.33 (C17); 166.34 (C12); idation inhibitory activity, is called total antioxidant
167.19 (C11); MS (m/z) (%): 556.8 [M+1]; UV-Vis
(DMSO, λmax, nm): 315, 279; Anal. calc. for
C29H24N4O6S: C, 62.58; H, 4.35; N, 10.07; S, 5.76%;
found: C, 61.75; H, 4.27; N, 10.16; S, 5.88%.
activity. In the DPPH, ABTS, and CUPRAC assays,
however, the antioxidant can transfer only electrons to
neutralize the radical.
The lipid peroxidation inhibitory activity of the
compounds was evaluated using the β-carotene-lin-
oleic acid assay [56]. For this test, 25 L of linoleic acid,
and 200 mg of Tween 40 emulsifier were added to 1 mL
of chloroform containing 0.5 mg β-carotene. The
chloroform solvent was evaporated under vacuum,
and 100 mL of distilled water saturated with oxygen
was added by vigorous shaking. One hundred and sixty
microliters of this mixture were delivered into each
well containing 40 μL of different concentrations of
the compounds dissolved in DMSO. The zero-time
absorbance was measured at 470 nm after the said
emulsion was added to each well in a microplate
reader. The emulsion system was kept at 50°C in an
oven. The absorbance was read until the color disap-
peared in control wells by controlling in every 30 min-
utes. A blank, devoid of β-carotene, was prepared for
background subtraction. BHT and α-tocopherol were
used as positive standards to compare the activity.
4-(4-(1-(2-(2-Hydroxy-2,2-diphenylacetyl)hydra-
zono)ethyl)phenylsulfonamido)benzoic acid (IIg). Yield
78%; orange solid, mp 111–113°C; IR (ν, cm–1):
3345 (N–H); 3057 (aromatic C–H); 2952 (aliphatic
C–H); 1682, 1606 (C=O); 1606 (C=N); 1335 (SO2
asymmetric stretching band); 1156 (SO2 symmetric
1
stretching band); H NMR (400 MHz) (DMSO-
d6/TMS) δ (ppm): 2.38 (s, 3H, HB); 7.19–7.24 (m,
3H, HF and HI); 7.29–7.44 (m, 10H, HL, HL', HK, HK',
HJ and HJ'); 7.80 (d, 2H, J = 8.4 Hz, HG); 7.86 (d, 2H,
J = 8.4 Hz, HC); 7.97 (d, 2H, J = 8.4 Hz, HD); 10.49
(s, 1H, HE); 10.86 (s, 1H, HA); 13C NMR (100 MHz)
(DMSO-d6/TMS) δ (ppm): 13.80 (C2); 118.84,
126.27, 127.33, 127.71, 127.84, 127.99, 128.23, 131.22
(C4, C5, C8, C9, C10, C15, C16, C17); 140.10, 142.25,
142.49, 143.86 (C3, C6, C7, C13); 152.72 (C1); 167.17
(C12); 169.37 (C11); MS (m/z) (%): 541.8 [M–2]; UV-
Vis (DMSO, λmax, nm): 295, 278; Anal. calc. for
C29H25N3O5S: C, 64.08; H, 4.64; N, 7.73; S, 5.90%;
found: C, 61.46; H, 4.59; N, 7.58; S, 5.81%.
The bleaching rate (R) of β-carotene was calcu-
lated according to the following equation:
Bleaching rate R : ln a b t ,
( )
(
)
where, ln = natural log, a = absorbance at time zero,
b = absorbance at time t (120 min). The antioxidant
activity (AA) was calculated in terms of percent inhibi-
tion relative to the control, using the following equa-
tion:
4-(4-(1-(2-(4-Bromobenzoyl)hydrazono)ethyl)phe-
nylsulfonamido)benzoic acid (IIh). Yield 70%; white
solid, mp 287–289°C; IR (ν, cm–1): 3262 (N–H);
3073 (aromatic C–H); 2951, 2829 (aliphatic C–H);
1674, 1662 (C=O); 1607 (C=N); 1336 (SO2 asymmet-
ric stretching band); 1160 (SO2 symmetric stretching
Antioxidant activity AA :
(
)
1
band); H NMR (400 MHz) (DMSO-d6/TMS) δ
A
− Acompound Acontrol × 100.
(
[
)
]
control
(ppm): 2.36 (s, 3H, HB); 7.22 (d, 2H, J = 7.6 Hz, HF);
7.73 (d, 2H, J = 6.8 Hz, HJ); 7.81–8.01 (m, 8H, HC,
HD, HI and HG); 10.92 (s, 2H, HA and HE); 12.77 (s,
1H, HH); 13C NMR (100 MHz) (DMSO-d6/TMS) δ
(ppm): 14.90 (C2); 118.77, 126.25, 126.54, 127.34,
127.66, 130.68, 131.24 (C4, C16, C5, C8, C9, C10, C14,
C15); 131.7, 139.99, 142.29, 142.80 (C3, C6, C7, C13);
148.02 (C1); 164.01 (C12); 167.18 (C11); MS (m/z) (%):
Then results were given as IC50 μg/mL corresponding
the concentration which protects 50% of β-carotene
amount [39–42, 57].
The ABTS cation radical scavenging activity was
determined spectrophotometrically [46] with slight
modifications [39–42, 57]. The ABTS•+ was obtained
by the reaction of 7 mM ABTS dissolved in H2O with
2.45 mM potassium persulfate. The mixture was
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 46 No. 5 2020

712
BOZKURT et al.
stored in the dark at room temperature for 12 h. The prine, and NH4Ac buffer (1 M, pH 7.0) solutions were
radical cation was stable in this form for more than
2 days if stored in the dark at room temperature. To
test the activity of the compounds, the prepared radi-
cal solution was diluted to get an absorbance of
0.700 0.025 at 734 nm with ethanol for one cm cell
length. Then, 160μL of the radical solution was added
to 40 μL of compound solution dissolved in DMSO at
different concentrations (5–50 μg/mL). After 10 min,
the percentage inhibition at 734 nm was calculated for
each concentration relative to a blank absorbance
(methanol). Lower absorbance of the reaction mixture
indicated higher free radical-scavenging activity. A
blank, devoid of ABTS solution was prepared for back-
ground subtraction. BHT and α-tocopherol were used
as positive standards to compare the activity.
added to 40 μL ml of compound solutions dissolved in
DMSO at different concentrations. The 96 well plates
were kept at room temperature for one hour. Then the
absorbance at 450 nm was recorded against a blank.
BHT and α-tocopherol were used as positive stan-
dards to compare the activity.
Then results of CUPRAC assay were given as
A
0.50 μg/mL corresponding to the concentration of
0.500 absorbances at graph drawn absorbance versus
concentration.
Determination of anticholinesterase activity. The
Ellman method was used to measure acetylcholines-
terase (AChE) and butyrylcholinesterase (BChE)
inhibitory activity [57, 58]. The commercial AChE
from electric eel and BChE from horse serum were
employed. As substrates of the enzymes, acetylthio-
choline iodide and butyrylthiocholine chloride were
utilized. 5,5′-dithio-bis(2-nitrobenzoic)acid (DTNB)
was made use of for measurement of activity as a col-
oring reagent. To dissolve the compounds, ethanol
solvent was used. To test the activity, 10 μL of sample
solution dissolved in ethanol at different concentra-
tions and 20 μL AChE (5.32 × 10–3 U) or BChE
(6.85 × 10–3 U) enzyme dissolved in buffer were added
to 150 μL of 100 mM sodium phosphate buffer
(pH 8.0). Then incubated for 15 min at 25°C. After
incubation, 10 μL of 0.5 mM DTNB were added, and
the reaction was started by addition of 10 μL of acet-
ylthiocholine iodide (0.71 mM) or butyryl-thiocho-
line chloride (0.2 mM). The measurement was moni-
tored spectrophotometrically by the formation of yel-
low 5-thio-2-nitrobenzoate anion, as the result of the
reaction of DTNB with thiocholine at 412 nm wave-
length using a 96-well microplate reader (SpectraMax
PC340, Molecular Devices, USA). Percentage of
inhibition of AChE or BChE was determined by com-
parison of reaction rates of samples relative to blank
sample (ethanol in phosphate buffer, pH 8) using the
formula (E – S)/E × 100, where E is the activity of
enzyme without test compound, and S is the activity of
the enzyme with a test compound. Galantamine was
used as a reference compound [58].
The ability to scavenge the ABTS cation radical was
calculated by using the following equation:
ABTS cation radical scavenging activity AA :
(
)
A
− Acompound Acontrol × 100.
(
[
)
]
control
Then results ABTS assay was given as IC50 μg/mL cor-
responding to the concentration, which scavenges
50% of ABTS cation radical.
DPPH free radical-scavenging activity of the
extracts of compounds was determined using the
DPPH radical [39, 44]. DPPH absorbs at 517 nm in its
radical form. However, if reduced by an antioxidant
transfer electron, its absorption at 517 nm decreases.
To test the compounds in this assay, 0.1 mM DPPH
solution was prepared in ethanol. 160 μL of this solu-
tion was added to 40 μL of compound solutions dis-
solved in DMSO at different concentrations. The 96
well plates were kept in the dark place. Thirty minutes
later, the absorbance was measured at 517 nm. Lower
absorbance of the reaction mixture indicated higher
free radical-scavenging activity. A blank, devoid of
DPPH solution was prepared for background subtrac-
tion. BHT and α-tocopherol were used as positive
standards to compare the activity.
The ability to scavenge the DPPH radical was cal-
culated by using the following equation:
DPPH free radical scavenging activity AA :
(
)
A
− Acompound Acontrol × 100.
(
[
)
]
control
Then results of DPPH assay were given as IC50 μg/mL
corresponding to the concentration, which scavenges
50% of DPPH free radical.
COMPLIANCE WITH ETHICAL STANDARDS
The cupric reducing antioxidant capacity of the
compounds was determined to see the reduction
potential of the compounds. This method is based on
the measurement of absorbance at 450 nm by the for-
mation of a stable complex between neocuproine and
copper (I), which is formed by the reduction of copper
(II) in the presence of antioxidant [39, 48]. To test the
cupric reducing antioxidant capacity of the com-
pounds, 40 μL each of 10 mM Cu (II), 7.5 mM neocu-
This article does not contain any studies involving
human participants performed by any of the authors and
does not contain any studies involving animals performed
by any of the authors.
Conflict of Interests
The authors declare that they have no conflicts of interests.
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DESIGN AND BIOEVALUATION
713
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