Synthesis and Anticholinesterase Activity Evaluation of Asymmetric Azines

Article abstract of DOI:10.1007/s11094-019-02035-7

Azines or di-imines are compounds containing R₁R₂C=N-N=CR₃R₄ fragment in its structure. These compounds have received special attention due to their importance as intermediates in the synthesis of drugs and substances with diverse pharmacological activities. In this research, twelve compounds derived of asymmetric azines of aryl aldehydes and aryl ketones were synthesized by microwave assisted irradiation, and their ability to inhibit acetylcholinesterase (AChE) enzyme was tested using the Ellman method. Among the evaluated azines, five compounds were able to inhibit AChE, especially compounds (7E, 8E)-1-benzylidene-2-(1-(4-nitrophenyl)ethylidene)hydrazine (7) and (7E,8E)-2-(1-(4-nitrophenyl)ethylidene)-1-((pyridin-2-yl)methylene)hydrazine (14), which showed percentage inhibition of 18.34% and 17.43% at concentrations of 22.46 and 10 μM, respectively.

Full text of DOI:10.1007/s11094-019-02035-7

DOI 10.1007/s11094-019-02035-7
Pharmaceutical Chemistry Journal, Vol. 53, No. 6, September, 2019 (Russian Original Vol. 53, No. 6, June, 2019)
SYNTHESIS AND ANTICHOLINESTERASE ACTIVITY
EVALUATION OF ASYMMETRIC AZINES
Andreza S. Figueredo,1 Jaqueline E. Queiroz,2
Lucas D. Dias,3 Hérika D. A. Vidal,2 Ingrid V. Machado,2
Giuliana M. Vila Verde,1 and Gilberto L. B. Aquino2,3,*
Original article submitted November 19, 2018.
Azines or di-imines are compounds containing R1R2C=N-N=CR3R4 fragment in its structure. These com-
pounds have received special attention due to their importance as intermediates in the synthesis of drugs and
substances with diverse pharmacological activities. In this research, twelve compounds derived of asymmetric
azines of aryl aldehydes and aryl ketones were synthesized by microwave assisted irradiation, and their ability
to inhibit acetylcholinesterase (AChE) enzyme was tested using the Ellman method. Among the evaluated az-
ines, five compounds were able to inhibit AChE, especially compounds (7E, 8E)-1-benzylidene-2-(1-(4-nitro-
phenyl)ethylidene)hydrazine (7) and (7E,8E)-2-(1-(4-nitrophenyl)ethylidene)-1-((pyridin-2-yl)methylene)-
hydrazine (14), which showed percentage inhibition of 18.34% and 17.43% at concentrations of 22.46 and
10 mM, respectively.
Keywords: azine, synthesis, microwave assisted irradiation, inhibition, acetylcholinesterase.
1. INTRODUCTION
action between hydrazine derivatives and aldehydes and/or
ketones catalyzed by acids or bases [4].
Azines or di-imines are compounds containing
R R C=N-N=CR R fragment in the structure, which are
In recent years, some series of azines have been prepared
by applying microwave assisted irradiation which can dra-
matically reduce the preparation time and provide good
yields [5, 6]; on the other hand, it is in consonance with
green chemistry. These compounds have received special at-
tention due to their importance as intermediates in the syn-
thesis of drugs and substances with diverse pharmacological
properties. The known biological activities include, among
others, anticonvulsant, antibacterial, antiparasitic, insecti-
cidal, anti-inflammatory, antioxidant and anti-tumor [7 – 15].
In view of this broad spectrum of activities described in
literature and in continued search for compounds capable of
inhibiting acetylcholinesterase (AChE) enzyme that plays a
key role in the evolution of Alzheimer’s disease, in this paper
we report the synthesis, characterization and evaluation of
1
2
3 4
usually synthesized by reactions between a primary amine
and carbonyl groups of aldehydes and/or ketones [1, 2]. De-
pending on the nature of R substituents, the azines can be
classified into symmetric (R R = R R ), asymmetric
1
2
3 4
(R R ¹ R R ),
aldazines
(R = R = H),
ketazines
1
2
3
4
1
3
(R R R R ¹ H), mixed azines (R = H; R R R ¹ H), and
aromatic, aliphatic, cyclic and acyclic azines [3]. For exam-
ple, Fig. 1 shows the structure of aromatic hydrazines.
1
2
3
4
1
2
3
4
The classical method to obtain azines is based on the re-
action of aldehydes with hydrazine. From the synthetic point
of view, there are two strategies in obtaining these com-
pounds: (1) heating the hydrazine with aldehydes and/or ke-
tones in solvents such as ethanol, methanol, tetrahydrofuran,
butanol. and glacial acetic acid and (2) the condensation re-
1
Faculdade de Farmácia, Universidade Estadual de Goiás (UEG),
Anápolis, Goiás, 75132-400 Brazil.
2
Laboratório de Pesquisa em Bioprodutos e Síntese, Campus Anápolis de
Ciências Exatas e Tecnológicas, Universidade Estadual de Goiás (UEG),
Anápolis, Goiás, 75132 – 400 Brazil.
3
Centro de Química de Coimbra (CQC), Departamento de Química da
Universidade de Coimbra, Rua Larga, Coimbra, 3004 – 535 Portugal.
*
e-mail: gilberto.benedito@ueg.br
Fig. 1. Aromatic azines.
544
0091-150X/19/5306-0544 © 2019 Springer Science+Business Media, LLC

Synthesis and Anticholinesterase Activity Evaluation
545
Scheme 1. Synthesis of hydrazones 1 and 2.
the anticholinesterase activity of a series of asymmetric azine
derivatives of aryl aldehydes and aryl ketones.
Synthesis of (E)-1-(4-nitrobenzylidene)hydrazine 1
(C). Hydrazine monohydrate (1 mmol, 40 mL) and 4-nitro-
benzaldehyde (1 mmol, 151 mg) were dissolved in anhy-
drous ethanol and the solution was stirred at room tempera-
ture for 10 min. The formation of hydrazone was confirmed
by GC-MS data.
2. MATERIALS AND METHODS
All reagents were purchased from Sigma-Aldrich and
used without additional purification. Thin layer chromatog-
raphy (TLC) analysis was performed on Merck silica gel
60F254 plates using hexane/ethyl acetate as the solvent and
iodine for developing TLC plates. Microwave irradiation
was carried out by using Discover DC-7196 CEM Corpora-
tion reactor. The melting points were determined in open
capillaries using Microquímica MQAPF-301 electrical appa-
ratus and were uncorrected. The characterization was per-
formed by gas chromatograph/mass spectrometry (GC-MS),
infrared (IR) spectrophotometry, and nuclear magnetic reso-
nance (NMR) spectroscopy.
Synthesis of (E)-1-(1-(4-nitrophenyl)ethylidene)hyd-
razine 2 (D). Hydrazine hydrate 70% solution (1 mmol) was
added to 5 ml of anhydrous ethanol, followed by adding of
4-nitroacetophenone (1 mmol, 165 mg). The mixture was
heated in a microwave reactor at 300 W in two cycles:
2.5 min at 120°C and 1.5 min at 100°C. Then, the reaction
mixture was cooled, and the precipitate was filtered and
washed with ethanol.
Synthesis of Series 1 (compounds 3 – 6) (E). Equiva-
lent amounts (1 mmol) of hydrazone 1 and various substi-
tuted benzaldehyde were added to a reaction tube with etha-
nol (2 mL) and kept under constant stirring for 2 h at room
temperature. The precipitate was filtered and washed with
cold ethanol.
The IR spectra were recorded in KBr pellets on the
FT-IR PerkinElmer Spectrum Frontier instrument. The NMR
spectra were recorded on Bruker Avance 500 instrument
working at 500 MHz (1H) and 125 MHz (13C) frequencies,
Synthesis of Series 2 (compounds 7 – 14) (F). Equiva-
lent amounts (1 mmol) of hydrazone 2 and various substi-
tuted benzaldehydes were added to a reaction tube contain-
ing ethanol (2 mL). The tube was sealed and the mixture was
heated in a microwave reactor at 300W for 3 cycles: first be-
ing 1.5 min at 80°C, followed by two cycles of 2.5 min at
100°C. The precipitate was filtered and washed with cold
ethanol.
using CDCl -d as the solvent and tetramethylsilane (TMS) as
3
an internal standard with the chemical shifts (d values) ex-
pressed in ppm units. The mass spectra were recorded in
Shimadzu GC-MS QP-2010 Ultra spectrometer.
3. EXPERIMENTAL SECTION
3.1. Synthesis
(7E,8E)-2-(4-nitrobenzylidene)-1-benzylidenehydrazi-
ne (3): C H N O (253.09 g/mol); yield 58.8%; m.p.
14 11
3
2
Twelve derived asymmetric azines of aryl aldehydes and
aryl ketones were synthesized, with p-nitro substituent on A
ring and other groups on B ring (Fig. 1). The synthesis of
these compounds passed via the initial formation of hydra-
zones 1 and 2 (Scheme 1) obtained by the reaction between
4-nitrobenzaldehyde or 4-nitroacetophenone with hydrazine
in ethanol under conditions C or D.
1
158.6°C; IR (KBr; n, cm-1): 1600 (C=N), 1520 (NO ); H
2
NMR (500 MHz, CDCl ; d, ppm): 7.47 (m, 3H, Ar-H), 7.87
(d, 2H, Ar-H), 8.02 (d, 2H, Ar-H), 8.31 (d, 2H, Ar-H), 8.68
(s, 1H, N=CH), 8.7 (s, 1H, N=CH); 13C NMR (125 MHz,
3
CDCl ; d, ppm): 124.04, 128.87, 129.13, 131.79, 133.59,
3
136.59, 140.03 and 163.57 (Ar), 159.21 (N=C); MS (m/z):
253 [M + H]+, 176, 131, 104, 77.
The following series of four azines were synthesized by
the reaction of hydrazones 1 or 2 with various benzaldehydes
or acetophenones substituted under different heating condi-
tions (E or F) in ethanol according to Scheme 2.
(7E,8E)-1-(4-methylbenzylidene)-2-(4-nitrobenzylide-
ne)hydrazine (4): C H N O (267.1 g/mol); yield 60.37%;
15 13
3
2
m.p. 163.8°C; IR (KBr; n, cm-1): 1600 (C=N), 1520 (NO );
2

546
Andreza S. Figueredo et al.
Scheme 2. Synthesis of asymmetric azines.
1H NMR (500 MHz, CDCl ; d, ppm): 2.43 (s, 3H, CH ), 7.28
(Ar-CH ), 123.57, 127.66, 128.55, 129.56, 131.64, 141.83,
3
3
3
144.24 and 162.18 (Ar), 148.62 and 159.29 (N=C); MS
(m/z): 281 [M + H]+, 266, 190, 159, 118.
(d, 2H, Ar-H), 7.76 (d, 2H, Ar-H), 8.0 (d, 2H, Ar-H), 8.31 (d,
1H, Ar-H), 8.66 (s, 1H, N=CH), 8.69 (s, 1H, N=CH); 13C
NMR (125 MHz, CDCl ; d, ppm): 21.9 (CH ), 124.21,
(7E,8E)-1-(4-isopropylbenzylidene)-2-(1-(4-nitrophe-
nyl)ethylidene)hydrazine (9): C H N O (309.15 g/mol);
3
3
128.79, 129.1, 129.7, 129.89, 140.33, 142.67 and 164 (Ar),
159.21 (N=C); MS (m/z): 267 [M + H]+, 176, 145, 118, 91.
(7E,8E)-1-(4-ethylbenzylidene)-2-(4-nitrobenzylidene)-
hydrazine (5): C H N O (281.3 g/mol); yield 78%; m.p.
18 19
3
2
yield 65.5%; m.p. 96.9 °C; IR (KBr; n, cm-1): 1600 (C=N),
1
1510 (NO ); H NMR (500 MHz, CDCl ; d, ppm): 1.3 (d,
6H, CH ), 2.56 (s, 3H, CH ), 2.99 (m, 1H, CH), 7.99 (d, 2H,
2
3
16 15
3
2
3
3
1
162.5°C; IR (KBr; n, cm-1): 1600 (C=N), 1510 (NO ); H
Ar), 8.07 (d, 2H, Ar), 8.28 (d, 2H, Ar), 8.3 (d, 2H, Ar), 8.43
(s, 1H, N=CH); 13C NMR (125 MHz, CDCl ; d, ppm): 15.24
(CH ), 23.79 (CH ), 34.26 (CH(CH ) ), 123.59, 126.98,
2
NMR (500 MHz, CDCl ; d, ppm): 1.28 (t, 3H, CH ), 2.73 (q,
2H, –CH ), 7.3 (d, 2H, Ar-H), 7.79 (d, 2H, Ar-H), 8.0 (d, 2H,
3
3
3
3
3
3 2
2
127.5, 128.7, 132, 144.24 and 162,14 (Ar), 148.61, 152.7 and
159.15 (N=C); MS (m/z): 309 [M + H]+, 294, 190, 91.
(7E,8E)-1-(4-methoxybenzylidene)-2-(1-(4-nitrophe-
nyl)ethylidene)hydrazine (10): C H N O (297.31 g/mol);
Ar-H), 8.31 (d, 2H, Ar-H), 8.66 (s, 1H, N=CH), 8.69 (s, 1H,
N=CH); 13C NMR (125 MHz, CDCl ; d, ppm): 15.25 (CH ),
29 (CH CH ), 123.99, 128.48, 128.78, 129.03, 131.18,
3
3
2
3
140.11, 148.72 and 163.7 (Ar), 158.29 (N=C); MS (m/z): 281
[M + H]+, 252, 176, 159, 132.
16 15
3
3
yield 44,3%; m.p. 134.3°C; IR (KBr; n, cm-1): 1600 (C=N),
1
(7E,8E)-2-(4-nitrobenzylidene)-1-((pyridin-2-yl)me-
thylene)hydrazine (6): C H N O (254.24 g/mol); yield
1510 (NO ), 1250 (O–C); H NMR (500 MHz, CDCl ; d,
ppm): 2.56 (s, 3H, CH ), 3.88 (s, 3H, CH ), 6.98 (d, 2H, Ar),
2
3
13 10
4
2
3
3
34%; m.p. 159 °C; IR (KBr; n, cm-1): 1600 (C=N), 1520
(NO ), 1460 (Py); 1H NMR (500 MHz, CDCl ; d, ppm): 7.38
(m, 1H, Ar), 7.81 (m, 1H, Ar), 8.04 (d, 2H, Ar), 8.1 (d, 1H,
7.82 (d, 2H, Ar), 8.08 (d, 2H, Ar), 8.28 (d, 2H, Ar), 8.42 (s,
1H, N=CH); 13C NMR (125 MHz, CDCl ; d, ppm): 15.21
(CH ), 58.44 (OCH ), 114.34, 123.59, 127.11, 127.66,
2
3
3
3
3
Ar), 8.32 (d, 2H, Ar), 8.69 (s, 1H, N=CH), 8.7 (s, 1H,
N=CH), 8,75 (d, 1H, Ar); 13C NMR (125 MHz, CDCl ; d,
ppm): 122.66, 124.05, 125.35, 129.38, 136.67, 149.39,
150.1, 150.17 and 162.85 (Ar), 159.8 (N=C); MS (m/z): 254
[M + H]+, 176, 105, 79.
130.33, 144.36, 160.65 and 162.24 (Ar), 148.56 and 159.21
(N=C); MS (m/z): 297 [M + H]+, 208, 190, 134, 104, 77.
(7E,8E)-1-(3,4,5-trimethoxybenzylidene)-2-(1-(4-nit-
3
rophenyl)ethylidene)hydrazine
(11):
C H N O
18 19 3 5
(357.36 g/mol); yield 67.4; m.p. 142.5°C; IR (KBr; n, cm-1):
1590 (C=N), 1510 (NO ), 1230 (O–C); 1H NMR (500 MHz,
(7E,8E)-1-benzylidene-2-(1-(4-nitrophenyl)ethylide-
ne)hydrazine (7): C H N O (267.28 g/mol); yield 68%;
2
CDCl ; d, ppm): 2.56 (s, 3H, CH ), 3.92 (s, 3H, CH ), 3.95
(s, 6H, CH ), 7.1 (s, 2H, Ar), 8.07 (d, 2H, Ar), 8.29 (d, 2H,
15 13
3
2
3
3
3
m.p. 147°C; IR (KBr; n, cm-1): 1598 (C=N), 1520 (NO ); 1H
2
3
Ar), 8.33 (s, 1H, N=CH); 13C NMR (125 MHz, CDCl ; d,
ppm): 15.28 (CCH ), 56.26 (OCH ), 60.98 (OCH ), 105.66,
NMR (500 MHz, CDCl ; d, ppm): 2.55 (s, 3H, CH ), 7.47
(m, 3H, Ar), 7.86 (d, 2H, Ar), 8.08 (d, 2H, Ar), 8.28 (d, 2H,
3
3
3
3
3
3
Ar), 8.43 (s, 1H, N=CH); 13C NMR (125 MHz, CDCl ; d,
ppm): 15.26 (CCH ), 123.57, 127.7, 128.5, 128.81, 131.25,
123.57, 127.66, 129.64, 141.08, 144.08, 153.57 and 158.95
(Ar), 148.6 and 158.87 (N=C); MS (m/z): 357 [M + H]+, 326,
208, 190, 131.
3
3
134.3, 144.11 and 162.3 (Ar), 148.66 and 159.02 (N=C); MS
(m/z): 267 [M + H]+, 190, 104, 77.
(7E,8E)-1-(3-methoxy,4-hidroxybenzylidene)-2-(1-(4-
nitrophenyl)ethylidene)hydrazine
(12):
C H N O
16 15 3 4
(7E,8E)-1-(4-methylbenzylidene)-2-(1-(4-nitrophenyl)-
ethylidene)hydrazine (8): C H N O (281,12 g/mol);
(313.31 g/mol); yield 67%; m.p. 144.9°C; IR (KBr; n, cm-1):
16 15
3
2
3500 (OH), 1590 (C=N), 1520 (NO ), 1200 (O–C); 1H NMR
yield 74%; m.p. 122.9°C; IR (KBr; n, cm-1): 1600 (C=N),
2
1
(500 MHz, CDCl ; d, ppm): 1.58 (s, 1H, OH), 2.56 (s, 3H,
CH ), 3.99 (s, 3H, CH ), 5.98 (s, 1H, Ar), 6.98 (d, 1H, Ar),
1520 (NO ); H NMR (500 MHz, CDCl ; d, ppm): 2.42 (s,
3H, CH ), 2.55 (s, 3H, CH ), 7.27 (d, 2H, Ar), 7.77 (d, 2H,
3
2
3
3
3
3
3
Ar), 8.08 (d, 2H, Ar), 8.27 (d, 2H, Ar), 8.42 (s, 1H, N=CH);
13C NMR (125 MHz, CDCl ; d, ppm): 15.22 (CCH ), 21.62
7.52 (d, 1H, Ar), 8.06 (d, 2H, Ar), 8.26 (d, 2H, Ar), 8.37 (s,
1H, N=CH); 13C NMR (125 MHz, CDCl ; d, ppm): 15.22
3
3
3

Synthesis and Anticholinesterase Activity Evaluation
547
(CCH ), 56,06 (OCH ), 108.74, 114.49, 123.59, 124.55,
control were analyzed by ANOVA test and P < 0.05 was con-
sidered to be significant (*P < 0.05; **P < 0.01;
***P < 0.001).
3
3
127.63, 144.29, 147, 147.7 and 161.95 (Ar), 148.97 and
159.41 (N=C); MS (m/z): 313 [M + H]+, 296, 190, 150.
(7E,8E)-1-(4-N,N-dimethylbenzylidene)-2-(1-(4-nitro-
phenyl)ethylidene)hydrazine
(13):
C H N O
17 18 4 2
4. RESULTS AND DISCUSSION
(310.35 g/mol); yield 68,4%; m.p. 194.9°C; IR (KBr; n,
cm-1): 1600 (C=N), 1525 (NO ), 1500 (C–N); 1H NMR
4.1. Synthesis of Azines
2
(500 MHz, CDCl ; d, ppm): 2.58 (s, 3H, CH ), 3.06 (s, 6H,
CH ), 6.72 (d, 2H, Ar), 7.76 (d, 2H, Ar), 8.06 (d, 2H, Ar),
8.27 (d, 2H, Ar), 8.43 (s, 1H, N=CH); 13C NMR (125 MHz,
3
3
A total of 14 asymmetric azines were synthesized. These
compounds were obtained by reaction between aromatic al-
dehydes/ketones and hydrazine according to procedures C,
D, E and F. The nitro group is fixed at para position on ring
A and the substituents are varied on the other ring B (Scheme
2). The structures of the synthesized compounds as well as
the percentage conversion of the reactants to expected prod-
ucts are presented in Table 1.
3
CDCl ; d, ppm): 15 (CCH ), 40.12 (NCH ), 111.68, 122.05,
3
3
3
123.51, 127.48, 130.32, 144.73, 160 and 161.33 (Ar), 152.53
and 160.68 (C=N); MS (m/z): 310 [M + H]+, 190, 147, 77.
(7E,8E)-2-(1-(4-nitrophenyl)ethylidene)-1-((pyridin-2-
yl)methylene)hydrazine (14): C H N O (268.1 g/mol);
14 112
4
2
yield 77.9%; m.p. 157 – 158°C; IR (KBr; n, cm-1): 1600
In comparison of the percentage formation of asymmet-
ric azines, Series 2 showed higher conversion to expected
products than Series 1. This fact can be explained by the
greater tendency of symmetric hydrazone to form dimers
(Series 1), due to their high reactivity. Hydrazones can react
with each other forming dimers and releasing hydrazine in
the reaction medium. The released hydrazine can then react
with a second aldehyde present in the medium to form a new
hydrazone and therefore a new dimer [18]. These competing
reactions decrease the percentage of formation of the ex-
pected product, which may therefore explain the smaller per-
centage of formation of asymmetric azines in Series 1. In
such cases, addition of the compound was provided for the
formation of corresponding dimers.
1
(C=N), 1510 (NO ); H NMR (500 MHz, CDCl ; d, ppm):
2.5 (s, 3H, CH ), 7.37 (m, 1H, Py), 7.8 (m, 1H, Py), 8.09 (d,
2
3
3
2H, Ar), 8.15 (d, 1H, Py), 8.28 (d, 2H, Ar), 8.38 (s, 1H, Py),
8.7 (s, 1H, N=CH); 13C NMR (125 MHz, CDCl ; d, ppm):
15.43 (CCH ), 121.87, 123.63, 124.97, 127.86, 136.63,
3
3
143.71, 153.23, 157.8 (Ar), 148.77 and 161.64 (C=N); MS
(m/z): 268 [M + H]+, 239, 190, 144, 78.
3.2. In Vitro Anti-Acetylcholinesterase Activity
The ability of the synthesized compounds to inhibit
AChE enzyme was tested using the Ellman method as modi-
fied by Rhee, et al. [16]. Tests were performed in 96-well
microplates with automatic reader. To each well in
microplate were added 125 mL of 5,5’-dithio-bis-2-nitroben-
zoic acid (DTNB) 3 mM in Tris buffer at pH 8 (50 mL) con-
taining 0.1% of bovine serum albumin and 25 mL of 1 mM
sample solution in methanol. The absorbance was monitored
at 412 nm every 10 sec during 230 sec. After this period,
25 mL of AChE enzyme aqueous solution 0.226 U/mL were
added and the absorbance was monitored at 412 nm every 10
sec during 230 sec. Physostigmine (eserine) was used as pos-
itive control. All reactions were carried out in triplicate.
The enzyme activity was expressed as percentage inhibi-
tion (%I) of AChE enzyme, by comparing the rates of test re-
actions in microplate wells with samples and test reaction us-
ing buffer without any inhibitor. The calculation was made
according to the following formula:
Few reports like the synthesis of asymmetric compounds
obtained in this study were found in the literature and none
described the use of microwave assisted irradiation as the
heating source. However, the synthesis of symmetric azines
TABLE 1. Synthesis of Asymmetric Azines under Microwave Irra-
diation
Com-
pound
Proce-
dure
% Con-
version
R1
R2
B ring
3
4
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
E
E
E
E
F
F
F
F
F
F
F
F
62.8
70.3
55.0
67.2
98.1
94.0
97.0
89.0
79.0
90.3
98.7
93.15
4-CH3-C6H4
4-CH2CH3-C6H4
2-pyridine
H
5
H
%I = [V (enzyme without sample) – V (enzyme
6
H
with sample)] / [V (enzyme without sample)] ´ 100.
7
Ph
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
8
4-CH3-C6H4
4-CH(CH3)2-C6H4
4-OCH3-C6H4
3,4,5-OCH3
3-OCH3, 4-OH
4-NMe2-C6H4
2-pyridine
Any increase in the absorbance before addition of the en-
zyme due to the spontaneous hydrolysis of the substrate is
properly corrected by subtracting the rate of the reaction be-
fore addition of the enzyme, obtained after addition of the
enzyme. The samples were analyzed in triplicate [16, 17].
Results presented in this study are the average of three repli-
cates (n = 3) and expressed as mean with standard error
( SEM). Statistical differences between the treatment and
9
10
11
12
13
14

548
Andreza S. Figueredo et al.
Scheme 3. Sequence of reactions according to the Ellman method
has frequently been described both under conventional heat-
ing conditions and by microwave assisted irradiation using
different reagents [3 – 5, 19]. A good example is this series
was reported by Kurteva, et al. [3], where 21 asymmetric az-
ines were synthesized by reaction of aromatic aldehydes and
hydrazine sulfate in ethanol/DMF, with yields between 84
and 98%. The highest yield in obtaining dimers is expected
since it involves only one step instead of two as in the prepa-
ration of asymmetric azines.
4.2. In Vitro Activity Assays
The ability to inhibit AChE enzyme by synthesized com-
pounds was evaluated in vitro. Compounds with several sub-
stituents at different positions (ortho, meta and para) of ring
B were evaluated in respect of the influence of structural fac-
tors on the activity. Initially, the objective was to verify de-
pendence of the activity on alkyl radicals in ring B (com-
pounds 3, 4, 5), as well as the effect of changes caused by the
insertion of a methyl group at carbon adjacent to ring A
(compounds 7, 8 and 9). Since compounds 6 and 14 were se-
lected for bioisosteric substitution (CH ® N) at the ortho po-
sition of ring B over compounds 3 and 7. The analysis of the
TABLE 2. Percentage Inhibition of AChE by Synthetized Azines
Compound
% Inhibitiona
Concentration (mM)
3
100
10
0.50 ± 0.03**
4
NIb
influence of oxygen radicals (-OH and -OCH ) determined
3
the choice of compounds 10, 11 and 12. Finally, compound
13 was chosen to introduce amino substituent. The tests were
carried out based on the Ellman method [17]. In this case, the
enzyme hydrolyzes a substrate, resulting in the production of
thiocholine that reacts with 5,5’-dithio-bis(2-nitrobenzoic
acid, known as the Ellman reagent, to produce 2-nitrobenzo-
ato-5-mercaptothiocholine/5-thio-2-nitrobenzoate which can
be detected at 412 nm (Scheme 3). The absorbance indicat-
ing inhibition of the enzyme decreases when substances ca-
pable of inhibiting AChE are added.
5
NIb
10
6
NIb
10
7
22.5
100
10
18.34 ± 1.02***
NIb
8
9
NIb
10
NIb
100
100
100
100
10
11
NIb
12
2.99 ± 0.11**
15.56 ± 0.87**
17.43 ± 0.93*
27.23 ± 0.82
13
14
This photometric method requires high solubility of all
reactants and especially aqueous samples, since the solutions
are prepared in aqueous buffers. This problem was partially
solved by Rhee, et al. [16] by adapting a method that allowed
using non-aqueous solutions and monitoring the production
of colored marker compounds in 96-well plates with
Physostigmine
100
a Data are expressed as mean ± SEM (triplicate); b NI = no inhibi-
tion; *P < 0.05, **P < 0.01, ***P < 0.001.

Synthesis and Anticholinesterase Activity Evaluation
549
2. S. Dayagi and Y. Degani, in: The Chemistry of the Carbon-Ni-
trogen Double Bond: S. Patai (ed.), Inter-Science, New York
(1970), pp. 61 – 147.
microplate reader. This adaptation offered a very sensible
and efficient method due to analysis of many substances in a
short time at very low concentrations, making it the most
widely used assessment of natural and synthetic compounds
[20]. The percentage of AChE inhibition by azines and the
maximum concentrations of drugs tested are shown in Ta-
ble 2.
3. V. B. Kurteva, S. P. Simeonov and M. Stoilova-Disheva,
Pharm. Pharmacol., 2, 1 – 9 (2011).
4. S. Poojary, D. Sunil, D. Kekud, et al., Opt. Mater., 85, 1 – 7
(2018).
5. J. Lasri, M. M. Aly, N. E. Eltaye, et al., J. Mol. Struct., 1164,
1 – 8 (2018).
The anti-AChE activity tests revealed low levels of the
enzyme inhibition or lack of activity. Compounds 3, 7, 12, 13
and 14 showed the ability to inhibit the enzyme in vitro.
Among these, the maximum percentages of inhibition were
shown by com pounds 7 (18.24%) and 13 (17.53%), which
did not have substituents on ring B. Compound 13 (with
dimethylamino group in p-position) exhibited 15.56% en-
zyme inhibition. Compounds 3 and 12 showed percentage in-
hibition of 0.5 and 2.99%, respectively, which were at the
lowest inhibition level and could can be classified as false
positive [21, 22].
6. S. P. Simeonov, V. B. Kurteva and R. P. Bontchev, Bulg. Chem.
Commun., 40, 409 – 417 (2008).
7. J. Safari and S. Gandomi-Ravandi, Synthetic. Commun., 41,
645 – 651 (2011).
8. J. O. Bauer, G. Leitus, Y. Ben-David, et al., ACS Catal., 6,
8415 – 8419 (2016).
9. J. Jayabharathi, A. Thangamani, M. Padmavathy, et al., Med.
Chem. Res., 15, 431 – 442 (2007).
10. D. Q. Li, X. J. Luo and M. J. Tan, Adv. Mater. Res., 634,
918 – 921 (2013).
11. W. W. Wardakhan, M. Sherif, R. M. Mohareb, et al., Int. J. Org.
Chem., 2, 321 – 331 (2012).
Further studies, like IC and bioavailability estimations
50
are needed to establish the best structure that influences the
anticholinesterase activity.
12. H. I. Gul, H. Suleyman and M. Gul, Pharm. Biol., 47, 968 – 972
(2009).
13. D. Q. Li, M. J. Tan and L. Jie, Adv. Mater. Res., 396,
2366 – 2369 (2012).
ACKNOWLEDGEMENTS
14. G. Gurkok, T. Coban and S. Suzen, J. Enzyme Inhib. Med.
Chem., 24, 506 – 515 (2009).
The authors are thankfull to Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq), Coorde-
nação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES), and Fundação de Amparo a Pesquisa do Estado de
Goiás (FAPEG) for financial support and to Universidade
Estadual de Goiás for technical support. LDD thanks CNPq
for his PhD grant (232620/2014-8 GDE). GLBA is grateful
for his postdoctoral grant Projeto “MATIS–Materiais e
Tecnologias Sustentáveis – CENTRO-01-0145-FEDER-
000014”.
15. N. Haider, T. Kabicher, J. Kaferbock, et al.; Molecules, 12,
1900 – 1909 (2007).
16. I. K. Rhee, M. Van De Meent, K. Ingkaninan, et al., J.
Chromatogr. A, 915, 217 – 223 (2001).
17. G. L. Ellman, K. D. Courtney, V. Andres, et al., Biochem.
Pharmacol., 7, 88 – 95 (1961).
18. R. Van Deun, T. N. Parac-Vogt, K. Van Hecke, et al., J. Mater.
Chem., 13, 1639 – 1645 (2003).
19. J. Fathi, V. Masand, R. Jawarkar, et al., J. Comput. Meth. Mol.
Design, 3, 57 – 68 (2011).
20. S. Di Giovanni, A. Borloz, A. Urbain, et al., Eur. J. Pharm. Sci.,
33, 109 – 119 (2008).
REFERENCES
21. A. Khalid, Z. Ul-Haq, M. N. Ghayur, et al., J. Steroid.
Biochem., 92, 477 – 484 (2004).
1. S. Upadyaya, P. Bhagavath and D. Sunil, J. Mol. Liq., 269,
22. I. Orhan, Q. Naz, M. Kartal, et al., Z. Naturforsch., 62,
684 – 688 (2007).
354 – 370 (2018).