Design, synthesis, and anticonvulsant evaluation of 4-GABA-3-nitrocoumarines, 1-thiocoumarines, quinolone-2-ones, and their derivatives

Article abstract of DOI:10.1007/s00044-019-02422-5

The novel group of 4-GABA-3-nitrocoumarines, 1-thiocoumarines, quinolone-2-ones, and their derivatives was designed as potential anticonvulsants using GABA pharmacophore and corresponding heterocyclic moieties. A number of compounds of this group were synthesized and studied in the maximum electroshock seizure (MES) test and in the model of primary-generalized convulsions caused by subcutaneous pentylenetetrazole (scPTZ) in mice. The most active compound in the MES test was found to be 1a (N-(3-nitrocoumarin-4-yl)-4-aminobutyric acid) at a dose range of 60–80 mg/kg that increased the number of survived animals up to 60% in comparison with the control group, whose survival rate was 10%. Compounds 1d (N-(3,6-dinitrocoumarin-4-yl)-4-amino-butyric acid methyl ester) at doses of 10–40 mg/kg and 3a (N-(3-nitro-2-oxo-1,2-dihydroquinolin-4-yl)-4-amino-butyric acid methyl ester) at a dose of 12.5 mg/kg had the most pronounced anticonvulsant effect in scPTZ test. [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00044-019-02422-5

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02422-5
ORIGINAL RESEARCH
Design, synthesis, and anticonvulsant evaluation of 4-GABA-3-
nitrocoumarines, 1-thiocoumarines, quinolone-2-ones, and their
derivatives
G. V. Mokrov1 S. A. Litvinova T. A. Voronina L. N. Nerobkova I. S. Kutepova I. G. Kovalev
1 ●
1 ●
1 ●
1 ●
1 ●
●
T. A. Gudasheva1 A. D. Durnev
1
●
Received: 12 February 2019 / Accepted: 7 August 2019
©
Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
The novel group of 4-GABA-3-nitrocoumarines, 1-thiocoumarines, quinolone-2-ones, and their derivatives was designed as
potential anticonvulsants using GABA pharmacophore and corresponding heterocyclic moieties. A number of compounds of
this group were synthesized and studied in the maximum electroshock seizure (MES) test and in the model of primary-
generalized convulsions caused by subcutaneous pentylenetetrazole (scPTZ) in mice. The most active compound in the MES
test was found to be 1a (N-(3-nitrocoumarin-4-yl)-4-aminobutyric acid) at a dose range of 60–80 mg/kg that increased the
number of survived animals up to 60% in comparison with the control group, whose survival rate was 10%. Compounds 1d
(
N-(3,6-dinitrocoumarin-4-yl)-4-amino-butyric acid methyl ester) at doses of 10–40 mg/kg and 3a (N-(3-nitro-2-oxo-1,2-
dihydroquinolin-4-yl)-4-amino-butyric acid methyl ester) at a dose of 12.5 mg/kg had the most pronounced anticonvulsant
effect in scPTZ test.
Graphical Abstract
●
●
●
●
Keywords Anticonvulsant Coumarines 1-Thiocoumarines Quinolone-2-ones GABA derivatives
Introduction
chronic disease characterized by repeated unprovoked
attacks of motor disorders, sensory, autonomic, or mental
functions, resulting from excessive neural discharges in the
gray matter of the cerebral cortex. The prevalence of epi-
lepsy in the population is high and affects nearly 1% of the
world’s population (World Health Organization 2005).
Seizures are the same for one patient but significantly differ
between patients. According to the international classifica-
tion of epilepsy, epileptic syndromes and similar diseases,
epilepsy is not considered as one single disease with various
types of attacks, but it is divided into separate types (epi-
leptic syndromes) with their own electroclinical features and
differ in their prognosis and approaches to therapy (Fisher
et al. 2017).
The creation of highly active anticonvulsant drugs is an
important problem of modern pharmacology. Epilepsy is a
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02422-5) contains supplementary
material, which is available to authorized users.
*
G. V. Mokrov
g.mokrov@gmail.com
1
Federal State Budgetary Institution “Zakusov Research Institute of
Pharmacology”, Ramenki str., 11-1-156, Baltiyskaya str., 8,
Moscow 125315, Russia

Medicinal Chemistry Research
Fig. 1 GABA-containing drugs with anticonvulsant activity
The derivatives of gamma-aminobutyric acid (GABA)
can be allocated in a special group among the existing
anticonvulsants. The most widely used drugs among them
are: gabapentin, pregabalin, vigabatrin, and progabide
Fig. 2 Design of 4-GABA-3-nitrocoumarines, 1-thiocoumarines, qui-
nolone-2-ones, and their derivatives
(
Fig. 1). The compounds of this group have significantly
different mechanisms of action, and for some of them are
not fully clear.
Gabapentin and pregabalin are the GABA analogues, but
they do not bind to the GABA receptors and do not directly
modulate GABA transport or metabolism (Uchitel et al.
create compounds with anticonvulsant activity (Srikrishna
et al. 2018, Proisl et al. 2017). It was assumed that the
combination of GABA and these heterocyclic systems
would give compounds with pronounced anticonvulsant
effect.
2
010). These compounds are the ligands of the auxiliary
α2δ subunit site of certain voltage-dependent calcium
channels (VDCCs), and thereby act as an inhibitors of α2δ
subunit-containing VDCCs (Calandre et al. 2016). Viga-
batrin is an irreversible mechanism-based inhibitor of
gamma-aminobutyric acid aminotransferase, the enzyme
responsible for the catabolism of GABA, which increases
the level of GABA in the brain (Rogawski and Löscher
Chemistry
The corresponding starting halogen-substituted heterocyclic
compounds were required to obtain the desired compounds.
4-Chloro-3-nitrocoumarin 4a is a commercially available
reagent. 4-Chloro-3,6-dinitrocoumarin 4b was obtained in
accordance with the literature method (Kuleš et al. 1984). 3-
Nitro-4-chloro-1-thioumarin 5 was obtained on the basis of
thiophenol 7 (Scheme 1). On the first stage thiophenol 7
reacted with malonic acid 8 to give malonic acid dithio-
phenyl ester 9. Cyclocondensation of the obtained com-
2
004). Progabide is an analogue and prodrug of GABA. Via
conversion into GABA progabide behaves as an agonist of
the GABA receptors (Fromm et al. 1985).
Thus, existence of many drugs with GABA pharmaco-
phore suggests that its use is very attractive for designing of
new compounds with a potential anticonvulsant activity.
pound 9 occurs in the presence of AlCl as a Lewis acid on
3
the second stage resulted in 4-hydroxy-1-thioumarin 10.
The nitration of this compound was carried out with nitric
acid in the presence of sulfuric and acetic acids to give 4-
hydroxy-3-nitro-1-thioumarin 11 (Savel’ev et al. 1978).
Then hydroxyl group in compound 11 was replaced by a
chlorine atom by the action of phosphoryl chloride in DMF,
it gave the target compound 5 (Savel’ev et al. 1978).
3-Nitro-4-chloroquinolin-2-one 6 was obtained accord-
ing to a similar scheme based on aniline 12 (Scheme 2). On
the first stage the reaction of malonic acid diethyl ester 11
with aniline 10 gave malonic acid danilide 12. On the
second stage cyclocondensation of the obtained dianilide 12
in 4 hydroxyquinolin-2-one 13 occurs in the presence of
Result and discussion
Design
The purpose of the research was synthesis and study of
anticonvulsant activity of compounds with general formula
1
–3, containing unsubstituted GABA pharmacophore or its
methyl ester, linked through an amino group to coumarin,
thioumarin, or quinolin-2-one heterocycle with one or two
nitro groups (Fig. 2). According to the literature, coumarin
and quinolin-2-one heterocycles are often used as a core to

Medicinal Chemistry Research
Scheme 1 Reagents and conditions: (i) POCl3, reflux, 40 min; (ii) AlCl3, NaCl, 195 °C, 25 min; (iii) HNO3, H2SO4, AcOH, RT., 3 h; (iv) POCl3,
DMF, 2 h
Scheme 2 Reagents and
conditions: (i) 210 °С, 5 h; (ii)
polyphosphoric acid, 150 °C,
5
1
h; (iii) HNO3 (54%), 75 °C,
2 min; (iv) POCl3, TBBAB,
MeCN, 40 °C, 30 min, then
reflux, 22 min
freshly prepared polyphosphoric acid when heated for 5 h
corresponding products ranged from 50 to 99%. Jung pre-
sented the interaction of 3-nitro-4,7-dichloroquinolin-2-one
with beta-alanine ethyl ether by refluxing in pyridine for 2 h
(Jung et al. 2003). A heterocyclic product with an amino
acid substituent was formed in 68% yield.
The target 4-GABA-3-nitrocoumarines, 1-thiocoumar-
ines, quinolone-2-ones, and their derivatives was obtained
by reacting the corresponding chlorine-substituted hetero-
cyclic compounds 4–6 with GABA or its methyl ester
(Scheme 3).
(
Shobana et al. 1989). The nitration of 4-hydroxyquinolin-
2
7
-one 13 was carried out in 54% nitric acid for 20 min at
5 °C to give 4-hydroxy-3-nitroquinolin-2-one 14 (Dolle
et al. 1995). 3-Nitro-4-chloroquinolin-2-one 15 was
obtained by replacing the hydroxyl group of 4-hydroxy-3-
nitroquinolin-2-one 14 with a chlorine atom by the action
with phosphoryl chloride in the presence of a quaternary
ammonium salt tri-n-butyl benzylammonium bromide
(
Dolle et al. 1995).
There is scant information in the literature regarding
Several reaction conditions have been developed to
obtain compounds 1–3 with unsubstituted carboxyl group
(R =Н). The first method consisted in the interaction of 4-
reactions of nitrocoumarines, 1-thiocoumarines, quinolone-
-ones like 4–6 with amino acids, and their derivatives to
date. Stunic described the interaction of 4-chloro-3-
nitrocoumarin 4a with glycine and alanine and their esters
Stunic et al. 1981). Reactions with unsubstituted amino
2
2
́
chlorocoumarins 4 and 4-chlorothycoumarin 5 with a 2.2-
fold excess of GABA when boiling in dioxane. Selection of
this solvent based on the fact that it dissolves the starting
materials and poorly dissolves the reaction product. As the
second method, it was proposed to interact equimolar
amounts of 4-chlorocoumarins 2 and 4-chlorothycoumarin
5 with GABA in the presence of triethylamine in boiling
ethanol. Regardless of the proposed conditions, reactions
come with high yields. It should be noted that the reaction
(
́
acids were carried out either by boiling in absolute ethanol
for 2 h, or in ethanol in the presence of triethylamine at
room temperature with stirring for 6 h. Reactions with
hydrochlorides of amino acids esters were carried out in
benzene in the presence of ammonia or triethylamine at
room temperature or heated for 30 min. The yields of the

Medicinal Chemistry Research
Scheme 3 Reagents and
conditions: (i) Dioxane, reflux, t
min; (ii) TEA, EtOH, reflux, t
min; (iii) TEA, toluene, RT, t
min; (iv) Py, reflux, t min; (v)
NaOH, H2O, MeOH, reflux,
4
0 min
Compound Conditions t, min
Х
R1
R2
i
ii
iii
i
iii
i
ii
iii
iv
v
120
90
30
60
25
120
180
30
120
-
1
а
O
Н
Н
1
b
O
O
O
H
NO
NO
CH
H
CH
3
3
1
c
2
2
1
d
2
a
S
Н
H
2
b
S
NH
NH
Н
Н
Н
CH
CH
Н
3
3
3
a
3
b
of 3-nitro-4-chloroquinolin-2-one 6 with GABA did not
lead to the desired product both under these conditions and
when carrying out in other solvents.
Anticonvulsant activity in maximal electroshock
seizure test (MES)
The interaction of 4-chlorocoumarins
4
and 4-
Initially the compounds were screened in the MES test
(Voronina and Nerobkova 2012, Löscher et al. 1991;8(2)).
Compound’s protection in the MES test indicates the ability
of a substance to prevent seizure spread. The MES test
caused tonic extension in 87.5–100% and death in 75–90%
of mice in control group. Compound 1a at a dose range of
20–40 mg/kg did not demonstrate protection from seizure
and did not increase survival rate in the MES test, while at
dose of 60–80 mg/kg it possessed significant protective
effect against MES in comparison with the control group
(Table 1). Compounds 1b and 2a had a dome-shaped dose
dependence of activity. Thus, compound 1b at a dose range
of 1–60 mg/kg had the weakest protection, whereas the
maximum number of survived animals was recorded at
doses of 10 mg/kg and 30 mg/kg. Compound 2a at doses of
chlorothycoumarin 5 with GABA methyl ester to form
2
compounds 1–2 (R =CH ) occurred much faster than with
3
unsubstituted GABA and under milder conditions: at room
temperature in toluene in the presence of triethylamine.
Pyridine was chosen for the reaction of 3-nitro-4-chlor-
oquinolin-2-one 6 with GABA ester as the solvent due to
the low solubility of the starting material in other solvents.
This reaction successfully proceeded by boiling in pyridine
for 2 h with the formation of the target ester 3a. N-(3-
nitroquinolin-2-one-4-yl)-4-aminobutyric acid 3b was
obtained by ether 3a saponifying with alkali action in
aqueous methanol.
Biological evaluation
20 and 40 mg/kg increased the survival rate of mice up to
The compounds obtained were evaluated in the in vivo
rodent models of epilepsy within the order of the Ministry
of Health of Russian Federation “Statement of the proper
laboratory practice rules” of April 1, 2016, N 199 and
according to Anticonvulsant Screening Program (ASP) of
the National Institute of Neurological Disorders and Stroke
50% and 63% relatively in comparison with the control
group, while at a dose range of 60–120 mg/kg the effec-
tiveness of 2a compound decreased to 38% in terms of
survival. The results obtained are presented in Table 1.
Compound 3a at a dose of 12.5 mg/kg increased the sur-
vival rate by 63% in comparison with the control group and
reduced the percentage of animals with tonic extensions up
o
(
NINDS).

Medicinal Chemistry Research
Table 1 Anticonvulsant activity of coumarin, thiocoumarin, and
quinolinone derivatives in the maximum electroshock test in mice
3
2–320 mg/kg (mice, i.p.) (Lotarski et al. 2014), vigabatrin
significant increase survival of mice (i.p.) at the dose of
0 mg/kg (Sills et al. 1999), and gabapentin at the dose of
Group
Dose, mg/kg
Number of
animals in
the group
% of animals % of
5
with tonic
extensions
survived
animals
419 mg/kg (Tutka et al. 2019). Thus the activity of com-
pound 1a in the MES test at least not inferior the actions of
known GABA derivatives.
Control, MES
–
10
10
100
10
Compound 1а
80
60
60*
+
MES
(р = 0087)
6
0
10
70
60*
Anticonvulsant activity in the model of primary-
generalized convulsions caused by subcutaneous
pentylenetetrazole (scPTZ)
(р = 0211)
4
2
0
0
10
10
8
80
30
10
14
100
100
63
Compound 1b
MES
60
+
3
0
8
38
Anticonvulsant activity of the tested compounds was eval-
uated against scPTZ to identify the agents rising the seizure
threshold (Voronina and Nerobkova 2012, Löscher et al.
1991;8(3)). Administration of scPTZ at a dose of 100 mg/kg
to control group developed convulsive manifestations in the
following sequence:
(р = 0069)
(р = 0275)
1
0
8
75
38
(
р = 0275)
5
1
–
8
8
10
8
8
8
8
8
8
8
8
8
8
100
100
90
13
13
20
25
25
25
25
13
0
Control, MES
Compound 1c + 40
75
MES
2
1
0
0
75
●
One or more myoclonic twitches of the whole body—
88
100% of mice.
Compound 1d
MES
40
75
+
●
Repeated clonic seizures of the fore and/or hind limbs
with a duration of more than 3 s without loss of the
inversion reflex—100% of mice.
2
1
–
0
0
100
100
87, 5
50
Control, MES
25
38
38
63
Compound 2а
120
●
●
Generalized clonic seizures of the fore and hind limbs
with the loss of the inversion reflex—90% of mice.
The death of animals—100% of mice.
+
MES
6
4
0
0
50
75
(
p = 0315)
2
1
0
0
8
8
63
50
25
25
0
75
Compound 1a at a dose range of 10–80 mg/kg and
Compound 2b
MES
60
8
88
compound 1c at a dose range of 10–40 mg/kg did not pre-
vent the development of chemical-induced seizures and
animal’s death (Table 2). Compound 1b increased the mice
survival only at a dose of 40 mg/kg (up to 38%) in com-
parison with the control group. Compound 1d showed an
outstanding protective effect at all tested doses (10–40 mg/
kg), preventing death in 50–63% of animals. Compounds
+
4
2
1
–
0
0
0
8
100
100
88
8
25
25
20
0
8
Control, MES
10
8
90
Compound 3а
50
100
100
50
+
MES
2
1
5
8
13
2, 5
8
63
(p = 0118)
(p = 0145)
2
a and 2b exhibited a maximum anticonvulsant effect at a
Asterisk indicates significant difference between the test group and the
control group, with p ≤ 0.05 (Fisher’s exact test)
dose of 10 mg/kg via increasing the survival of mice by
maximum 50%. Compound 3a showed anticonvulsant
activity at a dose of 12.5 mg/kg, increasing the survival rate
of mice up to 56% (Table 2). Dose increase of compounds
2a, 2b, and 3a diminished the anticonvulsant activity.
Thus, it can be noted that compound 1d at doses of
10–40 mg/kg and 3a at a dose of 12.5 mg/kg had the most
pronounced anticonvulsant effect in the test with scPTZ.
Compounds 1b, 2a, and 2b demonstrated activity in the test
at the trend level.
In accordance with the literature data, gabapentin exhi-
bits anticonvulsant activity in scPTZ test in the doses of
25–200 mg/kg, pregabalin in the doses of 10–40 mg/kg, and
vigabatrin in the doses of 200–600 mg/kg (mice, i.p.)
(Akula et al. 2009, Nieoczym et al. 2012). Synthesized
compounds 1d and 3a not inferior in its effect to
these drugs.
to 50%, but with the dose increase (up to 25 and 50 mg/kg)
its anticonvulsant effect was not recorded (Table 1). Com-
pounds 1c, 1d at doses of 10–40 mg/kg, and compound 2b
at doses of 10–60 mg/kg were identified as MES inactive
displaying no significant protection.
Thus, it was identified that in the MES test 1a at a dose
range of 60–80 mg/kg increased the number of survived
animals up to 60% in comparison with the control group,
whose survival rate was 10%. Compounds 1b, 2a, and 3a
showed activity at doses of 10–30, 40, and 12.5 mg/kg at
the trend level, respectively.
Among known GABA-derived anticonvulsants in the
MES test pregabalin shows activity in the doses of

Medicinal Chemistry Research
Table 2 Anticonvulsant effect of coumarin, thiocumarin, and
quinolinone derivatives in test with subcutaneous pentylenetetrazole
in mice
while it was the only compound in this derivatives that
possessed the activity with the presence of an ester group.
In the test with scPTZ, compound 1d was the most
active, it contains GABA pharmacophore in the form of
methyl ester and a coumarin heterocycle with two nitro
groups. The existence of an ester group in this compound
was critical for the presence of activity, since such a com-
pound without an ester group (1c) was not active. The
activity of compound 1b, that differs from 1d by the
absence of one nitro group, decreased. Coumarin derivative
with one nitro group and unsubstituted GABA moiety
Compound
Dose, mg/kg Number of
animals in
% of
survived
animals
the group
Subcutaneous
pentylenetetrazole
100
80
10
0
1
1
1
1
2
2
3
а
b
c
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
6
9
0
6
2
0
0
0
0
40
38
0
(compound 1a) was inactive in the screening test. Replacing
2
1
0
0
of heteroatom in the heterocyclic core with a sulfur atom led
to the fact that the compounds with unsubstituted GABA
pharmacophore (2a) and with methyl ester of GABA
pharmacophore (2b) had the moderate activity. The qui-
noline compound 3a with GABA methyl ester had even
more pronounced activity.
As a result, we identified the following trends: com-
pounds with a coumarin heterocyclic core and with
unsubstituted GABA pharmacophore were more active in
the MES test; compounds with GABA methyl ester
exhibited more activity in the test with scPTZ.
0
40
13
13
0
2
1
0
0
d
а
b
а
40
63*
63*
50#
25
2
1
0
0
40
2
1
0
0
38
50#
40
38
38
50#
0
2
1
0
0
Conclusion
50
The novel group of 4-GABA-3-nitrocoumarines, 1-thio-
coumarines, quinolone-2-ones, and their derivatives was
designed with GABA pharmacophore and corresponding
heterocyclic moieties as potential anticonvulsants. A num-
ber of compounds of this group were synthesized and stu-
died in the MES test and in the model of primary-
generalized convulsions caused by scPTZ in mice. The most
active compound in the MES test was found to be 1a at dose
range of 60–80 mg/kg that increased the number of survived
animals up to 60% in comparison with the control group,
whose survival rate was 10%. Compounds 1d at doses of
10–40 mg/kg and 3a at a dose of 12.5 mg/kg had the most
pronounced anticonvulsant effect in scPTZ test.
2
1
5
17
56*
2, 5
Asterisk indicates significant difference between the test group and the
control group, with p ≤ 0.05 (Fisher’s exact test); The hash symbol
indicates the tendency to reliability of the difference between the test
group and the control group, with p ≤ 0.08 (Fisher’s exact test)
Structure activity relationship analysis of synthesized
compounds was conducted based on the obtained data of
the anticonvulsant activity screening in mice and it indi-
cated that substance’s activity essentially depended on their
structure. In the MES test, compound 1a showed the highest
activity, it contains a coumarin heterocycle with one nitro
group in the 3rd position and GABA pharmacophore. The
change of 1a to its methyl ester (1b) resulted in a decrease
of anticonvulsant activity. The additional nitro group in
compounds 1a and 1b (compounds 1c and 1d) led to a
complete disappearance of the anti-seizure protection. The
change of the heteroatom in the coumarin heterocycle
demonstrated the decrease in the activity of the compounds.
In addition, thiocumarin derivative with GABA pharma-
cophore 2a had the anticonvulsant activity, whereas this
compound with methyl ester 2b did not demonstrate effi-
cacy. The activity of the quinolone derivative 3a was weak,
Experimental section
Chemistry
The chemical reagents used in the synthesis were obtained
from commercial supplier and used without purification.
TLS was performed on silica gel F254 plates (Merck).
Melting points are uncorrected and were measured in open
1
capillary tubes, using OptiMelt melting point apparatus. H
NMR spectral data were recorded on Bruker Fourier
300 spectrometer (300 MHz) in DMSO-d6 and CDCl using
3

Medicinal Chemistry Research
1
the signals of the residual protons of the solvents (δ 2.50
and 7.25, respectively) as the internal standard. High-
resolution mass spectra (HRMS) were recorded on a
M.p. 142–143 °C (Savel’ev et al. 1978)). Н NMR (DMSO,
δ, ppm, J/Hz): 7.38 (m, 2H, HC(6), HC(8)); 7.44 (t, 1H, HC
(7), J = 8.1); 8.17 (d, 1H, HC(5), J = 8.1).
3
3
«
Bruker micrOTOF II» MS equipment.
We used commercial 4-chloro-3-nitrocoumarin 4а
3-Nitro-4-chloro-1-thiocumarin (5) (Savel’ev et al.
(
AlfaAesar). 4-Chloro-3,6-dinitrocoumarin 4b was obtained
1978)
in accordance with the literature method (Kuleš et al. 1984).
4
mmol (0.373 ml) of phosphoryl chloride was dissolved in
2.5 ml of dimethylformamide and mixed for 30 min.
mmol (0.89 g) of 4-hydroxy-3-nitrothiocoumarin (11) in
Malonic acid dithiophenyl ester (9)
4
The mixture of 75 mmol (7.81 g) malonic acid 8, 80 mmol
9.6 ml of dimethylformamide was added to the solution, and
the mixture was stirred for 2 h. 40 ml of cold water are
added to the reaction mixture, the precipitate was filtered
off, washed three times with 10 ml of distilled water, and
recrystallized from 10 ml of ethanol to obtain 0.85 g of dark
yellow crystals (yield 88%). M.p. 171–173 °C (Lit: M.p.
(
(
12.27 g, 7.46 ml) phosphoryl chloride, and 150 mmol
16.52 g, 15.35 ml) thiophenol 7 was refluxed for 40 min.
Eighty milliliters of ethanol was added to the cooled mix-
ture. The precipitate formed was filtered and washed with
5
0 ml of ethanol to obtain a pale blue powder. After
1
recrystallization from 30 ml of ethanol, 17.9 g of a white
crystalline substance was obtained (yield 82%). M.p.
176–177 °C (Savel’ev et al. 1978)). Н NMR (DMSO, δ,
ppm, J/Hz): 7.77, 7.89, 7.94 (three m, each 1H, HC(6), НС
2
9
2
7
4–95 °С. (Lit: M.p. 94.6–95.3 °C (Matsuo and Shindo
(7), НС(8)); 8.38 (d, 1H, HC(5), J = 8.1).
1
011)). Н NMR (DMSO, δ, ppm, J/Hz): 4.33 (s, 2H, СН );
2
.36–7.54 (m, 10H, 2 Ph).
Malonic acid bis-anilide (14) (Shobana et al. 1989)
4-Hydroxy-1-thioumarin (10)
A mixture of 200 mmol (18.64 g) aniline (12) and
100 mmol (16.02 g) of malonic acid diethyl ester (13) was
6
0 mmol (8.00 g) aluminum chloride and 40 mmol (2.34 g)
heated at 210 °C for 5 h. After cooling the reaction mixture,
the precipitate formed was filtered and recrystallized from
ethanol to obtain 19.3 g of a white powder (yield 76%). M.
sodium chloride were added to 20 mmol (5.76 g) dithio-
phenyl malonic acid ester 9 and mixed thoroughly. The
mixture was heated to 195 °C, and the temperature was
maintained for 25 min. After cooling, 100 ml of 1%
hydrochloric acid and 100 ml of ethyl acetate were poured
into the reaction mass. The organic layer was separated, and
the product was extracted with 100 ml of a 10% sodium
hydroxide solution. The aqueous solution was separated and
acidified to pH = 4, the precipitate was filtered and washed
twice with 20 ml of distilled water, obtaining 1.85 g of gray
p. 227–229 °С (Lit: M.p. 226–227 °С (Shobana et al.
1
1989)). Н NMR (DMSO, δ, ppm, J/Hz): 3.48 (s, 2H, СН );
2
3
3
7.07 (dd, 2H, 2H(4), J = 7.6, J = 7.4); 7.32 (dd, 4H, 2H
(3), 2 Н(5), J = 7.6, J = 8.1); 7.60 (d, 4H, 2H(2), 2 Н
(6), J = 8.1), 10.17 (s, 2 Н, 2 NH).
1
3
2
3
1
2
3
4-Hydroxyquinolin-2-(1H)-one (15) (Shobana et al.
1989)
powder (yield 73%). M.p. 211–212 °С (Lit: M.p. = 210 °C
1
(
Jamkhandi and Rajagopal 1963)). Н NMR (DMSO, δ,
A mixture of 50 mmol (12.72 g) of malonic acid bis-anilide
(14) and 80 g of polyphosphoric acid was heated at 150 °C
for 5 h and poured into 400 g of ice. The precipitate formed
was filtered, washed with water until neutral and dried,
giving 5.88 g of a pale yellow powder (yield 73%). M.p. >
ppm, J/Hz): 6.07 (s, 1H, HC(3)); 7.48 (m, 2H, HC(6), HC
8)); 7.60 (m, 1H, HC(7)); 8.13 (d, 1H, HC(5), J = 8.1);
2.26 (br.s, 1H, OH).
3
(
1
1
4
-Hydroxy-3-nitro-1-thioumarin (11) (Savel’ev et al.
300 °С (Lit: M.p. > 300 °С (Shobana et al. 1989)). Н NMR
1
978)
(DMSO, δ, ppm, J/Hz): 5.74 (s, 1H, HC(3)); 7.13 (dd, 1H,
3
3
3
H(6), J = 8.0, J = 7.1); 7.27 (d, 1H, Н(8), J = 8.0);
1
2
3
3
A mixture of 19.8 mmol (1.4 ml) of 65% nitric acid and
9.8 mmol (1.14 ml) of 96% sulfuric acid was added to a
suspension of 18 mmol (3.21 g) 4-hydroxy-1-thiocoumarin
0 in 80 ml of glacial acetic acid. The mixture was stirred at
7.48 (dd, 1H, H(7), J = 8.0, J = 7.1); 7.77 (d, 1H, H(5),
J = 8.0); 11.96 (s, 1 Н, NH).
1
2
3
1
1
4-Hydroxy-3-nitroquinolin-2-(1H)-one (16) (Dolle
et al. 1995)
room temperature for 3 h. The precipitation was filtered off,
washed twice with 10 ml of distilled water, dissolved in
boiling toluene, passing the hot solution through activated
carbon and evaporated to dryness, yielding 3.78 g of pro-
duct as a yellow powder (yield 92%). M.p. 149–152 °С (Lit:
A suspension of 20 mmol (3.22 g) 4-hydroxyquinolin-2-
(1H)-one (15) in 30 ml of nitric acid (d = 1.33 g/ml) was
stirred at room temperature for 10 min and at a temperature

Medicinal Chemistry Research
3
3
3
of 75 °C for 20 min The resulting solution was poured into
50 ml of ice-cold water. The precipitate formed was fil-
tered, washed with water until neutral and dried, yielding
.83 g of an orange powder (yield 93%). M.p. 243–245 °С
NHCH CH , J = J = 7.1); 2.30 (t, 2 Н, СН СООН, J
2
2
1
2
2
3
1
= 7.1); 3.18 (t, 2H, NHCH , J = 7.1); 7.41 (d, 1 Н, Н(8),
J = 8.2); 7.45 (dd, 1H, HC(6), J = J = 8.2); 7.73 (dd,
2
3
3
3
1
2
3
3
3
3
1H, HC(7), J = J = 8.2); 8.33 (d, 1H, HC(5), J = 8.2);
1 2
1
(
Lit: M.p. 250 °С (Dolle et al. 1995)). Н NMR (DMSO, δ,
8.44 (br.s, 1H, NH); 12.00 (br.s, 1 Н, СООН). Anal. Calcd
for С Н N О : C, 53.63; H, 4.14; N, 9.59. Found: С,
53.46; Н, 4.24; N, 9.61. HRMS (ESI+) m/z calcd for
ppm, J/Hz): 7.27 (m, 2H, H(6), H(8)); 7.65 (m, 1H, H(7));
1
3
13
2
6
3
8
.02 (d, 1H, H(5), J = 7.6); 11.96 (s, 1 Н, NH).
+
С Н N О [M+H] 293.0768; found 293.0764.
1
3
13
2
6
3
1
-Nitro-4-chloroquinolin-2-(1H)-one (6) (Dolle et al.
995)
N-(3,6-dinitrocoumarin-4-yl)-4-aminobutyric acid (1c)
1
(
5 mmol (3.09 g) 4-hydroxy-3-nitroquinolin-2-(1H)-one
16) and 36 mmol (5.52 g) phosphoryl chloride were added
to solution of 60 mmol (21.38 g) of tri-n-
Obtained using method A, t = 60 min, yield 81%. Yellow
powder. M.p. 183–185 °С. Н NMR (DMSO, δ, ppm, J/
1
3
3
a
Hz): 1.91 (tt, 2 Н, NHCH CH , J = 7.2, J = 6.8); 2.30
2 2 1 2
3
3
butylbenzylammonium bromide in 60 ml of acetonitrile.
The mixture was stirred at 40 °C for 30 min, then heated
under reflux for 22 min and evaporated to dryness. 60 ml of
water was added to the residue and the mixture was stirred
at room temperature for 3 h. The precipitate formed was
filtered, washed with water until neutral, and recrystallized
(t, 2 Н, СН СО, J = 7.2); 3.17 (dt, 2 Н, NHCH , J = 6.8,
J = 5.0); 7.62 (d, 1 Н, Н(8), J = 9.1); 8.49 (dd, 1 Н, Н(7),
J = 9.1, J = 2.3); 8.83 (br.s, 1 Н, NH); 9.39 (d, 1 Н, Н(5),
J = 2.3), 12.14 (br.s, 1 Н, СООН). Anal. Calcd for
2
2
1
3
3
4
3
2
4
С Н N О : C, 46.30; H, 3.29; N, 12.46. Found: С, 46.21;
1
3
11
3
8
Н, 3.50; N, 12.62.
from 20 ml of acetone to give 2.02 g of a yellow powder
1
(
yield 60%). M.p. 231–233 °С. Н NMR (DMSO, δ, ppm,
N-(3-nitro-1-thioumarin-4-yl)-4-aminobutyric acid (2a)
J/Hz): 7.46 (m, 2H, H(6), H(8)); 7.80 (m, 1H, H(7)); 8.01
3
(
d, 1H, H(5), J = 7.9); 11.03 (s, 1 Н, NH).
Obtained using method A (t = 120 min, yield 60%) and
method B (t = 180 min, yield 74%). Yellow powder. M.p.
1
Synthesis of 4-GABA-3-nitrocoumarines and 1-
thiocoumarines (1 and 2, R =H). General method
215–216 °С. Н NMR (DMSO, δ, ppm, J/Hz): 1.90 (m,
2
3
2 Н, NHCH CH ); 2.30 (t, 2 Н, СН СООН, J = 7.1); 3.18
2
2
2
(
m, 2H, NHCH ); 7.62 (m, 3H, HC(6), HC(7), HC(8)); 8.40
2
3
Method A
(d, 1H, HC(5), J = 8.1); 8.43 (br.s, 1H, NH); 12.21 (br.s,
1
Н, СООН). Anal. Calcd for С Н N О S: C, 50.65; H,
13 11 2 5
2
1
2 mmol (2.27 g) of GABA was added to a solution of
0 mmol of substituted 4-chlorocoumarin (4) or nitro-4-
3.92; N, 9.09; S, 10.40. Found: С, 50.64; Н, 3.69; N, 9.33;
S, 10.41.
chloro-1-thioumarin (5) in 20 ml of dioxane. The reaction
mass was refluxed for t minutes, then cooled. The resulting
precipitate was filtered, washed with 10 ml of dioxane, and
three times with 10 ml of distilled water. The resulting
substance was recrystallized from ethanol.
Synthesis of 4-GABA-3-nitrocoumarines and 1-
thiocoumarines methyl esters (1 and 2, R =CH ).
General method
2
3
A thoroughly mixed mixture of 10 mmol (1.54 g) GABA
methyl ester hydrochloride solution in 8 ml of water and
Method B
20 mmol (2.02 g) of triethylamine was added to a solution
1
0 mmol (1.03 g) of GABA and 11 mmol (1.11 g) of trie-
of 10 mmol of substituted 4-chlorocoumarin (4) or nitro-4-
chloro-1-thioumarin (5) in 80 ml of toluene with stirring.
The mixture was stirred at room temperature for t minutes.
The precipitate was filtered, washed with 50 ml of 1% HCl
and 100 ml of distilled water and dried.
thylamine was added to a solution of 10 mmol of substituted
4
2
minutes, then evaporated to dryness. The residue was
washed three times with 50 ml of distilled water and
recrystallized from ethanol.
-chlorocoumarin (4) or nitro-4-chloro-1-thioumarin (5) in
00 ml of ethanol. The reaction mass was refluxed for t
N-(3-Nitrocoumarin-4-yl)-4-amino-butyric acid methyl ester
(
1b)
N-(3-nitrocoumarin-4-yl)-4-aminobutyric acid (1a)
Yield 79%. t = 30 min. Light yellow powder. M.p.
1
Obtained using method A (t = 120 min, yield 59%) and
125–126 °С. Н NMR (DMSO, δ, ppm, J/Hz): 1.92 (tt, 2 Н,
3
3
3
method B (t = 90 min, yield 69%). Yellow powder. M.p.
NHCH CH , J = J = 7.1); 2.38 (t, 2 Н, СН СООН, J
2 2 1 2 2
1
2
10–211 °С. Н NMR (DMSO, δ, ppm, J/Hz): 1.90 (tt, 2 Н,
= 7.1); 3.19 (m, 2H, NHCH ); 3.55 (s, 3Н, OCH ); 7.39 (d,
2 3

Medicinal Chemistry Research
3
3
1
1
Н, Н(8), J = 8.7); 7.44 (t, 1H, HC(6), J = 8.0); 7.73 (t,
N-(3-nitro-2-oxo-1,2-dihydroquinolin-4-yl)-4-amino-butyric
acid (3b)
3
3
H, HC(7), J = 7.8); 8.36 (d, 1H, HC(5), J = 8.1); 8.47
(
br.s, 1H, NH). Anal. Calcd for С Н N О : C, 54.90; H,
15 15 2 6
4
.61; N, 9.15. Found: С, 54.78; Н, 4.72; N, 9.06. HRMS
A mixture of 0.66 mmol (0.2 g) of N-(3-nitro-2-oxo-1,2-
dihydroquinolin-4-yl)-4-amino-butyric acid methyl ester (3a),
1.3 mmol (52 mg) sodium hydroxide, 20 ml water, and 20 ml
of methanol was refluxed for 40 min, cooled to room tem-
perature and acidified with 1% hydrochloric acid until acidic.
The precipitate was filtered, washed with water and dried,
yielding 0.12 g of a dark yellow powder (yield 64%).M.p.
119–120 °С. 1Н NMR (DMSO, δ, ppm, J/Hz): 1.85 (m, 2 Н,
+
(
ESI+) m/z calcd for С Н N О [M+H] 307.0925;
13 13 2 6
found 307.0921.
N-(3,6-dinitrocoumarin-4-yl)-4-amino-butyric acid methyl
ester (1d)
Yield 79%. t = 25 min. Light yellow powder. M.p.
1
3
1
30–131 °С. Н NMR (DMSO, δ, ppm, J/Hz): 1.94 (tt, 2 Н,
NHCH CH ); 2.28 (t, 2 Н, СН СОOCH , J = 7.2); 3.08 (m,
2
2
2
3
3
3
3
3
NHCH CH , J = 7.3, J = 6.8); 2.40 (t, 2 Н, СН СО, J
=
3
2H, NHCH ); 7.24 (m, 1H, HC(6)); 7.27 (d, 1 Н, Н(8), J =
2
2
1
2
2
2
3
3
7.3); 3.17 (dt, 2 Н, NHCH , J = 6.8, J = 5.0); 3.57 (s,
7.6); 7.43 (m, 1 Н, NHСН ); 7.58 (m, 1H, HC(7)); 8.21 (d,
2
1
3
2
2
3
Н, ОСН ); 7.62 (d, 1 Н, Н(8), J = 9.2); 8.49 (dd, 1 Н, Н
1H, HC(5), J = 8.2); 11.53 (br.s, 1H, NH(quinoline)), 12.16
3
3
4
3
(
7), J = 9.2, J = 2.5); 8.81 (t, 1 Н, NH, J = 5.0); 9.39 (d,
(br.s, 1 Н, СООН). Anal. Calcd for C H N O : C, 53.61; H,
1
3 13 3 5
4
1
Н, Н(5), J = 2.5). Anal. Calcd for С Н N О : C, 47.87;
4.50; N, 14.43. Found: С, 53.58; Н, 4.45; N, 14.40.
14
13
3
8
H, 3.73; N, 11.96. Found: С, 48.08; Н, 3.62; N, 12.11.
Pharmacology
N-(3-nitrothiocoumarin-4-yl)-4-amino-butyric acid methyl
ester (2b)
Animals
Yield 77%. t = 30 min. Light yellow powder. M.p.
Experiments were performed on white outbred male mice
weighing 20–26 g. Experimental animals were obtained
from the Stolbovaya nursery of the Federal State Budgetary
Institution of Science “Scientific Center for Biomedical
Technologies of the Federal Medical-Biological Agency”
(Moscow Region). The maintenance of animals complied
with the guidelines of laboratory practice and the regulatory
documents “Sanitary guidelines for vivarium organization,
1
1
25–126 °С. Н NMR (DMSO, δ, ppm, J/Hz): 1.92 (tt, 2 Н,
3
3
3
NHCH CH , J = J = 7.1); 2.38 (t, 2 Н, СН СООН, J
2
2
1
2
2
=
7.1); 3.19 (m, 2H, NHCH ); 3.55 (s, 3Н, OCH ); 7.58 (t,
2 3
3
1
Н, Н(6), J = 8.0); 7.64 (m, 1H, HC(6)); 7.68 (m, 1H, HC
3
(
8)); 8.40 (d, 1H, HC(5), J = 8.5); 8.36 (br.s, 1H, NH).
Anal. Calcd for С Н N О S: C, 52.17; H, 4.38; N, 8.69;
1
4
14
2
5
S, 9.95. Found: С, 52.06; Н, 4.22; N, 8.87; S, 9.87. HRMS
+
o
(
ESI+) m/z calcd for С Н N О [M+H] 323.0696;
equipment and maintenance” N 708n.
13
13
2
6
found 323.0694.
The maximal electroshock seizure test
N-(3-nitro-2-oxo-1,2-dihydroquinolin-4-yl)-4-amino-butyric
acid methyl ester (3a)
In the MES test, an electrical stimulus of 0.3 s in duration
(144 mA, 500/300 V/mA) was delivered via corneal elec-
5
mmol (0.77 g) of 4-aminobutyric acid methyl ester
hydrochloride was added to a solution of 1.25 mmol
0.28 g) 3-nitro-4-chloroquinolin-2(1H)-one (6) in 10 ml of
trodes (Rodent Shocker RS installation, type 221, Harvar-
dApparatus, GmbH, Germany) primed with an electrolyte
solution containing an anesthetic agent. Mice were tested in
40 min after intraperitoneal (ip) administration of test
compound (Voronina and Nerobkova 2012, Löscher et al.
1991;8(2)). Each dose of the compound was tested on 8–10
animals. The following indicators were recorded: the tonic
extension of the hind and forelimbs and the death of ani-
mals. The anticonvulsant effect of the tested compounds
was assessed by their ability to prevent the development of
tonic extension and animal death.
(
pyridine. The mixture was heated under reflux for 2 h. The
solvent was evaporated to dryness, 15 ml of water was
added to the residue, and the product was extracted twice
with 15 ml of dichloromethane. The organic layer was
evaporated to dryness, and the residue was recrystallized
from ethanol to give 0.20 g of a dark yellow powder (yield
1
6
1
7
1
1
3%). M.p. 208–210 °С. Н NMR (DMSO, δ, ppm, J/Hz):
3
.98 (m, 2 Н, NHCH CH ); 2.36 (t, 2 Н, СН СООН, J =
2
2
2
.1); 3.11 (m, 2H, NHCH ); 3.55 (s, 3 Н, OCH ); 7.24 (t,
2
3
3
3
Н, Н(6), J = 7.6); 7.28 (d, 1 Н, Н(8), J = 7.6); 7.42 (t,
Н, NHСН , J = 4.3); 7.58 (t, 1H, HC(7), J = 7.5); 8.20
The model of primary-generalized convulsions caused by
subcutaneous pentylenetetrazole
3
3
2
3
(
d, 1H, HC(5), J = 8.2); 11.53 (br.s, 1H, NH(quinoline)).
Anal. Calcd for C H N O : C, 55.08; H, 4.95; N, 13.76.
Found: С, 55.01; Н, 4.78; N, 13.61.
Each dose of test compound was tested on groups of at least
eight animals. Tested compounds were suspended in saline
1
4
15
3
5

Medicinal Chemistry Research
and ip administrated 40 min before the introduction of
subcutaneous pentylenetetrazole (Sigma-Aldrich, USA).
Control group of animals was ip pretreated with saline in an
equivalent volume. To obtain a convulsive seizure, penty-
lenetetrazole was administered subcutaneously to the cer-
vical region of the back at a dose of 100 mg/kg, what
produced clonic seizures lasting for a period of at least 5 s in
Jung J, Oh S, Kim W et al. (2003) Synthesis and biological properties
of 4-substituted quinolin-2(1H)-one analogues. J Heterocycl
Chem 40(4):617–623. https://doi.org/10.1002/jhet.5570400410
Kuleš M, Trkovnik M, Jurić A (1984) Synthesis and testing of 4-
chloro-3,6-dinitrocoumarin, a new derivatizing agent useful in
thin layer chromatography of amines and amino acids. Acta
Pharm Jugosl 34:81
Löscher W, Fassbender CP, Nolting B (1991) The role of technical,
biological and pharmacological factors in the laboratory evalua-
tion of anticonvulsant drugs. II. Maximal electroshock seizure
models. Epilepsy Res 8(2):79–94. https://doi.org/10.1016/0920-
9
7% (CD ) of mice in the control group. Animals were
97
observed during 30–60 min after subcutaneous pentylene-
tetrazole introduction. The number of dead animals was
recorded (Voronina and Nerobkova 2012, Löscher et al.
1
211(91)90075-Q
Löscher W, Hönack D, Fassbender CP, Nolting B (1991) The role of
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1
016/0920-1211(91)90062-K
Statistical analysis
Lotarski S, Hain H, Peterson J, Galvin S, Strenkowski B, Donevan S,
Offord J (2014) Anticonvulsant activity of pregabalin in the
maximal electroshock-induced seizure assay in α2δ1 (R217A)
and α2δ2 (R279A) mouse mutants. Epilepsy Res 108
Statistical processing of the results was performed using MS
Excel 2010 and BioStat 2009 (Analyst Soft Inc.). The
normal distribution of the data was determined by the
Shapiro–Wilk criterion. The significance of differences
between groups was determined using nonparametric cri-
teria: Kruskal–Walis and Fisher’s exact test.
(5):833–842. https://doi.org/10.1016/j.eplepsyres.2014.03.002
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malonic acid S,O-esters via monoalcoholysis of symmetric
dithiomalonates under neutral conditions. Org Lett 13
(16):4406–4409. https://doi.org/10.1021/ol201744u
Nieoczym D, Socała K, Luszczki JJ, Czuczwar SJ, Wlaź P (2012)
Sildenafil influences the anticonvulsant activity of vigabatrin and
gabapentin in the timed pentylenetetrazole infusion test in mice.
Prog Neuropsychopharmacol Biol Psychiatry 39(1):129–135.
https://doi.org/10.1016/j.pnpbp.2012.05.020
Proisl K, Kafka S, Košmrlj J (2017) Chemistry and applications of 4-
Hydroxyquinolin-2-one and Quinoline-2,4-dionebased com-
pounds. Curr Org Chem 21:1949–1975. https://doi.org/10.2174/
Acknowledgements The work was carried out according to the state
order of 2019–2021, topic No. 0521-2019-0008 in Zakusov Research
Institute of Pharmacology “Design and synthesis of new heterocyclic
compounds with potential pharmacological activity”.
Compliance with ethical standards
1
385272821666170711155631
Conflict of interest The authors declare that they have no conflict of
interest.
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jurisdictional claims in published maps and institutional affiliations.
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