Synthesis, in vivo and in silico anticonvulsant activity studies of new derivatives of 2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)acetamide

Article abstract of DOI:10.1016/j.ejmech.2019.06.085

In order to expand the arsenal of biologically active substances of anticonvulsive action by the interaction of 2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)acetic acid with the corresponding amines in the presence of N,N′-carbonyldiimidazole in the dioxan

Full text of DOI:10.1016/j.ejmech.2019.06.085

Accepted Manuscript
Synthesis, in vivo and in silico anticonvulsant activity studies of new derivatives of 2-
(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)acetamide
El Kayal Wassim Mokhamad, Sergiy Yu Shtrygol, Sergiy V. Zalevskyi, Amjad abu
Shark, Vadim V. Tsyvunin, Sergiy M. Kovalenko, Natalya D. Bunyatyan, Lina O.
Perekhoda, Hanna I. Severina, Victoriya A. Georgiyants
PII:
S0223-5234(19)30612-9
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.085
EJMECH 11488
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 April 2019
Revised Date: 26 June 2019
Accepted Date: 28 June 2019
Please cite this article as: E.K. Wassim Mokhamad, S.Y. Shtrygol, S.V. Zalevskyi, A.a. Shark, V.V.
Tsyvunin, S.M. Kovalenko, N.D. Bunyatyan, L.O. Perekhoda, H.I. Severina, V.A. Georgiyants,
Synthesis, in vivo and in silico anticonvulsant activity studies of new derivatives of 2-(2,4-dioxo-1,4-
dihydroquinazolin-3(2H)-yl)acetamide, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.06.085.
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7 m
Protection

ACCEPTED MANUSCRIPT
Synthesis, in vivo and in silico anticonvulsant activity studies of new derivatives
of 2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)acetamide
El Kayal Wassim Mokhamada, Sergiy Yu. Shtrygol’a, Sergiy V. Zalevskyia, Amjad abu Sharka,
Vadim V.Tsyvunina, Sergiy M. Kovalenkob,c, Natalya D. Bunyatyanc,d, Lina O. Perekhodaa, Hanna
I. Severinaa, Victoriya A. Georgiyantsa
a National University of Pharmacy, 53, Pushkinskaya st., Kharkiv, 61002, Ukraine
b V.N.Karazin Kharkiv National University, 4 Svobody sq., Kharkiv, 61077, Ukraine
c Federal State Autonomous Educational Institution of Higher Education I.M. Sechenov First
Moscow State Medical University, 8 Trubeckaya, Moscow, 119991, Russia
d Federal State Budgetary Institution "Scientific Centre for Expert Evaluation of Medicinal
Products", Petrovsky boulevard 8, bld. 2, Moscow, 127051, Russia
ABSTRACT
In order to expand the arsenal of biologically active substances of
anticonvulsive action by the interaction of 2-(2,4-dioxo-1,4-dihydroquinazolin-
3(2H)-yl)acetic acid with the corresponding amines in the presence of N,N'-
carbonyldiimidazole in the dioxane medium, a systematic series of 2-(2,4-dioxo-1,4-
dihydroquinazolin-3(2H)-yl)-N-R-acetamides was obtained. A novel approach to
synthesis of the key intermediate - 2-(2,4-dioxo-1,4-dihydro-quinazolin-3(2H)-
yl)acetic acid was developed. The structure and purity of the resulting substances was
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confirmed by elemental analysis, H NMR, C NMR spectroscopy and LC/MS.
Based on the results of docking studies using SCIGRESS software, selected
compounds with the best affinity for anticonvulsant protein biomes (PDB codes:
4COF, 3F8E and 1 EOU) are promising for experimental studies of anticonvulsant
activity. A comparative analysis of the results of molecular docking and in vivo
results suggests that there is a positive correlation between scoring protein inhibition
and experimental data. Pharmacological studies have revealed the leader compound
2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-[(2,4-dichlorophenyl)methyl]acet-
amide, which improved all the experimental convulsive syndrome rates in mice
without motor coordination impairment and may be recommended for further
research. The lowest values of the scoring function of the ligand-peptide interaction
are obtained for the synthesized compound and сarbonic anhydrase II (gene name
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CA2) (PDB code 1 EOU), so its inhibition is proposed by us as the most probable
mechanism of the anticonvulsive effect of the leader compound.
Key
words:
synthesis,
2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-
yl)acetamide, molecular docking, anticonvulsant activity.
INTRODUCTION
Epilepsy takes one of the leading places among neurological diseases, which
greatly affects life quality and life expectancy. According to Lancet [1], mortality
from epilepsy today is 0.22% of global mortality. Disability-adjusted life years for
epilepsy is 0.5% of total, and this disease is in the top 5 of neurological diseases for
years of healthy life lost as a result of disability [1]. That is why the expansion of the
range of antiepileptic drugs is necessary for improving this situation. In world
neurological practice as in Ukraine, anticonvulsants of different generations and
different chemical structures are used [2, 3]. Unfortunately, at the present time, there
are no drugs that are equally effective at different forms and degrees of convulsive
disorders [4, 5]. It should be noted that despite the emergence of new modern
anticonvulsants, the first generation drugs – phenobarbital (I), phenytoin (II), which
have certain significant disadvantages – remain in demand in the treatment of
convulsive conditions. New anticonvulsant drugs: stiripentol (III), pregabalin (IV),
zonisamide (V), lamotrigine (VI), tiagabine (VII), topiramate (VIII) – do not give a
long-term therapeutic result, are dose-dependent and toxic (Fig. 1) [6-9].
Fig. 1. Chemical structures of known anticonvulsants
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One of the most promising directions in modern medical chemistry used for
purposeful synthesis of potential new APIs is the structural modification of known
drugs in order to improve their pharmacological properties. In view of this our
attention is attracted by the heterocyclic system of pyrimidine as the basis of the most
"old" anticonvulsant – phenobarbital. By modification of its structure we have
already received substances with promising anticonvulsant properties [10].
Continuing the research on the modification of the pyrimidine ring, we drew attention
to derivatives of quinazoline, which actively search for potential anticonvulsants [11],
and compound (IX) has previously been used in medical practice, called
methaqualone, as a hypnotic and anticonvulsant drug [12]. The versatility of synthetic
approaches to the production and chemical modification of quinazoline derivatives,
the presence of anticonvulsant activity in the spectrum of their pharmacological
activity is an indisputable justification for further studies of this group of compounds
[13-15]. We decided to modify the structure of methaqualone, replacing the aryl
substituent on the acetic acid residue, thus combining quinazoline scaffold with one
of the major neurotropic mediators, glycine. To block the hydrophilic carboxyl group,
its amination was planned using amines of different nature: alkyl, aryl-alkyl and aryl
(Fig. 2).
Fig. 2. Modification of the structure of methaqualone in the target compounds
MATERIALS AND METHODS
Reagents manufactured by Sigma-Aldrich, USA were used in this work. The
required reagents were purified using standard techniques. Control of the reactions
was carried out using thin-layer chromatography (eluent-ethyl acetate-hexane 1 : 2)
on the plates "Sorbfil UV-254". Manifestations of chromatograms were carried out in
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the UV rays of the "Chromatographic UVF 254/365 Illuminator" (mode 254 nm).
The melting points were determined using the Kofler block. Elemental analysis was
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determined by the Dumas method. H NMR spectra and C NMR spectra were
recorded on the Varian Gemini 400 MHz spectrometers and the Varian Gemini 100
MHz spectrometers. The solvent was dimethyl sulfoxide (DMSO-d6). Chemical
shifts are shown on a scale (m.ch.). LC/MS spectra were recorded with a PE SCIEX
API 150EX liquid chromatograph equipped with a UV detector (λmax 215 and 254
nm) and using a Luna-C18 column, Phenomenex (100 × 4 mm). Elution was started
with water and stopped with acetonitrile / water (95:5, v/v) using a linear gradient at a
flow rate of 0.15 mL/min and an analysis cycle time of 25 min.
The docking simulations were performed with the SCIGRESS software
package (Fujitsu, Fukuoka, Japan (license 742F6852C191)). Docking studies were
conducted with the methods of a fast and quantum molecular docking in order to
search the affinity of synthesized chemical structures to targets and to determine the
possible mechanisms of their anticonvulsive effect. The stages of molecular docking
with the help of the SCIGRESS software package were as follows:
1. The choice of bio-targets and determination of their active sites.
In the present study, to carry out the docking studies the proteins type-A γ-ami-
nobutyric acid receptors (GABAARs) (code 4COF), сarbonic anhydrase II (gene
name CA2) (codes 3F8E and 1OEU) domains were chosen as a targets for
anticonvulsant activity. The structure of proteins of the Human GABAA Receptor
(code 4COF) and сarbonic anhydrase II (gene name CA2) (code 3F8E and 1OEU)
were chosen from Protein Data Bank (PDB) [16].
Type-A γ-aminobutyric acid receptors (GABAAR) are the principal mediators
of rapid inhibitory synaptic transmission in the human brain [17]. Because the
reversal potential for chloride in most neurons is close to or more negative than the
resting membrane potential, activation of GABAA receptors tends to stabilize or
hyper polarize the resting potential, and can make it more difficult for excitatory
neurotransmitters to depolarize the neuron and generate an action potential. The net
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effect is typically inhibitory, reducing the activity of the neuron. A decline in
GABAAR signalling triggers hyperactive neurological disorders such as epilepsy.
The carbonic anhydrase inhibition has demonstrated interesting pharmacologic
applications such as anticonvulsant agents. These enzymes have shown a potential
target for designing anticonvulsant drugs with a novel mechanism of action. The
enzymes play an important role in the anion exchange processes [18]. Carbonic
anhydrase II (gene name CA2), is one of fourteen forms of human α carbonic
anhydrases, which catalyses reversible hydration of carbon dioxide. Inhibition of
carbonic anhydrase in the brain leads to the accumulation of CO2 in the brain and
inhibition of excessive paroxysmal discharges of the neurons, which causes the
antiepileptic activity of the drug. [19-21]. The protein type-A γ-aminobutyric acid
receptors, сarbonic anhydrase II (gene name CA2) have been modeled using the
electron crystallographic structure at 3,5Å resolution and were downloaded from the
RCSB Protein Data Bank (PDB ID: 4COF, 3F8E and 1OEU respectively)
(www.pdb.org). Water molecules were removed and hydrogens were added to crystal
structure of protein before docking. After assigning charge and protonation state final
refinement (energy minimization) was done using MM3 force field runs.
2. The 3D-optimization of all structures.
All chemical structures were generated using ISIS DRAW 4.0 software using
standard bond, lengths and angles. All structures were designed using the ISIS
DRAW 4.0 software and saved as .mol. files. Then these structures were imported
into the SCIGRESS software and saved in .csf format. The next step was the
optimization of all structures by the ММ3 molecular mechanics method.
3. The Fast dock and Fast quantum Dock into Active Site.
The docking study was performed using Scigress Explorer 7.7 installed in a
single machine running on a 3.4 GHz Intel Core 2 Duo Processor with 1GB RAM
and 160 GB Hard Disk with Windows XP as the Operating System. Fast docking
method, in which receptor is rigid and ligands are flexible, was adopted and binding
energy values were compared with each other. The main achievement of our
approach is a combination of fast search with a special account for overlooked
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physical interactions (the implementation of sophisticated quantum methods). At the
end of the docking study, the minimum Consensus scores for the best ligand position
for each of ligand was obtained. Energy minimization or complex optimization was
done for molecular docking calculations and to optimize geometries within the
binding site. Complex optimization gave ligands with minimum energy pose within
the active site cavity of the protein. The geometry of the molecules was optimized
using MO-G computational application that computes and minimizes an energy
related to the heat of molecule formation. The augmented Molecular Mechanics
(MM3) parameter was used for optimizing the molecules up to its lowest stable
energy state. This energy minimization was done until the energy change was less
than 0.001 kcal/mol or the molecules were updated almost 500 times. The valency
and hydrogen bonding of the ligands, as well as target proteins were subsequently
corrected using the Workspace module. Hydrogen atoms were added to protein
targets for correct ionization. For automated docking of ligands into the active sites
we used the genetic algorithm with a fast and simplified Potential of Mean Force
(PMF) scoring scheme. PMF uses types of atoms, which are similar to the empirical
force fields used in mechanics and dynamics. The minimization is performed by the
Fast-Dock engine, which uses a Lamarkian genetic algorithm (LGA), so that
individuals adapt to the surrounding environment (Population size: 500; Crossover:
0.2; Elitism: 50; Max Generations: 50 000; Mutation rate: 0.0; Convergence 0.1; Max
iterations: 1000; Rate: 0.01). Visual analysis of the complexes of compounds from
the active site of proteine (PDB ID: 4COF, 3F8E and 1OEU) was performed using
Discovery Studio Visualizer 4.0 program.
Pharmacological screening for anticonvulsant activity was performed on a
baseline model of convulsions in mice [22, 23]. Corazole (pentylenetetrazole) is a
classic proconvulsant, the effect of which is caused by inhibition of the GABA site of
the benzodiazepine receptor complex and the decrease in the intensity of GABA-
ergic inhibitory processes in the central nervous system. 72 adult random-bred albino
mice of either sex weighing 18-25 g were used. Animals were kept in conditions in
accordance with the hygiene rules and principles of the EU Directive 2010/10/63 EU
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for animal experiments. Mice were randomly divided into 12 groups (each was
formed from animals of either sex): (1) control (n = 12) – a model of convulsions
induced by pentylenetrazole, (Sigma, USA) at a dose of 90 mg / kg subcutaneously in
form of aqueous solution; (2) a reference drug group (n = 6) – sodium valproate
(Depakin, Sanofi-Aventis, France) at a dose of 300 mg / kg intragastrically 30
minutes prior to the administration of pentylenetetrazole; (3-12) groups of
experimental animals (n = 5-9), which were administered the test compounds at a
dose of 100 mg / kg into the stomach in the form of a suspension on twin-80 30
minutes before the pentylenetetrazole. The observation lasted 60 minutes. Latency of
the convulsions, the number of clonic-tonic convulsions in one mouse, % of animals
with clonic and tonic convulsions, the duration of the convulsive period (from the
first to the last attack) and the lifetime of the animals before death (in mice with
lethal outcome) were calculated. The severity of seizures was evaluated according to
a scale ranging from 1 to 6: 1 – trembling; 2 – circus movement; 3 – clonic seizures;
4 – clonic-tonic seizures with a lateral position; 5 – tonic extension; 6 – tonic
extension leading to the animal’s death. [24].
To evaluate the influence of compounds on motor coordination the rotarod
test was used [22, 23]. 72 adult random-bred albino mice of either sex (weighing 18-
22 g) were randomly divided into 12 groups (each was formed from animals of either
sex): (1) control – intact mice (n = 6); (2) a reference drug group (n = 6) – sodium
valproate (Depakin, Sanofi-Aventis, France) at a dose of 300 mg / kg intragastrically
30 minutes prior to the test; (3-12) groups of experimental animals (n = 6), which
were administered the compounds at a dose of 100 mg / kg into the stomach in the
form of a suspension on twin-80 30 minutes before the test. Number of mice that fall
from the rotating rod before 1, 2, 3 and 5 minutes has been determined.
Study of acute toxicity of a leader compound. To determine acute toxicity
using the express method by T. Pastushenko [25] the leader compound as a
suspension on tween-80 was administered into the mice stomach (3 animals per dose)
once in a wide range of doses (1000, 3000, 5000 mg / kg dose which are respectively
10, 30 and 50 times higher than the ED50). The volume of the suspension was 40 ml /
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kg, corresponding to 0.8 ml per mouse weighing 20 g. The animals were observed for
14 days, assessing their behaviour and physical condition and overall survival after
30 min, 1 h, 3 h, 6 h, 12 h, 24 h, 7 days and 14 days.
Statistical analysis. STATISTICA 8.0 for Windows has been used. Statistically
significant intergroup differences were evaluated by parametric Student’s t-test for
normal distribution and non-parametric Mann-Whitney U-test in case of his absence
[26, 27]. Quantitative data were presented as mean ± standard error of mean (SEM),
and for results in the alternative form (death – survival) – as the % of the observed
effect in the group. In this case, for the statistical significance determination the
Fisher’s angular transformation was used. Differences were considered statistically
significant as p < 0.05.
RESULTS AND DISCUSSIONS
The study described in the article was aimed at a targeted search for substances
with anticonvulsant activity. At the same time, for the synthesis of potential APIs, it
was necessary to develop an approach that allows to synthesize the aim products with
good yields and acceptable purity. Authors [28] have used for synthesis of analogous
substances the approach described by Bogert and Scatchard [29] via preparation
benzoyleneurea, allowed to synthesize anilide (compound 7h) with a yield 19%.
There are data in the literature about other approaches and attempts to introduce
amino acid residues into the quinazoline fragment. ‘Direct Amide Cyclization’ to
Peptides Containing an Anthranilic Acid Residue was used by Philipova et al. [30]
and other аuthors [31]; the interaction of methyl 2-isocyanatobenzoate with glycine
described in literature too [32]. However, the methods described do not allow to
obtain key intermediate -2-(2,4-dioxo-1,4-dihydro-quinazolin-3(2H)-yl)acetic acid
with appropriate yield. Therefore, we decided to develop a different approach for the
synthesis of planned substances in order to increase the yields of both key
intermediate and aim products.
Previously, we proposed an approach to the synthesis of combinatorial libraries
of condensed heterocycles containing the 2-mercapto-4-(3H)-pyrimidinone fragment
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[33], which consists in the cyclization of substituted 2-(methylcarboxy)benzene-
isothiocyanates with primary amines. This method advantageously differs from the
traditional synthesis of such compounds by cyclization of substituted methyl
anthranilates with isothiocyanates, since it allows the use as primary amines of
compounds such as amino acids for which it is not possible to obtain isothiocyanates,
therefore the traditional pathway of synthesis is unacceptable.
A starting material for the synthesis of a 2-(2,4-dioxo-1,4-dihydroquinazolin-3
(2H)-yl)-N-R-acetamide 7a-7n systematic series was used 2-(2,4-dioxo-1,4-dihydro-
quinazolin-3(2H)-yl)acetic acid 4 which was prepared by oxidation of 2-(4-oxo-2-
thioxo-1,4-dihydroquinazolin-3(2H)-yl) acetic acid 3 hydrogen peroxide heating to
70°C for 30 minutes. 2-(4-Oxo-2-thioxo-1,4-dihydroquinazolin-3(2H)-yl) acetic acid
3 was synthesized by adding methyl 2-isothiocyanatobenzoate 1 glycine 2 in the
presence of triethylamine in a medium of 2-propanol during boiling for 30 minutes
(Scheme 1).
Scheme 1
New derivatives of 2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl) acetamide
7a-7n were prepared by sequential interaction of 2-(2,4-dioxo-1,4-dihydroquinazolin-
3(2H)-yl) acetic acid 4 with N,N'-carbonyldiimidazole 5 and the corresponding
amines 6 in dioxane (Scheme 2).
2-(2,4-Dioxo-1,4-dihydroquinazolin-3(2H)-yl) acetic acid 4, in reaction with N,N'-
carbonyldiimidazole, forms an appropriate acylimidazole. The carbon dioxide
released during the reaction is a reaction catalyst for amines [34, 35], with the
resultant target 2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-R-acetamides.
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Scheme 2
6
5
4
7a-7n
7a R=CH3
7b R=C2H5
7c R=C3H7
7d R=C4H9
7h R=C6H5
7i R=CH2-C6H5
7j R=(CH2)2-C6H5
7k R=(CH2)3-C6H5
7l R=CH2-4-ClC6H5
7m R=CH2-2,4-diClC6H5
7n R=CH2-2-OCH3C6H5
7e R=sec. butyl
7f R=CH2-CH=CH2
7g R= cyclohexyl
Synthesized substances 7a-7n – white crystalline substances, easily soluble in
2-propanol, dioxane, dimethylformamide, not soluble in water.
The structure and purity of the compounds obtained were confirmed by
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elemental analysis, H NMR, C NMR spectroscopy and LC/MS. The reaction was
monitored by thin layer chromatography.
One of the reasons for the effective search for anticonvulsants is the high death
rate of animals in a screening experiment [36]. As a result, in order to pre-evaluate
the anticonvulsant potential of substances in silico methods, which allow us to select
not only the most promising substances, but also to predict the mechanism of action
and to correctly select the screening model, are prevalent [37, 38]. The most
commonly used method for such purposes is molecular docking.
Molecular docking gives possibility to reduce efforts and time due to carrying
out the procedure that is similar to high-performance biological screening. Knowing
the structure of the target (receptor or enzyme) and the structure of the ligand it is
possible to explain the mechanism of interaction at the molecular level and calculate
the strength of binding between them (affinity) [39]. The aim of the docking is the
search of the most suitable positions and orientations of the ligands in the ligand
binding centre of the receptor or enzyme, as well as identification of factors that may
lead to improvement of the ligand-receptor interaction.
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The target protein structures of 4COF, 3F8E and 1EOU were docked with new
quinazoline derivatives, which provided excellent results as were seen by the least
values of the binding energy in Tab. 1. The scores of docking results are based on the
free binding energies and the hydrogen bond interactions involved in the binding
mode. The obtained docking scores of results of synthesized derivatives are presented
in Table 1.
Table 1
Compound Dock scoring functions
Com-
pound
7a
1
EOU
4COF
The scores of Fast Dock
3F8E
-84.7
-94.8
-96.9
-68.5
-64.8
-75.0
-68.3
-64.6
-66.7
-70.4
-71.6
-72.6
-66.9
-75.6
-72.2
-82.9
-83.0
-28.1
-28.9
-26.6
-29.2
-21.4
-30.5
-21.4
-33.5
-33.8
-48.6
-54.3
-35.9
-55.3
-32.8
7b
7c
-96.8
-93.6
7d
7e
-93.3
7f
7g
-100.7
-110.8
-107.5
-106.9
-108.8
-104.0
-117.6
-115.5
7h
7i
7j
7k
7l
7m
7n
The results of docking studies have shown that utilization of benzyl, phenyl,
phenoxymethyl, 2,4-dihalogenbenzyl residue for the structure optimization of
quinazoline scaffolds is effective approach in novel anticonvulsant agents design and
may be taken as the variant of hybrid pharmacophore approach.
Based on the results of docking studies, 10 compounds with the highest affinity
for anticonvulsant biotargets - proteins (PDB codes: 4COF, 3F8E and 1 EOU) were
selected and are promising for experimental studies of anticonvulsant activity.
Molecule 7m showed better binding energies (the highest negative dock score
-117) to the protein 1 EOU than the others. The stability of this complex is due
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mainly to the energetically favourable geometric location of the ligands in the
active centres of these receptors, the formation of hydrogen bonds between them,
intermolecular electrostatic and donor-receptor interactions. This means that it can
fit well in the receptor cavity (1 EOU), forming an energetically most stable drug
receptor complex, thus inhibition of this protein is proposed by us as the most
probable mechanism for the implementation of the anticonvulsant effect (Fig.1)
Molecule 7m form enzyme-ligand complex with 1 EOU protein due to the
favorable acceptor and hydrogen bond between the Oxygen atoms of the quinazoline
fragment, and amide fragment with the aminoacid moieties of Asn 67, His64,
Thr199, Thr198. The 2,4-dichlorophenyl ring of 7m forms a hydrophobic
interaction (π-π interaction) with Asn 67, His 64 and almost occupies the same
hydrophobic pocket. Pi-Cationic and Pi-anionic interactions are formed between the
phenyl fragments of the molecules and the moietiy of Gln 92. π-Н interactions arise
with the participation of aromatic and quinazoline fragments of molecules with
aminoacid moieties of His64. Additional stabilization of complexes occurs as result
of π-π interaction of the quinazoline fragments of the molecules with aminoacid
moiety His119 (Fig. 1).
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Fig. 1. Complex of the ligand 7m with the binding site of protein (PDB ID:
1EOU).
The following colour codes have been employed: red, green and light blue
represent oxygen, chlorine and nitrogen atoms respectively; ashen gray forms the
carbon backbone. Active site amino acid residues are represented as sticks coloured
according to residue type (Sequence protocol-Karplus and Schultz Flexibility).
The variation of binding energy of the stable enzyme-ligand complexes is in
good agreement with the observed anticonvulsant activities of 2-(2,4-dioxo-1,4-
dihydroquinazolin-3(2H)-yl)-N-R-acetamides.
Analysis of the results of pharmacological screening for 10 selected
compounds shows that four compounds caused statistically significant anticonvulsant
effect according to the criterion of the integral protective index – reduction of
lethality compared with control (Table 2). They can be considered highly effective
anticonvulsants.
In addition, the aim of this study is to investigate the influence of alkyl chain
length and electronic effect of the electron donating- and withdrawing groups at
phenyl ring in amide fragment of 2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-R-
acetamides on the anticonvulsant activity and hence deduce the structure activity
relationship.
The presence of an aromatic ring in the structure of synthesized compounds
7h-7n increases the lipophilicity of molecules and thus facilitates their penetration
through the blood-brain barrier. Obviously, therefore, all these compounds have the
best anticonvulsant activity. The enhancement of activity is also facilitated by the
presence of electron-acceptor substituents in the 2-position of the aromatic nucleus.
The simultaneous presence of two chlorine atoms in the 2 and 4 positions of the
benzyl substituent brings the compound 7m into absolute leaders. Compound 7m
provided a complete protective effect lethality 0% vs. 91.7% in control, p < 0.01) and
not inferior to the standard anticonvulsant medicine sodium valproate without any
indication of positive effect on the course of the convulsions. Satisfactory indices
were in compounds 7m.
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Absolute leader-compound 7m improved all the experimental convulsive
syndrome indicators. It was 3.5 times longer (p < 0.05), lengthened the latency of the
convulsions, contributed to the maximum reduction in the number of clonic and tonic
paroxysms (severity ≥ 3 points) to 1.00 ± 0.32 versus 3.58 ± 0.42 in the control (p
<0.05) per animal. For animals with clonic (severity of 3 points) and tonic (≥ 4
points) attacks – 80% vs. 100% (p < 0.05) and up to 40% vs. 100% (p < 0.01). The
median severity of the convulsions against the background of compound 7 (13)
decreased by 2.1 times and was 2.80 ± 0.73 points against 5.83 ± 0.17 range in the
control (p < 0.01). The duration of the convulsive period decreased by 26.7 times and
was 0.45 ± 0.38 minutes versus 12.00 ± 1.34 min, (p < 0.01) in the control.
Two other "hit compounds" 7h and 7k, in addition to decreasing the lethality,
are improved by 5 indicators of convulsive syndrome, and compound 7c – 4
indicators (Table. 2). However, all of them, unlike compound 7m, only tendentiously
increased the latency of the convulsions.
An additional advantage of compound 7h is a significant decrease in the
number of the most dangerous tonic convulsions (the latter only occurred in 55.5%,
p < 0.01 vs. control, against the background of compound 7k – 80%, p < 0.05 vs.
control). Compound 7с did not affect this indicator.
Three compounds (7f, 7g and 7 n) showed a weak anticonvulsant effect. By
decreasing the lethality rate by 60-80%, they improved only 1-2 indicators of the
severity of the convulsive syndrome. In particular, compound 7g reduced the total
number of attacks per animal, compound 7f reduced only the number of tonic attacks,
and compound 7n reduced the number of both clonic and tonic attacks per animal
(Table. 2).
Two compounds 7g and 7i were indifferent to the pentylenetetrazole
convulsions, since they did not have the desired effect on any of their rates of
lethality at 80% and yielded to this indicator of sodium valproate group (Table 2).
One compound 7l revealed moderate proconvulsive properties: the lethality of
animals at the background of its application was equal to 100%, while the time of
death decreased by 1.9 times compared to control (p < 0.05).
14

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The results obtained confirm the results of molecular docking and show
GABA-positive properties of compounds 7m, 7h, 7c and 7k, since the convulsive
action of pentylenetetrazole is due to depressant effects on GABA-ergic inhibitory
processes.
Analysis of the results of rotarod test for 10 selected compounds (Tabl. 3)
shows that there are no substances impairing motor coordination of mice among 2-
(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)acetamide derivatives studied. However,
sodium valproate revealed mild motor impairment, significantly increasing the
number of mice that fell down from the rotating rod before 3 and 5 minutes.
In the study of acute toxicity, the median death dose (LD50) of leader
compound 7m could not be established, since administration of any study dose
(including a maximum dose of 5000 mg / kg) was not accompanied by death of
animals for 14 days. The assessment of the general condition of the animals did not
reveal manifestations of intoxication, such as lateral position, blepharoptosis,
hypersalivation, diarrhoea, etc. In addition, normal coordination of the movements
and tone of the skeletal muscles was maintained. When the test compound was
administered at doses of 3000 and 5000 mg / kg in 30 minutes and 1 hour, a
temporary decrease in motor activity of mice was detected due to overloading of the
volume of the administered solution of compound 7m.
Consequently, compound 7m is classified for toxicity class V according to
Hodge and Sterner [40] as practically non-toxic substance (LD50> 5000 mg / kg).
15

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Table 2
Influence of the investigated substances on the pentylenetetrazole-induced seizures n mice (M ± m)
% of mice with
Number of
clonic-tonic
convulsions
in 1 mouse
Severity of
the
convulsions,
points
Duration of
the
convulsive
period, min
Lifetime of
the animal Lethal
convulsions
Group of
animals
Dose,
mg/kg
n
Latency , min
to death,
min
ity, %
clonic tonic
Control
Sodium
valproate
7c
12
6
–
5.79±0.58
3.58±0.42
100
100
5.83±0.17
12.00±1.34
7.64±3.62
17.45±1.85
20.90
91.7
300
26.65±10.61**
2.00±0.82*
66.7**
50 **
2.83±0.98**
16.7**
5
5
5
9
5
5
5
5
5
5
100
100
100
100
100
100
100
100
100
100
7.50±3.20
6.60±3.69
3.80±0.55
1.40±0.24**
2.20±0.58
3.20±0.97
100 #
100 #
100 #
100 ##
100 #
100 #
80 *
100 #
80 *
5.00±0.45*
5.00±0.63
5.60±0.40
2.20±1.26**
9.80±7.83
13.11±6.49
1.24±1.06**#
14.05±2.98
9.98±5.44
3.05±2.76 **
4.54±1.89 **
0.45±0.38 **
7.25±2.93
6.89±0.70*
16.95±12.89
11.53±5.60
40.0*
60.0
80.0 #
7f
7g
7h
7i
100 #
55.5**
100 #
100 #
80 *
10.57±3.32
4.96±1.37
1.22±0.22**
3.00±0.55
4.22±0.46**
5.60±0.40
7.14±3.45* 33.3**
17.50±3.82
17.79±2.93
80.0 #
80.0 #
7j
7k
7l
11.43±4.06
15.51±11.13
4.82±1.30
20.38±10.00*
15.14±11.31
1.80±0.37*
1.20±0.37**
2.00±0.32*
1.00±0.32**
2.40±0.93
5.60±0.40
4.00±1.10 *
6.00±0.00 #
2.80±0.73 **
4.40±1.17
12.16±6.42 40.0 *
100 #
80 *
100 #
40 **
80 *
9.36±2.06*
–
12.45±5.91
100 ##
0 **
7m
7n
80 *
60.0
Note. Statistically significant differences:
1. compared with control group indicators: * – p<0.05; ** – p<0.01;
2. compared with indicators of sodium valproate group: # – p<0.05; ## – p<0.01.
16

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Table 3
Influence of the investigated substances on motor coordination (rotarod test)
Number of mice that fall from the rod, amount / percentage
before 1 min before 2 min before 3 min before 5 min
Group of animals n
Control
Sodium valproate 6
6
1 / 17%
3 / 50%
2 / 33%
2 / 33%
1 / 17%
1 / 17%
1 / 17%
2 / 33%
1 / 17%
3 / 50%
1 / 17%
3 / 50%
2 / 33%
4 / 67%
3 / 50%
3 / 50%
2 / 33%
2 / 33%
2 / 33%
3 / 50%
2 / 33%
4 / 67%
3 / 50%
4 / 67%
2 / 33%
5 / 83% *
3 / 50%
3 / 50%
3 / 50%
3 / 50%
3 / 50%
3 / 50%
3 / 50%
4 / 67%
3 / 50%
4 / 67%
3 / 50%
6 / 100% *
3 / 50% #
4 / 67% #
3 / 50% #
4 / 67% #
3 / 50% #
4 / 67% #
3 / 50% #
5 / 83%
6
6
6
6
6
6
6
6
6
6
7c
7f
7g
7h
7i
7j
7k
7l
7m
7n
4 / 67% #
5 / 83%
Note. Statistically significant differences:
1. compared with control group indicator: * – p<0.05;
2. compared with indicator of sodium valproate group: # – p<0.05.
General method of synthesis of 2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-
R-acetamides 7а-7n
Glycine (0.1 mol) 7.5 g dissolved in a mixture of 150 ml of water and 14 ml
(0.1 mol) of triethylamine. The resulting solution is added with stirring to a warm
solution of 19.2 ml (0.1 mol) of methyl ester of 2-isothiocyanatobenzoic acid in 200
ml of 2-propanol. The solution was boiled for 30 minutes and acidified with
hydrochloric acid (10-12.5 ml) to pH = 2. The formed precipitate was filtered,
washed with water and boiled under stirring in 200 ml of acetone. After cooling, the
precipitate is filtered off and washed twice with 20 ml of acetone. To a solution (0.3
mol) of sodium hydroxide in 400 ml of water at a temperature of 50°C and stirring,
add 2-(4-oxo-2-sulfanilidene-1,2,3,4-tetrahydroxy-quinazolin-3-yl) acetic acid (0.1
mol). Then, at intensive stirring at 5°C, hydrogen peroxide (35 ml of 50% (1 mol)
diluted 1 : 1 with water) is carefully added (drops or small portions of 5 ml) at a rate
such that the temperature of the reaction mixture does not exceed 70°C. The reaction
mixture is stirred for 30 minutes. After cooling to room temperature, add 9 ml of
17

ACCEPTED MANUSCRIPT
acetic acid. The formed precipitate is filtered, washed with 100 ml of water.
Recrystallize from a mixture of 50 ml of DMF and 200 ml of ethanol. To a
suspension of (0.1 mol) of 2-(2,4-dioxo-1,2,3,4-tetrahydroxyquinazolin-3-yl) acetic
acid in 200 ml of anhydrous dioxane is added (0.12 mol) of N,N'-
carbonyldiimidazole. The mixture is boiled for 2-4 hours, controlling the reaction
through thin-layer chromatography (eluent-ethyl acetate-hexane 1 : 2). After passing
the reaction, add (0.12 mol) of the corresponding amine and boil for 2 more hours,
controlling the passage of the TLC reaction (eluent - ethyl acetate-hexane 1 : 2). After
cooling, brew 500 ml of water. The formed precipitate is filtered off, washed with 50
ml of 2-propanol and recrystallized from a mixture of 50 ml of DMF and 200 ml of 2-
propanol.
2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-methylacetamide 7а
o
1
80% yield, T. melt. = 292-294 C (dimethylformamide-2-propanole 1:4). H NMR
(400 MHz, DMSO-d6) δ: 2.6 (d, 3H, -NH-CH3 J = 6,4 Hz ), 5.0 (s, 2H, -CH2-CO-),
7.32 (t, 1H, Ar–H, J = 6 Hz), 7.4 (d, 1H, Ar–H, J = 8 Hz), 7.73 (t, 1H, Ar–H, J = 6
Hz), 7.92 (d, 1H, Ar–H, J = 8 Hz), 7.96 (t, 1H, -CO-NH, J = 4 Hz), 13.0 ( s, 1H, NH
(het)). 13C NMR (100 MHz, DMSO-d6) δ: 167.08, 161.89, 150.11, 139.56, 135.08,
127.40, 122.54, 115.19, 113.85, 42.64, 25.52. 55.50% С, 4.50% Н, 18.00% N,
Сalculated for C11H11N3O3 Found,%: С 56.65, Н 4.75, N 18.02, LC-MС m/z: 234.0
[(M+H)+].
2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-ethylacetamide 7b
o
1
84% yield, T. melt. = 268-270 C (dimethylformamide-2-propanole 1:4). H NMR
(400 MHz, DMSO-d6) δ: 1.00 (t, 3H, -CH2-CH3, J = 8 Hz), 3.05 (quintet, 2H, -CH2-
CH3 ), 4.45 (s, 2H, -CH2-CO-), 7.19 (t, 2H, Ar–H, J = 9.3 Hz), 7.65 (t, 1H, Ar–H, J
= 8 Hz), 7.9 (d, 1H, Ar–H, J = 9.3 Hz), 8.03 (t, 1H, -CO-NH, J = 6.4 Hz), 11.5 ( s,
1H, NH (het)). 13C NMR (100 MHz, DMSO-d6) δ: 166.29, 161.87, 150.10, 139.53,
135.06, 127.40, 122.53, 115.19, 113.82, 42.58, 33.35, 14.71. 58.05% С, 5.17% Н,
17.29% N Сalculated for C12H13N3O3 Found,%: С 58.29, Н 5.30, N 17.09, LC-MС
m/z: 248.0 [(M+H)+]
2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-propylacetamide 7c
18

ACCEPTED MANUSCRIPT
o
1
82% yield, T. melt. = 312-314 C (dimethylformamide-2-propanole 1:4). H NMR
(400 MHz, DMSO-d6) δ: 0.8 (t, 3H, -CH2-CH3 J = 9.3 Hz ), 1.35 (kv, 2H, -CH2-CH3
J = 6.4 Hz ), 3.0 (kv, 2H, -NH-CH2- J = 2.6 Hz ), 4.45 (s, 2H, -CH2-CO), 7.18 (t, 2H,
Ar–H, J = 7 Hz), 7.63 (t, 1H, Ar–H, J = 7 Hz), 7.9 (d, 1H, Ar–H, J = 7 Hz), 8.05 (t,
1H, -CO-NH, J = 4 Hz), 11.4 ( s, 1H, NH (het)). 13C NMR (100 MHz, DMSO-d6) δ:
166.48, 161.87, 150.11, 139.54, 135.06, 127.41, 122.53, 115.18, 111.83, 42.58,
38.75, 22.36, 11.37. 59.46% С, 5.65% Н, 16.03% N Сalculated for C13H15N3O3
Found,%: С 59.76, Н 5.79, N 16.08, LC-MС m/z: 262.0 [(M+H)+].
2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-butylacetamide 7d
o
1
75% yield, T. melt. = 292-294 C (dimethylformamide-2-propanole 1:4). H NMR
(400 MHz, DMSO-d6) δ: 0.8 (t, 3H, -CH2-CH3 J = 8 Hz ), 1.15-1.4 (m, 4H, -CH2-
CH2-CH3 ), 3.0 (kv, 2H, -NH-CH2- J = 6.4 Hz ), 4.41 (s, 2H, -CH2-CO), 7.18 (t,
2H, Ar–H, J = 8 Hz), 7.65 (t, 1H, Ar–H, J = 7.6 Hz), 7.89 (d, 1H, Ar–H, J = 7.6 Hz),
8.0 (t, 1H, -CO-NH, J = 5 Hz), 11.4 ( s, 1H, NH (het)). 13C NMR (100 MHz, DMSO-
d6) δ: 166.41, 161.87, 150.10, 139.54, 135.08, 127.42, 122.54, 115.19, 113.84, 42.52,
38.32, 31.24, 19.51, 13.69. 61.02% С, 6.17% Н, 15.03% N Сalculated for
C14H17N3O3 Found,%: С 61.08, Н 6.22, N 15.26, LC-MС m/z: 276.2 [(M+H)+].
2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-(2-methylpropyl)acetamide 7e
1
82% yield, T. melt. = 270-272oC (dimethylformamide-2-propanole 1:4). H NMR
(400 MHz, DMSO-d6) δ: 0.8 (d, 6H, 2×CH3 J = 6.4 Hz ), 1.6-1.7 (m, 1H, -CH-
(CH3)2), 2.85 (t, 2H, -NH-CH2- J = 6.4 Hz ), 4.45 (s, 2H, -CH2-CO), 7.19 (t, 2H,
Ar–H, J = 6 Hz), 7.63 (t, 1H, Ar–H, J = 6 Hz), 7.88 (d, 1H, Ar–H, J = 6 Hz), 8.05 (t,
1H, -CO-NH, J = 4 Hz), 11.4 ( s, 1H, NH (het)). 13C NMR (100 MHz, DMSO-d6) δ:
166.55, 161.88, 150.12, 139.54, 135.07, 127.42, 122.54, 115.19, 113.85, 46.16,
42.58, 28.13, 20.10. 61.17% С, 6.36% Н, 15.06% N Сalculated for C14H17N3O3
Found,%: С 61.08, Н 6.22, N 15.26, LC-MС m/z: 276.0 [(M+H)+].
2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-(prop-2-en-1-yl)acetamide 7f
1
80% yield, T. melt. = 272-274oC (dimethylformamide-2-propanole 1:4). H NMR
(400 MHz, DMSO-d6) δ: 3.65 (t, 2H, -NH-CH2- J = 5 Hz ), 4.48 (s, 2H, -CH2-CO-),
5.05 and 5.13 (dd, 2H, -CH=CH2- J = 12 Hz, J = 16 Hz), 7.18 (t, 2H, Ar–H, J = 10
Hz), 7.65 (t, 1H, Ar–H, J = 10 Hz), 7.9 (d, 1H, Ar–H, J = 10 Hz), 8.25 (t, 1H, -CO-
NH-, J = 4 Hz), 11.4 ( s, 1H, NH (het)). 13C NMR (100 MHz, DMSO-d6) δ: 166.55,
19

ACCEPTED MANUSCRIPT
161.91, 150.12, 139.56, 135.09, 127.42, 122.55, 115.20, 115.12, 113.87, 42.63,
40.92. 60.11% С, 5.12% Н, 16.36% N Сalculated for C13H13N3O3 Found,%: С
60.23, Н 5.05, N 16.21, LC-MС m/z: 260.1 [(M+H)+]
2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-cyclohexylacetamide 7g
1
86% yield, T. melt. = 308-310oC (dimethylformamide-2-propanole 1:4). H NMR
(400 MHz, DMSO-d6) δ: 1.0-1,25 (m, 5H, cyclohexyl), 1.5 (d, 1H, cyclohexyl, J =
13.3 Hz ), 1.7 (t, 4H, cyclohexyl, J = 20 Hz ), 3.4-3.55 (m, 1H, -CH- (cyclohexyl)),
4.45 (s, 2H, -CH2-CO-), 7.19 (t, 2H, Ar–H, J = 8 Hz), 7.64 (t, 1H, -CO-NH-, J = 8
Hz), 7.9 (t, 2H, Ar–H, J = 10 Hz), 11.4 ( s, 1H, NH (het)). 13C NMR (100 MHz,
DMSO-d6) δ: 165.48, 161.83, 150.10, 139.50, 135.09, 127.42, 122.56, 115.19,
113.77, 47.80, 42.48, 32.47, 25.25, 24.57. 63.63% С, 6.18% Н, 13.82% N
Сalculated for C16H19N3O3 Found,%: С 63.77, Н 6.36, N 13.94, LC-MС m/z: 302.2
[(M+H)+].
2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-phenylacetamide 7h
o
1
75% yield, T. melt. = 294-296 C (dimethylformamide-2-propanole 1:4). H NMR
(400 MHz, DMSO-d6) δ: 5.26 (s, 2H, -CH2-CO), 7.02 (t, 1H, Ar–H, J = 8 Hz), 7.28
(t, 2H, Ar–H, J = 8 Hz), 7.35 (t, 1H, Ar–H, J = 8 Hz), 7.42 (d, 1H, Ar–H, J = 8 Hz),
7.53 (d, 2H, Ar–H, J = 8 Hz), 7.77 (t, 1H, Ar–H, J = 8 Hz), 7.97 (d, 1H, Ar–H, J =
8 Hz), 8.44 (t, 1H, -CO-NH-, J = 6 Hz), 11.55 ( s, 1H, NH (het)). 13C NMR (100
MHz, DMSO-d6) δ: 165.50, 161.89, 150.12, 139.52, 138.80, 135.29, 128.82, 127.71,
127.47, 126.22, 122.73, 115.30, 113.67, 43.12. 61.55% С, 4.20% Н, 13.20% N
Сalculated for C16H13N3O3 Found,%: С 61.72, Н 4.21, N 13.50, LC-MС m/z: 296.0
[(M+H)+].
2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-benzylacetamide 7i
o
1
84% yield, T. melt. = 294-296 C (dimethylformamide-2-propanole 1:4). H NMR
(400 MHz, DMSO-d6) δ: 4.25 (d, 2H, -NH-CH2- J = 5.7 Hz ), 4.5 (s, 2H, -CH2-
CO), 7.15-7.32 (m, 7H, Ar–H), 7.64 (t, 1H, Ar–H, J = 10 Hz), 7.9 (d, 1H, Ar–H, J =
10 Hz), 8.58 (t, 1H, -CO-NH, J = 8 Hz), 11.5 ( s, 1H, NH (het)). 13C NMR (100 MHz,
DMSO-d6) δ: 166.85, 161.95, 150.15, 139.57, 139.28, 135.11, 128.27, 127.44,
127.11, 126.77, 122.56, 115.21, 113.89, 42.74, 42.12. 66.12% С, 4.78% Н, 13.36%
N Сalculated for C17H15N3O3 Found,%: С 66.01, Н 4.89, N 13.58, LC-MС m/z: 310.0
[(M+H)+].
20

ACCEPTED MANUSCRIPT
2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-(2-phenylethyl)acetamide 7j
o
1
69% yield, T. melt. = 300-302 C (dimethylformamide-2-propanole 1:4). H NMR
(400 MHz, DMSO-d6) δ: 2.7 (t, 2H, -CH2-C6H5 J = 8 Hz ), 3.25 (s, 2H, -NH-CH2-),
4.45 (s, 2H, -CH2-CO-), 7.15-7.30 (m, 7H, Ar–H), 7.64 (t, 1H, Ar–H, J = 8 Hz), 7.92
(d, 1H, Ar–H, J = 8 Hz), 8.18 (t, 1H, -CO-NH, J = 5.3 Hz), 11.5 ( s, 1H, NH (het)).
13C NMR (100 MHz, DMSO-d6) δ: 166.69, 161.88, 150.12, 139.55, 139.44, 135.24,
128.68, 128.34, 127.42, 126.10, 122.70, 122.53, 115.29, 113.83, 42.63, 41.73, 35.16.
66.63% С, 5.22% Н, 13.08% N Сalculated for C18H17N3O3 Found,%: С 66.86, Н
5.30, N 13.00, LC-MС m/z: 324.2 [(M+H)+].
2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-(3-phenylpropyl)acetamide 7k
o
1
82% yield, T. melt. = 315-317 C (dimethylformamide-2-propanole 1:4). H NMR
(400 MHz, DMSO-d6) δ: 1.7 (kvin, 2H, -CH2-C6H5 ), 2.45-2.6 (m, 2H, -CH2-CH2-
CH2-), 3.05 (kv, 2H, -NH-CH2- , J = 5.3 Hz), 5.0 (s, 2H, -CH2-CO-), 7.1-7.2 (m,
3H, Ar–H), 7.24 (t, 2H, Ar–H, J = 9.3 Hz), 7.32 (t, 1H, Ar–H, J = 9.3 Hz), 7.39 (d,
1H, Ar–H, J = 10.6 Hz), 7.73 (t, 1H, Ar–H, J = 10.6 Hz), 7.93 (d, 1H, Ar–H, J = 10.6
Hz), 8.07 (t, 1H, -CO-NH-, J = 6.4 Hz), 12.95 (s, 1H, NH (het)). 13C NMR (100
MHz, DMSO-d6) δ: 166.61, 161.90, 150.13, 141.77, 139.55, 135.06, 128.32, 128.27,
127.41, 125.72, 122.62, 122.53, 115.20, 113.85, 42.67, 38.28, 32.45, 30.96. 63.00%
С, 6.10% Н, 17.00% N Сalculated for C19H19N3O3 Found,%: С 63.68, Н 6.12, N
17.09, LC-MС m/z: 338.2 [(M+H)+].
2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-[(4-chlorophenyl)methyl]
acetamide 7l
o
1
88% yield, T. melt. = 300-302 C (dimethylformamide-2-propanole 1:4). H NMR
(400 MHz, DMSO-d6) δ: 4.3 (d, 2H, -NH-CH2- , J = 5 Hz), 5.05 (s, 2H, -CH2-CO),
7.26 (d, 2H, Ar–H, J = 8.9 Hz), 7.34 (d, 3H, Ar–H, J = 8 Hz), 7.4 (d, 1H, Ar–H, J =
8.9 Hz), 7.73 (t, 1H, Ar–H, J = 8 Hz), 7.94 (d, 1H, Ar–H, J = 8 Hz), 8.6 (t, 1H, -CO-
NH-, J = 5 Hz), 13.0 (s, 1H, NH (het)). 13C NMR (100 MHz, DMSO-d6) δ: 167.03,
161.95, 150.15, 139.56, 138.38, 135.09, 131.37, 128.96, 128.19, 127.43, 122.56,
115.22, 113.87, 42.78, 41.49. 66.00% С, 5.15% Н, 13.20% N, 9.8% Cl Сalculated
for C17H14 ClN3O3 Found,%: С 66.01, Н 5.30, N 13.00, О 17.84, 9.85 Cl, LC-MС
m/z: 344.1 [(M+H)+].
2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-[(2,4-dichlorophenyl)methyl]-
acetamide 7m
21

ACCEPTED MANUSCRIPT
o
1
78% yield, T. melt. = 310-312 C (dimethylformamide-2-propanole 1:4). H NMR
(400 MHz, DMSO-d6) δ: 4.3 (d, 2H, -NH-CH2- , J = 6.4 Hz), 5.2 (s, 2H, -CH2-
CO-), 7.35 (t, 1H, Ar–H, J = 8 Hz), 7. 4 (t, 3H, Ar–H, J = 4.8 Hz), 7.58 (s, 1H, Ar–
H), 7.75 (t, 1H, Ar–H, J = 8 Hz), 7.95 (d, 1H, Ar–H, J = 8 Hz), 8.65 (t, 1H, -CO-NH,
13
J = 4.8 Hz), 13.0 (s, 1H, NH (het)). C NMR (100 MHz, DMSO-d6) δ: 167.27,
161.96, 150.15, 139.55, 135.44, 135.17, 132.86, 132.26, 130.05, 128.56, 127.45,
127.28, 122.61, 115.24, 113.84, 42.75, 41.93. 58.00% С, 6.12% Н, 15.20% N,
17.90% Cl Сalculated for C17H13 Cl2N3O3 Found,%: С 58.30, Н 6.20, N 15.26, 17.98
Cl, LC-MС m/z: 378.0 [(M+H)+].
2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-[(2-methoxyphenyl)methyl]
acetamide 7n
o
1
85% yield, T. melt. = 292-294 C (dimethylformamide-2-propanole 1:4). H NMR
(400 MHz, DMSO-d6) δ: 3.7 (s, 3H, -OCH3 ), 4.2 (d, 2H, -NH-CH2- , J = 6.4 Hz),
5.05 (s, 2H, -CH2-CO-), 6.84 (d, 2H, Ar–H, J = 8 Hz), 7.17 (d, 2H, Ar–H, J = 8 Hz),
7.34 (t, 1H, Ar–H, J = 8 Hz), 7.4 (d, 1H, Ar–H, J = 8 Hz), 7.74 (t, 1H, Ar–H, J = 8
Hz), 7.95 (d, 1H, Ar–H, J = 8 Hz), 8.5 (t, 1H, -CO-NH-, J = 6 Hz), 12.95 (s, 1H, NH
(het)). 13C NMR (100 MHz, DMSO-d6) δ: 166.91, 161.95, 156.60, 150.17, 139.56,
135.09, 128.03, 127.62, 127.43, 126.61, 122.55, 120.13, 115.20, 113.87, 110.43,
55.34, 42.72, 37.24. 60.00% С, 4.55% Н, 11.60% N Сalculated for C18H17N3O4
Found,%: С 60.83, Н 4.82, N 11.82, LC-MС m/z: 340.2 [(M+H)+].
CONCLUSIONS
A synthesis of new derivatives of 2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-
yl) acetamide 7a-7n was performed with good yields by amination of 2-(2,4-dioxo-
1,4-dihydro-quinazolin-3(2H)-yl)acetic acid 4 in anhydrous dioxane at CDI
presence. A novel approach for synthesis key intermediate 4 has been developed. It
was prepared by oxidation of previously synthesized 2-(4-oxo-2-thioxo-1,4-
dihydroquinazolin-3(2H)-yl) acetic acid 3 by hydrogen peroxide at 70°C. 2-(4-Oxo-
2-thioxo-1,4-dihydroquinazolin-3(2H)-yl) acetic acid was synthesized by interaction
of methyl 2-isothiocyanatobenzoate 1 with glycine 2 in the presence of triethylamine
in 2-propanol. The structure and purity of the all resulting substances were confirmed
by elemental analysis, 1H NMR, 13C NMR spectroscopy and LC/MS.
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Docking studies were performed to estimate anticonvulsant potential and to
select more prospective substances for pharmacological investigations. Among 10
compounds selected four compounds 7c, 7h, 7k, 7m caused statistically significant
anticonvulsant effect on pentyltetrazole seizures at animals. Compound 7m provided
a complete protective effect lethality and found to be superior to sodium valproate.
Experimental studies have found that it is a non-toxic substance, devoid of motor
coordination impairment. Taken together our docking results and in vivo results show
that there is a positive correlation between the dock scores and the inhibition of
protein сarbonic anhydrase II (gene name CA2) (PDB code 1 EOU).
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Acknowledgments
The authors gratefully acknowledge the financial support from the National University of
Pharmacy.
The authors have declared no conflict of interest.
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• New 2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)acetamides were
synthesized.
• A novel effective approach to synthesis of key intermediate was
developed.
• Docking studies was performed to select compounds for
pharmacological screening
• A leader compound 7m was found to be superior to sodium valproate.