Synthesis, computational and biological activity of heteroatomic compounds based on phenylthiourea and acetophenone

Article abstract of DOI:10.1016/j.molstruc.2020.128844

The presented article is devoted to a promising direction of organic chemistry - the synthesis of new sulfur- and nitrogen-containing thiourea and acetophenone derivatives that have physiological properties. So at first time on the base of phenylthiourea,

Full text of DOI:10.1016/j.molstruc.2020.128844

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Synthesis, computational and biological activity of heteroatomic compounds based on
phenylthiourea and acetophenone
Vagif Farzaliyev, Aleksey Shuriberko, Afsun Sujayev, Sabiya Osmanova, Sevinj
Gojayeva, Konul Gahramanova
PII:
S0022-2860(20)31169-8
DOI:
https://doi.org/10.1016/j.molstruc.2020.128844
MOLSTR 128844
Reference:
To appear in: Journal of Molecular Structure
Received Date: 19 January 2020
Revised Date: 30 April 2020
Accepted Date: 5 July 2020
Please cite this article as: V. Farzaliyev, A. Shuriberko, A. Sujayev, S. Osmanova, S. Gojayeva, K.
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phenylthiourea and acetophenone, Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/
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Synthesis, Computational and Biological Activity
of Heteroatomic Compounds based on Phenylthiourea and Acetophenone
Vagif Farzaliyeva, Aleksey Shuriberkob, Afsun Sujayeva*, Sabiya Osmanovaa,c,
Sevinj Gojayevaa, Konul Gahramanovaa
aLaboratory of Theoretical Bases of Synthesis and Action Mechanism of Additives,
Institute of Chemistry of Additives, Azerbaijan National Academy of Sciences, 1029, Baku,
Azerbaijan
b Laboratory of Bioregulator, Institute of Biochemistry of Biologically Active Compounds
of the NAS of Belarus, 230030, Grodno, Belarus
cDepartment of Natural Sciences, Western Caspian University,1001, Baku, Azerbaijan

Synthesis, Computational and Biological Activity
of Heteroatomic Compounds based on Phenylthiourea and Acetophenone
Vagif Farzaliyeva, Aleksey Shuriberkob, Afsun Sujayeva*, Sabiya Osmanovaa,c,
Sevinj Gojayevaa, Konul Gahramanovaa
aLaboratory of Theoretical Bases of Synthesis and Action Mechanism of Additives, Institute of Chem-
istry of Additives, Azerbaijan National Academy of Sciences, 1029, Baku, Azerbaijan
b Laboratory of Bioregulator, Institute of Biochemistry of Biologically Active Compounds of the
NAS of Belarus, 230030, Grodno, Belarus
cDepartment of Natural Sciences, Western Caspian University,1001, Baku, Azerbaijan
Address for Correspondences:
*Dr. Afsun Sujayev
aLaboratory of Theoretical Bases of Synthesis and
Action Mechanism of Additives
Institute of Chemistry of Additives 1029-Baku, Azerbaijan
Phone : +994503978801
Fax : +994125149210
E-mails : sucayevafsun@gmail.com
1

ABSTRACT
The presented article is devoted to a promising direction of organic chemistry - the syn-
thesis of new sulfur- and nitrogen-containing thiourea and acetophenone derivatives that have
physiological properties. So at first time on the base of phenylthiourea, acetylacetone and 37%
water solution of formaldehyde (formaline) in the presence of trifluoracetic acid has been elabo-
rated new effective synthesic method of 1-(6-Methyl-3-phenyl-2-sulfanylidene-1,2,3,4-tetra-
hydropyrimidin-5-yl)ethanone. Also, according to the reaction of thiylation of acetophenone and
some of its p-substituted derivatives Synthesis of 2,2'-((1-phenylethane-1,1-
diyl)bis(sulfanediyl))diacetic acid were obtained by us. A direct amidation reaction of Synthesis
of 2,2'-((1-phenylethane-1,1-diyl)bis(sulfanediyl))diacetic acid with primary amines was con-
ducted. The structure of the new compounds was interpreted by NMR spectra and X-ray
structure analysis.
As a result of studies, it was found that the greatest biological activity that can be useful
in creating drugs is compound 1-(6-Methyl-3-phenyl-2-sulfanylidene-1,2,3,4-tetrahydro-
pyrimidin-5-yl)ethanone. This compound is characterized by antioxidant effect significant effect
on the functioning of biological membranes, and in low concentrations (below 7.5 mg/l) it stabi-
lizes the membranes with an erythrocyte. The effect of an increase in the membrane potential of
mitochondria was discovered, probably associated with the ability to bind to carrier proteins. In
addition, when virtually predicting the spectrum of biological activity, a number of properties are
shown that have promise when creating new drugs.
Key words: phenylthiourea, acetophenone, heterocyclic, computer prediction, biological mem-
branes, antioxidant effect.
Running Head: Biological effects of heteroatomic compounds based on phenylthiourea and
acetophenone.
2

1. INTRODUCTION
In recent years, the study of the synthesis and properties of substances containing thiourea
fragments has been gained special attention in the chemistry of heterocyclic compounds. Appar-
ently, this is due to the fact that at present, these compounds have found and continue to find
broader areas of application in various branches of medicine, technology, industry and agricul-
ture. As it is seen from the literature, this field contains a large number of research works con-
ducted in various directions [1-4]. However, when analyzing the most recent literature, it be-
comes clear that the formulation of heterocyclic compound of thiourea by three-component con-
densation reactions mainly covers the synthesis of dihydropyrimidinethiones [5-8].
The dihydropyrimidinethiones display many pharmacological properties [9], as part of our
interest in this kind of materials, we report here the synthesis and crystal structure determination
of the title compound. Our synthesis is based in the Bijinelli reaction, which consists on a three-
component condensation of an aldehyde, a methyl-ene active compound and an thiourea deriva-
tive in acidic media. This procedure is the most simple and useful for the preparation of 3,4-
dihydropyrimidene-2(1H) thiones [10-13].
Despite the differences in the methodology used for the synthesis of dihydropyrimidine
2(1H)-ones by the Bijinelli reaction, these methods are not excluded in particularly substituted
aromatic and aliphatic aldehydes due to certain difficulties such as long reaction time and low
efficiency. However, despite these difficulties and also 125 years having passed after the first
application of single-phase three-component condensation method, interest in scientific research
in this area has not been reduced, on the contrary, it has increased and the above mentioned area
has been expanded much using all three components. Merely, the number of patent and scientific
articles published in recent years related to “Bijinelli condensation” is quite large.
At the same time, compounds containing amide groups in the molecule exhibit high
biological activity and are used in medicine, chemical technology, and other fields [14-18]. In
this regard, the development of effective methods for the synthesis of compounds with an amide
group is one of the important directions of modern organic synthesis. It should be noted that the
literature describes the amidation reactions of the simplest organic monoacids [19-21] and some
diacids [22-24]. However, there is completely no information on the amidation of bis-acids
containing heteroatoms in the molecule, in particular sulfur atoms.
2. MATERIALS AND METHODS
2.1. Measurements
3

The chemical structure of the synthesized compounds (M, M-1, M-2) was confirmed using
1H, 13C NMR spectra. At the same time, the structure of M and M-1 compounds was confirmed
by the X-ray structure analysis. NMR experiments have been performed on a Bruker FT NMR
1
13
spectrometer AVANCE 300 (300 MHz for H and 75 MHz for C) with BVT 3200 variable-
1
temperature unit in 5mm sample tubes using Bruker Standard software (Topspin 3.1). The H
and 13C chemical shifts were referenced to internal tetramethylsilane (TMS). NMR grade
DMSO-d6 (99.7%, containing 0.3% H2O) was used for the synthesized compounds.
2.2. Crystallography
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are
estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in
the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in
cell parameters are only used when they are defined by crystal symmetry. An approximate (iso-
tropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and
goodness of fit S are based on F2 , conventional R-factors R are based on F, with F set to zero
for negative F2. The threshold expression of F2 > σ(F2 ) is used only for calculating R-
factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on
F2 are statistically about twice as large as those based on F, and R- factors based on ALL data
will be even larger.
Data collection: The reaction time and the purity of the substances were controlled by the
NTX method (Sorbfil) (ethyl acetate/hexane = 1:2). The structure of the compounds was
confirmed by the X-ray analysis: “Bruker APEX II CCD” (T = 100 K, λMoKα radiation,
graphite monochromatic, φ- and ω-scanning, 2θmax = 56°). SAINT-Plus [25] was exploited for
cell refinement and data collection. SHELXTL [26] software was utilized to solve and refine the
structures structure.
2.3.Experimental
2.3.1. 1-(6-methyl-3-phenyl-2-sulfanilden-1,2,3,4-tetrahidropirimidin-5) etanon (M-1)
Phenylthiourea, 15.2 g (0.1 mol), 37% water solution of formaldehyde (formaline), 3 g (0.1
mol) and 13 g (0.1 mol) of acetylacetone were dissolved in 10 ml of ethanol and then 0.5 ml of
trifluoroacetic acid was added. The mixture was vigorously stirred during 4–5 h at room temper-
ature and then cooled and kept one day at 0°C. The white crystals of 1-(6-Methyl-3-phenyl-2-
4

sulfanylidene-1,2,3,4-tetrahydropyrimidin-5-yl)ethanone (M-1) were filtered and washed with
dichloromethane. Suitable single crystals for X-ray analysis were obtained by slow evaporation
from ethanol. H atoms were placed in calculated position and refined using a riding model, with
C—H distances in the range 0.95-0.99 Å and N—H distance of 0.88 Å, with Uiso(H) =
1.2Ueq(N,Cmethylene and Caromatic) and 1.5 Ueq (Cmethyl ). M-1 possesses orthorhombic
orientation (Pna21). Unit cell data, and bond lengths are given in Supporting Information (SI).
1-(6-methyl-3-phenyl-2-sulfanilden-1,2,3,4-tetrahidropirimidin-5) etanon (M-1): yield 19.2 g
(70%), m.p. =180°C, Rf = 0.35. Eluent - ethanol:hexane (5:2).1H NMR (300 MHz, DMSO-d6, δ)
13
1.35 (s, 3H, CH3), 6.8–7.1 (m, H, Ar), 7.4 (m, H, Ar), 9.35 (s, 1H, NH). C NMR (75 MHz,
DMSO-d6, δ) 24, 29, 37, 51, 86, 117, 122, 129, 132, 141, 151, 179 (C=S), 205 (C=O).
2.3.2. Synthesis of 2,2'-((1-phenylethane-1,1-diyl)bis(sulfanediyl))diacetic acid (M).
To the acetophenone (12 g, 0.1 mol) in C6H6, mercaptoacetic acid (23 g, 0.25 mol) was
added dropwise at room temperature. The mixture was stirred at 80°C for 9 h. The synthesized
2,2'-((1-phenylethane-1,1-diyl)bis(sulfanediyl))diacetic acid (M) is a white crystalline substance,
which is separated by recrystallization from water. M dissolve well in alcohol, acetone, and
poorly in hexane and benzene. Single crystal of M was developed with the same procedure
mentioned above for M-1. Space group of M is observed to be P-1 with triclinic orientation.
Bond lengths and dehedral angles were given in SI.
2,2'-((1-phenylethane-1,1-diyl)bis(sulfanediyl))diacetic acid (M): yield 23.2 g (81.1%), m.p.
1
136–137°C; HNMR (300 MHz, DMSO-d6, ꢀ): 1.92 (t, 3H, CH3); 3.38 (q, 2H, CH2); 7.12 (m,
5H, CH-Ar); 12.34 (m, 1H, OH). 13C NMR (75 MHz, DMSO-d6): 25.4; 38.3; 56.6; 127.2; 128.7;
139.2; 173.5.
2.3.3. Synthesis of 2,2'-((1-phenylethane-1,1-diyl)bis(sulfanediyl))bis(N-phenylacetamide)
(M-2)
M-2 was syntheszied from M according to the following procedure: Mixture of 2,2'-((1-
phenylethane-1,1-diyl)bis(sulfanediyl))diacetic acid (M) (10.0 g, 0.035 mol) and 2-
aminopyridine (6.6 g, 0.07 mol) was stirred in benzene solution at 80°C for 10 h. The reaction
was carried out in the presence of a catalytic amount of boric acid. Re-crystallization from EtOH
gave white crystal substance: When bis-acid (M) and amine were mixed, an exothermic effect
was observed, which can be explained by salt formation. The synthesized bis-amide was a
5

crystalline substance with a melting point of 92 °C, readily soluble in polar and insoluble in non-
polar solvents.
2,2'-((1-phenylethane-1,1-diyl)bis(sulfanediyl))bis(N-phenylacetamide) (M-2): yield, 14.5 g
1
(95.0%), m.p. 92°C, H NMR (300 MHz, DMSO-d6, ꢀ): 1.92 (s., 3H, CH3); 2.02 (s., 3H, CH3);
3.33 (d., 2H, CH2); 7.45; 8.12; 8.25; 8.96 (m., 4H, CH-Ar); 7.12; 7.26 (t., 2H, CH-Ar); 10.01
13
(m., 1H, NH). C NMR (75 MHz, DMSO-d6, ꢀ): 24.6; 35.6; 55.8; 117.8; 123.8; 128.7; 129.0;
141.6; 143.6; 168.2.
2.4. Biochemical Studies
2.4.1. Effects on biological membranes
The study used compounds M-1, M-2 and an extract of phenolic compounds of ginger
root (Zingiber officinale). Solutions of compounds M-1 and M-2 were prepared by dissolving in
DMSO; the range of studied concentrations was 240–1.88 mg/L in the final volume of the incu-
bation solution.
Ginger phenolic compounds were extracted from ginger root crushed and dried at 45 °С.
A Soxhlet apparatus was used, 96% ethanol and 0.01% HCl as a solvent, extraction temperature
of about 50 °C (reduced pressure) for 2 hours. The resulting extract was filtered, partially evapo-
rated on a rotary evaporator at a temperature of 45 °C. The resulting solution was cooled to -20,
the precipitated crystalline mass was separated and dried in a vacuum oven. The resulting prod-
uct is a concentrate of phenolic compounds, with a predominance mainly of shogaols and gin-
gerols. In studies, the extract was also dissolved in DMSO; the range of studied concentrations
for hemolysis was 30-0.47 mg/L in the final volume of incubation.
Red blood cells were obtained from heparinized blood of outbred white rats. The first
erythrocyte sedimentation from the blood was carried out at 600g for 10 minutes, then it was
washed twice with 10-fold saline solution (0.9% NaCl) using the same centrifugation modes.
The final mass of red blood cells (1 ml) for experiments was resuspended in 7.5 ml of 0.9%
NaCl.
The incubation medium contained 0.5 ml of a suspension of red blood cells, 1 ml of 0.9%
NaCl and 10 μl of the test substance in DMSO. As a positive hemolysis sample, 1 ml of dH2O
was added instead of 1 ml of NaCl. A control sample without hemolysis of 0.5 ml of a suspen-
sion of red blood cells, 1 ml of 0.9% NaCl and 10 μl of DMSO. The degree of hemolysis was
calculated in% according to the formula:
6

Х%=Е ꢂЕ ꢄ∗ꢅꢆꢆ
ꢁ
ꢃ
ꢄ∗Е
ꢇ
Where E1 is the optical density of the control (NaCl) sample.
E2 - optical density of the test sample.
E3 - optical density of hemolysis (H2O) sample.
2.4.2. Antioxidant activities
For the preparation of brain homogenate, the freshly isolated brain of outbred white rats
was homogenized with PBS at a tissue/buffer ratio of 1:9 per glass/Teflon homogenizer, 1300
rpm, 5-9 strokes. The resulting homogenate was clarified by centrifugation at 1500 g for 10
minutes. The supernatant was used in the experiment.
The incubation medium contained 200 μl of brain homogenate, 800 ml of PBS and 10 μl of
sample. We studied M-1 and M-2 at a concentration of 1.88-120 mg/L, ginger extract 1.88-240
mg/L and Trolox at a concentration of 3.3 mg/L as a reference substance. Solutions were pre-
pared in DMSO medium; the control contained 10 μl of DMSO. The mixture was incubated for 1
hour at 37 °C with constant stirring. At this time, another such mixture was prepared, but which
was not incubated and was used as a zero point. After that, the MDA content was determined by
the method with thiobarbituric acid.
2.4.3. The influence of M-1 and M-2 on the membrane potential of mitochondria of
brown adipose tissue and rat liver.
Tissue for isolation of mitochondria (interscapular brown adipose (BF) and liver) was re-
moved from white outbred rats in a cold room. All procedures were carried out at + 4 °C. Mito-
chondria were isolated by differential centrifugation; for this, the tissues were homogenized in a
Potter-Elveyem type homogenizer, tissue/extraction medium ratio 1:9, homogenization mode:
1300 rpm, 15 strokes for BWT and 5 strokes for the liver. The isolation medium of the composi-
tion was used: 0.25 M sucrose, 1 mM EGTA, 20 mM HEPES, pH 7.4. The resulting homogenate
was centrifuged at 600 g for 10 min, + 4 °C. The supernatant was collected and reprecipitated at
12000g, 20 min. The mitochondrial sediment was repeatedly washed with a 10-fold volume of
isolation medium.
The membrane potential was recorded by the change in fluorescence of the penetrating saf-
ranin O cation at an excitation wavelength of 495 nm and an emission of 586 nm. The membrane
potential recording medium (1 ml) consisted of: 0.125 M sucrose, 0.02 M HEPES, 0.05 M KCl,
1 mM EGTA, 20 mM KH2PO4, 5 mM MgCl2, 6 μM safranin O, pH 7.4, 300 μg mitochondrial
7

protein, incubation temperature 28 °C. To prevent the action of fatty acids adsorbed on mito-
chondria, 0.01% fat-free bovine serum albumin was added to the incubation medium. As a sub-
strate for oxidation (SUB), a mixture of pyruvate, malate, and succinate is 5.1.5 μM, respective-
ly. To block the action of UCP1 protein and other antiporters, 3 mM guanosine diphosphate
(GDP) was added, phosphorylation was started by adding 100 μmol ADP. The membrane poten-
tial was discharged (point 0) by administering 1 μM FCCP.
Samples of substances were prepared as previously indicated, as a control, 10 μl of DMSO
was added, which at this concentration did not lead to visible changes.
3. RESULTS AND DISCUSSION
3.1. Chemistry
The dihydropyrimidinethiones display many pharmacological properties, as part of our in-
terest in this kind of materials, we report here the synthesis and crystal structure determination of
the title compound [27]. Our synthesis is based in the Bijinelli reaction, which consists on a
three-component condensation of an aldehyde, a methylene active compound and an thiourea
derivative in acidic media. In other words, 1-(6-methyl-3-phenyl-2-sulfanilden-1,2,3,4-
tetrahidropirimidin-5) etanon was formed as a result of the interaction of phenylcyanocarbamide,
37% water formaldehyde and acetylacetone [28]. (M-1) compound synthesized (Scheme 1):
Scheme 1
The mechanism of this reaction can be predicted by Kappe's theory. Thus, initially, a
proton is formed by the combination of a proton. This cation reacts with the enol form of β-
diketoefir and forms thioureid. In the latter phase, cycling occurs with the separation of the water
molecule.
The effect of aldehydes on the release of the reaction product in the synthesis processes
was investigated. It has been shown that the reaction product yield is higher when the aromatic
aldehydes are absorbed than the aliphatic aldehydes. This is explained by the fact that in the
aromatic aldehydes the carbonyl group has a high activity due to the electron-acceptor properties
of phenyl radicals, which increases its reactivity.
The synthesized amides were prepared in two steps. By the thiolation reaction of aceto-
phenone with mercaptoacetic acid,
bis-acid,
namely 2,2'-((1-phenylethane-1,1-
diyl)bis(sulfanediyl))diacetic acid (M), was obtained in the first step (Scheme 2):
Scheme 2
8

The reaction was carried out by heating a mixture of components in a benzene solution in
the presence of catalytic amounts of benzenesulfonic acid. Acetophenones were found to react
with mercaptoacetic acid to produce 2,2'-((1-phenylethane-1,1-diyl)bis(sulfanediyl))diacetic acid
(M). The synthesized 2,2'-((1-phenylethane-1,1-diyl)bis(sulfanediyl))diacetic acid (M) is a white
crystalline substance, which is separated by recrystallization from water. M dissolve well in
alcohol, acetone, and poorly in hexane and benzene.
In
the
second
step,
the
reactions
of
2,2'-((1-phenylethane-1,1-
diyl)bis(sulfanediyl))diacetic acid (M) with 2-aminopyridine [29-33] were carried out and we
obtained
a
new
bis-amide
-
2,2'-((1-phenylethane-1,1-diyl)bis(sulfanediyl))bis(N-
phenylacetamide) (M-2) (Scheme 3).
Scheme 3
3.2. X-ray structural determination
Crystals for X‐ray spectroscopic analysis or X‐ray crystallographic analysis are obtained by
double crystallization of M-1 and M-2 compoundes. The structure and crystalline form of (M-1)
compound have been shown in the figure 1, but the selected difference for bond length and
valence angle has been described in SI. (M-1) Crystalline compound holds crystallographical
independent molecules in the central bicyclic fragment. (M-1) molecule holds complex three-
organic compound system consisting of tetrahydropyrimidine and benzol ring. In bicyclic
fragment the cycles of tetrahydropyrimidine take a shape of symmetric platform. In this case
benzol cycle in practice directs to the cycle of hexahydropyrimidine prependicularly. The
relevant conformation of three cyclic fragment were determined in the previously learned
relative compounds.
As can be seen from SI (M-1) molecule is placed at phenylthiourea fragment in
compressed form– N(1)-C(2) və N(3)-C(2) bond length becomes short, in C(2)=S(1) bond
according to it becomes long in comparison with statistical C-N single and C=S double bonds.
Single crystal X‐ray diffraction of compound M-1 was carried out on a Bruker APEX II
CCD diffractometer (T = 296 K, λMoKα‐radiation, graphite monochromator, φ‐ and ω‐scanning,
θmax = 28.4°, θmin = 1.7° ). The crystals of (M1) (Figure 1). compound (C13H14N2OS, Mr =
246.32) are colourless crystal, with cube structured, sizes 0.30 × 0.30 × 0.30 mm, one striped: a
= 24.3527 (10) Å, b = 7.2374 (3) Å, c = 7.0063 (3) Å, θ = 2.9–28.3°, Z = 4, Mo Kα radiation μ
= 0.25 mm-1, T = 100 K, 0.30 x0.30x0.30 mm, 13745 measured reflections 3089 independent
reflections 2877 reflections with I >2σr(I) Rint = 0.016 19 restraints H-atom parameters con-
strained ∆ρ_max=0.40 e Å^(-3) ∆ρ_min=-0.35 e Å^(-3). In the compound, the C8, C9, C11 and
9

C12 atoms of the phenyl ring are disordered over two sets of sites in a 0.60 (3):0.40 (3) ratio.
The heterocycle ring is essentially planar (r.m.s.= 0.017 Å) and form a dihedral angle of 82.0
(2)° with the phenyl ring. The crystal packing is stabilized by intermolecular N3—H3N···O1
hydrogen bonds (Table 1), which link the molecules into chains running parallel to the b axis,
with graph-set notation C(6). Intermolecular hydrogen bonds are formed with NH groups acting
as donors and O atoms as acceptors, N-H…O.
(M-1) compound is diastereoisomer asymmetrical centre of carbon atom. The crystal of the
studied compound shows racemate in chiral atoms.
Figure 1
The crystals of 2,2'-((1-phenylethane-1,1-diyl)bis(sulfanediyl))diacetic acid (M) compound
(C12H14O4S2, Mr=286.35) are white crystal, with triclinic structured, sizes 0.260 x 0.240 x 0.210
3
mm , one striped: a = 6.5374(16) Å, b = 7.1581(17) Å, c = 14.551(4) Å, β = 77.273(4)°, V =
3
3
-1
654.1(3) Å , space group P-1, Z= 2, ds = 1.454 Mg/m , μ = 0.410 mm . 5712 reflection
intensity has been measured (2396 nonarbitrary reflections [R(int) = 0.0278]) and semiempirical
correction against absorption by the help of SADABS program has been made for them. The
crystal packing is stabilized by intermolecular O(1)-H(1)...O(4)#1 and O(3)-H(3)...O(2)#1 hy-
drogen bonds (Figure 2, Table 2).
3.3. BIOCHEMICAL RESULTS
3.3.1. Study of the effect of M-1 and M-2 on the amount of hemolysis of red blood
cells
The hemolysis value shows how much the substance interacts with the biological mem-
brane, in this case, the erythrocyte membrane. High hemolytic activity indicates the ability of a
substance to form pores in the membrane or to completely destroy it, leading to the release of
intramembrane contents. This test is also important when testing drugs for parenteral administra-
tion, since many medicinal substances have this activity and lead to the so-called drug hemolytic
anemia.
The experimental results are presented in Figure 3.
Figure 3
As can be seen from the graph, the compound M-2 has an extremely low hemolytic activ-
ity, the maximum effects are at a concentration of 120 ml/l (table 3) where 6.80% of erythrocyte
hemolysis is observed. The M-1 substance is characterized by greater activity on the hemolysis
10

index, so at a concentration of 120 mg/l, 80.03% hemolysis was detected. These values are not
significant, since such a high concentration in the blood (120 mg/l) is difficult to achieve and the
actual concentration of drugs in the blood is much lower. In addition, concentrations of sub-
stances M-1 and M-2 below 15-7.5 mg/l even exhibit some membrane-stabilizing effects, where
the level of hemolysis was lower than in the control samples.
At the same time, the relatively harmless natural components of ginger root had signifi-
cant hemolytic activity, so at a concentration of 28-30 mg/l, complete hemolysis of red blood
cells was observed in the incubation medium.
Table 3
3.2.2 Study of antioxidant properties.
The study of antioxidant properties was carried out on an in virto model using clarified
brain homogenate. During homogenate incubation, a working complex of enzymes and autooxi-
dation of biological substrates leads to the appearance of free radicals, including reactive oxygen
species. The occurring free radical processes initiate the oxidation of the unsaturated fatty acids
contained in the homogenate and increase the level of malondialdehyde (MDA). This model is
more acceptable from the point of view of antioxidants of biological significance since it is as
close as possible to processes occurring in the body, unlike, for example, studies based on the
reduction of Fe3+→ Fe2+, Cu2+ → Cu+, DPPH, ABTS etc.
Data on testing the test substances for antioxidant activity are presented in Figure 4.
Figure 4
Trolox at a concentration of 3.3 mg / L was chosen as a known molecule with a powerful
antioxidant effect for comparison. The M-2 substance in this test behaved as a prooxidant in the
entire concentration range. The maximum prooxidant effect was achieved at a concentration of
M-2 in solution of 60 mg/L and amounted to 31.38% increase in the level of MDA production
from the control, where pure DMSO was used (Table 4). The substance M-1 has some antioxi-
dant effect, although it is relatively small in relation to the same concentrations of the phenolic
extract of ginger and Trolox. In small doses (below 3 mg/L), slight induction of MDA formation
was observed, and the maximum antioxidant effect was at a concentration of M-1 of 60 mg/L,
which reduced the production of malondialdehyde by 19.09%. Ginger phenolic compounds have
quite good antioxidant properties and, at concentrations above 30 mg/L, they almost completely
suppress oxidative reactions in this test.
Table 4
11

Thus, M-2 is a prooxidant, M-1 is an antioxidant 21.2 times weaker than Trolox, ginger
phenolic compounds are 1.84 times weaker than Trolox.
3.3.2. The effect of M-1 and M-2 on the mitochondrial membrane potential of
brown adipose tissue and rat liver.
The study of the effect of substances on the mitochondrial membrane potential (MP) al-
lows one to judge the effect of the test substance on the mitochondrial membrane and on the ac-
tivity of the electron transport chain, which includes enzymes for substrate oxidation, electron
transfer, and ATP synthesis. An effect on specific transmembrane proteins (carriers, ion chan-
nels) can also be detected. In addition, the studied substances themselves may turn out to be
sources of ion channel formation and induce the transition of both metal ions and protons, which
will affect the proton transmembrane gradient (membrane potential).
Characteristic of the control line. The first minute the incubation medium containing the
substrate and BSA is prescribed, after the introduction of mitochondria (Mito), the probe is ab-
sorbed by the mitochondria and the MT accumulates. At this stage, the effect of separation by
fatty acids was removed, but the active ion channels and mainly UCP1 in BZhT lead to MP dis-
sipation. The following is the introduction of GDF, which blocks UCP1 and other channels, at
this stage the MP has maximum value and stability. The increase in MP after HDF indirectly
characterizes the activity of UCP1. If MP after the introduction of mitochondria in the presence
of BSA almost immediately reached its maximum value and subsequent HDF leads to a slight
increase, this indicates a low activity of UCP1. The presence of ADP causes a slight decrease in
MP spent on ATP synthesis. FCCP causes a complete reset of the polarization of the membrane
and is used as a zero point in the calculations.
It was found that compound M-1 has a more significant effect than M-2, manifested in an
increase in membrane potential in the state of substrate stimulation on mitochondria of BZhT
(30.34% higher than the control sample at a concentration of 30 mg/l) (Figure 5). At the same
time, significant effects on the mitochondria of the liver were not revealed over the entire range
of tested concentrations (1.88-120 mg/l) (Figure 6). An increase in membrane potential at the
stage of substrate stimulation is associated with blocking of ion channels. Taking into account
the fact that M-1 does not influence a similar stage in liver mitochondria, it should be assumed
that M-1 binds and blocks UCP1, since this channel is most significant for BJT mitochondria and
is absent in liver mitochondria. A slight increase in MP was also observed after the administra-
tion of HDF. Thus, compound M-1 confirms its stable action on the membrane. In addition, it
can probably bind to some proteins on the surface of the membrane, which requires further
12

study. An M-1 concentration of 60 mg/L has the same effect as 30, but 120 mg/L leads to MP
inhibition and side effects when measuring the fluorescence of a voltage-sensitive probe.
Figure 5
In the range of tested concentrations (1.88–240 mg/L), the M-1 compound did not cause
significant changes in the membrane potential of the liver mitochondria, insignificant line vibra-
tions are associated with optical effects and their effect on fluorescence at high concentrations of
the substance (Figure 6).
Figure 6
In similar studies of the M-2 substance, no significant effects were found on both mito-
chondria of BWT and the liver (Figure 7 and Figure 8).
Figure 7 and Figure 8
3.3.3. Computer prediction of the spectrum of biological activity
Computer prediction was performed by the PASS program (Prediction of Activity Spec-
tra for Substances). Initial computer screening allows the selection of promising compounds
based on their structure and analogy of biological activity with known substances.
The data on the prediction of the biological activity of compound M-1 are presented in table 4.
Of the 537 predicted activities, 44 were selected with a forecast probability of over 40%.
Table 5
As can be seen from the table, some types of biological activity (membrane permeability
inhibitor - 66.6%, calcium channel blocker -33.6%) are confirmed by the studies above, where
M-1 stabilized red blood cells at low concentrations, leading to an increase in the membrane
potential of mitochondria. In addition, the spectrum contains predictive activities that are im-
portant in medicine, for example, with a high degree of probability M-1 will have an analgesic
effect, antiviral and an agent for coronary artery disease.
The following table 4 presents the data on the prediction of the biological activity of
compound M-2. 744 types of activity were detected, the bulk of which is the inhibition of vari-
ous enzymes, 58 of these activities, a positive prognosis of which above 40% are presented in
table 6.
Table 6
13

According to prediction, M-2 is an inhibitor of many enzymes, including those important
for metabolism (flavoprotein dehydrogenases). However, some effects may still be useful in bi-
ology and medicine (esterase inhibitor, TRPA1 agonist, virus attachment inhibitor).
4. CONCLUSİONS
As a result of studies, it was found that the greatest biological activity that can be useful
in creating drugs is compound M-1. This compound is characterized by antioxidant effect signif-
icant effect on the functioning of biological membranes, and in low concentrations (below 7.5
mg/l) it stabilizes the membranes with an erythrocyte. The effect of an increase in the membrane
potential of mitochondria was discovered, probably associated with the ability to bind to carrier
proteins. In addition, when virtually predicting the spectrum of biological activity, a number of
properties are shown that have promise when creating new drugs.
Compound M-2 is characterized by prooxidant activity, inertness with respect to biologi-
cal membranes. Although the predicted biological effect based on the structure of the compound
is also of interest for further study. The data obtained indicate the promise of this area of re-
search, the need to verify other biological properties of the studied compounds. By varying other
substituents in the structure of the compounds, it is possible to obtain preparations with a given
biological activity.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
This work was carried out as part of international collaborative research of the Institute of Chem-
istry of Additives of ANAS and the Institute of Biochemistry of Biologically Active Compounds
of the NAS of Belarus.
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17

Scheme 1. Synthesis of 1-(6-methyl-3-phenyl-2-sulfanilden-1,2,3,4-tetrahidropirimidin-5)
etanon (M-1)
Scheme 2. Synthesis of 2,2'-((1-phenylethane-1,1-diyl)bis(sulfanediyl))diacetic acid (M)
O
H
N
SCH2COOH
SCH2C
N
N
+
H2N
CH3
CH3
-H2O
N
SCH2COOH
N
SCH2C
O H
Scheme 3. Synthesis of 2,2'-((1-phenylethane-1,1-diyl)bis(sulfanediyl))bis(N-phenylacetamide)
(M-2)
18

0
Table1. Hydrogen-bond geometry (Å, ) of 1-(6-Methyl-3-phenyl-2-sulfanylidene-1,2,3,4-
tetrahydropyrimidin-5-yl)ethanone (M-1)
D - H...A
D—H
H... A
D . . . A
D —H . . . A
N3—H3N... O11
0.88
2.05
2.920 (2)
168
Table 2. Hydrogen bonds for c [Å and °] of 2,2'-((1-phenylethane-1,1-diyl)bis(sulfane-
diyl))diacetic acid (M)
____________________________________________________________________________
D-H...A
d(D-H)
d(H...A)
d(D...A)
<(DHA)
____________________________________________________________________________
O(1)-H(1)...O(4)#1
O(3)-H(3)...O(2)#1
0.85(2)
0.85(2)
1.83(9)
1.79(3)
2.643(8)
2.635(8)
160(25)
174(14)
____________________________________________________________________________
Symmetry transformations used to generate equivalent atoms:
#1 -x,-y+1,-z
Table 3. Data on the degree of hemolysis of red blood cells with substances
M-1, M-2 and phenolic extract of ginger.
The degree of hemolysis (%)
Concentration
Ginger
(mg/ml)
М-1
М-2
extract
240
120
60
108,03 5,54
-
80,03
58,82
23,66
0,66
6,80
2,83
-0,62
-0,29
0,54
1,21
-
-
30
112,76
70,87
17,89
9,17
15
7,5
3,75
0,00
-1,50
19

1,88
0,94
0,47
-3,56
1,00
4,96
3,64
3,34
-
-
-
-
Table 4. Data on testing the antioxidant activity of substances M-1, M-2, ginger extract and
Trolox. Negative values indicate the induction of MDA production (prooxidant effect).
The suppression of the operating time
Concentration
MDA (%)
mg/l
М-1
М-2
Ginger extract
1,875
3,75
7,5
-5,48
4,03
-28,93
-9,37
31,54
46,53
8,16
-3,07
70,47
15
13,86
15,42
19,09
15,69
-10,46
-16,72
-31,38
-30,90
91,18
30
95,14
60
96,89
120
240
97,94
99,90
Table 5. Probable predicted activity of the substance M-1 (reliability of the forecast above
40%)
Probability
Mistake
Predicted Activity
Analgesic, non-opioid
0,728
0,708
0,707
0,682
0,005
0,009
0,009
0,017
Analgesic
Antianginal
Apoptosis agonist
5-O-(4-coumaroyl)-D-quinate
inhibitor
3'-monooxygenase
0,659
0,032
0,636
0,666
0,655
0,584
0,572
0,013
0,054
0,062
0,029
0,019
Centromere associated protein inhibitor
Membrane permeability inhibitor
Antieczematic
Chloride peroxidase inhibitor
tRNA-pseudouridine synthase I inhibitor
20

0,558
0,589
0,006
0,053
Antimitotic
Phosphatase inhibitor
Mannotetraose
2-alpha-N-
0,548
0,055
acetylglucosaminyltransferase inhibitor
Acute neurologic disorders treatment
Leukopoiesis inhibitor
Antiviral (Adenovirus)
Antiviral (Rhinovirus)
2-Hydroxymuconate-semialdehyde
inhibitor
0,547
0,501
0,478
0,491
0,057
0,018
0,009
0,028
hydrolase
0,478
0,026
0,518
0,473
0,454
0,468
0,521
0,507
0,464
0,464
0,458
0,46
0,068
0,025
0,012
0,027
0,083
0,073
0,053
0,056
0,051
0,055
0,043
0,065
0,026
0,119
0,085
0,047
0,043
0,062
0,029
0,042
0,065
0,007
0,076
Oxidoreductase inhibitor
Anesthetic general
Flavin-containing monooxygenase substrate
Chemosensitizer
Glutamyl endopeptidase II inhibitor
Calcium channel (voltage-sensitive) activator
Nitrate reductase (cytochrome) inhibitor
Arginine 2-monooxygenase inhibitor
Antihypoxic
Fragilysin inhibitor
0,445
0,458
0,411
0,504
0,47
Dermatologic
Insulysin inhibitor
Laccase inhibitor
Fibrinolytic
Gastrin inhibitor
0,427
0,42
CYP2F1 substrate
Lysostaphin inhibitor
0,435
0,387
0,387
0,4
Histamine release stimulant
Loop diuretic
Retinoic acid metabolism inhibitor
3-Hydroxybenzoate 6-monooxygenase inhibitor
Calcium channel (voltage-sensitive) blocker
Radiosensitizer
0,339
0,404
21

0,336
0,408
0,427
0,008
0,088
0,11
Calcium channel blocker
1,4-Lactonase inhibitor
General pump inhibitor
Table 6. Probable predicted activity of the substance M-2 (reliability of the forecast above
40%)
Probability
0,694
Mistake
Predicted Activity
Electron-transferring-flavoprotein dehydrogenase
0,013
inhibitor
0,611
0,596
0,028
0,043
Fragilysin inhibitor
Pro-opiomelanocortin converting enzyme inhibi-
tor
0,572
0,547
0,559
0,556
0,541
0,542
0,538
0,536
0,542
0,514
0,481
0,483
0,019
0,01
Chenodeoxycholoyltaurine hydrolase inhibitor
Polyneuridine-aldehyde esterase inhibitor
Biotinidase inhibitor
0,028
0,028
0,025
0,03
Endopeptidase So inhibitor
Histidine N-acetyltransferase inhibitor
Formaldehyde transketolase inhibitor
Oxygen scavenger
0,039
0,045
0,069
0,05
Glutathione thiolesterase inhibitor
Pseudolysin inhibitor
Dehydro-L-gulonate decarboxylase inhibitor
Transcription factor STAT3 inhibitor
Antiulcerative
0,02
0,026
Nicotinic
alpha6beta3beta4alpha5
receptor
0,549
0,094
antagonist
0,481
0,481
0,486
0,526
0,471
0,451
0,031
0,031
0,039
0,08
Naphthalene 1,2-dioxygenase inhibitor
Ferredoxin-NAD+ reductase inhibitor
Sulfur reductase inhibitor
Kidney function stimulant
0,027
0,01
Transcription factor STAT inhibitor
Botulin neurotoxin A light chain inhibitor
22

0,51
0,075
0,018
0,061
0,051
Nicotinic alpha4beta4 receptor agonist
Glucuronate isomerase inhibitor
Thioredoxin inhibitor
0,447
0,49
0,462
Fibrolase inhibitor
Gamma-guanidinobutyraldehyde dehydrogenase
inhibitor
0,445
0,04
0,424
0,435
0,507
0,448
0,448
0,394
0,396
0,428
0,458
0,424
0,393
0,37
0,04
tRNA-pseudouridine synthase I inhibitor
Peptidyl-dipeptidase Dcp inhibitor
Mucomembranous protector
0,051
0,132
0,075
0,076
0,023
0,024
0,057
0,093
0,065
0,035
0,018
0,099
0,022
Carboxypeptidase Taq inhibitor
Limulus clotting factor B inhibitor
Thiopurine S-methyltransferase inhibitor
PfA-M-1 aminopeptidase inhibitor
Manganese peroxidase inhibitor
Phthalate 4,5-dioxygenase inhibitor
Nitrate reductase (cytochrome) inhibitor
Carbon-monoxide dehydrogenase inhibitor
HIV attachment inhibitor
0,442
0,363
Complement factor D inhibitor
Allyl-alcohol dehydrogenase inhibitor
Glycosylphosphatidylinositol phospholipase D
inhibitor
0,457
0,344
0,385
0,118
0,01
Allantoin racemase inhibitor
Glutamine-phenylpyruvate
inhibitor
transaminase
0,052
0,433
0,378
0,104
0,05
Nicotinic alpha2beta2 receptor antagonist
Cyanoalanine nitrilase inhibitor
NAD(P)+-arginine ADP-ribosyltransferase inhib-
itor
0,387
0,062
0,406
0,382
0,363
0,41
0,083
0,06
Lysine 2,3-aminomutase inhibitor
CYP2F1 substrate
0,041
0,09
Focal adhesion kinase 2 inhibitor
Fructose 5-dehydrogenase inhibitor
23

0,4
0,08
Pterin deaminase inhibitor
Lysase inhibitor
0,411
0,407
0,391
0,41
0,093
0,09
2-Hydroxyquinoline 8-monooxygenase inhibitor
5 Hydroxytryptamine release inhibitor
Chloride peroxidase inhibitor
MAP3K5 inhibitor
0,075
0,095
0,047
0,092
0,362
0,405
Antiseborrheic
FIGURE LEGENDS
Figure 1. X-ray structure of 1-(6-methyl-3-phenyl-2-sulfanilden-1,2,3,4-tetrahidropirimidin-5)
etanon (M-1).
Figure 2. X-ray structure of synthesis of 2,2'-((1-phenylethane-1,1-diyl)bis(sulfanediyl))-
diacetic acid (M).
Figure 3. The degree of hemolysis of red blood cells with substances M-1, M-2 and phenolic
extract of ginger.
Figure 4. Antioxidant activity of substances M-1, M-2, ginger extract and Trolox. Negative val-
ues indicate the induction of MDA production (prooxidant effect).
Figure 5. The effect of the substance M-1 in various concentrations on the membrane potential
of mitochondria of brown adipose tissue of rats.
Figure 6. Effect of M-1 substance in various concentrations on the membrane potential of rat
liver mitochondria.
Figure 7. Effect of substance M-2 in various concentrations on the membrane potential of mito-
chondria of brown adipose tissue of rats.
Figure 8. Effect of M-2 substance in various concentrations on the membrane potential of rat
liver mitochondria.
24

Figure 1.
Figure 2.
25

Figure 3.
Figure 4.
Figure 5.
26

Figure 6.
Figure 7.
27

Figure 8.
28