Evaluation of N-Hydroxy-, N-Metoxy-, and N-Acetoxybenzoyl-Substituted Derivatives of Thymine and Uracil as New Substances for Prevention and Treatment of Long-Term Complications of Diabetes Mellitus

Article abstract of DOI:10.1134/S1068162019010163

New uracil and thymine derivatives, N¹-,N³- and N¹,N³-(RO-benzoyl)-(1H,3H)-pyrimidine- 2,4-diones, were synthesized (RO- is hydroxy, acetoxy- or methoxy-group). The compounds were studied in a complex of in vitro tests for the ability to inhibit the development of long-term complications of diabetes. Their ability to cleave cross-links of proteins has been evaluated. The most significant ways of pharmacological correction of thrombosis, angio-, nephro-, encephalo-, and cardiopathy, antiglycation, chelating, and antiplatelet activities, have been established. The most active compound in terms of antiplatelet action, N¹- hydroxybenzoyluracil, exceeded acetylsalicylic acid by ~44%. In terms of their ability to chelate copper (II) cations, all compounds (with the exception of 1,3-bis(3-hydroxybenzoyl)-(1H,3H)-pyrimidine-2,4-dione that was not not studied in this test) showed the activity, whose IC₅₀ fell in the range between that for pioglitazone (44.1 μM) and pyridoxamine (136.7 μM) comparison drugs. The best antiglycation effect at the 1 mM concentration was observed for N¹,N³-bismethoxy- and N¹,N³-bisacetoxybenzoyl derivatives of thymine. The maximum activity to cleave cross-links of proteins (C = 1 mM), comparable to that of alagebrium, was established for 1,3-bis(4-methoxybenzoyl)uracil, for which also high rates of other estimated activities were noted. Thus, the N¹-,N³- and N¹,N³-(RO-benzoyl) derivatives of uracil and thymine are promising basiсs for creating drugs that suppress the development of long-term complications of diabetes.

Full text of DOI:10.1134/S1068162019010163

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2018, Vol. 44, No. 6, pp. 769–777. © Pleiades Publishing, Ltd., 2018.
Evaluation of N-Hydroxy-, N-Metoxy-, and N-Acetoxybenzoyl-
Substituted Derivatives of Thymine and Uracil as New Substances
for Prevention and Treatment of Long-Term Complications
of Diabetes Mellitus
A. A. Spasova, b, c, A. K. Brela, R. A. Litvinova, c, 1 , S. V. Lisinaa, A. F. Kucheryavenkoa, Yu. N. Budaevaa,
O. A. Salaznikovaa, A. I. Rashchenkoa, D. D. Shamshinaa, V. V. Batrakova, and A. V. Ivanova
aVolgograd State Medical University, Volgograd, 400131 Russia
bVolgograd Medical Scientific Center, Volgograd, 400081 Russia
cScientific Institute of Pharmacology of VSMU, Antioxidant Lab., Volgograd, 400001 Russia
Received June 25, 2018; in final form, July 9, 2018
Abstract⎯New uracil and thymine derivatives, N1-,N3- and N1,N3-(RO-benzoyl)-(1H,3H)-pyrimidine-
2,4-diones, were synthesized (RO- is hydroxy, acetoxy- or methoxy-group). The compounds were studied in
a complex of in vitro tests for the ability to inhibit the development of long-term complications of diabetes.
Their ability to cleave cross-links of proteins has been evaluated. The most significant ways of pharmacolog-
ical correction of thrombosis, angio-, nephro-, encephalo-, and cardiopathy, antiglycation, chelating, and
antiplatelet activities, have been established. The most active compound in terms of antiplatelet action, N1-
hydroxybenzoyluracil, exceeded acetylsalicylic acid by ~44%. In terms of their ability to chelate copper (II)
cations, all compounds (with the exception of 1,3-bis(3-hydroxybenzoyl)-(1H,3H)-pyrimidine-2,4-dione
that was not not studied in this test) showed the activity, whose IC50 fell in the range between that for piogli-
tazone (44.1 μM) and pyridoxamine (136.7 μM) comparison drugs. The best antiglycation effect at the 1 mM
concentration was observed for N1,N3-bismethoxy- and N1,N3-bisacetoxybenzoyl derivatives of thymine.
The maximum activity to cleave cross-links of proteins (C = 1 mM), comparable to that of alagebrium, was
established for 1,3-bis(4-methoxybenzoyl)uracil, for which also high rates of other estimated activities were
noted. Thus, the N1-,N3- and N1,N3-(RO-benzoyl) derivatives of uracil and thymine are promising basiсs for
creating drugs that suppress the development of long-term complications of diabetes.
Keywords: diabetes mellitus, long-term complications of diabetes mellitus, pyrimidine, thymine, uracil
DOI: 10.1134/S1068162019010163
INTRODUCTION
by cardiovascular diseases [2]. However, there are no
reliable drugs that can prevent the development of
long-term effects of DM, the treatment and preven-
tion of which for a long time faded into insignificance
compared to the development of means for correcting
glucose levels [3].
The basis of the pathogenesis of the most life-
threatening distant complications of DM (damage to
the cardiovascular system with the development of
acute ischemia), as well as a number of polysystemic
pathologies, lies in the general mechanism of disrupt-
ing the structural organization of proteins (collagens,
elastin, crystallins, beta-amyloids, etc.) due to the for-
mation of covalent bonds between them [4–8]. The
process of formation of protein-protein cross-links
actively occurs in the stroma of organs and vessels in
addition to prolonged hyperglycemia and oxidative
stress, observed in DM [4, 5, 9]. Similar cross-linking
DM is a complex medical and social problem
worldwide, including in the Russian Federation. The
primary disease is accompanied by the development of
numerous complications. According to global medical
and economic analyses, the annual cost of combating
DM and the complex of its complications, including
nephropathy, cardiopathy, angiopathy, polyneuropa-
thy, encephalopathy, etc., account for about 12% of
total public health financing [1]. Most of the funds are
indirect costs due to the disability of the population
associated with the development of long-term compli-
cations, the largest contribution among which is made
Abbreviations: DM, diabetes mellitus; ROS, reactive oxygen
species; GEP, glycation-end products; NMR, nuclear magnetic
resonance; BSA, bovine serum albumin.
Corresponding author: phone: +7 (902) 361-87-70; e-mail: lit-
vinov.volggmu@mail.ru.
1
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770
SPASOV et al.
processes are noted during aging of the body, Alzhei-
mer’s, Parkinson’s and other diseases [6]. The forma-
tion of cross-linked proteins proceeds along the glu-
cose-dependent pathway (glycation of amino acid res-
idues Schiff bases Amadori products Suyama
Hemodynamics under conditions of the develop-
ment of angiopathy in DM is prevented by increased
atherogenesis and increased thrombus formation in
addition to a decrease in fibrinolysis [12]. Disruption
of hemostasis is associated with dysglycemia, and ath-
erogenesis, that complicates the course of the disease,
is mediated by pathological inflammation, partly
determined by the impaired antioxidant status of the
organism. Currently, antiplatelet agents (including
cyclooxygenase inhibitors, for example, acetylsalicylic
acid) are widely used to combat pathological throm-
bosis. In opposition to the development of atheroscle-
rosis, a certain efficacy in clinical practice, especially
in DM, was achieved by using substances with an anti-
oxidant effect, for example, probucol, which protects
lipoproteins from peroxidation and suppresses the for-
mation of foam cells [13]. Considering the potential
for creating new compounds that affect the processes
of thrombus formation, it should be noted that the
presence of antithrombotic activity is established for
isomers of hydroxybenzoic acid [14], therefore, its
derivatives can be the basis for creating agents with
antiplatelet activity. In addition, pyrimidine-based
substances are also reported to exhibit antiplatelet
effect. For example, this has been established for
derivatives of 2-aminopyrimidine, 2-substituted
derivatives of 4,6-diaminopyrimidine, as well as for
polycyclic compounds based on it [15, 16]. And as
shown in [11], nitrazolopyrimidines have signs of an
antioxidant effect, which is expressed in their inhibi-
tion of the autoxidation reaction of ascorbic acid.
These data give additional medical and pharmacolog-
ical significance to pyrimidine derivatives, as well as
compounds-analogues of hydroxybenzoic acid, and
the development of new antidiabetic agents on their
basis.
⇒
⇒
⇒
or Namiki, or Hodge reaction formation of amino-
⇒
adipate
cross-linking) or the oxidative pathway
⇒ ⇒
⇒
(formation of ROS Stadtman path formation of
aminoadipate (or other product of oxidative degrada-
tion of amino acids) cross-linking) [7]. Both path-
⇒
ways include redox reactions catalyzed directly by
transition metal cations (Cu2+, Fe2+, Fe3+, etc.) or
accelerated by ROS formed by metals [7, 8]. Thus,
ROS and the ways of their formation, transition met-
als, including the effect on their electronic properties
causing catalysis, the stages of reactions of formation
of GEP, as well as covalent protein-protein bonds
available for cleavage can serve as new targets of
potential medicinal substances capable of slowing
down the development of DM complications. It can
be assumed that the effect on the listed targets and
reactions can slow the development of long-term com-
plications of DM. Moreover, taking into account the
interrelation of the process of oxidative degradation of
amino acids, dependent on the production of ROS
and the presence of transition metals, with the forma-
tion of GEP, as mentioned above, the quantitative
expression of the activity of compounds in their influ-
ence on the catalytic and ROS-producing functions of
transition metals will likely be correlated with the abil-
ity of the same substances to prevent the glycation
reaction from proceeding, i.e., with their antiglycation
activity. That is, the ability of substances to bind tran-
sition metal cations and their antiglycation action
should probably be related quantitatively. According to
some literature data [10], the ability of compounds to
bind transition metals and simultaneously inactivate
the ROS they produce is called “multifunctional anti-
oxidant action with chelating.” Drawing a conclusion
from the above, it can be assumed that the ability of
compounds to be antioxidants and/or bind transition
metals can be used in the fight against long-term com-
plications of DM.
The purpose of our work is the synthesis and study
of the biological properties of new N-(RO-ben-
zoyl)pyrimidines (thymine and uracil derivatives,
where RO is a methoxy, acetoxy or hydroxy group) as
the basis for development of potential drugs for the
treatment of complications of DM.
Many researches in modern medical chemistry are
associated with the search for new biologically active
structures as the basis for the creation of medicines
and the development of effective methods for their
synthesis. Pyrimidine derivatives, thymine and uracil,
are of interest because some of their derivatives have
the abovementioned properties. For example, nitrazo-
lopyrimidine derivatives destroy cross-links of gly-
cated proteins, inhibit the formation of GEP, and also
bind copper ions [11].
RESULTS AND DISCUSSION
The target compounds were synthesized by the
acylation of the corresponding nitrogenous bases with
substituted hydroxybenzoyl chlorides in pyridine at
room temperature (Scheme 1).
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EVALUATION OF N-HYDROXY-, N-METOXY-
771
Table 1. N-(RO-benzoyl)-derivatives of (1H,3H)-pyrimidine-2,4-diones (I)–(XIV) (Scheme 1)
RO-substituent in Bz
Compound 5-X
Name
N1
N3
(I)
(II)
(III)
(IV)
(V)
H
H
H
H
H
H
3-OH
3-ОН
H
4-OH
H
H
4-OMe
3-OH
H
1,3-Bis(3-hydroxybenzoyl)-(1H,3H)-pyrimidine-2,4-dione
1-(4-Hydroxybenzoyl)-(1H,3H)-pyrimidine-2,4-dione
1,3-Bis(4-hydroxybenzoyl)-(1Н,3Н)-pyrimidine-2,4-dione
1-(2-Acetoxybenzoyl)-(1Н,3Н)-pyrimidine-2,4-dione
1-(4-Acetoxybenzoyl)-(1Н,3Н)-pyrimidine-2,4-dione
1,3-Bis(4-methoxybenzoyl)-(1Н,3Н)-pyrimidine-2,4-dione
1,3-Bis(3-hydroxybenzoyl)-5-methyl-(1H,3H)-pyrimidine-2,4-dione
1-(4-Hydroxybenzoyl)-5-methyl-(1Н,3Н)-pyrimidine-2,4-dione
1,3-Bis(4-hydroxybenzoyl)-5-methyl-(1Н,3Н)-pyrimidine-2,4-dione
1,3-Bis(2-acetoxybenzoyl)-5-methyl-(1Н,3Н)-pyrimidine-2,4-dione
1,3-Bis(3-acetoxybenzoyl)-5-methyl-(1Н,3Н)-pyrimidine-2,4-dione
1,3-Bis(4-acetoxybenzoyl)-5-methyl-(1Н,3Н)-pyrimidine-2,4-dione
1-(4-Methoxybenzoyl)-5-methyl-(1Н,3Н)-pyrimidine-2,4-dione
1,3-Bis(4-methoxybenzoyl)-5-methyl-(1Н,3Н)-pyrimidine-2,4-dione
4-OH
4-OH
2-OAc
4-OAc
4-OMe
(VI)
(VII)
(VIII)
(IX)
(X)
CH3 3-OH
CH3 4-OH
CH3 4-OH
CH3 2-OAc
CH3 3-OAc
CH3 4-OAc
CH3 4-OMe
CH3 4-OMe
4-OH
2-OAc
3-OAc
4-OAc
H
(XI)
(XII)
(XIII)
(XIV)
4-OMe
OR
O
C
X
O
O
O
O
N
Cl
C
O
X
X
NH
NH
N
NH
N
O
O
N
H
O
N
H
O
X
C
C
RO
O
O
C5H5N
C5H5N
RO
RO
(COCl2)
(II) X = H, OR = 4-OH
(I) X = H, OR = 3-OH
(IV) X = H, OR = 2-OAc
(V) X = H, OR = 4-OAc
(VIII) X = CH3, OR = 4-OH
(III) X = H, OR = 4-OH
(VI) X = C, OR = 4-OMe
(VII) X = CH3, OR = 3-OH
HO
O
C
RO
(XIII) X = CH3, OR = 4-OMe (IX) X = CH3, OR = 4-OMe
(X) X = CH3, OR = 2-OAc
(XI) X = CH3, OR = 3-OAc
(XII) X = CH3, OR = 4-OAc
(XIV) X = CH3, OR = 4-OMe
Scheme 1.
N1-Derivatives of uracil and thymine were synthe-
sized by the acylation of the corresponding nitroge-
nous bases with an equimolar amount of a RO-substi-
tuted benzoyl chloride. To obtain the N1,N3-bis-deriv-
atives, the initial reagents were used in a ratio of 2 : 1;
the reaction was carried out in pyridine at room tem-
(Table 1), whose structure was confirmed by the
1H NMR spectroscopy (Table 2).
The described experimental models for evaluation
of biological activity imitate different stages of the
pathogenesis of long-term complications of DM.
Methods for assessing the copper-binding, antiglyca-
perature. Thus, we synthesized the new compounds tion and cleaving cross-links activities of compounds
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772
Table 2. Characterization of the obtained N-(RO-benzoyl)-substituted pyrimidines
Compound Yield,% mp, °С
SPASOV et al.
Data of 1Н NMR spectra (DMSO-d6), δ, ppm
(I)
71
321–323 8.25–8.23 (1 H, d, Pyr), 7.86–7.82 (2 H, m, С6Н4), 7.75–7.72 (2 H, m, С6Н4),
7.30–7.26 (2 H, t, С6Н4), 7.08 (2 H, s, OH), 6.95–6.94 (2 H, m, С6Н4),
5.48–5.45 (1 H, d, Pyr)
(II)
(III)
(IV)
(V)
81
76
72
77
82
355–356 6.86–7.89 (4 Н, m, С6Н4), 5.87–5.89 (1 H, d, Pyr), 7.90–7.92 (1H, d, Pyr),
1.86 (3Н, s, СН3), 10.27 (1 Н, s, OH), 11.48 (1 Н, s, NH)
348–350 8.16–8.14 (1H, d, Pyr), 8.19 (2H, s, OH), 7.39–7.37 (4H, d, С6Н4),
6.82–6.80 (4H, m, С6Н4), 5.47–5.45 (1H, d, Pyr)
338–339 7.14–7.81 (4 Н, m, С6Н4), 5.86–5.88 (1 Н, d, Pyr), 7.85–7.88 (1 Н, d, Pyr),
2.38 (3Н, s, ОС(О)СН3), 11.48 (1 Н, s, NH)
319–320 7.28–7.85 (4 Н, m, С6Н4), 5.87–5.89 (1 Н, d, Pyr), 7.87–7.89 (1 Н, d, Pyr),
2.31 (3Н, s, ОС(О)СН3), 11.48 (1 Н, s, NH)
(VI)
319–322 7.92–7.88 (1Н, d, Pyr), 7.73–7.68 (4H, m, С6Н4), 7.33 (1Н, d, Pyr),
7.05–7.01 (4H, m, С6Н4), 3.85 (6H, s, –CH3)
(VII)
80
73
313–314 6.93–8.22 (4 Н, m, С6Н4), 8.22 (1Н, s, Pyr), 1.80 (3Н, s, СН3), 9.72 (2 Н, s, OH)
(VIII)
382–383 6.84–7.62 (4 Н, m, С6Н4), 8.07 (1Н, s, Pyr), 1.86 (3Н, s, Pyr, СН3),
10.27 (1 Н, s, ОH), 11.48 (1 Н, s, NH)
(IX)
(X)
77
86
79
384–385 6.78–7.76 (8 Н, m, С6Н4), 8.06 (1Н, s, Pyr), 1.80 (3Н, s, СН3), 10.27 (2Н, s, ОH)
331–332 7.16–7.96 (8 Н, m, С6Н4), 7.89 (1Н, s, Pyr), 2.38 (6Н, s, ОС(О)СН3), 1.91 (3Н, s, СН3)
(XI)
309–310 7.38–7.99 (8 Н, m, С6Н4), 8.04–8.05 (1Н, d, Pyr) , 2.28 (6Н, s, ОС(О)СН3),
1.80 (3Н, s, СН3)
(XII)
84
88
69
312–313 7.28–7.91 (8 Н, m, С6Н4), 8.06–8.07 (1Н, d, Pyr), 2.31 (6Н, s, ОС(О)СН3),
1.80 (3Н, s, СН3)
(XIII)
328–329 7.02–7.88 (4 Н, m, С6Н4), 8.07 (1Н, s, Pyr), 3.86 (3Н, s, ОСН3), 1.86 (3Н, s, СН3),
11.48 (1 Н, s, NH)
323–324 6.94–7.90 (8 Н, m, С6Н4), 8.06 (1Н, s, Pyr), 3.86 (6Н, s, ОСН3), 1.80 (3Н, s, СН3)
(XIV)
Pyr, pyrimidine.
simulate the individual stages of the metabolic path- one of the important parts in the pathogenesis of DM
way for the cross-linked proteins formation. The sum-
mary data from studies of biological activities are given
in Table 3.
complications [17], and the pharmacological correc-
tion of this pathological process is an important con-
tribution to the cumulative antidiabetic effect.
As a result of evaluation of the antiplatelet effect of
N-(RO-benzoyl)pyrimidine derivatives, we found that
they are characterized by suppression of ADP-
induced platelet functional activity at an inducer con-
centration of 5 μM. The analysis of results showed that
the derivative (I) had shown the highest activity, sig-
nificantly exceeding the result of acetylsalicylic acid
(single factor analysis, Newman–Keuls posttest, p <
0.05). The compound is an uracil derivative, and is
1,3-bis(3-hydroxybenzoyl)-(1H,3H)-pyrimidine-2,4-
dione. The antiplatelet activity of all other synthesized
compounds was comparable to that of acetylsalicylic
acid and, therefore, it can be concluded that the RO-
benzoyl residue in the pyrimidine heterocycle is a
promising substituent for targeted search of agents
with antiplatelet activity. The hemodynamic disorder
When evaluating copper-binding activity, we found
that all compounds were sufficiently active in the cop-
per-induced ascorbic acid autoxidation model. The
exception was compound (I), the activity level of
which could not be determined due to the extremely
high optical density of the substance at 265 nm, which
is a limitation for the corresponding technique. Their
IC50 values were in the range between those for the
control substances, pyridoxamine (136.7 μM) and
pioglitazone (44.1 μM). At the same time, none of the
compounds statistically significantly exceeded piogli-
tazone in activity (Table 3). Thus, all the investigated
substances can be considered as copper chelators, hav-
ing a fairly high level of this activity, corresponding to
induced by the mechanism of increased thrombosis is the activity of pioglitazone and/or pyrodoxamine.
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EVALUATION OF N-HYDROXY-, N-METOXY-
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Table 3. Pharmacological activity of derivatives of uracil (I)–(VI) and thymine (VII)–(XIV)
Antiplatelet activity
Antiglycation, ∆%
Cleavage of cross-links,
∆% (M m), C = 1 μM
Chelation, IC50, μM
Compounds/derivatives
(platelets) ∆%
(M m), C = 1 μM
(M m), C = 0.1 μM
(I)
(II)
(III)
(IV)
(V)
(VI)
(VII)
(VIII)
(IX)
(X)
(XI)
(XII)
(XIII)
(XIV)
Aminoguanidine
Acetylsalicylic acid
EDTA
Pioglitazone
Lipoic acid
Pyridoxamine
Alagebrium (ALT-711)
N.A.
–7.40 2.40
24.40 2.10*
12.00 4.90
–5.20 4.00
–9.70 0.80
13.50 2.00*
14.50 4.00
–18.50 7.10
2.40 7.80
25.60 6.90
27.80 2.30*
33.70 1.80*
35.60 1.10*
8.00 3.80
43.40 2.40*
N.A.
25.10 2.30
21.70 1.80
18.12 1.52
15.90 1.14
20.08 1.64
18.68 1.90
17.21 1.18
21.35 2.70
15.28 0.79
19.38 2.50
13.59 0.74
14.14 2.30
20.43 1.43
19.15 2.10
N. A.
5.13 9.69
117.5 (R2 = 0.98)
107.2 (R2 = 0.99)
148.2 (R2 = 0.99)
174.8 (R2 = 0.98)
67.5 (R2 = 0.93)
108.1 (R2 = 0.98)
120.5 (R2 = 0.98)
128.3 (R2 = 0.99)
50.7 (R2 = 0.99)
73.9 (R2 = 0.98)
71.0 (R2 = 0.96)
32.6 (R2 = 0.99)
45.6 (R2 = 0.98)
>1000.0
–
–
–
6.32 6.76
22.19 3.28
–
–
–
–
–
–
–
–
N. A
N.A.
N.A.
N.A.
N.A.
N.A.
23.02 8.79
N.A.
17.38 1.37
N.A.
0.7 (R2 = 0.95)
44.1 (R2 = 0.98)
68.9 (R2 = 0.96)
136.7 (R2 = 0.98)
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A., the compound was not tested or, due to limitations of the method, the activity could not be established; “–”, the compound
did not exhibit activity in the test; Univariate analysis of variance, Newman–Keuls posttest, p < 0.05; M m correspond to the arithmetic
2
mean (M) and the standard error of the arithmetic mean (m); R value corresponds to the coefficient of determination in the IC estima-
50
tion by the linear regression method and indicates how much the conditional dispersion of the model (line) differs from the dispersion
of real values.
Evaluating the results established by this method, rection of oxidative stress under conditions of DM, in
we carried out a structural and functional analysis of particular, accompanied by disordered transition met-
the compounds. As a result, we found a correspon- als homeostasis. This approach is potentially promis-
ing in the treatment of long-term complications of
DM, taking into account the characteristics of their
pathogenesis [9].
dence between the presence of a higher chelating
activity and the presence of a methoxy group (com-
pound (VI), (XIII) and (XIV)) as a substituent in the
benzoyl radical, but not hydroxy or acetoxy groups
whose carriers had lower activity. However, we also
noted that the molecular core probably affects the
manifestation of this activity. Uracil-based com-
pounds were able to bind copper more actively in the
autoxidation reaction of ascorbic acid than thymine
derivatives, and the activity level of uracil-based com-
pounds was higher in IC50.
Evaluating the antiglycation activity of N-(RO-
benzoyl)-pyrimidine derivatives at a concentration of
1 mM, we noted the presence of statistically signifi-
cant activity for compounds (II), (VI), (XII), (XIII),
and (XIV). The compounds differ both in the substit-
uent in the RO-benzoyl residue and in the number of
N-substitutions (mono- and disubstituted) in the
pyrimidine ring. However, by visual comparison, it
was noted that higher antiglycation activity is inherent
in compounds with uracil core than in ones with thy-
mine core, similar to the fact that the chelating activity
of the tested compounds was also more pronounced in
uracil derivatives.
In general, taking into account the peculiarities of
the ascorbic acid autoxidation model, the ability of
compounds to exert antioxidant and chelating effects
in a system in which the oxidation inducer is a transi-
tion metal cation makes it possible to additionally con-
sider (RO-benzoyl)-containing pyrimidine derivatives
Based on the fact that the ability of compounds to
as compounds capable of giving pharmacological cor- eliminate the catalytic and ROS producing abilities of
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774
SPASOV et al.
copper cations correlates with the antiglycation activ- the formation of long-term complications of DM is
ity [7, 8], the level of these activities was compared. accompanied by a shift in the pH value of the intersti-
Between the two activities, the Spearman’s rank cor- tial fluid towards acidification [20]. Thus, the same
relation coefficient r(s) = –0.7 was determined at a groups, apparently, are able to realize several variants
significance level of p ≤ 0.05 (the negative value of r(s) of activity, which is associated with their biophysical
is due to the comparison of the IC50 values of the che- features.
lating activity and the Δ indicator, % of the antiglyca-
tion action). Compound (I) was excluded from the
correlation analysis, which is associated with its high
light absorption at λ 265 nm and the associated inabil-
ity to establish copper-binding activity.
The correlation of the two activities can be associ-
ated with:
(a) The presence of the required target in both
experimental models and its role in the key reaction
processes.
(b) Common functional groups of compounds
involved in both reactions.
(c) Common factors affecting the course of both
reactions.
Discussing item (a), it should be mentioned that
the presence of copper (II) cations permanently
bonded to BSA affects the glycation kinetics [18], so
BSA itself can act as a source of Cu2+ during the reac-
tion.
For item (b) the commonality of the groups implies
that the same pharmacophore groups of the com-
pound are involved in the manifestation of both activ-
ities, that is, the fragments of the molecule responsible
for the manifested action. In one case, these groups
act as binding sites for the transition metal; in the
other, they participate in the inactivation of carbonyl
intermediates. At the same time, the submolecular
mechanisms of the participation of functional groups
in reactions are different, and the tendency for the
realization of one or other of the two mentioned reac-
tions may vary under the influence of environmental
conditions. pH is one of the factors on which these
pharmacophore groups depend on metal binding or
inactivation of carbonyl intermediates. This is noted
for the drug metformin, which is widely used in treat-
ment of diabetes of the second type. The drug simul-
taneously exhibits antiglycation and chelating proper-
ties [8], and at the same time, it has two potential
binding sites for metals, which under certain condi-
tions are also involved in dicarbonyl binding reactions.
At a neutral pH, metformin is in a partially protonated
(monoprotonated) state [19], which determines its
multiple action, the ability to act as an antiglycation
Item (c) implies that ascorbic acid, due to its sensi-
tivity to the direct oxidative effect of transition metals,
as well as the ROS formed by them, is very convenient
in studying the effect of substances on the oxidative
agents of the redox reactions, which are included in
the metabolic pathway of the formation of cross-
linked proteins (clearly presented in [7]) in order to
determine whether substances are capable of inhibit-
ing the oxidative action of metals or highly reactive
oxygen compounds. GEP formation reactions are one
of the steps in this metabolic pathway. Besides this, the
autoxidation reaction of ascorbic acid seems to be sim-
ilar to the autoxidation of glucose, since both reactions
proceed in the presence of transition metals in a simi-
lar way. When the glycation reaction proceeds, glucose
autoxidation, which is one of the stages of this reac-
tion, serves as one of the sources of carbonyl interme-
diates with high reactivity [21], which contributes to
an increase in the rate of the reaction process. How-
ever, due to the similarity of the two reactions, it can
be assumed that substances inhibiting autoxidation of
ascorbic acid may slow down the autoxidation of glu-
cose, which will also contribute to slowing down the
reaction of formation of GEP while reducing the pool
of highly reactive carbonyl intermediates that will be
formed from less glucose.
Thus, the three arguments presented allow us to
interpret the possible causes of the ratio of the two
pharmacological activities of the compounds, as well
as the two inextricably connected pathways of patho-
genesis of the development of long-term complica-
tions of DM.
We found that only one of the synthesized com-
pounds is able to cleave effectively proteins’ cross-
links. Compound (VI) (Table 3) was comparable in
level to the control compound, alagebrium (ALT-711).
Two more compounds, (I) and (V), proved to be of low
activity. For all other studied compounds, cleaving
cross-links activity was not found. For compound (VI)
and alagebrium, the IC50 values determined were of
1.32 and 1.89 mM, respectively (Fig. 1). Compounds (I),
(V), and (VI) have a uracil core.
Summarizing the evaluation of the pharmacologi-
substance, directly binding carbonyl intermediates, cal properties of the studied compounds, we can con-
and the ability to bind transition metals. This is due to clude that this class of compounds is promising as a
the biophysical features, the ability to participate in basis for developing substances capable of suppressing
the ion interaction, to act as a donor/acceptor of pro- the development of diabetes-associated pathias. All
tons, to enter into π–π interactions [19]. At the same compounds exhibit antiplatelet effect to varying
time, a more acidic environment, favorable for the degrees (the highest activity was observed in com-
preservation of groups in the protonated state, is also pounds (I), (II), and (VII). This property is possibly
favorable for the occurrence of exchange reactions related to the structural features of the benzoyl substit-
with dicarbonyls, as described in [8]. It is known that uent. All synthesized compounds with the exception of
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EVALUATION OF N-HYDROXY-, N-METOXY-
775
120
70
60
50
40
30
20
10
(а)
(b)
100
80
60
40
20
y = 22.70x – 6.991
y = 38.54x – 1.218
R2 = 0.945
R2 = 0.900
0
0.5
1.0
С, mМ
1.5
2.0
0
2
4
6
C, mМ
Fig. 1. The dependence of the activity to cleave cross-links of proteins of the compound (VI) (a) and alagebrium (b) on concen-
trations.
(I) possess copper (II) chelating activity with signs of
an antioxidant effect. Compound (I) had an extremely
high optical density at λ 265 nm, which limited the
opportunity in its study. The chelating activity is prob-
ably related and partially determines the presence of
the antiglycation action (compounds (II), (VI), (XI),
(XII), (XIII)).
EXPERIMENTAL
Melting points were determined by the capillary
method on a StuartSMP-30 instrument at a heating
rate of 10°C/min. The purity and identity of the com-
pounds was confirmed by thin-layer chromatography
on Silufol UV-254 plates; the mobile phase was 2-pro-
panol–water, 7 : 3; the spots were developed in iodine
vapor and UV light. The 1H NMR spectra of the deriv-
atives were recorded on a Bruker DRX500 spectrome-
ter in DMSO-d6, hexamethyldisiloxane as an internal
standard. To interpret the spectra, a licensed software
product of the company Advanced Chemistry Devel-
opment Inc. under the commercial name
ACD/HNMR Predictor Pro v.3 was used.
For evaluation of the antiplatelet activity, a two-
channel laser analyzer of platelet aggregation 230 LA
(Biola, Russia) was used. For evaluation of antiglycation
activity, the fluorescence of GEP was measured on a
TECAN M 200 Pro spectrofluorometer (λ(excitation)
370 nm, λ(emission) 440 nm). The ability of compounds
to cleave cross-links in proteins was determined using
TECAN M 200 Pro in terms of light absorption
(λ 450 nm). Chelating activity was determined on a spec-
trophotometer APEL PD 303 UV, Japan (λ 265 nm).
In the series of all considered thymine and uracil
derivatives, the best activity profile was established for
compound (VI), which is due to its ability to cleave
protein-protein covalent cross-links. The formation of
these cross-links is the final stage in the pathway of
metabolic transformations occurring in the stroma in
patients with DM, which is the basis of the pathogen-
esis of the development of some diabetes-associated
pathias. In addition, giving an assessment of the
potential medical significance of the established activ-
ity of the compound (VI), it can be said that among
the activities of chelation of transition metals, suppres-
sion of the formation of GEP and cleavage of protein-
protein cross-links, the latter is of particular impor-
tance.
Synthesized N-(RО-benzoyl)-substituted deriva-
tives of thymine and uracil can be a promising basis for
development of agents that slow down the develop-
ment of diabetes-associated pathias and long-term
complications of the disease. However, it is assumed
that the additional cleaving cross-links activity of
compound (VI), comparable to that of alagebrium,
will not only slow down the development of long-term
complications of DM, but also probably partially
restore the biophysical properties of the finely orga-
nized stroma ultrastructure, which undergoes changes
in this disease. Thus, the class of substances described
has a high medical and pharmacological potential.
All solvents and reagents were obtained from com-
mercial sources and were used without purification.
RO-substituted benzoyl chlorides were synthesized
according to the reported procedures [22, 23].
General procedure for the synthesis of 1,3-bis(RO-
benzoyl)-(1H,3H)-pyrimidine-2,4-diones (I), (III),
(VI), (VII), (IX), (X) derivatives ), (XI), (XII), (XIV)
[24]. A solution of 200 mmol of RO-benzoyl chloride
in 10 mL of chloroform was added dropwise over
15 min to a solution of 100 mmol of uracil or thymine
in 30 mL of dry pyridine. A solid product and pyridine
hydrochloride were precipitated. The mixture was
stirred for 1 h at room temperature. Then the solution
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 44 No. 6 2018

776
SPASOV et al.
was filtered under vacuum; the precipitate was washed recorded spectrophotometrically in a low-tempera-
with cold water and recrystallized from benzene.
ture quartz cuvette (λ 265 nm). The IC50 of test sub-
stances were determined by the linear regression
method (Microsoft Excel 2007). Statistical data pro-
cessing was performed using Microsoft Excel 2007
software, as well as GraphPad Prism 5.0 software
using the Mann-Whitney pairwise comparison crite-
rion, as well as a single-factor variational analysis at
p < 0.05.
N1-Derivatives of uracil and thymine (II), (IV),
(V), (VIII), (XIII) were obtained similarly at a molar
ratio of starting materials of 1 : 1.
Yields, melting points and NMR spectral data of
compounds (I)–(XIV) are given in Table 1.
Experimental (Biology)
The ability to cleave the cross-links of glycated pro-
teins was investigated by measuring the optical absorp-
tion of solutions previously obtained by incubating a
BSA solution (50 mg/mL) with glucose (0.5 M) for
three months, introduced into the medium containing
collagen to form cross-links with this protein [31].
Collagen was isolated from the tails of rats three to five
months old weighing about 200 g, devitalized by
decapitation under ether anesthesia. The material of
the connective tissue of the tail, collected by the oper-
ational method, was immediately transferred to a 0.1%
solution of acetic acid for acid dissolution of collagen
and its extraction for seven days. Then the contents of
the vessels were centrifuged at 8000 g (10 min) to pre-
cipitate residual fibers and the collagen-rich superna-
tant was collected. The resulting collagen-containing
gel was used to coat the bottom of flat-bottomed,
transparent 96-well microplates (70 μL of gel per
well). Microplates were carefully treated with a phos-
phate buffer (pH 7.4) for 1 h to neutralize acidic colla-
gen. The plates were blocked with Superblock
(PIERCE) at 37°C for 1 h. Then, a solution of BSA
(fraction V) was added and incubation was carried out
at 37°C for 4 h to form cross-links. A solution of the
compound in DMSO (10 μL) at a final concentration
of 1 mM was added to the wells with glycated protein
and to the wells with BSA. The plate was incubated at
37°C for 16 h. Then, 80 μL/well of antibodies against
BSA (1 : 500) were added to the wells, and the plate
was incubated at 37°C for 50 min. Next, 80 μL/well of
horseradish peroxidase-labeled goat anti-rabbit IgG
(1 : 1000) were added to the wells. The wells were incu-
bated at 37°C for 50 min. Subsequently, 3,3′,5,5′-
tetramethylbenzidine (100 μL/well) was added. The
plate was incubated at room temperature in the dark-
ness for 20 min. To stop the reaction, a 2 M solution of
H2SO4 was used. Within 10 min after the reaction, the
optical absorption was recorded at 450. Alagebrium
(Sigma-Aldrich) was used as a control substance. The
results were processed in Microsoft Excel (Microsoft,
United States) with the calculation of basic statistical
indicators of descriptive statistics.
Antiplatelet activity of substances was investigated
at a concentration of 100 μM on a model of ADP–
induced (Reanal, Hungary) aggregation of rabbit
platelets by changing the degree of light transmission
of plasma rich in platelets [25]. The activity of the sub-
stances was evaluated by a decrease in platelet aggrega-
tion relative to the control. Acetylsalicylic acid (Shan-
dong Xinhua Pharmaceutical Co., Ltd, China) was
used as a control drug. Statistical data processing was
performed using Microsoft Excel 2007 software, as
well as GraphPadPrism 5.0 software using univariate
variational analysis with the Newman–Keuls posttest
(p < 0.05) and preliminary verification of the normal-
ity of the distribution using the Kolmogorov–Smirnov
criterion.
Antiglycation activity was determined by the
method reported in [16]. The reaction medium for the
glycation reaction contained 500 mM of glucose and
1 mg/mL of BSA (fraction V), dissolved in a phos-
phate buffer solution (pH 7.4). Test compounds were
dissolved in 99% DMSO. The final concentration of
substances after their addition to the reaction medium
was 1 mM. Control samples contained an equivalent
volume of DMSO. Samples were incubated for 24 h at
60°C. Aminoguanidine hydrochloride (Sigma-
Aldrich) was used as a control substance. Statistical
data processing was performed using Microsoft Excel
2007 software, as well as GraphPadPrism 5.0 software
using univariate variational analysis with the New-
man–Keuls posttest (p < 0.05) and preliminary verifi-
cation of the normality of the distribution using the
Kolmogorov–Smirnov criterion.
Chelating activity of the compounds was studied in
a concentration range of 10–200 μM by the method of
suppressing the copper-dependent autoxidation of
ascorbic acid [26, 27]; the IC50 value were estimated.
Lipoic acid, pioglitazone and pyridoxamine [8] were
used as controls, for which a combination of chelating
and antipathia properties was shown [28–30]. The
compounds were dissolved in 99% DMSO. The indi-
cator of the activity of copper(II) cations was 100 μM
ascorbic acid solution; the source of Cu2+ was CuSO4 ⋅
5H2O in a final concentration of 150 μM (95 μM in
Quantitatively, the activity of the compounds in the
tests for evaluation of the antiplatelet and antiglycation
terms of the same mass of anhydrous salt). Complex- actions, as well as actions to cleave the cross-links of
ation was carried out by preliminary mixing and incu- proteins, was evaluated by the value of Δ%, reflect-
bation for 5 min at 37°C of a solution of copper sulfate ing the ratio of the results obtained for the sample
and the test substance or DMSO. The mixture was with the substance and the results of the control
added to a solution of ascorbic acid. Autoxidation was group. The value was determined by the formula:
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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EVALUATION OF N-HYDROXY-, N-METOXY-
777
Fedotov, V.V., Slepukhin, P.A., Chupakhin, O.N.,
Charushin, V.N., and Rusinov, V.L., Archiv der Phar-
mazie, 2017, vol. 350, no. 12.
Δ% = (1 – datatest/Mdatacontrol) × 100%; where the
datatest is the absolute value of the trait registered in the
test (the level of fluorescence, optical absorption or
light scattering) in each individual sample with the
substance, the Mdatacontrol is the average level of the
same trait in the control samples without substance.
For a visual representation of the ∆% values, we used
the definition of indicators M m, corresponding to
the arithmetic mean (M) and the standard error of the
arithmetic mean (m).
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The authors declare that they have no conflict of
interest. This article does not contain any studies
involving animals or human participants performed by
any of the authors.
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Translated by E. Stepanova
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 44 No. 6 2018