Uracil Hydroxybenzamides as Potential Antidiabetic Prodrugs

Article abstract of DOI:10.1007/s11094-019-02029-5

A series of N¹, N³-bis-hydroxybenzoyl, -acetoxybenzoyl, and -methoxybenzoyl uracil derivatives were synthesized. All compounds were screened for the ability to rupture protein cross links and antiglycating, chelating, and antiaggregant properties, which are most significant for pharmacological treatment of thrombosis and angio-, nephro-, encephalo-, and cardiopathies. 1,3-bis-(4-Methoxybenzoyl)pyrimidine-2,4(1H,3H)-dione was a promising antidiabetic agent with all studied activities.

Full text of DOI:10.1007/s11094-019-02029-5

DOI 10.1007/s11094-019-02029-5
Pharmaceutical Chemistry Journal, Vol. 53, No. 6, September, 2019 (Russian Original Vol. 53, No. 6, June, 2019)
URACIL HYDROXYBENZAMIDES AS POTENTIAL
ANTIDIABETIC PRODRUGS
A. K. Brel’,1 A. A. Spasov,1 S. V. Lisina,1,* S. S. Popov,1 A. F. Kucheryavenko,1
R. A. Litvinov,1 O. A. Salaznikova,1 and A. I. Rashchenko1
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 53, No. 6, pp. 28 – 32, June, 2019.
Original article submitted January 14, 2019.
A series of N1, N3-bis-hydroxybenzoyl, -acetoxybenzoyl, and -methoxybenzoyl uracil derivatives were syn-
thesized. All compounds were screened for the ability to rupture protein cross links and antiglycating, chelat-
ing, and antiaggregant properties, which are most significant for pharmacological treatment of thrombosis and
angio-, nephro-, encephalo-, and cardiopathies. 1,3-bis-(4-Methoxybenzoyl)pyrimidine-2,4(1H,3H)-dione
was a promising antidiabetic agent with all studied activities.
Keywords: hydroxybenzamides, uracil, antidiabetic activity, chelating activity.
Hydroxybenzoic acids and nitrogenous bases exhibit var-
ious types of pharmacological activity and include
5-(thien-2-yl)-, 5-(furan-2-yl)-, and (E)-5-(2-bromovinyl)-
hydroxybenzamide substituents as platforms for designing
potential drugs for therapy of diabetes complications.
Target compounds were synthesized via acylation of the
2¢-deoxyuridine (Brivudin) (antiviral activity); 5-[alkoxy-
(4-nitrophenyl)]substituted uracils (antitumor activity) [1];
6-(aryl)alkylaminouracil derivatives (immunotropic activity)
[2], etc. The antidiabetic potential of pyrimidine derivatives
seems interesting because they were capable of rupturing
cross links of glycated proteins and displayed antiglycating,
multifunctional antioxidant, and chelating activity [3 – 5].
Diabetic patients have an increased risk of developing
cardiovascular pathologies (elevated platelet aggregation
combined with atherogenesis and reduced fibrinolysis) [6].
Antiplatelet activity was found for hydroxybenzoic acid iso-
mers [7]. This made it a promising platform for designing
antiplatelet agents.
appropriate nitrogenous bases by hydroxybenzoyl chlorides
in Py (Scheme 1) [9].
Scheme 1
OR
O
O
HO
O
Cl
O
C
C
C
NH
O
C
N
(COCl)
N
H
O
2
RO
RO
O
N
2:1
O
RO
I – IX
RO = 2-OH (I), 3-OH (II), 4-OH (III), 2-OAc (IV), 3-OAc (V), 4-OAc
(VI), 2-OMe (VII), 3-OMe (VIII), 4-OMe (IX).
N1, N3-Uracils were prepared via acylation of the corre-
sponding nitrogenous base by the acid chloride
(base—acid-chloride ratio 1:2) in Py at room temperature.
Pyrimidine derivatives can act as multifunctional antiox-
idants by reducing the risk of developing inflammations and
pathologies [3, 8]. Binding of excesses of redox-active metal
cations (Cu2+, Fe2+, etc.) that disturb the in vivo antioxidant
balance is an important criterion of overall antidiabetic activ-
ity [8].
EXPERIMENTAL CHEMICAL PART
The goals of the present work were to synthesize and
study the biological activity of new uracil derivatives with
Melting points were determined in capillaries on a Stuart
SMP-30 apparatus at heating rate 10°C/min. The purity and
identity of compounds were confirmed by TLC on Silufol
UV-254 plates using i-PrOH—H O (7:3) mobile phase and I
1
Volgograd State Medical University, Ministry of Health of the RF, 1
Pavshikh Bortsov Sq., Volgograd, 400131 Russia; tel: (8442) 38-50-05.
2
2
*
e-mail: svlisina@gmail.com
vapor and UV light for detection. PMR spectra of the com-
511
0091-150X/19/5306-0511 © 2019 Springer Science+Business Media, LLC

512
A. K. Brel’ et al.
a
b
70
60
50
40
30
20
10
0
120
100
80
60
40
20
0
y = 22.70x – 6.991
R2 =0.900
y = 38.54x – 1.2185
R2 = 0.9455
0
0.5
1.0
C, mM
1.5
2.0
0
2
4
6
C, mM
Fig. 1. Concentration dependences of rupturing activity of IX (a) and Alagebrium (ALT-711) (b ).
pounds in DMSO-d with HMDS internal standard were re-
(AGEs). The reaction mixture contained glucose (500 mM)
and bovine serum albumin (BSA, 1 mg/mL) dissolved in
phosphate buffer (pH 7.4). All tested compounds were dis-
solved in DMSO. The resulting solutions of test compounds
at a final concentration of 10–3 M or the corresponding vol-
ume of solvent (control) were added to the samples, which
were incubated for 24 h at 60°C. The specific fluorescence of
the glycation products was determined in a TECAN M
200Pro plate reader (for l 370 nm and l 440 nm). Activ-
6
corded on a Bruker DRX500 spectrometer. Spectra were in-
terpreted using Advanced Chemistry Development Inc. li-
censed software under the commercial name ACD/HMNR
Predictor Pro v. 3.
All solvents and reagents were purchased commercially
and used without further purification. Table 1 presents the
mps, yields, and spectral data.
Hydroxybenzoyl chloride was synthesized by the litera-
ture method [10, 11].
ex
em
ity was calculated as the ratio to the fluorescence of control
General method for synthesizing 1,3-bis-(hydroxy-
benzoyl)pyrimidine-2,4(1H,3H)-diones (I-IX) [12]. A so-
lution of heterocyclic base (100 mmol) in anhydrous Py
(30 mL) was treated slowly and dropwise over 15 min with
samples as parameter D, %. The reference compound was the
well-known
non-enzymatic
glycosylation
inhibitor
aminoguanidine (Sigma Aldrich) [8]. Results were processed
statistically using Microsoft Excel 2007 software and the
GraphPad Prism 5.0 program with Mann—Whitney pairwise
comparisons.
hydroxybenzoyl chloride (200 mmol) in CHCl (10 mL). As
3
a result, the product and pyridinium chloride precipitated.
The mixture was stirred for 1 h at room temperature and fil-
Chelating activity was studied using metal-dependent
tered. The precipitate was rinsed with cold H O and
autoxidation of ascorbic acid (AA) in the range
4.0 × 10–5 – 1.5 × 10–4 M with determination of the IC [14].
The reference compounds were known chelators, i.e., lipoic
2
recrystallized from C H .
6
6
50
1,3-bis-(4-Methoxybenzoyl)pyrimidine-2,4(1H,3H)-
dione (IX), yield 31.19 g (82%), R 0.360 (i-PrOH—H O,
acid and pyridoxamine, with IC values for the chelating ac-
f
2
50
7:3).
tivity in the aforementioned concentration range [8]. An AA
solution (100 mM) was used to evaluate the chelating proper-
ties. The autoxidation catalyst was CuSO ·5H O solution.
EXPERIMENTAL BIOLOGICAL PART
4
2
The complexation involved preliminary mixing of
autoxidation catalyst solution (final concentration 150 mM,
equivalent to 95 mM of anhydrous salt) and test-compound
solution or solvent (DMSO, 99%) followed by incubation for
5 min at 37°C on a water bath. Autoxidation was initiated by
adding the incubated mixture to the AA solution. AA oxida-
tion was recorded spectrophotometrically using the optical
density (APEL PD 303 UV spectrophotometer, Japan;
Antiaggregant activity of the compounds at a concentra-
tion of 10–4 M was studied in vitro using the ADP-induced
rabbit platelet aggregation model (Reanal, Hungary) and the
change of light transmittance of platelet-rich plasma [13]. A
230 LA dual-channel laser analyzer (NPF Biola, Russia) of
platelet aggregation was used. Activities of compounds were
estimated from the decrease of platelet aggregation vs. the
control. The reference drugs was acetylsalicylic acid (ASA,
Shandong Xinhua Pharmaceutical Co., Ltd., China). Results
were processed statistically using Microsoft Excel 2007 soft-
ware and the GraphPad Prism 5.0 program with
Mann—Whitney pairwise comparisons.
265 nm, 10 mm pathlength). IC values of test compounds
50
were determined by linear regression if the activity was sta-
tistically significant. Results were processed statistically us-
ing Microsoft Excel 2007 software and the GraphPad Prism
5.0 program with Mann—Whitney pairwise comparisons.
The ability of the new compounds at 10–3 M to rupture
cross links of glycated proteins was studied in vitro by mea-
suring the optical density of solutions containing glycated
Antiglycating activity was determined by the published
method [3] using the ability of the compound to inhibit for-
mation of fluorescent advanced glycation end products

Uracil Hydroxybenzamides as Potential Antidiabetic Prodrugs
513
a
b
80
70
60
50
40
30
20
10
0
90
80
70
60
50
40
30
y = 1.4279x – 46.034
R2 =0.9337
y = 0.8293x – 38.764
R2 = 0.9998
20
10
0
20
40
60
80
100
40
60
80
100
120
140
Concentration, mM
Concentration, mM
Fig. 2. Concentration dependences of chelating activity of III (a) and IX (b).
BSA, which was prepared by incubating BSA (1 mg/mL)
and glucose solutions (500 mM) for two months. A colla-
gen-coated 96-well plate was treated carefully with phos-
phate buffer (pH 7.4) for 1 h to neutralize acidic collagen.
The plate was blocked with Superblock (Pierce) at 37°C for
1 h, treated with BSA solution, and incubated at 37°C for
4 h. The test compound was added to wells (100 mL/well)
with glycated BSA and with untreated BSA. The plates were
action on a TECAN M 200 Pro plate reader. The reference
drug was Alagebrium (ALT-711), the activity of which was
evaluated at the same concentration as for the compounds,
i.e., 10–3 M. IC values were determined to characterize
50
quantitatively the concentration dependences of the com-
pounds and reference drug. The concentration range for this
was 0.5 × 10–3 – 5 × 10–3 M. Results were processed using
Microsoft Excel software (Microsoft, USA) to calculate the
descriptive statistics.
incubated at 37°C for 16 h, treated with rabbit antibody
against BSA (1:500, 80 mL/well), incubated at 37°C for
50 min, treated with labeled horseradish peroxidase IgG
against rabbit antibodies (1:1000), incubated at 37°C for
RESULTS AND DISCUSSION
50 min,
(100 mL/well), and incubated at room temperature in the dark
for 20 min. The reaction was stopped using H SO (2 M).
treated
with
3,3¢,5,5¢-tetramethylbenzidine
Table 2 summarizes the experimental results.
Antiaggregant activities of the N-hydroxybenzoyl uracils
found that they all possessed varying abilities to inhibit
platelet functioning induced by ADP at a final concentration
2
4
Optical density at 450 nm was measured 10 min after the re-
TABLE 1. Characteristics of bis-(Hydroxybenzoyl)pyrimidine-2,4(1H,3H)-diones
Compound
-OR
Yield, %
87
mp, °C
PMR spectra (DMSO-d6,), d, ppm
I
2-OH
328 – 330
11.82 (s, 2H, -OH), 8.12 – 8.10 (d, 2H, uracil), 7.44 – 7.40 (m, 4H, C6H4), 7.04 – 6.98 (m,
2H, C6H4), 6.90 – 6.84 (m, 2H, C6H4), 5.48 – 5.44 (d, 2H, uracil)
II
3-OH
71
321 – 323
8.25 – 8.23 (d, H, uracil), 7.86 – 7.82 (m, 2H, C6H4), 7.75 – 7.72 (m, 2H, C6H4),
7.30 – 7.26 (t, 2H, C6H4), 7.08 (s, 2H, -OH), 6.95 – 6.94 (m, 2H, C6H4), 5.48 – 5.45 (d, H,
uracil)
III
IV
4-OH
2-OAc
3-OAc
4-OAc
2-OCH3
3-OCH3
4-OCH3
76
91
85
87
71
69
82
348 – 350
305 – 307
334 – 336
311 – 313
327 – 329
301 – 303
319 – 322
8.16 – 8.14 (d, H, uracil), 8.19 (s, 2H, -OH), 7.39 – 7.37 (d, 4H, C6H4), 6.82 – 6.80 (m,
4H, C6H4), 5.47 – 5.45 (d, H, uracil)
8.13 – 8.11 (d, H, uracil), 7.43 – 7.41 (m, 2H, C6H4), 7.32 – 7.28 (m, 2H, C6H4),
6.93 – 6.85 (m, 4H, C6H4), 5.47 – 5.45 (d, H, uracil), 2.33 (s, 6H, -CH3)
V
8.37 – 8.35 (d, H, uracil), 7.85 – 7.83 (m, 2H, C6H4), 7.39 – 7.35 (t, 2H, C6H4), 7.27 – 7.25
(t, 2H, C6H4), 6.99 – 6.96 (m, 2H, C6H4), 5.47 – 5.45 (d, H, uracil), 2.26 (s, 6H, -CH3)
VI
8.47 – 8.45 (d, H, uracil), 7.39 – 7.37 (d, 4H, C6H4), 6.94 – 6.91 (d, 4H, C6H4), 5.47 – 5.45
(d, H, uracil), 2.25 (s, 6H, -CH3)
VII
VIII
IX
8.14 – 8.11 (d, H, uracil), 7.47 – 7.45 (m, 2H, C6H4), 7.25 – 7.23 (m, 2H, C6H4),
6.99 – 6.96 (m, 4H, C6H4), 5.55 – 5.52 (d, H, uracil), 4.02 (s, 6H, -CH3)
8.32 – 8.29 (d, H, uracil), 7.89 – 7.86 (m, 2H, C6H4), 7.38 – 7.34 (t, 2H, C6H4), 7.29 – 7.26
(t, 2H, C6H4), 6.98 – 6.94 (m, 2H, C6H4), 5.48 – 5.46 (d, H, uracil), 4.02 (s, 6H, -CH3)
7.88 (d, 1H, uracil), 7.73 – 7.68 (m, 4H, C6H4), 7.33 (d, 1H, uracil), 7.05 – 7.01 (m, 4H,
C6H4), 3.85 (s, 6H, -CH3).

514
A. K. Brel’ et al.
of 5 × 10–6 M. Compounds III, VI, and IX had antiplatelet
activity equal to that of the reference drug ASA. Compounds
I and II, which were 1.3 (36%) and 1.4 (44%) times, respec-
tively, more active than ASA, had the highest antiaggregant
activity for the used model. These hydroxybenzamide sub-
stituents both had OH groups in the 2- and 3-positions, re-
spectively. However, III (4-hydroxybenzamide) had antiag-
gregant activity comparable to that of the reference drug.
Acetoxybenzamides IV and VI and methoxybenzamide IX
had low antiaggregant activity. Thus, the studied series con-
tained compounds with antiaggregant activity greater than or
equal to that of ASA. This confirmed that a compartment
comprising an N-hydroxybenzamide as a uracil substituent
was promising. Compounds with hydroxybenzoyl substitu-
ents with 2- and 3-OH groups were most active.
Chelating activity at initial concentration 4.0 × 10–5
M
was not statistically significant for any of the compounds
(Table 2). Compounds I, III, IV, and VI at the maximum
concentration of 1.5 × 10–4 M were unsuitable for determin-
ing the half-active concentrations because of excessive light
absorption at operating wavelength 265 nm at concentrations
>80 mM. The IC values for III and IX were 107.1 and
50
67.5 mM, respectively (Fig. 2).
The IC values of the chelating activity found for the
50
reference drugs capable of inhibiting growth of diabetes-as-
sociated pathologies, i.e., pyridoxamine and lipoic acid [8],
were 136.7 and 68.9 mM, respectively. The ability of III and
IX to inhibit Cu-catalyzed AA autoxidation suggested that
these compounds were multifunctional antioxidants under in
vivo conditions. Compound IX could be proposed to have the
ability to inhibit the development of diabetic pathologies
through several mechanisms given the observed ability to
rupture protein cross links and exhibit antiglycating activity.
Thus, the research indicated that modifying the uracil
core by incorporating hydroxybenzoyl substituents was ad-
vantageous because the direct substituents of the hydroxy-
benzoyl radical such as a 4-OMe or 2- and 3-OH could con-
tribute to the rupturing, antiglycating, and antiaggregant ac-
tivity according to a pharmacological analysis of the
molecular isomers. The new 1,3-bis-(hydroxybenzoyl)pyri-
midine-2,4(1H,3H)-diones were promising for developing
antidiabetic drugs of complex action. Compound IX
[1,3-bis-(4-methoxybenzoyl)pyrimidine-2,4(1H,3H)-dione]
had the most pronounced efficiency for rupturing glycated
protein cross links. This type of activity was combined with
antiplatelet, antiglycating, and chelating activities, which
was important for preventing vascular complications of dia-
The investigation of the ability of the compounds to rup-
ture cross links of glycated proteins found that IX was the
most active and comparable to ALT-711 [15, 16]. Compound
II was weakly active. The other compounds were inactive.
The dose—activity (to rupture cross links) relationship was
determined and used to find the IC value (1.32 mM)
50
(Fig. 1). The IC of ALT-711 was 1.89 mM. Thus, IX was
50
30% more active than ALT-711. It could be concluded that
uracil derivatives with hydroxybenzamides could potentially
be platforms for designing a new class of antidiabetic com-
pounds that rupture cross links of glycated proteins and are
more active than ALT-711.
Assessment of the antiglycating activity of these com-
pounds established that 4-OH III and 4-OMe IX exhibited
similar although 3.6 and 3.2 times, respectively, less activity
than aminoguanidine. However, 4-acetoxybenzoyl derivative
VI did not exhibit this type of activity.
TABLE 2. Biological Activity of N-Hydroxybenzoyl Uracils
Compound
Antiaggregant activity D, %
Cross-link rupture D, %
Chelating activity D, %
Antiglycating activity D, %
I
–
N. A.
23.58 ± 2.3
25.10 ± 1.43
18.12 ± 1.5
15.38 ± 0.9
18.82 ± 1.8
18.68 ± 1.9
17.38 ± 1.37
–
19.00 ± 9.32
— 9.85 ± 10.82
— 6.91 ± 4.85
15.29 ± 4.45
0.50 ± 3.47
3.85 ± 8.56
–
II
5.13 ± 9.69
— 7.40 ± 2.40
III
–
–
–
12.00 ± 4.90*
IV
2.60 ± 10.20
VI
3.10 ± 12.40
IX
22.19 ± 3.28
13.50 ± 2.00*
ASA
–
–
–
–
–
Alagebrium (ALT-711)
Pyridoxamine
Lipoic acid
Aminoguanidine
–
23.02 ± 8.79
–
–
–
–
26.56 ± 6.92
37.82 ± 4.70
— 5.0 ± 2.1
–
–
43.4 ± 2.4*
Note: N. A. is the inability to study the activity because intrinsic fluorescence of the compound limited the method; * differences statistically
significant from the control group, Mann—Whitney criterion (p < 0.05).

Uracil Hydroxybenzamides as Potential Antidiabetic Prodrugs
515
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