5-Заміщені N-(9Н-пурин-6-іл)-1,2-оксазол-3-карбоксаміди як інгібітори ксантиноксидази
Synthetic 6-substituted purine derivatives are known to exhibit diverse bioactivity. In this paper, a series of N-(9H-purin-6-yl)-1,2-oxazole-3-carboxamide derivatives were synthesized and evaluated in vitro against xanthine oxidase, an enzyme of purine catabolism. The introduction of aryl substitue...
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V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry of the National Academy of Sciences of Ukraine
2020
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Ukrainica Bioorganica Acta| _version_ | 1871193587504381952 |
|---|---|
| author | Muzychka, Oksana V. Kobzar, Oleksandr L. Shablykin, Oleh V. Brovarets, Volodymyr S. Vovk, Andriy I. |
| author_facet | Muzychka, Oksana V. Kobzar, Oleksandr L. Shablykin, Oleh V. Brovarets, Volodymyr S. Vovk, Andriy I. |
| author_institution_txt_mv | [
{
"author": "Oksana V. Muzychka",
"institution": "V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry of the NAS of Ukraine, 1 Murmanska St., Kyiv, 02094, Ukraine"
},
{
"author": "Oleksandr L. Kobzar",
"institution": "V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry of the NAS of Ukraine, 1 Murmanska St., Kyiv, 02094, Ukraine"
},
{
"author": "Oleh V. Shablykin",
"institution": "V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry of the NAS of Ukraine, 1 Murmanska St., Kyiv, 02094, Ukraine"
},
{
"author": "Volodymyr S. Brovarets",
"institution": "V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry of the NAS of Ukraine, 1 Murmanska St., Kyiv, 02094, Ukraine"
},
{
"author": "Andriy I. Vovk",
"institution": "V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry of the NAS of Ukraine, 1 Murmanska St., Kyiv, 02094, Ukraine"
}
] |
| author_sort | Muzychka, Oksana V. |
| baseUrl_str | https://bioorganica.com.ua/index.php/journal/oai |
| collection | OJS |
| datestamp_date | 2026-07-19T14:56:52Z |
| description | Synthetic 6-substituted purine derivatives are known to exhibit diverse bioactivity. In this paper, a series of N-(9H-purin-6-yl)-1,2-oxazole-3-carboxamide derivatives were synthesized and evaluated in vitro against xanthine oxidase, an enzyme of purine catabolism. The introduction of aryl substituent at position 5 of the oxazole ring was found to increase the inhibition efficiency. Some of the inhibitors containing 5-substituted isoxazole and purine moieties were characterized by IC50 values in the nanomolar range. According to the kinetic data, the most active N-(9H-purin-6-yl)-5-(5,6,7,8-tetrahydronaphthalen-2-yl)-1,2-oxazole-3-carboxamide demonstrated a competitive type of inhibition with respect to the enzyme-substrate. Molecular docking was carried out to elucidate the mechanism of enzyme-inhibitor complex formation. The data obtained indicate that xanthine oxidase may be one of the possible targets for the bioactive purine carboxamides. |
| doi_str_mv | 10.15407/bioorganica2020.01.020 |
| first_indexed | 2025-07-17T12:19:41Z |
| format | Article |
| fulltext |
ISSN 1814-9758. Ukr. Bioorg. Acta, 2020, Vol. 15, N 1
UDC 547.78+577.152
DOI: https://doi.org/10.15407/bioorganica2020.01.020
20
RESEARCH ARTICLE
5-Substituted N-(9H-purin-6-yl)-1,2-oxazole-3-carboxamides as xanthine
oxidase inhibitors
Oksana V. Muzychka, Oleksandr L. Kobzar, Oleh V. Shablykin, Volodymyr S. Brovarets,
Andriy I. Vovk*
V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry of the NAS of Ukraine, 1 Murmanska St., Kyiv, 02094, Ukraine
Abstract: Synthetic 6-substituted purine derivatives are known to exhibit diverse bioactivity. In this paper, a series of N-(9H-purin-6-yl)-
1,2-oxazole-3-carboxamide derivatives were synthesized and evaluated in vitro against xanthine oxidase, an enzyme of purine catabolism.
The introduction of aryl substituent at position 5 of the oxazole ring was found to increase the inhibition efficiency. Some of the inhibitors
containing 5-substituted isoxazole and purine moieties were characterized by IC50 values in the nanomolar range. According to the kinetic
data, the most active N-(9H-purin-6-yl)-5-(5,6,7,8-tetrahydronaphthalen-2-yl)-1,2-oxazole-3-carboxamide demonstrated a competitive type
of inhibition with respect to the enzyme-substrate. Molecular docking was carried out to elucidate the mechanism of enzyme-inhibitor
complex formation. The data obtained indicate that xanthine oxidase may be one of the possible targets for the bioactive purine
carboxamides.
Keywords: N-(9H-purin-6-yl)-1,2-oxazole-3-carboxamides, synthesis, bioactivity, xanthine oxidase.
Introduction
Purine derivatives are known to possess a range of
biological properties as inhibitors of kinases,
sulfotransferases, phosphodiesterases, and other enzymes as
well as ligands of some proteins [1]. It was reported that
derivatives of 6-(N-benzoylamino)purine can be potent
inhibitors of bromodomain-containing protein 4 (BRD4),
which control the expression of genes related to
inflammation, apoptosis, and cell proliferation [2-3].
Structural analogs of this compound with bulky biaryl
substituent were found to be potential inhibitors of the
cytosolic 5'-nucleotidase II, which regulates intracellular
nucleotide pools and has been recognized as a therapeutic
target for hematological cancers [4]. At the same time,
Received:
Revised:
Accepted:
Published online:
15.05.2020
22.05.2020
25.05.2020
30.06.2020
Corresponding author. Tel.: +380-44-558-5388;
e-mail: vovk@bpci.kiev.ua (A. I. Vovk)
ORCID: 0000-0001-6167-076X
6-(N-benzoylamino)purine was described as an inhibitor of
the purine catabolizing enzyme, xanthine oxidase [5]. This
enzyme catalyzes the oxidation of hypoxanthine and
xanthine to uric acid with the generation of superoxide
radicals. The increased uric acid levels lead to
hyperuricemia and gout, and overproduction of superoxide
radicals and other reactive oxygen species can promote
chronic inflammatory and cardiovascular diseases, cancer,
and diabetes [6].
The inhibitors of xanthine oxidase can be represented by
two groups, which include purine derivatives [7-8] and non-
purine compounds. The purine analog allopurinol is widely
used in clinical practice [9]. More effective non-purine
inhibitors of xanthine oxidase have also been developed,
such as derivatives of imidazole [10], pyrazole [11],
isoxazole [12], selenazole [13], and thiazole [14]. Among
them, febuxostat, with inhibition constants in the nanomolar
range, was approved for the treatment of hyperuricemia and
gout [15]. However, allopurinol and febuxostat are known
to induce side effects [9, 16], and there is thus interest in
new bioactive compounds targeting xanthine oxidase.
Introducing isoxazole fragment into organic molecules is
considered as a strategy for designing bioactive compounds
with anticancer, antimicrobial, anti-inflammatory, and other
© Muzychka O. V. et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Ukrainica Bioorganica Acta
www.bi oorgan ica .org .ua
mailto:vovk@bpci.kiev.ua
https://orcid.org/0000-0001-6167-076X
ISSN 1814-9758. Ukr. Bioorg. Acta, 2020, Vol. 15, N 1
21
activities [17, 18]. Many of such compounds are
represented by 3,5-substituted isoxazoles bearing other
heterocyclic rings [19]. As an example, the hybrid
molecules containing isoxazole, purine, and coumarin
moieties were synthesized and tested in vitro as antioxidants
and enzyme inhibitors [20]. In this paper, we synthesized
substituted isoxazole-purine conjugates structurally similar
to bioactive 6-(N-benzoylamino)purine. The 5-substituted
derivatives of N-(9H-purin-6-yl)-1,2-oxazole-3-carboxami-
des were evaluated in vitro as inhibitors of xanthine
oxidase.
Results and discussion
The synthesis of 5-substituted isoxazole acids was
carried out using known synthetic methods [21] by the
reaction of commercially available ketones with diethyl
oxalate in the presence of sodium ethoxide [22]. The
synthetic route included cyclization of ethyl 2,4-di-
oxobutanoates into ethyl isoxazole-3-carboxylates by the
addition of hydroxylamine hydrochloride in ethanol at
reflux [23] followed by saponification of the ester function
with sodium hydroxide in ethanol. Corresponding acyl
chlorides 3a-g were synthesized in the reaction of the
isoxazole acids and thionyl chloride in benzene and used
without purification in acylation of adenine (1) or 8-amino-
quinoline (2) (Scheme 1). All compounds were obtained in
moderate to good yield. After crystallization of the crude
products, the compounds 4a-g and 5f were characterized by
1H NMR, IR spectra, and MS.
The inhibition activities of compounds 4a-g against
xanthine oxidase were assayed by monitoring the rate of
enzymatic conversion of xanthine to uric acid. The IC50
values were defined as the concentration of the tested
compound causing 50% inhibition of the enzyme with
50 µM xanthine as a substrate [24]. Allopurinol and
6-(N-benzoylamino)purine were used as reference
inhibitors. Given the potential interest of the structures
containing isoxazole and purine moieties, compound 4f was
also evaluated in vitro against purine nucleoside
phosphorylase, however, no effect was observed on this
enzyme.
Experimental data (Table 1) showed that compounds 4a
with 5-methyl-1,2-oxazole fragment displayed slightly
decreased inhibitory activity as compared with
6-(N-benzoylamino)purine. The introduction of the
aromatic group at 5-position of the isoxazole ring
substantially increased the inhibitory potency of compounds
4b and 4e. Further increasing of xanthine oxidase inhibition
was observed in the case of methyl or methoxy substituent
at para-position of the phenyl ring of inhibitors 4c and 4d,
respectively. Modification of the isoxazole ring by
tetrahydronaphthalene fragment led to significant enzyme
inhibition by compound 4f with IC50 value of 14 nM which
is approximately 280-fold more effective than that of
allopurinol. Compound 4g with more hydrophilic
benzodioxinyl substituent demonstrated lower inhibitory
potency. The importance of the purine part of the hybrid
molecules in the inhibition mechanism was supported by
compound 5f which showed no activity.
Table 1. Xanthine oxidase inhibitory activity of N-(9H-
purin-6-yl)-1,2-oxazole-3-carboxamides 4a-g.
Compound IC50, µMa
6-(N-benzoylamino)purine 0.55±0.04
4a 0.75±0.18
4b 0.074±0.011
4c 0.048±0.013
4d 0.037±0.002
4e 0.078±0.011
4f 0.014±0.004
4g 0.044±0.003
Allopurinol 4.03±0.27
aIC50 values were calculated as the mean of 2-3 assays ± standard
deviation.
Kinetic studies were performed for the most active
compounds 4f at different concentrations of substrate and
the inhibitor to characterize the mechanism of inhibition.
The double reciprocal Lineweaver-Burk plots indicated a
competitive type of inhibition (Figure 1). This reveals that
the inhibitor interacts with free enzyme competing with the
substrate for the binding site. The calculated Ki value for
compound 4f was 7.46 ± 0.36 nМ. It is known that
allopurinol in complex with xanthine oxidase provides
electron transfer to molecular oxygen with a generation of
superoxide, but 6-(N-benzoylamino)purine does not exhibit
such effect [5]. Compound 4f was also not able to generate
superoxide radical that was confirmed by a test with
xanthine oxidase and reduced 2,6-dichlorophenolindo-
phenol, controlled by absorbance at 605 nm. This result
suggests that the purine part of the inhibitor with bulky
5-substituted isoxazole fragment is located near the
molybdopterin center without electron transfer.
Figure 1. Lineweaver-Burk plots for inhibition of xanthine
oxidase by compound 4f. The inhibitor concentrations were 0 (○),
5 nМ (●), 10 nМ (□), and 15 nМ (Δ).
0
5
10
15
20
25
30
35
40
-50 0 50 100 150
1
/V
0
,
µ
M
-1
∙m
in
1/[S]0, mM-1
O. V. Muzychka,, O. L. Kobzar, O. V. Shablykin et al.
22
N
N N
H
N
NH2
+
4h, reflux, Py
R = Me (a); (c); (f);
O
OS
(b);
R
NO
O
H
N
N
4a-g
(d);4-MeC6H4 (e);4-MeOC6H4 (g)
R
NO
O
H
N
N N
NH
N
or
N
NH2
5f
R
NO
O
Cl
1
2
Ph
3a-g
Scheme 1. Synthesis of N-(9H-purin-6-yl)-1,2-oxazole-3-carboxamides 4a-g and compound 5f.
To elucidate the possible mechanism of the enzyme-
inhibitor complex formation, computer modeling was
performed. Molecular docking calculations using a
modified version of Autodock 4.2 [25] showed that the
N-7-protonated tautomer of purine inhibitor can be
preferred for the formation of the enzyme-inhibitor
complex. The calculated docking energy for N-7- and
N-9-protonated tautomeric forms of compound 4f were
-9.09 kcal/mol and -8.02 kcal/mol, respectively. The
docking results (Figure 2) showed that two NH groups of
N-7-protonated tautomeric form participate in the formation
of hydrogen bonds with carboxylate group of Glu802 which
can be involved in protonation of the enzyme-substrate
[26, 27]. At the same time, HOH1365 provides the
interaction of N-9 atom of the purine ring of the inhibitor
with Glu1261 which can act as a general base in the
enzymatic reaction [28]. Purine fragment of the inhibitor is
also stabilized by aromatic-aromatic interaction with
Phe914 and Phe1009. The isoxazole ring can form
hydrophobic and van der Waals contacts with Leu873 and
Val1011. Tetrahydro-naphthalene substituent shows
aromatic-sigma interaction with Val1011 as well as
hydrophobic and van der Waals contacts with His875 and
Phe649 at the site exit.
Figure 2. Possible binding mode of inhibitor 4f in the active site
of bovine milk xanthine oxidase.
Conclusions
In the present paper, 5-substituted N-(9H-purin-6-yl)-1,2-
oxazoles were synthesized, and their inhibitory properties
were evaluated in vitro against xanthine oxidase. The
incorporation of a nonpolar bulky substituent at the
isoxazole ring provided a better binding affinity to the
enzyme active site. The most active compound, N-(9H-
purin-6-yl)-5-(5,6,7,8-tetrahydronaphthalen-2-yl)-1,2-oxa-
zole-3-carboxamide, was a competitive inhibitor of the
enzyme with an inhibition constant in the nanomolar range.
These data are helpful to consider xanthine oxidase as one
of the possible targets for isoxazole-containing purine
derivatives with diverse bioactivity.
Experimental section
The xanthine oxidase from bovine milk, bacterial purine
nucleoside phosphorylase and xanthine were purchased
from Sigma-Aldrich. Spectrophotometric measurements
were performed on a Specord M-40. NMR spectra were
obtained on a Bruker Avance DRX-500 instrument (1H,
500 MHz) in a solution of DMSO-d6 relative to internal
TMS. The IR spectra were recorded on a Vertex 70
spectrometer from KBr pellets. The melting points were
determined on a Fisher-Johns instrument. The LC/MS
spectra were recorded on an Agilent 1100 series high-
performance liquid chromatograph equipped by a diode
matrix with an Agilent LC/MSD SL mass selective
detector. The LC/MS parameters were set as follows:
column, Zorbax SBC18 1.8 μm, 4.6х15 mm (PN 821975-
932); solvents A, acetonitrile-water mixture (95:5), 0.1%
trifluoroacetic acid and B, 0.1% aqueous trifluoroacetic
acid; eluent flow rate, 3 ml/min; injection volume, 1 μl;
UV detection, 215, 254, 265 nm; atmospheric-pressure
chemical ionization (APCI) was used; scanning range, m/z
80-1000.
ISSN 1814-9758. Ukr. Bioorg. Acta, 2020, Vol. 15, N 1
23
Synthesis
General procedure for the acylation of adenine
A suspension of adenine (1) or 8-aminoquinoline (2)
(7.4 mmol) and corresponding acyl chloride (8 mmol) in
pyridine (10 ml) was mixed at room temperature and then
heated under reflux for 4 hours. The reaction mixture was
cooled and water (20 ml) was added, the precipitate was
filtered, washed with ethanol (5 ml). All the products 4a-g
and 5f were purified by recrystallization from a mixture of
DMF and water (1:1).
6-(N-Benzoylamino)purine was synthesized as described
previously [29].
Yield 86%, mp 242-242.5 °С [29]. IR (KBr) ν 1521,
1552, 1581, 1599, 1621, 1686, 3256, 3369. 1H NMR (500
MHz, DMSO-d6) 7.56-7.65 (m, 3H), 8.03-8.15 (m, 2H),
8.51 (s, 1H), 8.75 (s, 1H), 11.09 (br s, 0.1H, NН), 11.52
(br s, 0.9H, NН), 12.37 (br s, 0.9H, NН), 13.47 (br s, 0.1H,
NН). LC/MS (CI) m/z 240,2 (M+H)+. Anal. Calcd. for
С12Н9N5О: C, 60.25; H, 3.79; N, 29.27. Found: C, 60.13;
H, 3.81; N, 29.19.
5-Methyl-N-(9H-purin-6-yl)-1,2-oxazole-3-carboxamide
(4a).
Yield 60%, mp 270-271 °C. ІR (KBr) ν 1556, 1597,
1632, 1704, 2900, 3111, 3215. 1H NMR (500 MHz,
DMSO-d6) 2.35 (s, 3H), 7.40 (s, 1H), 8.52 (s, 1H), 8.74 (s,
1H), 11.00-12.25 (br s, 1H, NН), 12.25-14.00 (br s, 1H,
NН). LC/MS (CI) m/z 245.2 (M+H)+. Anal. Calcd. for
C10H8N6O2: C, 49.18; H, 3.30; N, 34.41. Found: С, 49.02;
H, 3.05; N, 34.23.
3-Phenyl-N-(9H-purin-6-yl)-1,2-oxazole-5-carboxamide
(4b).
Yield 91%, mp 256-257 °C. ІR (KBr) ν 1526, 1627,
1713, 3341. 1H NMR (500 MHz, DMSO-d6) 7.50-7.65 (s,
4H), 7.90-8.00 (m, 2H), 8.55 (s, 1H), 8.75 (s, 1H), 10.95-
11.40 (br s, 0.2H, NН), 11.40-12.00 (br s, 0.8H, NН),
12.30-12.80 (br s, 0.8H, NН), 13.30-13.90 (br s, 0.2H, NН).
LC/MS (CI) m/z 307.3 (M+H)+. Anal. Calcd. for
C15H10N6O2: C, 58.82; H, 3.29; N, 27.44. Found: C, 58.64;
H, 3.18; N, 27.23.
5-(4-Methylphenyl)-N-(9H-purin-6-yl)-1,2-oxazole-3-
carboxamide (4c).
Yield 86%, mp 255-256 °C. IR (KBr) ν 1523, 1559,
1610, 1718, 3354. 1H NMR (500 MHz, DMSO-d6) 2.37
(s, 3H), 7.37 (d, J 7.3, 2H), 7.51 (s, 1H), 7.84 (d, J 7.3, 2H),
8.54 (s, 1H), 8.75 (s, 1H), 10.80-12.10 (br s, 1H, NН),
12.10-13.00 (br s, 1H, NН). LC/MS (CI) m/z 321.3 (M+H)+.
Anal. Calcd. for C16H12N6O2: C, 60.00; H, 3.78; N, 26.24.
Found: C, 60.01; H, 3.54; N, 26.21.
5-(4-Methoxyphenyl)-N-(9H-purin-6-yl)-1,2-oxazole-3-
carboxamide (4d).
Yield 86%, mp 265-266 °C. IR (KBr) ν 1468, 1508,
1552, 1610, 1704, 3128, 3366. 1H NMR (500 MHz,
DMSO-d6) 3.83 (s, 3H), 7.11 (d, J 7.3, 2H), 7.46 (s, 1H),
7.89 (d, J 7.3, 2H), 8.56 (s, 1H), 8.76 (s, 1H), 10.90-11.10
(br s, 0.2H, NН), 11.50-11.80 (br s, 0.8H, NН), 12.40-12.70
(br s, 0.8H, NН), 13.40-13.70 (br s, 0.2H, NН). LC/MS (CI)
m/z 337.4 (M+H)+. Anal. Calcd. for C16H12N6O3: C, 57.14;
H, 3.60; N, 24.99. Found: C, 57.01; H, 3.34; N, 25.68.
N-(9H-Purin-6-yl)-5-(thiophen-2-yl)-1,2-oxazole-3-
carboxamide (4e).
Yield 86%, mp 252-253 °C. IR (KBr) ν 1505, 1524,
1557, 1608, 1628, 1666, 1718, 3107, 3367. 1H NMR (500
MHz, DMSO-d6) 7.24-7.32 (m, 1H), 7.40 (s, 1H), 7.80-
7.86 (m, 1H), 7.86-7.92 (m, 1H), 8.55 (s, 1H), 8.75 (s, 1H),
11.00-11.30 (br s, 0.2H, NН), 11.50-11.90 (br s, 0.8H, NН),
12.35-12.75 (br s, 0.8H, NН), 13.40-13.70 (br s, 1H, NН).
LC/MS (CI) m/z 312.4 (M+H)+. Anal. Calcd. for
C13H8N6O2S: C, 50.00; H, 2.58; N, 26.91. Found: C, 49.88;
H, 2.13; N, 26.67.
N-(9H-Purin-6-yl)-5-(5,6,7,8-tetrahydronaphthalen-2-
yl)-1,2-oxazole-3-carboxamide (4f).
Yield 86%, mp 229-230 °C. IR (KBr) ν 1523, 1621,
1714, 2932, 3359. 1H NMR (500 MHz, DMSO-d6) 1.65-
1.80 (m, 4H), 2.70-2.85 (m, 4H), 7.21 (d, J 8.4, 1H), 7.42
(s, 1H), 7.62 (br s, 2H), 8.47 (s, 1H), 8.69 (s, 1H), 10.50-
13.50 (br s, 0.5H partially in exchange). LC/MS (CI) m/z
361.4 (M+H)+. Anal. Calcd. for C19H16N6O2: C, 63.33;
H, 4.48; N, 23.32. Found: C, 63.17; H, 4.21; N, 23.27.
5-(2,3-Dihydro-1,4-benzodioxin-6-yl)-N-(9H-purin-6-yl)-
1,2-oxazole-3-carboxamide (4g).
Yield 86%, mp 269-270 °C. IR (KBr) ν 1506, 1556,
1576, 1624, 1716, 3127, 3356. 1H NMR (500 MHz,
DMSO-d6) 4.27-4.35 (m, 4H), 7.02 (d, J 8.1, 1H), 7.40-
7.50 (m, 3H), 8.55 (s, 1H), 8.75 (s, 1H), 10.95-11.15 (br s,
0.2H, NН), 11.50-11.75 (br s, 0.8H, NН), 12.40-12.65 (br s,
0.8H, NН), 13.45-13.65 (br s, 0.2H, NН). LC/MS (CI) m/z
365.4 (M+H)+. Anal. Calcd. for C17H12N6O4: C, 56.05;
H, 3.32; N, 23.07. Found: C, 56.10; H, 3.11; N, 23.14.
N-(Quinolin-8-yl)-5-(5,6,7,8-tetrahydronaphthalen-2-yl)-
1,2-oxazole-3-carboxamide (5f).
Yield 78%, mp 166-167 °C. ІR (KBr) ν 1498, 1577,
1615, 1729, 2937, 3129, 3411. 1H NMR (500 MHz,
DMSO-d6) 1.65-1.75 (m, 4H), 2.7-2.85 (m, 4H), 7.18-
7.77 (m, 7H), 8.44 (d, J 8.4, 1H), 8.71 (d, J 7.5, 1H), 8.98
(d, J 3.9, 1H), 10.95 (s, 1H, NН). LC/MS (CI) m/z 370.3
(M+H)+. Anal. Calcd. for C23H19N3O2: C, 74.78; H, 5.18;
N, 11.37. Found: С, 74.55; H, 5.05; N, 11.13.
Inhibition of xanthine oxidase
The enzymatic reaction was studied in sodium phosphate
buffer (50 mM, pH 7.4) at 25 °C. The mixture contained
xanthine (50 µM), inhibitor (from 2.5 nM to 50 µM), EDTA
(0.1 mM), and 1% DMSO was incubated for 5 min and the
reaction was initiated by addition of xanthine oxidase. The
enzyme concentration was 0.008 units/mL. The reaction
rate was monitored by the change in optical density at
293 nm. The IC50 values were calculated from the plot of
the inhibition percentage against inhibitor concentrations.
O. V. Muzychka,, O. L. Kobzar, O. V. Shablykin et al.
24
Purine nucleoside phosphorylase test
The reaction mixture contained sodium phosphate buffer
(0.1 M, pH 7.4), guanosine (0.1 mM), bacterial purine
nucleoside phosphorylase (0.071 units/mL), inhibitor
(50 µM), EDTA (0.1 mM) and 0.5% DMSO. The
enzymatic reaction was investigated at 25 °C. The reaction
rate was monitored by the change in optical density at
258 nm.
Reduction of 2,6-dichlorophenolindophenol
The reaction mixture contained 100 mM sodium
phosphate buffer (pH 7.4), 15 µM 2,6-dichlorophenol-
indophenol, and 10 µM xanthine or inhibitor. The reaction
was initiated by the addition of xanthine oxidase
(0.008 units/mL) to the reaction mixture and the absorbance
change was monitored at 605 nm.
Molecular docking
The docking simulation was performed to analyze the
probable binding mode of inhibitors at the active site of
xanthine oxidase by using a modified version of
AutoDock 4.2 [25]. The inhibitors were docked into the
active site of xanthine oxidase using chain C (PDB code
3B9J) [30]. Before the docking calculations, other chains,
ligand (2-hydroxy-6-methylpurine), and water molecules,
with the exception of important for catalytic mechanism
HOH1365 [31], were removed from the initial structure of
the enzyme. The oxygen atom of the molybdopterin
cofactor was replaced on a water molecule HOH1334. The
structures of inhibitors were converted into three-
dimensional ones and optimized in the MMFF94s force
field by using program Avogadro [32]. AutoDock Tool
(MGLTools 1.5.6) was used to prepare the docking files.
The constraint position for the C8 atom of the purine
fragment was added to docking parameter files using the
ATPOSCONSTR keyword [25]. A ligand's atom number
from PDBQT file and its constrained coordinates (-57.055,
-18.200 and 19.928 for x, y, and z, respectively) were
included in this parameter with a maximal allowed distance
of 3 Å. The Lamarckian genetic algorithm was applied to
search for the optimum binding pose of the ligands [33].
The analysis of the binding mode of the inhibitors was
performed using Discovery Studio 3.5 visualizer.
Notes
Acknowledgments and finances. The work was
financed by the National Academy of Sciences of Ukraine
(project CPDF 1-17).
The authors declare no conflict of interest.
Author contributions. O. V. M: the investigation of
bioactivity, writing. O. L. K: molecular docking
simulations, analysis of the experimental results, writing.
O. V. S: synthesis of compounds, writing experimental
section. V. S. B: synthesis of compounds,
conceptualization. A. I. V: conceptualization, writing, and
editing.
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5-Заміщені N-(9Н-пурин-6-іл)-1,2-оксазол-3-карбоксаміди як інгібітори
ксантиноксидази
О. В. Музичка, О. Л. Кобзар, О. В. Шабликін, В. С. Броварець, А. І. Вовк*
Інститут біоорганічної хімії та нафтохімії ім. В.П. Кухаря НАН України, вул. Мурманська, 1, Київ, 02094, Україна
Резюме: У цій роботі нами синтезовано серію похідних N-(9Н-пурин-6-іл)-1,2-оксазол-3-карбоксаміду та оцінено їх інгібувальну здатність щодо
ксантиноксидази, ферменту пуринового катаболізму. Синтез 5-заміщених ізоксазолкарбонових кислот здійснено за допомогою відомих
синтетичних методів. Для ацилювання аденіну використовували відповідні ацилхлориди, отримані реакцією ізоксазолкарбонових кислот з
тіонілхлоридом. За результатами досліджень in vitro наявність фенільного замісника в положенні 5 оксазольного кільця підвищує ефективність
інгібування ксантиноксидази. Подальше зростання інгібувального впливу спостерігалося при введенні метильної або метокси-групи в пара-
положення фенільного кільця. Деякі з інгібіторів, що містять 5-заміщені ізоксазолові та пуринові фрагменти, характеризувались наномолярними
значеннями IC50. Згідно кінетичних даних, найбільш активний N-(9Н-пурин-6-іл)-5-(5,6,7,8-тетрагідронафтален-2-іл)-1,2-оксазол-3-карбоксамід
демонстрував конкурентний тип інгібування щодо субстрату з константою інгібування 7,46 ± 0,36 нМ. Для з’ясування механізму формування
комплексу фермент-інгібітор було проведено молекулярний докінг. Результати моделювання показали, що N-7-таутомерна форма інгібітора
може забезпечувати формування водневих зв’язків, гідрофобних і Ван-дер-Ваальсових контактів та донорно-акцепторних взаємодій. Отримані
результати вказують на те, що ксантиноксидаза може бути однією з можливих мішеней для біоактивних карбоксамідних похідних пурину.
Ключові слова: N-(9Н-пурин-6-іл)-1,2-оксазол-3-карбоксаміди, синтез, біоактивність, ксантиноксидаза.
http://cccc.uochb.cas.cz/61/3/0389/
http://cccc.uochb.cas.cz/61/3/0389/
http://cccc.uochb.cas.cz/61/3/0389/
1. Legraverend, M.; Grierson, D. S. The Purines: Potent and Versatile Small Molecule Inhibitors and Modulators of Key Biological Targets. Bioorg. Med. Chem. 2006, 14, 3987-4006.
12. Wang, S.; Yan, J.; Wang, J.; Chen, J.; Zhang, T.; Zhao, Y.; Xue, M. Synthesis of some 5-phenylisoxazole-3-carboxylic acid derivatives as potent xanthine oxidase inhibitors. Eur. J. Med. Chem. 2010, 45, 2663-2670.
13. Guan, Q.; Cheng, Z.; Ma, X.; Wang, L.; Feng, D.; Cui, Y.; Bao, K.; Wu, L.; Zhang, W. Synthesis and bioevaluation of 2-phenyl-4-methyl-1,3-selenazole-5-carboxylic acids as potent xanthine oxidase inhibitors. Eur. J. Med. Chem. 2014, 85, 508-516.
|
| id | oai:ojs2.bioorganica.com.ua:article-4 |
| institution | Ukrainica Bioorganica Acta |
| keywords_txt_mv | keywords |
| language | English |
| last_indexed | 2026-07-20T01:01:05Z |
| publishDate | 2020 |
| publisher | V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry of the National Academy of Sciences of Ukraine |
| record_format | ojs |
| resource_txt_mv | bioorganicacomua/80/8d3c8766dc5ba8cb39995136ee9c8980.pdf |
| spelling | oai:ojs2.bioorganica.com.ua:article-42026-07-19T14:56:52Z 5-Substituted N-(9H-purin-6-yl)-1,2-oxazole-3-carboxamides as xanthine oxidase inhibitors 5-Заміщені N-(9Н-пурин-6-іл)-1,2-оксазол-3-карбоксаміди як інгібітори ксантиноксидази Muzychka, Oksana V. Kobzar, Oleksandr L. Shablykin, Oleh V. Brovarets, Volodymyr S. Vovk, Andriy I. N-(9H-purin-6-yl)-1,2-oxazole-3-carboxamides synthesis bioactivity xanthine oxidase N-(9Н-пурин-6-іл)-1,2-оксазол-3-карбоксаміди синтез біоактивність ксантиноксидаза Synthetic 6-substituted purine derivatives are known to exhibit diverse bioactivity. In this paper, a series of N-(9H-purin-6-yl)-1,2-oxazole-3-carboxamide derivatives were synthesized and evaluated in vitro against xanthine oxidase, an enzyme of purine catabolism. The introduction of aryl substituent at position 5 of the oxazole ring was found to increase the inhibition efficiency. Some of the inhibitors containing 5-substituted isoxazole and purine moieties were characterized by IC50 values in the nanomolar range. According to the kinetic data, the most active N-(9H-purin-6-yl)-5-(5,6,7,8-tetrahydronaphthalen-2-yl)-1,2-oxazole-3-carboxamide demonstrated a competitive type of inhibition with respect to the enzyme-substrate. Molecular docking was carried out to elucidate the mechanism of enzyme-inhibitor complex formation. The data obtained indicate that xanthine oxidase may be one of the possible targets for the bioactive purine carboxamides. У цій роботі нами синтезовано серію похідних N-(9Н-пурин-6-іл)-1,2-оксазол-3-карбоксаміду та оцінено їх інгібувальну здатність щодо ксантиноксидази, ферменту пуринового катаболізму. Синтез 5-заміщених ізоксазолкарбонових кислот здійснено за допомогою відомих синтетичних методів. Для ацилювання аденіну використовували відповідні ацилхлориди, отримані реакцією ізоксазолкарбонових кислот з тіонілхлоридом. За результатами досліджень in vitro наявність фенільного замісника в положенні 5 оксазольного кільця підвищує ефективність інгібування ксантиноксидази. Подальше зростання інгібувального впливу спостерігалося при введенні метильної або метокси-групи в пара-положення фенільного кільця. Деякі з інгібіторів, що містять 5-заміщені ізоксазолові та пуринові фрагменти, характеризувались наномолярними значеннями IC50. Згідно кінетичних даних, найбільш активний N-(9Н-пурин-6-іл)-5-(5,6,7,8-тетрагідронафтален-2-іл)-1,2-оксазол-3-карбоксамід демонстрував конкурентний тип інгібування щодо субстрату з константою інгібування 7,46 ± 0,36 нМ. Для з’ясування механізму формування комплексу фермент-інгібітор було проведено молекулярний докінг. Результати моделювання показали, що N-7-таутомерна форма інгібітора може забезпечувати формування водневих зв’язків, гідрофобних і Ван-дер-Ваальсових контактів та донорно-акцепторних взаємодій. Отримані результати вказують на те, що ксантиноксидаза може бути однією з можливих мішеней для біоактивних карбоксамідних похідних пурину V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry of the National Academy of Sciences of Ukraine 2020-06-30 Article Article application/pdf application/pdf https://bioorganica.com.ua/index.php/journal/article/view/4 10.15407/bioorganica2020.01.020 Ukrainica Bioorganica Acta; Vol. 15 No. 1 (2020): Ukrainica Bioorganica Acta; 20-25 Ukrainica Bioorganica Acta; Том 15 № 1 (2020): Ukrainica Bioorganica Acta; 20-25 1814-9766 1814-9758 10.15407/bioorganica2020.01 en https://bioorganica.com.ua/index.php/journal/article/view/4/3 https://bioorganica.com.ua/index.php/journal/article/view/4/6 Copyright (c) 2020 Oksana V. Muzychka, Oleksandr L. Kobzar, Oleh V. Shablykin, Volodymyr S. Brovarets, Andriy I. Vovk https://creativecommons.org/licenses/by/4.0 |
| spellingShingle | N-(9Н-пурин-6-іл)-1,2-оксазол-3-карбоксаміди синтез біоактивність ксантиноксидаза Muzychka, Oksana V. Kobzar, Oleksandr L. Shablykin, Oleh V. Brovarets, Volodymyr S. Vovk, Andriy I. 5-Заміщені N-(9Н-пурин-6-іл)-1,2-оксазол-3-карбоксаміди як інгібітори ксантиноксидази |
| title | 5-Заміщені N-(9Н-пурин-6-іл)-1,2-оксазол-3-карбоксаміди як інгібітори ксантиноксидази |
| title_alt | 5-Substituted N-(9H-purin-6-yl)-1,2-oxazole-3-carboxamides as xanthine oxidase inhibitors |
| title_full | 5-Заміщені N-(9Н-пурин-6-іл)-1,2-оксазол-3-карбоксаміди як інгібітори ксантиноксидази |
| title_fullStr | 5-Заміщені N-(9Н-пурин-6-іл)-1,2-оксазол-3-карбоксаміди як інгібітори ксантиноксидази |
| title_full_unstemmed | 5-Заміщені N-(9Н-пурин-6-іл)-1,2-оксазол-3-карбоксаміди як інгібітори ксантиноксидази |
| title_short | 5-Заміщені N-(9Н-пурин-6-іл)-1,2-оксазол-3-карбоксаміди як інгібітори ксантиноксидази |
| title_sort | 5-заміщені n-(9н-пурин-6-іл)-1,2-оксазол-3-карбоксаміди як інгібітори ксантиноксидази |
| topic | N-(9Н-пурин-6-іл)-1,2-оксазол-3-карбоксаміди синтез біоактивність ксантиноксидаза |
| topic_facet | N-(9H-purin-6-yl)-1,2-oxazole-3-carboxamides synthesis bioactivity xanthine oxidase N-(9Н-пурин-6-іл)-1,2-оксазол-3-карбоксаміди синтез біоактивність ксантиноксидаза |
| url | https://bioorganica.com.ua/index.php/journal/article/view/4 |
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