A study about excellent xanthine oxidase inhibitory effects of new pyridine salts

Article abstract of DOI:10.1007/s00706-021-02831-6

A series of pyridine salts were synthesized containing vitamin B3 (niacin), isonicotinonitrile, and non-substituted pyridine fragment from reactions of 1,3-dibromopropane, 1,4-dibromobutane, and 4,4′-bis(chloromethyl)-1,1′-biphenyl reactants with pyridine

Full text of DOI:10.1007/s00706-021-02831-6

Monatshefte für Chemie - Chemical Monthly
https://doi.org/10.1007/s00706-021-02831-6
ORIGINAL PAPER
A study about excellent xanthine oxidase inhibitory efects of new
pyridine salts
Ülkü Yılmaz1 · Samir Abbas Ali Noma2 · Tuğba Taşkın Tok3,4 · Betül Şen5 · Yetkin Gök2 · Aydın Aktaş2,6
Burhan Ateş2 · Muhittin Aygün5
·
Received: 7 April 2021 / Accepted: 1 August 2021
© Springer-Verlag GmbH Austria, part of Springer Nature 2021
Abstract
A series of pyridine salts were synthesized containing vitamin B3 (niacin), isonicotinonitrile, and non-substituted pyridine
fragment from reactions of 1,3-dibromopropane, 1,4-dibromobutane, and 4,4′-bis(chloromethyl)-1,1′-biphenyl reactants with
pyridines. The builds of pyridine salts were identifed by dint of IR, 1H, and 13C NMR spectroscopic techniques, and CHN
analyses. Therewithal, the build of a compound was assigned via the X-ray single-crystal difraction method. 1,3-Bis(3-
cyanopyridine-1-ium-1-yl)propane dibromide crystallizes in the orthorhombic space group Ccce, with half of the cation
molecule and one bromide anion in the asymmetric unit. Enzyme inhibitory properties of pyridine compounds were tested on
the activities of xanthine oxidase (XO) and determined in the range from 0.394 to 0.623 μM. All of the compounds inhibited
enzyme more efectively than allopurinol that which is a standard drug. In addition, docking calculations were applied to
investigate the binding properties and interactions of pyridine salts towards XO.
Graphic abstract
n
inhibition
interactio
Pyridine salts
Keywords Pyridine salt · Xanthine oxidase · Molecular docking · Crystal structure · Enzyme inhibition
* Ülkü Yılmaz
ulku.yilmaz@ozal.edu.tr
Introduction
1
Department of Engineering Basic Sciences, Faculty
of Engineering and Natural Sciences, Malatya Turgut Özal
Xanthine oxidase, a highly sophisticated flavoprotein
University, Malatya, Turkey
enzyme, is found in most kinds of bacteria in humans [1].
XO is an enzyme responsible for uric acid formation from
purines [2]. In addition, this enzyme is the cause of the
peroxide and superoxide released during this process, the
excessive work of this enzyme causes oxidative stress [3].
Oxidative stress and hyperuricemia on account of the XO
enzyme are important factors that give rise to gout illness.
The uric acid is formed in the liver and removed with the
urine. Excessive uric acid in the blood causes urate forma-
tion and shows symptoms with pain and swelling in the
2
3
4
Department of Chemistry, Faculty of Science and Arts, İnönü
University, Malatya, Turkey
Department of Chemistry, Faculty of Arts and Sciences,
Gaziantep University, 27310 Gaziantep, Turkey
Department of Bioinformatics and Computational Biology,
Institute of Health Sciences, Gaziantep University,
27310 Gaziantep, Turkey
5
6
Department of Physics, Faculty of Science, Dokuz Eylul
University, 35390 Izmir, Turkey
Health Services Vocational School, Inonu University,
44280 Malatya, Turkey
Vol.:(0123456789)
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Ü. Yılmaz et al.
joints. In addition, this condition can cause kidney disorders
and even cancer [4–9].
been approved as an inhibitor drug (febuxostat and topirox-
ostat) for the XO enzyme in recent years, was considered.
This similarity includes both structural functional groups
and bulky properties. To discover the binding modes and
interactions of strong pyridine salts with the binding area
of xanthine oxidase (XO), one of the molecular modelling
techniques, molecular docking method was used. In addition,
we have explored the single crystal of one compound with
X-ray difraction method.
Gout is a disease that occurs as a result of excessive pro-
duction of uric acid and causes particularly severe joint pain.
Uric acid accumulates in the joints and causes rheumatic
pain [8, 10–14]. Gout is the most common cause of infam-
matory rheumatism. Causes of increased uric acid level are
known as foods containing high amounts of purines, obe-
sity, sugar, and excessive alcohol consumption [15–18]. The
drugs used in the treatment of this disease are anti-hyper-
uricemia and anti-infammatory agents that reduce uric acid
levels and infammation [19–22].
Results and discussion
Allopurinol is the frst XO inhibitor to be used clinically
and has been used in hyperuricemia and gout therapy for
nearly ffty years. Although allopurinol is a highly efective
XO inhibitor as a purine analog, it has signifcant and serious
side efects. Especially skin rashes, gastrointestinal prob-
lems, liver dysfunction, allergic response bone marrow sup-
pression, renal failure, jaundice, and hepatitis are the most
important known side efects. The NSAIDs (nonsteroidal
anti-infammatory drugs) are used as acute gout drugs other
than allopurinol, but they have also serious side efects such
as nausea, diarrhea, and vomiting [2, 7, 9, 23–28]. There-
fore, the discovery of efective gout medications that have
no or fewer side efects is important. Non-purines comprise
structurally versatile compounds that are further classifed
as synthetically derived or naturally occurring compounds.
Febuxostat was the frst non-purine FDA-approved drug in
2009 for the treatment of hyperuricemia. Also, topiroxostat
is a recently (in 2013) approved XO inhibitor for the treat-
ment of gout and hyperuricemia in Japan [29–34].
Synthesis
In this study, we synthesized nine pyridine salts. Compound
2c was prepared according to the literature [43]. All the pyri-
dine salts were synthesized by reactions of alkyl dihalides
and pyridines (Fig. 1). The structures of all the pyridine
compounds were characterized by IR, H, and 13C NMR
1
techniques, elemental analysis, and melting point. The 1H
NMR spectra of new derivatives showed peaks in the feld
of 9.66–7.59 ppm these belong to aromatic protons. In the
13C spectra of pyridinium dihalides, characteristic pyridine
aromatic carbon peaks were assigned between 147.3 and
127.5 ppm. The alkyl group peaks were observed between
64.2 and 27.3 ppm. In addition, the peaks of C=N groups
were observed between 1451 and 1495 cm−1 in the IR
spectra.
Structural description of compound 1b
On the other hand, aromatic compounds, particularly
nitrogen-containing heterocyclic compounds, are present
in most natural bioactive species. Similarly, the pyridinium
core is widely found in various pharmaceuticals as an impor-
tant functional part [35–37]. The pyridinium nucleus, which
has a positive charge, is used to interact with anionic cell
components through electrostatic interaction. Therefore,
pyridine structures dissolve well in polar solvents and are
capable of secondary interactions. This may provide pref-
erable binding with enzymes or bacterial receptors. Phys-
icochemical properties and the water solubility can be also
adjusted of the planned compound [38–41]. In addition to
these, the cyclostellettamines and njaoaminiums (macro-
cycles containing 3-alkylpyridinium salts) have exhibited
cytotoxic activitiy against A549 cancer cell lines and strong
antibacterial activity against a series of Gram-positive and
Gram-negative bacteria [42].
The asymmetric unit of compound 1b consists of one half-
cation and one bromide anion. The other half of the cation is
generated by the application of twofold rotation symmetry,
with the rotation axis running through the central C1 atom of
the propane bridge (Fig. 2). The cyanopyridine ring is essen-
tially planar, with a maximum deviation of −0.029(4) Å for
atom N2 and an overall r.m.s. deviation of 0.017 Å. Moreo-
ver, the two cyanopyridine rings are nearly parallel to each
other with a dihedral angle of 1.56(2)° between their best-
ftplanes. The C≡N bond distance is 1.141(5) Å, and can be
compared with those corresponding to some other related
complexes [44–47]. The C5–C8 bond length is 1.447(6) Å,
which is a bit longer than those reported in other compounds
with 4-cyanopyridine [46, 47]. This observation may be a
consequence of a decrease in mesomeric interaction between
the pyridine ring and nitrile substituent.
In this study, based on the information summarized
above, a group of pyridine salts was synthesized by our
group and xanthine oxidase inhibition activities were inves-
tigated. While synthesizing the pyridine derivatives in the
study, the similarity to the non-purine structure, which has
The crystal packing view of 1b is shown in Fig. 3. The
neighbouring molecules in compound 1b are linked to one
another by a pair of C6–H6⋯N1ihydrogen bonds [H6⋯N1
2.57 Å, C6⋯N13.468(6) Å, C6–H6⋯N1 161°, symmetry
code: (i)x,1/2−y,3/2−z], forming centrosymmetric dimers
1 3

A study about excellent xanthine oxidase inhibitory efects of new pyridine salts
Fig. 1 Synthesis procedure of pyridine salts
Fig. 2 View of the molecular structure of 1b, depicting the atom
labelling and displacement ellipsoids drawn at the 50% probabil-
ity level. The asymmetric unit of compound 1b is given in darker
colours, and the symmetry-generated part (symmetry code: (i)
3/2−x,1−y,+z) of the molecule is given in lighter colours. H atoms
are shown as spheres of arbitrary radius. Selected bond parameters
(Å, o): N1–C8 1.141(5), N2–C2 1.485(4), N2–C3 1.338(5), N2–C7
1.344(5), C1–C2 1.506(4), C3–C4 1.375(5), C4–C5 1.380(5), C5–C6
1.376(6), C5–C8 1.447(6), C6–C7 1.368(6), C3–N2–C2 119.8(3),
C3–N2–C7 120.4(4), C2i–C1–C2 117.7(5), N2–C2–C1 113.3(3),
N2–C3–C4 121.6(4), C4–C5–C8 120.0(4), C5–C6–C 118.8(4),
N1–C8–C5 177.9(5), C2i–C1–C2–N2 −66.6(3), C2–N2–C3–C4
−179.2(3), C8–C5–C6–C7 177.7(4)
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Ü. Yılmaz et al.
Fig. 3 A partial view from c-axis of the crystal packing of compound 1b, showing the C–H⋯N and C–H⋯Br hydrogen-bonded supramolecular
architecture, which resembles a two-dimensional brick-wall motif propagating parallel to the (001) plane
with an R2(10) ring motif. The resulting dimers are almost
planar un2its which features a zigzagging layer structure
lying parallel to the (010) plane. The molecules are also
involved in intermolecular C3–H3⋯Br1ii and C4–H4⋯Br1iii
hydrogen bonds [H3⋯Br1 2.75 Å, C3⋯Br1 3.658(4) Å,
C3–H3⋯Br1 165°; H4⋯Br1 2.71 Å, C4⋯Br1 3.608(4)
Å, C4–H4⋯Br1 162° symmetry code: (ii)1 − x,y,1/2 − z;
(iii)1−x,1/2−y,z], producing in inversion dimmers through
cyclic R2(10) motifs. Adjacent dimers are further connected
through4C7–H7⋯Br1iv [H7⋯Br1 2.62 Å, C7⋯Br1 3.515(5)
Å, C7–H7⋯Br1 161°, symmetry code: (iv)1−x,1−y,1−z]
interactions, leading to infnite chains propagating along the
bc diagonal (Fig. 2).
inhibitors of xanthine oxidase. However, many researches
have also performed to get more potent non-purine XO
inhibitors [50]. Herein, we were tested inhibitory proper-
ties of pyridine salts on the activity of XO and found that
the IC50 values were in the ranges from 0.394 to 0.623 μM
as summarized in Table 1. The IC50 value of allopurinol,
standard drug, was founded as 1.364 μM. In this study, all
the pyridine salts showed stronger inhibition properties on
XO than allopurinol. In a recent study, 2-aminopyridine and
N-heterocyclic carbene (NHC) ligands exhibited IC50 for XO
in the range from 0.58 to 1.69 μM [51]. In another study,
new pyrrole carboxamide derivatives were reported that IC50
value range was in the 4.608–7.084 μM [52]. According to
Table 1 The inhibition values (IC50) of the pyridine salts on xanthine
XO inhibition results
oxidase enzyme (XO)
In vitro inhibition efect of pyridine salts on serum bovine,
XO was measured by the increase in the formation of uric
acid levels at 294 nm [48]. The high levels of XO activity
are related to diferent cancer types due to the production
of reactive oxygen species [49]. Therefore, control of XO
activity limits the production of uric acid or reactive oxygen
species. Allopurinol and oxypurinol are purine-like struc-
ture and the most used as XO inhibitors. Nevertheless, these
inhibitors massively cause serious side efect in the purine
metabolism due to the similarity of the purine metabolite.
Febuxostat is the frst compound as an emerging class of
antihyperuricaemic agents and the non-purine selective
Compound
IC50/μM
r2
1a
1b
1c
2a
2b
2c
3a
3b
0.441 0.007
0.524 0.046
0.623 0.029
0.575 0.046
0.514 0.031
0.541 0.024
0.394 0.021
0.603 0.042
0.413 0.026
1.364 0.058
0.982
0.932
0.969
0.997
0.998
0.981
0.989
0.971
0.948
0.987
3c
Allopurinol
1 3

A study about excellent xanthine oxidase inhibitory efects of new pyridine salts
our results, these pyridine salts may be a good alternative
for XO inhibition [53].
hydrophobic interactions with Glu879, Phe1009, and Ile714
are observed between the relative compound and the tar-
get protein. The second compound, 1,1′-([1,1′-biphenyl]-
4,4′-diylbis(methylene))bis(3-pyridin-1-ium) dichloride
(3c), forms three electrostatic interactions with Glu879 and
Phe1009 residues; and fve hydrophobic interactions with
Phe1009, Ala1078, Ala1079, Val1011 and Leu712 resi-
dues in the active site of XO. The last compound, 3b has
a third better afnity against XO target. A hydrogen bond
(Asn1145), two electrostatic (Glu879) and three hydropho-
bic interactions (Phe1009, Ile714, and Leu712) are formed
between 3b and XO. It showed the detail information about
applied docking studies in Table S2 of the supplementary
fle.
Docking results between pyridine salts
and xanthine oxidase enzyme (XO)
In the molecular docking process, the interaction mechanism
and types of the possible complex that will occur because of
the reaction of allopurinol as a reference compound with XO
have been defned. According to the results obtained later,
the same procedure was performed for the pyridine salts
(1a-1c, 2a-2c, and 3a-3c). At the end of docking, the bind-
ing energy values of the mentioned compounds, showing the
binding afnity against the XO target, were compared with
allopurinol individually and their potential as XO inhibitors
was defned.
The efect of the carboxylic acid groups in the meta posi-
tion of each pyridine ring on the binding energy values
becomes stronger due to the increase in electron distribution
in the structure, i.e. resonance; and as a result, the compound
3a (− 32.47 kJ mol−1) has better docking result data than
others. Besides, the absence of any side groups in the same
ring system for the compound 3c (− 31.30 kJ mol−1) and
the binding of nitrile groups at the 4th position of the ring
group for the compound 3b (−28.70 kJ mol−1) change the
binding afnities of the respective compounds against the
target protein, XO. These were confrmed visually in Fig. 5
and numerically in Table 2. Overall, the compound 3a, 3b,
and 3c have shown better binding afnity than allopurinol
(−25.52 kJ mol−1) as a reference XO inhibitor.
The reference compound has distant bond interactions
with important residues in the binding site of XO, including
three hydrogen bonds (Ser876, Glu879, and Arg880) and
fve hydrophobic interactions (Phe914, Phe1009, Ala1078,
and Ala1079 residues). We detail these data in Fig. S1 and
Table S1 in the supplementary fle.
In this part of the study, nine compounds (1a–1c, 2a–2c,
and 3a–3c) considered as pyridine salts were docked with
XO. The compounds 3a–3c have a better afnity than oth-
ers. 1,1′-([1,1′-Biphenyl]-4,4′-diylbis(methylene))bis(3-
carboxypyridin-1-ium) dichloride (3a) which is the best
one, has seven hydrogen bonds with Arg880, Asn1145,
Lys713, Thr1010, and Phe1009 residues of the target
protein (Fig. 4). In addition, two electrostatic and two
Besides the binding afnities of the compounds, their
orientations are quite signifcant for optimal potent docking
Fig. 4 The best docking pose of
compound 3a (orange and stick)
with XO
1 3

Ü. Yılmaz et al.
Fig. 5 The relative poses and superimposed forms of pyridine salts with the important residues in the binding region of XO as target protein [3a,
orange, stick; 3c, light pink, stick; 3b, light blue, stick and reference compound (allopurinol), green, ball-and-stick]
Table 2 The binding energy data and inhibition values (IC50) of the
(allopurinol). In this way, the docking numerical data can be
easily displayed and evaluated visually. At the same time,
these results reveal the importance of the poses and the size
of the molecular surface areas of the compound 3a, 3b, and
3c in the binding region of XO and, accordingly, why the
related compounds have better binding afnities against tar-
get protein than the reference compound.
pyridine salts and reference compound (allopurinol) against XO
Ligand name
Binding energy/kJ mol−1
IC50/μM
1a
1b
1c
2a
2b
2c
3a
3b
−20.67
−26.19
−22.18
−20.50
−26.69
−22.72
−32.47
−28.70
−31.30
−25.52
0.441
0.524
0.623
0.575
0.514
0.541
0.394
0.603
0.413
1.364
Conclusion
In brief, this study contains the synthesis and inhibitor appli-
cations of the nine pyridine salts. All of the new salts were
3c
Allopurinol
characterized by using FT-IR spectroscopy, 1H NMR, 13
C
NMR, and CHN analysis techniques. The characterization
data were consistent with builds of compounds. The new
pyridine salts were used to determine the efcient inhibi-
tion against XO enzymes. Therefore, these complexes may
be efective inhibitors of XO enzymes due to nano and
micromolar levels of IC50. In addition, as one of the visual
with the target. So that they display desired biological
activity against XO. Figure 5 exhibits three complexes (3a,
3b, and 3c) exposed to XO protein with the best docking
interaction and superimposed with the reference compound
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A study about excellent xanthine oxidase inhibitory efects of new pyridine salts
computational techniques, docking studies yielded appro-
priate interactions between the pyridine salts and the tar-
get. Molecular docking fndings based on the pyridine salts
regarding the target structure, XO, support the experimental
data in this research. The efect of the compounds on the
active surface of the target has been carefully studied and
described based on docking fndings such as types of interac-
tions and binding energy data. Additionally, it was suggested
which one active or not based on which condition in XO as a
target. The crystal structure determination of compound 1b
was performed by single crystal X-ray difraction method.
The bromide anion plays a signifcant role in the stabiliza-
tion and packing of the crystal structures of the 1b. The
supramolecular network is consolidated by hydrogen bonds
between the cation molecules and the bromide anions, as
well as by C–H⋯N hydrogen-bonding interactions between
the neighbouring molecules.
1,3-dibromopropane, nicotinic acid, 3-cyanopyridine, and
pyridine.
1,3‑Bis(3‑cyanopyridine‑1‑ium‑1‑yl)propane dibromide (1b,
C15H14Br2N4) Cream colored solid; yield 0.80 g (81%); m.p.:
284–285 °C. Anal. Calc. for C15H14Br2N4(410.11 g/mol):
C, 43.93; H, 3.44; N, 13.66. Found: C, 43.83; H, 3.32; N,
13.50; FT-IR: ꢀ=2241 (C≡N), 1469 (N=C) cm−1; 1H NMR
(400 MHz, DMSO-d6/D2O): δ=9.23 (d, 4H, J=8 Hz, Ar–
Hpyr), 8.58 (d, 4H, J=8 Hz, Ar–Hpyr), 4.78 (t, 4H, J=8 Hz,
N–CH2), 2.68 (quint, 2H, J =7 Hz, CH2) ppm; 13C NMR
(100 MHz, DMSO-d6/D2O): δ=146.6, 131.6, 128.1 (Ar–C),
115.0 (CN), 58.9 (N-CH2), 31.8 (CH2) ppm.
1,3‑Bis(3‑pyridine‑1‑ium‑1‑yl)propane dibromide (1c,
C13H16Br2N2) White solid; yield 0.68 g (76%); m.p.:
129–130 °C. Anal Calc. for C13H16Br2N2 (360.09 g/mol):
C, 43.36; H, 4.48; N, 7.78. Found: C, 43.21; H,4.34; N,
1
7.65; FT-IR: ꢀ = 1486 (N=C) cm−1; H NMR (400 MHz,
Experimental
DMSO-d6): δ=9.03 (d, 4H, J=8 Hz, Ar–Hpyr), 8.56 (d, 2H,
J=8 Hz, Ar–Hpyr), 8.10–8.08 (m, 4H, Ar–Hpyr), 4.76 (t, 4H,
J = 8 Hz, N–CH2), 2.70 (quint, 2H, J = 8 Hz, CH2) ppm;
13C NMR (100 MHz, DMSO-d6): δ = 146.3, 145.0, 128.7
(Ar–C),57.9 (N–CH2), 30.2 (CH2) ppm.
All chemicals were buy-in from Aldrich, Fluka, Merck, and
1
Acros Chemical Co. All NMR were performed H NMR
(400 MHz) and 13C NMR (100 MHz) spectra were recorded
using Bruker Avance III 400 MHz NMR spectrometer.
Infrared spectra were recorded as KBr pellets in the range
4000–400 cm−1 on a Perkin–Elmer Spectrum One FT-IR
spectrometer. CHN analysis was recorded by LECO CHNS-
932 elemental analyzer. Melting points were recorded using
an electrothermal melting point apparatus, Electrothermal
9100. The compound 2c was prepared according to the lit-
erature [43].
1,4‑Bis(3‑carboxypyridine‑1‑ium‑1‑yl)butane dibromide
(2a, C16H18Br2N2O4) White solid; yield 0.78 g (83%); m.p.:
242–243 °C. Anal. Calc. for C16H18Br2N2O4 (462.14 g/mol):
C, 41.58; H, 3.93; N, 6.06. Found: C, 41.30; H, 3.81; N,
6.00; FT-IR: ꢀ=1725 (C=O), 1461 (N=C) cm−1; 1H NMR
(400 MHz, DMSO-d6/D2O): δ=9.30 (s, 2H, Ar–Hpyr), 9.01
(d, 2H, J=4 Hz, Ar–Hpyr), 8.86 (d, 2H, J=8 Hz, Ar–Hpyr),
8.13–8.10 (m, 2H, Ar–Hpyr), 4.65 (bs, 4H, N–CH2), 1.99
(bs, 4H, CH2) ppm; 13C NMR (100 MHz, DMSO-d6/D2O):
δ=163.7 (COOH), 146.5, 145.8, 145.7, 134.7, 128.5 (Ar–
C), 60.8 (N–CH2), 27.5 (CH2) ppm.
1,3‑Bis(3‑carboxypyridine‑1‑ium‑1‑yl)propane dibro‑
mide (1a, C15H16Br2N2O4) A blend of 0.50 g nicotinic acid
(4.10 mmol), 0.41 g 1,3-dibromopropane (2.05 mmol) and
5 cm3 DMF was stirred and heated at 90 °C for 12 h. After
completion of the reaction, all evaporating components were
cast out by dint of vacuum. The crude product was crystal-
lised from EtOH/H2O (1:1). White solid; yield 0.72 g (79%);
m.p.: 289–290 °C. Anal. Calc. for C15H16Br2N2O4 (448.11
g/mol): C, 40.21; H, 3.60; N, 6.25. Found: C, 40.15; H,
3.52; N, 6.20; FT-IR: ꢀ =1712 (C=O), 1471 (N=C) cm−1;
1H NMR (400 MHz, DMSO-d6/D2O): δ=9.44 (s, 2H, Ar–
Hpyr), 9.11 (d, 2H, J=4 Hz, Ar–Hpyr), 8.94 (d, 2H, J=8 Hz,
Ar–Hpyr), 8.21–8.17 (m, 2H, Ar–Hpyr), 4.77 (t, 4H, J=8 Hz,
N–CH2), 2.72–2.64 (m, 2H, CH2) ppm; 13C NMR (100 MHz,
DMSO-d6/D2O): δ = 163.4 (COOH), 147.3, 146.4, 146.0,
133.4, 128.7 (Ar–C), 58.3 (N–CH2), 32.2 (CH2) ppm.
The other pyridinium salts 1–3 were synthe-
sized via a similar method for compound 1a using
4,4′-bis(chloromethyl)-1,1′-biphenyl, 1,4-dibromobutane,
1,4‑Bis(3‑cyanopyridine‑1‑ium‑1‑yl)butane dibromide (2b,
C16H16Br2N4) Cream colored solid; yield 0.83 g (83%); m.p.:
249–250 °C. Anal. Calc. for C16H16Br2N4 (424.14 g/mol):
C, 45.31; H, 3.80; N, 13.21. Found: C, 45.22; H, 3.75; N,
13.14; FT-IR: ꢀ=2246 (C≡N), 1460 (N=C) cm−1; 1H NMR
(400 MHz, DMSO-d6/D2O): δ=9.19 (d, 4H, J=8 Hz, Ar–
Hpyr), 8.49 (d, 4H, J=8 Hz, Ar–Hpyr), 4.69 (bs, 4H, N–CH2),
2.02 (bs, 4H, CH2) ppm; 13C NMR (100 MHz, DMSO-d6/
D2O): δ=146.3, 131.4, 131.3, 127.8 (Ar–C), 115.0 (CN),
61.6 (N–CH2), 27.3 (CH2) ppm.
1,4‑Bis(3‑pyridine‑1‑ium‑1‑yl)butane dibromide (2c,
C14H18Br2N2) White solid; yield 0.68 g (76%); m.p.: 244–
245 °C. Anal. Calc. for C14H18Br2N2 (374.12 g/mol):
C, 44.95; H, 4.85; N, 7.49. Found: C, 44.83; H, 4.80; N,
1
7.42; FT-IR: ꢀ = 1488 (N=C) cm−1; H NMR (400 MHz,
1 3

Ü. Yılmaz et al.
DMSO-d6/D2O): δ=9.99 (d, 4H, J=8 Hz, Ar–Hpyr), 8.56
(d, 2H, J=8 Hz, Ar–Hpyr), 8.09 (d, 4H, J=8 Hz, Ar–Hpyr),
4.64 (bs, 4H, N–CH2), 1.99 (bs, 4H, CH2) ppm; 13C NMR
(100 MHz, DMSO-d6/D2O): δ=146.1, 144.9, 128.6 (Ar–C),
60.5 (N–CH2), 27.5 (CH2) ppm.
added to start the reaction. Absorbance changes were kineti-
cally followed at 294 nm for 2 min. Allopurinol was used
as a positive control. The percent inhibition of compounds
on XO activity was calculated and then results were given
as IC50 values.
1,1′‑[[1,1′‑Biphenyl]‑4,4′‑diylbis(methylene)]bis(3‑car‑
boxypyridin‑1‑ium) dichloride (3a, C26H22Cl2N2O4) White
solid; yield 0.85 g (84%); m.p.: 240–241 °C. Anal. Calc. for
C26H22Cl2N2O4 (497.37 g/mol): C, 62.79; H, 4.46; N, 5.63.
Found: C, 62.63; H, 4.40; N, 5.54; FT-IR: ꢀ=1716 (C=O),
Molecular docking
Docking studies were executed with Auto Dock Vina [54]
to capture and explain important fndings at the molecular
level of all pyridine salts as ligands, with xanthine oxidase
(XO) as a target. Gaussian 09 (G09) [55] was used in the
preparation of the ligand(s) for molecular docking study. The
synthesised pyridine salts were drawn with help of G09 soft-
ware and minimised on the DFT/B3LYP/6-31G * base set
of the same software. Afterward, the target protein, XO, was
also prepared. Additionally, allopurinol was used a reference
compound to examine its interactions with XO.
1
1495 (N=C) cm−1; H NMR (400 MHz, DMSO-d6/D2O):
δ=9.37 (s, 2H, Ar–Hpyr), 9.11 (d, 2H, J=8 Hz, Ar–Hpyr),
8.88 (d, 2H, J=8 Hz, Ar–Hpyr), 8.15–8.11 (m, 2H, Ar–Hpyr),
7.73 (d, 4H, J=8 Hz, Ar–H), 7.59 (d, 4H, J=8 Hz, Ar–H),
5.88 (s, 4H, CH2) ppm; 13C NMR (100 MHz, DMSO-d6/
D2O): δ=163.7 (COOH), 146.3, 145.9, 145.5, 140.7, 135.7,
133.5, 130.1, 128.7, 128.1 (Ar–C), 63.8 (CH2) ppm.
1,1′‑[[1,1′‑Biphenyl]‑4,4′‑diylbis(methylene)]bis(3‑cyano‑
pyridin‑1‑ium) dichloride (3b, C26H20Cl2N4) Cream colored
solid; yield 0.84 g (87%); m.p.: 269–271 °C. Anal. Calc. for
C26H20Cl2N4 (459.37 g/mol): C,67.98; H, 4.39; N, 12.20.
Found: C, 67.79; H, 4.27; N, 12.12; FT-IR: ꢀ=2081 (C≡N),
Table 3 Crystal data and structure refnement details for the com-
pound 1b
1
1451 (N=C) cm−1; H NMR (400 MHz, DMSO-d6/D2O):
δ=9.66 (d, 2H, J=8 Hz, Ar–Hpyr), 8.76 (d, 4H, J=8 Hz,
Ar–Hpyr), 7.79–7.72 (m, 10H, Ar–H), 6.05 (s, 4H, CH2)
ppm; 13C NMR (100 MHz, DMSO-d6/D2O): δ = 146.7,
131.9, 130.4, 130.0, 128.0, 127.9, 127.5 (Ar–C), 115.3
(CN), 64.2 (CH2) ppm.
Experimental details
Compound 1b
Chemical formula
Formula weight
Temperature/K
Space group
Crystal system
a/Å
C15H14Br2N4
410.12
295(2)
Ccce
Orthorhombic
13.2777(11)
18.2691(13)
13.4725(8)
90
3268.0(4)
8
1,1′‑[[1,1′‑Biphenyl]‑4,4′‑diylbis(methylene)]bis(3‑pyri‑
din‑1‑ium) dichloride (3c, C24H22Cl2N2) White solid;
yield 0.70 g (85%); m.p.: 304–305 °C. Anal. Calc. for
C24H22Cl2N2 (409.35 g/mol): C, 70.42; H, 5.42; N, 6.84.
Found: C, 70.38; H, 5.41; N, 6.72; FT-IR: ꢀ=1485 (N=C)
cm−1; 1H NMR (400 MHz, DMSO-d6/D2O): δ=9.21 (d, 4H,
J=8 Hz, Ar–Hpyr), 8.61 (t, 2H, J=8 Hz, Ar–Hpyr), 8.16 (t,
4H, J=8 Hz, Ar–Hpyr), 7.74 (d, 4H, J=8 Hz, Ar–H), 7.64
(d, 4H, J=8 Hz, Ar–H), 5.90 (s, 4H, CH2) ppm; 13C NMR
(100 MHz, DMSO-d6/D2O): δ=146.5, 145.1, 140.6, 134.0,
130.0, 129.0, 128.1 (Ar–C), 63.5 (CH2) ppm.
b/Å
c/Å
Α=β=γ/°
Cell volume/Å3
Formula unit cell Z
ρcalc/g cm−3
1.667
F(000)
1616.0
4.959
μ/mm−1
Crystal size/mm3
Difractometer
Radiation/wavelength/Å
Refections measured
Range of h, k, l
0.38×0.28×0.23
Xcalibur, Eos
MoKα/0.71070
3638
−16≤h≤15
−22≤k≤15
−16≤l≤10
1667/0/96
R1 =0.0413, wR2 =0.0689
R1 =0.0847, wR2 =0.0836
1.027
In vitro inhibition of xanthine oxidase (XO) activity
The in vitro XO activity was assayed by measuring the uric
acid formation spectrophotometrically at 294 nm at 37 °C
[47]. This assay method includes XO (0.2 U), and xanthine
(1 mM) in phosphate bufer (50 mM, pH 7.4). To determine
IC50 values of the compounds on XO activity, pyridine salts
were added to the reaction mixture. Then, XO and com-
pounds were pre-incubated for 10 min, and xanthine was
Data/restraints/parameters
Final R indexes [I≥2σ (I)]
Final R indexes [all data]
Goodness-of-ft on F2
Largest dif. Peak/hole/e Å−3
0.350/−0.37
1 3

A study about excellent xanthine oxidase inhibitory efects of new pyridine salts
8. Johnson P, Loganathan C, Iruthayaraj A, Poomani K, Thayuma-
navan P (2018) Biochimie 154:1
Crystal structure determination
9. Aktas A, Ali Noma SA, Celepci DB, Erdemir F, Gok Y, Ates B
(2019) J Mol Struct 1184:487
Single-crystal data were collected on a Rigaku-Oxford
Xcalibur difractometer with an Eos CCD area detector at
room temperature, and were measured by ω-scan technique
using graphite–monochromated MoKα (λ = 0.71073 Å)
radiation from an enhance X-ray source. The data collec-
tion, cell refnement and data reduction were performed
using the CrysAlisPro program [56]. Solution, refnement,
and analysis of the structures were done using the OLEX2
system [57]. The crystal structure of 1b was solved and
the space group determined by the ShelXT [58] structure
solution program using Intrinsic Phasing. Refnement of
coordinates and anisotropic thermal parameters of non-
hydrogen atoms were carried out by the full-matrix least-
squares method based on F2 against all refections using
the SHELXL [59]. Anisotropic thermal parameters were
applied to all non-hydrogen atoms. Methylene H atoms
(C–H = 0.97 Å) and aromatic H atoms (C–H = 0.93 Å)
were positioned geometrically. The fgures were made
using OLEX2 [57]. The summary of the crystal data,
experimental details, and refnement results for compound
1b under consideration are listed in Table 3.
10. Suresh E (2005) Postgrad Med J 81:572
11. Adams JU (2009) Nat Biotech 27:309
12. Chen X, Zuo A, Deng Z, Huang X, Zhang X, Geng C, Li T, Chen
J (2017) Fitoterapia 122:144
13. Cheng X, Changgui L (2015) Gout Hyperuricemia 2:15
14. Hafez RM, Abdel-Rahman TM, Naguib RM (2017) J Adv Res
8:475
15. Kondo M, Hirano Y, Nishio M, Furuya Y, Nakamura H, Watanabe
T (2013) J Food Sci 78:1935
16. Emmerson BT (1996) N Engl J Med 334:445
17. Nakagawa T, Hu H, Zharikov S, Tuttle KR, Short RA, Glushakova
O, Ouyang X, Feig DI, Block ER, Herrera-Acosta J, Patel JM,
Johnson RJ (2006) Am J Physiol Renal Physiol 290:625
18. Gao X, Qi L, Qiao N, Choi HK, Curhan G, Tucker KL, Ascherio
A (2007) Hypertension 50:306
19. Rahmi EP, Kumolosasi E, Jalil J, Husain K, Buang F, Abd Razak
AF, Jamal JA (2020) Front Pharmacol 11:1
20. Kuo CF, Grainge MJ, Zhang W, Doherty M (2015) Nat Rev Rheu-
matol 11:649
21. Dalbeth N, Stamp L (2007) Semin Dial 20:391
22. Chen LY, Yin HF, Lan Z, Ma S, Zhang C, Yang Z, Li P, Lin B
(2011) J Ethnopharmacol 135:399
23. Havlika J, Gonzalez de la Huebraa R, Hejtmankova K, Fernandez
J, Simonova J, Melich M, Rada V (2010) J Ethnopharm 132:461
24. Lespade L, Bercion S (2010) J Phys Chem 114:921
25. Khuan CS, Shahid MSM, Sim YS, Hussein H, Gaik OS, Rosman
A, Liew CG, Goh E, Chyn GS (2008) CPG Manag Gout Health
13:21
The further crystallographic data for compound 1b has
been deposited to with the Cambridge Crystallographic
Data Centre, (www.ccdc.cam.ac.uk/data_request/cif, e-mail:
deposit@ccdc.cam.ac.uk) and is available on request quoting
the deposition number CCDC 2035762.
26. Leigh M, Castillo CE, Raines DJ, Duhme-Klair AK (2011) Chem
Med Chem 6:612
27. Gawlik-Dziki U, Dziki D, Swieca M, Nowak R (2017) Food Chem
225:138
28. Sathisha KR, Gopal S, Rangappa KS (2016) Eur J Pharmacol
776:99
Supplementary Information The online version contains supplemen-
tary material available at https://doi.org/10.1007/s00706-021-02831-6.
29. Singh JV, Bedi PMS, Singh H, Sharma S (2020) Expert Opin Ther
Pat 30:769
Acknowledgements The authors acknowledge to Inonu University for
the infrared, nuclear magnetic resonance (proton and carbon) spec-
troscopic reading, and CHN analyses of the pyridine compounds. We
also acknowledge to Inonu University Research Fund (I.U. B.A.P. No:
FOA-2021-2320) for support to carry out this study. Dokuz Eylül Uni-
versity for the use of the Oxford Rigaku Xcalibur Eos Difractometer
(purchased under University Research Grant no: 2010.KB.FEN.13) is
also greatly acknowledged.
30. Ojha R, Singh J, Ojha A, Singh H, Sharma S, Nepali K (2017)
Expert Opin Ther Pat 27:311
31. Kumar R, Darpan S, Sharma S, Singh R (2011) Expert Opin Ther
Pat 21:1071
32. Borges F, Fernandes E, Roleira F (2002) Curr Med Chem 9:195
33. Pacher P, Nivorozhkin A, Szabo C (2006) Pharm Rev 58:87
34. Kumar R, Joshi G, Kler H, Kalra S, Kaur M, Arya R (2018) Med
Res Rev 38:1073
35. Wang P-Y, Fang H-S, Shao W-B, Zhou J, Chen Z, Son B-A, Yang
S (2017) Bioorg Med Chem Lett 27:4294
36. Sambhy V, Peterson BR, Sen A (2008) Angew Chem Int Ed
47:1250
References
37. Messali M (2015) Molecules 20:14936
38. Zhou J, Tao Q-Q, Wang P-Y, Shao W-B, Wu Z-B, Li Z, Yang S
(2018) Bioorg Med Chem Lett 28:1742
1. Boban M, Kocic G, Radenkovic S, Pavlovic R, Cvetkovic T,
Deljanin-Ilic M, Ilic S, Bobana MB, Djindjic B, Stojanovic D,
Sokolovic D, Jevtovic-Stoimenov T (2014) Ren Fail 36:613
2. Pacher P, Nivorozhkin A, Szabo C (2006) Pharmacol Rev 58:87
3. Nakamura T, Murase T, Nampei M, Morimoto N, Ashizawa N,
Iwanaga T, Sakamoto R (2016) Eur J Pharm 780:224
4. Song JU, Jang JW, Kim TH, Park H, Park WS, Jung S-H, Kim GT
(2016) Bioorg Med Chem Lett 26:950
39. Mouradzadegun A, Elahi S, Abadast F, Motamedi H (2016) Res
Chem Intermed 42:1583
40. Jia RX, Duan YF, Fang Q, Wang XY, Huang JY (2016) Food
Chem 196:381
41. Haldar J, Kondaiah P, Bhattacharya S (2005) J Med Chem 48:3823
42. Althagbi HI, Alarif WM, Al-Footy KO, Abdel-Latef A (2020)
Mar Drugs 18:368
5. Dalbeth N, So A (2010) Ann Rheum Dis 69:1738
6. Onul N, Ertik O, Mermer N, Yanardağ R (2018) J Biochem Mol
Toxicol 32:e22021
43. Adonin SA, Gorokh ID, Samsonenko DG, Sokolov MN, Fedin VP
(2016) Chem Commun 52:5061
44. Kammer MN, Koplitz LV, Mague JT (2012) Acta Cryst E
68:o2514
7. Sharmaine C, Er LK (2020) Sains Malaysiana 49:405
1 3

Ü. Yılmaz et al.
45. Hardcastle KI, Laing MJ, Mcgauley TJ, Lehner CF (1974) J Cryst
Mol Struct 4:305
Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao
O, Nakai H, Vreven T, Montgomery JA Jr, Peralta JE, Ogliaro
F, Bearpark M, Heyd JJ, Brothers E, Kudin KN, Staroverov VN,
Kobayashi R, Normand J, Raghavachari K, Rendell A, Burant JC,
Iyengar SS, Tomasi J, Cossi M, Rega N, Millam JM, Klene M,
Knox JE, Cross JB, Bakken V, Adamo C, Jaramillo J, Gomperts
R, Stratmann RE, Yazyev O, Austin AJ, Cammi R, Pomelli C,
Ochterski JW, Martin RL, Morokuma K, Zakrzewski VG, Voth
GA, Salvador P, Dannenberg JJ, Dapprich S, Daniels AD, Farkas
Ö, Foresman JB, Ortiz JV, Cioslowski J, Fox DJ (2009) Gaussian
09, Revision E.01. Gaussian Inc, Wallingford
46. Fuquen RM, Arana C, De Simone CA (2012) Acta Cryst E
68:o2805
47. Laing M, Sparrow N, Sommerville P (1971) Acta Cryst B 27:1986
48. Springer J, Tschirner A, Hartman K, Palus S, Wirth EK, Ruis SB,
Möller N, von Haehling S, Argiles JM, Köhrle J, Adams V, Anker
SD, Doehner W (2012) Int J Cancer 131:2187
49. Özerkan D, Ertik O, Kaya B, Kuruca SE, Yanardag R, Ülküseven
B (2019) New Drugs 37:1187
50. Mao Q, Dai X, Xu G, Su Y, Zhang B, Liu D, Wang S (2019) Eur
J Med Chem 181:111558
56. Agilent (2014) Crys Alis PRO. Agilent Technologies, Yarnton
57. Dolomanov OV, Bourhis LJ, Gildea RJ, Howard JAK, Puschmann
H (2009) J Appl Cryst 42:339
51. Türker F, Noma SAA, Aktaş A, Al-Khafaji K, Tok TT, Ateş B,
Gök Y (2020) Monatsh Chem 151:1557
52. Kıbrız İE, Saçmacı M, Yıldırım İ, Noma SAA, Tok TT, Ateş B
(2018) Arch Pharm Chem Life Sci 351:e1800165
53. Sweeney AP, Wyllie SG, Shalliker RA, Markham JL (2001) J
Ethnopharm 75:273
58. Sheldrick GM (2015) Acta Crystallogr Sect A 71:3
59. Sheldrick GM (2015) Acta Crystallogr Sect C 71:3
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
54. Trott O, Olson AJ (2010) J Comput Chem 31:455
55. Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA,
Cheeseman JR, Scalmani G, Barone V, Mennucci B, Petersson
GA, Nakatsuji H, Caricato M, Li X, Hratchian HP, Izmaylov AF,
Bloino J, Zheng G, Sonnenberg JL, Hada M, Ehara M, Toyota K,
1 3