Design, synthesis, and biological evaluation of 5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitrile derivatives as xanthine oxidase inhibitors

Article abstract of DOI:10.1111/cbdd.13114

A series of 5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitrile derivatives (1a–p) was designed, synthesized, and identified as xanthine oxidase inhibitors with micromolar level potencies. Among them, the most promising compounds 1j and 1k were obtained with IC₅₀ values of 8.1 and 6.7?μm, respectively. The Lineweaver–Burk plot revealed that compound 1k acted as a mixed-type xanthine oxidase inhibitor. SAR analysis revealed that a carbon atom occupying the X³ position is not as effective as a nitrogen atom, and an iso-pentyloxy or a cyclopentyloxy at the 2-position of benzonitrile moiety will benefit the inhibitory potency. The basis of xanthine oxidase inhibition by 1k was rationalized by molecular modeling studies.

Full text of DOI:10.1111/cbdd.13114

PROFESSOR FAN-HAO MENG (Orcid ID : 0000-0002-1331-2164)
Article type
: Research Article
Design,
synthesis
and
biological
evaluation
of
5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitrile derivatives as xanthine oxidase
inhibitors
Ting-jian Zhang, Song-ye Li, Zhang Yi, Qing-xia Wu, Fan-hao Meng*
School of Pharmacy, China Medical University, 77 Puhe Road, North New Area, Shenyang
110122, China
* Corresponding author.
E-mail address: fhmeng@cmu.edu.cn (F-h. Meng).
Abstract
A series of 5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitrile derivatives (1a-p) was
designed, synthesized and identified as xanthine oxidase inhibitors with micromolar level
potencies. Among them, the most promising compounds 1j and 1k were obtained with IC50
values of 8.1 μM and 6.7 μM, respectively. The Lineweaver-Burk plot revealed that
compound 1k acted as a mixed-type xanthine oxidase inhibitor. SARs analysis revealed that a
carbon atom occupying the X3 position is not as effective as a nitrogen atom; and an
iso-pentyloxy or a cyclopentyloxy at the 2-position of benzonitrile moiety will benefit the
inhibitory potency. The basis of xanthine oxidase inhibition by 1k was rationalized by
molecular modeling studies.
Keywords: 1,2,3-Triazole; Topiroxostat; Xanthine oxidase inhibitor.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13114
This article is protected by copyright. All rights reserved.

Introduction
Xanthine oxidase (XO) is a critical, rate-limiting enzyme in purine metabolism. It
catalyzes the last two steps of purine catabolism in humans, i.e., hydroxylation of
hypoxanthine to xanthine and xanthine to uric acid.[1] In parallel with the hydroxylation
-
process, two kinds of reactive oxygen species (ROS), superoxide anion (O2 ) and hydrogen
peroxide (H2O2), are produced.[2] XO is therefore a critical source of uric acid and ROS.
Over-production of uric acid can lead to hyperuricemia, which is the key cause of gout. Thus,
XO is considered the most promising target for treating hyperuricemia and gout.[3, 4]
Meanwhile, an excess of ROS could cause various pathological states including inflammation,
metabolic disorders, atherosclerosis, cancer and chronic obstructive pulmonary disease.[5, 6]
Thus, inhibition of XO could reduce the formation of ROS and benefit the treatment of these
diseases.[7, 8] Allopurinol is a prototypical XO inhibitor and has been widely used in the
treatment of hyperuricemia and gout for several decades. However, in some cases, it has been
reported that allopurinol and its analogues, which possess a similar backbone of purine, can
cause severe life-threatening side effects.[9] Therefore, searching for novel non-purine XO
inhibitors with more potent XO inhibitory potency but fewer side effects has always been a
hotspot.
In the past two decades, numerous classical non-purine XO inhibitors characterized by a
five-membered ring linking a carboxyl group (febuxostat analogues) or a pyridine
(topiroxostat analogues) to an aryl nitrile moiety have been reported (Fig. 1). Febuxostat[10] is
a classical non-purine XO inhibitor, that bears a thiazole moiety as the five-membered ring
linker, has an outstanding inhibitory potency as well as acceptable side effects and was
approved by the US Food and Drug Administration (FDA) in 2009. Thereafter, a lot of
febuxostat analogues have been reported, such as Y-700,[11]
selenazoles,[12]
2-(indol-5-yl)thiazoles,[13] isoxazoles,[14] imidazoles,[15] and 1,2,3-triazoles.[16] Topiroxostat
(also known as FYX-051) is another typical non-purine XO inhibitor, which bears a
1,2,4-triazole as five-membered ring linker,[17] and was approved in Japan in 2013. The action
mechanism of topiroxostat is not only occupying the active pocket but also covalently
bonding with the receptor,[18] which makes it a powerful enzymatic inhibitor. In general,
topiroxostat analogues were not as abundant as febuxostat analogues, a typical representative
This article is protected by copyright. All rights reserved.

is the pyridyl-1,2,4-triazoles reported by Takahiro Sato et al[19] which possess excellent
potency (Fig. 1). Furthermore, other XO inhibitors with various structural classes have also
been recently published, including isocytosines,[5, 20-22] N-(1,3-diaryl-3-oxo-propyl)amides,[23]
N-acetyl pyrazolines,[24] hydroxylated chalcones,[25] 9-deazaguanines,[26] benzimidazoles,[27]
flavonoids,[28, 29] fraxamosides,[30] pyrano[3,2-d]pyrimidines,[31] 2-arylbenzo[b]furans[32] and
benzaldehydes.[33]
We have focused on the structural modifications of five-membered ring and the structure
activity relationship (SAR) investigations of classical XO inhibitors.[14-16] Our previous
studies disclosed that an oxime-like fragment fixing at the X1 position will be beneficial[15]
and a polar N atom occupying the X2 position will be disadvantage for the potency of
febuxostat analogues.[16] Since 1,2,3-triazole is a strictly isostere of 1,2,4-triazole as well as a
welcome molecular building block in drug design.[34, 35] in this work, we used a 1,2,3-triazole
to replace the 1,2,4-triazole of topiroxostat, and designed a series of
5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitrile derivatives (1a-p) as topiroxostat
analogues (Fig. 1) to investigate the structural modification of the X3 position. The target
compounds were prepared via a Huisgen 1,3-dipolar cycloaddition reaction under microwave
conditions to build the crucial 1,2,3-triazole ring. The in vitro activity was evaluated by
enzymatic inhibition assays. The synthesized compounds have been patented by our team.[36]
In addition, molecular modeling and the steady-state kinetic analysis were both carried out to
explore the inhibitory behaviors of these compounds.
Results and discussions
Chemistry
The synthesis of target compounds 1a-p was performed as outlined in Scheme 1.
Compounds 5 were considered as the key intermediates, of which synthetic method has been
reported in our previous studies.[16] In brief, commercially available 2-hydroxybenzonitrile
was nitrated by conc. HNO3 in an AcOH solution to provide 2-hydroxy-5-nitrobenzonitrile
(2). Compound 2 was alkylated with corresponding alkyl bromides or alkyl chlorides to
produced 2-alkoxy-5-nitrobenzonitriles (3), which followed by the nitro reduction to yield
2-alkoxy-5-aminobenzonitriles (4). The diazotization of 4 followed by the treatment with
This article is protected by copyright. All rights reserved.

sodium azide yielded the desired intermediates 2-alkoxy-5-azidobenzonitriles (5). Cyclization
of 5 with 4-ethynylpyridine hydrochloride in the presence of copper sulfate and vitamin C
under microwave conditions produced 5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitriles
1
13
1a-p. The structures of the synthesized compounds were elucidated by H NMR, C NMR
and MS. All the spectral data were in accordance with the assumed structures.
Biological activity
Bovine XO in vitro inhibitory potencies by target compounds 1a–p were determined by
37]
spectrophotometrically measuring uric acid levels at 294 nm.[17,
Allopurinol and
topiroxostat were included as reference compounds. In general, half of the synthesized
compounds possessed the micromolar level potencies, some of which were comparable to
allopurinol but much lower than the leading compound topiroxostat. Nevertheless, we were
interested in summarizing the SARs and investigating their inhibitory behaviors, and
expected to provide some insights for the further investigations.
Table 1. In vitro XO inhibitory potencies of compounds 1a-p.
Compound
R group
Methyl
IC50 (μM)
n.a. a
1a
1b
1c
1d
1e
1f
n-Propyl
n-Butyl
n.a.
20.8± 0.94 #
16.3± 1.96 #
n.a.
n-Pentyl
Allyl
Methoxyethyl
iso-Propyl
iso-Butyl
sec-Butyl
n.a.
1g
1h
1i
25.4± 1.11 #
n.a.
18.0± 2.12 #
This article is protected by copyright. All rights reserved.

iso-Pentyl
8.1± 1.16
6.7± 0.70 #
n.a.
1j
Cyclopentyl
Cyclohexyl
Benzyl
1k
1l
1m
45.0± 4.43 #
23.7± 2.12 #
n.a.
para-Methoxybenzyl
para-Chlorobenzyl
para-Cyanobenzyl
/
1n
1o
n.a.
1p
Allopurinol
Topiroxostat
8.5± 0.67
0.016± 0.003
/
a n.a.: not active (<50% inhibition at 50 μM).
# P＜0.05, versus allopurinol.
In the linear alkyl subseries of 1a-e as shown in Table 1, n-butyl derivative (1c) and
n-pentyl derivative (1d) were effective; especially, 1d exhibited a relatively high potency
with an IC50 value of 16.3 μM. The transition of the n-butyl (1c, IC50 =20.8 μM) to the
methoxyethyl (1h) accompanied by a loss of the potency, meaning that the inserted oxygen
atom could damage the potency and that a polar atom fusing in the R group may be not
welcome. This point was also true in the 1,2,3-triazole series.[16]
Generally speaking, the branched alkyl derivatives (1g-j) showed stronger potency than
their linear alkyl counterparts (e.g., 1g versus 1b; 1i versus 1c; 1j versus 1d), the exception
was 1h, bearing an iso-butyl but being totally inactive. The most potent compound in
branched alkyl subseries was 1j (R = iso-pentyl, IC50 = 8.1 μM), which showed a comparable
potency to allopurinol (IC50 = 8.5 μM). Surprisingly, a cyclopentyl derivative (1k, IC50 = 6.7
μM) presented the highest potency in the series and was slightly more effective than
allopurinol. However, expanding the ring to the cyclohexyl (1l), the potency completely lost.
In addition, some benzyl derivatives (1m-p) were also synthesized. Only 1m (R = benzyl,
IC50 = 45.0 μM) and 1n (R = para-methoxybenzyl, IC50 = 23.7 μM) exhibited weak potency,
but the rest were thoroughly inactive.
This article is protected by copyright. All rights reserved.

To sum up, compounds 1j and 1k were identified as the most promising XO inhibitors in
the series with comparable inhibitory effects to allopurinol. However, in comparison to
topiroxostat (IC50 = 0.016 μM) and the pyridyl-1,2,4-triazoles,[19] their potencies were much
lower, which may be a direct result of the structural changes at the X3 and X4 positions. The
X4 position fusing an N atom would be acceptable (e.g., Y-700); however, a carbon atom
occupying the X3 position may be not as welcome as a nitrogen atom, as nearly all the
classical XO inhibitors possessing an N atom at the X3 position.[9] In order to further
investigate their inhibitory behaviors in molecular level, the molecular modeling simulations
were performed in the following step.
Molecular modeling
To foresee the possible interactions and to rationalize the activities of the synthesized
compounds against XO, molecular modeling simulations of topiroxostat and the
representative compound 1k in the binding pocket of XO were performed with MOE
(Molecular Operating Environment, version 2016.08, Chemical Computing Group Inc.,
Canada) software. The crystal structure of bovine XO in complex with topiroxostat (PDB
code: 1V97) was used in the docking calculations.[18] In order to validate the docking
protocol, a self-docking of topiroxostat into the binding pocket was firstly performed. As
shown in Fig. 2A, topiroxostat stacked well with the crystallographic ligand, and the H-bonds
predicted by MOE were similar to those found in the crystal structure. Although the covalent
bonding, as co-crystal structure emerged,[18] didn’t be simulated, this protocol may provide
insights into the other interactions besides the covalent bonding.
As shown in Fig. 2B, the 1k docking conformation and the original ligand played a set
of similar interactions with XO binding pocket, such as pyridine N atom interacting with
Glu1261, the triazole ring linking to Arg880 and Thr1010 residues by a water bridge and the
cyano group accepting an H-bond from Asn768. Additionally, the cyclopentyloxy ether tail
was surrounded by several lipophilic amino acid residues such as Leu648, Phen649 and
Phen1013. The interactions provided a reasonable explanation for the potency of 1k.
However, some adverse conformation features were also observed. As shown in Fig. 2B
and 2C, the topiroxostat X3-N directly pointed toward the Glu802 terminal carboxyl by a
This article is protected by copyright. All rights reserved.

short distance of 2.84 Å. The transition from the X3-N of topiroxostat to the X3-CH of 1k
may induce a hydrophobic repulsion and lead the 1,2,3-triazole moiety twisted to keep away
from the Glu802 (Fig. 2C). We respectively measured the pyridine-triazole dihedral angles of
the 1k docking pose, the energy minimized 1k pose and the topiroxostat crystallographic
pose by MOE software; the former (20.3°) was much bigger than the latter two (1.2° and 0.6°,
respectively), meaning that the stability of 1k in XO binding pocket was not as well as that of
topiroxostat (Fig. 2C and 2D). This uncomfortable binding mode provided a possibly
explanation of the lower potency of 1k.
Steady-state kinetic analysis
The enzyme kinetic studies were carried out to further investigate the action mode of the
representative compound 1k with XO (Fig. 3). The Lineweaver-Burk plots analysis
demonstrated that 1k acted as a mixed-type XO inhibitor. This action mode was distinguished
with topiroxostat, but similar with the hydroxy-topiroxosat, a metabolite of the topiroxostat
possessing a micromolar level potency.[17] The different inhibition type may be another factor
for the lower potency of 1k than that of topiroxostat.
Conclusions
We
designed,
synthesized
and
identified
a
series
of
5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitrile derivatives as XO inhibitors (1a-p).
SARs analysis revealed that a carbon atom occupying the X3 position is not as potent as a
nitrogen atom; and an iso-pentyloxy or a cyclopentyloxy at the 2-position of benzonitrile
moiety will benefit the inhibitory potency. As a result, the most promising compounds 1j and
1k were obtained with IC50 values of 8.1 μM and 6.7 μM, respectively. The Lineweaver-Burk
plot showed that compound 1k acted as a mixed-type XO inhibitor and molecular modeling
provided a reasonable bonding mode for its inhibitory behaviors. Further investigations based
on 1j and 1k were in progress.
This article is protected by copyright. All rights reserved.

Experimental section
Chemistry
Unless otherwise indicated, reagents and solvents were purchased from commercial
sources and used without further purification. All reactions were monitored by TLC using
silica gel aluminum cards (0.2 mm thickness) with a fluorescent indicator at 254 nm. The
column chromatography was performed using silica gel (200-300 mesh) from Qingdao Ocean
Chemicals (Qingdao, Shandong, China). Melting points were recorded on an RD-1 melting
1
13
apparatus and were uncorrected. H NMR and C NMR spectra were recorded on a Bruker
600 MHz spectrometer. Chemical shifts were expressed in parts per million using DMSO-d6
as the solvent. ESI-HRMS data were gathered using a Bruker microTOF-Q instrument.
General
procedure
for
the
synthesis
of
2-alkoxy-5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitriles (1a-p)
A solution of 5 (742 μmol), 4-ethynylpyridine hydrochloride (1110 μmol), copper sulfate
pentahydrate (89 μmol) and vitamin C (89 μmol) in ethanol (10 mL) and water (10 mL) was
heated under microwave conditions at 50°C for 8 min. After the completed reaction, about
half of the solution was evaporated in vacuum. The residue was diluted with water. The
formed precipitate was collected by filtration and purified by column chromatography (ethyl
acetate: petroleum ether
=
1:
3
to 1:1) to provide the corresponding
2-alkoxy-5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitriles (1a-p).
2-Methoxy-5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitrile (1a)
1
A pink powder, yield 39.0%. H NMR (600 MHz, DMSO-d6) δ 9.51 (s, 1H), 8.71 (br s,
2H), 8.36 (d, J = 2.7 Hz, 1H), 8.27 (dd, J = 9.1, 2.7 Hz, 1H), 7.87 (d, J = 4.9 Hz, 2H), 7.53 (d,
J = 9.2 Hz, 1H), 4.02 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ 160.91, 150.53, 144.98,
137.25, 129.73, 127.16, 125.43, 122.01, 119.64, 115.36, 113.77, 101.13, 57.03. ESI-HRMS
calcd. for C15H12N5O [M + H]+ 278.1036, found: 278.1050.
2-Propoxy-5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitrile (1b)
This article is protected by copyright. All rights reserved.

1
A white powder, yield 41.2%. H NMR (600 MHz, DMSO-d6) δ 9.50 (s, 1H), 8.70 (d, J
= 4.6 Hz, 2H), 8.34 (d, J = 2.7 Hz, 1H), 8.23 (dd, J = 9.1, 2.7 Hz, 1H), 7.86 (d, J = 5.9 Hz,
2H), 7.52 (d, J = 9.2 Hz, 1H), 4.21 (t, J = 6.4 Hz, 2H), 1.87 – 1.75 (m, 2H), 1.03 (t, J = 7.4
Hz, 3H). 13C NMR (150 MHz, DMSO-d6) δ 160.29, 150.50, 144.95, 137.27, 129.58, 127.03,
125.33, 121.92, 119.56, 115.29, 114.45, 101.35, 70.85, 21.73, 10.15. ESI-HRMS calcd. for
C17H16N5O [M + H]+ 306.1349, found: 306.1352.
2-Butoxy-5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitrile (1c)
A yellow powder, yield 80.4%. 1H NMR (600 MHz, DMSO-d6) δ 9.50 (s, 1H), 9.15 (br s,
2H),8.33 (d, J = 2.7 Hz, 1H), 8.22 (dd, J = 9.1, 2.7 Hz, 1H), 8.10 (br s, 2H), 7.52 (d, J = 9.2
Hz, 1H), 4.24 (t, J = 6.4 Hz, 2H), 1.83 – 1.73 (m, 2H), 1.54 – 1.43 (m, 2H), 0.97 (t, J = 7.4
Hz, 3H). 13C NMR (150 MHz, DMSO-d6) δ 160.27, 152.25, 145.26, 136.46, 129.56, 126.95,
125.26, 121.76, 116.87, 115.28, 114.41, 101.33, 69.20, 30.32, 18.55, 13.62. ESI-HRMS calcd.
for C18H18N5O [M + H]+ 320.1506, found: 320.1521.
2-Pentyloxy-5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitrile (1d)
A white powder, yield 55.0%. 1H NMR (600 MHz, DMSO-d6) δ 9.50 (s, 1H), 8.76 (br s,
2H), 8.34 (d, J = 2.7 Hz, 1H), 8.23 (dd, J = 9.1, 2.7 Hz, 1H), 7.88 (s, 2H), 7.52 (d, J = 9.2 Hz,
1H), 4.24 (t, J = 6.5 Hz, 2H), 1.84 – 1.75 (m, 2H), 1.50 – 1.41 (m, 2H), 1.38 (m, 2H), 0.92 (t,
13
J = 7.2 Hz, 3H). C NMR (150 MHz, DMSO-d6) δ 160.29, 150.50, 145.01, 137.10, 129.57,
127.01, 125.32, 121.89, 120.01, 115.29, 114.44, 101.34, 69.48, 27.94, 27.45, 21.77, 13.89.
ESI-HRMS calcd. for C19H20N5O [M + H]+ 334.1662, found: 334.1674.
2-Allyloxy-5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitrile（1e）
1
An off-white powder, yield 65.0%. H NMR (600 MHz, DMSO-d6) δ 9.51 (s, 1H), 8.70
(d, J = 3.4 Hz, 2H), 8.37 (d, J = 2.7 Hz, 1H), 8.24 (dd, J = 9.1, 2.7 Hz, 1H), 7.86 (d, J = 5.8
Hz, 2H), 7.53 (d, J = 9.2 Hz, 1H), 6.10 (ddd, J = 22.3, 10.4, 5.1 Hz, 1H), 5.50 (m, 1H), 5.36
(dd, J = 10.6, 1.3 Hz, 1H), 4.86 (d, J = 5.1 Hz, 2H). 13C NMR (150 MHz, DMSO-d6) δ
159.76, 150.51, 144.95, 137.26, 132.30, 129.79, 126.99, 125.42, 121.97, 119.55, 118.41,
115.29, 114.78, 101.50, 69.70. ESI-HRMS calcd. for C17H14N5O [M + H]+ 304.1193, found:
This article is protected by copyright. All rights reserved.

304.1185.
2-(2-Methoxyethoxy)-5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitrile（1f）
1
A white powder, yield 64.8%. H NMR (600 MHz, DMSO-d6) δ 9.51 (s, 1H), 8.70 (s,
2H), 8.35 (d, J = 2.7 Hz, 1H), 8.23 (dd, J = 9.1, 2.7 Hz, 1H), 7.86 (d, J = 5.7 Hz, 2H), 7.54 (d,
J = 9.2 Hz, 1H), 4.44 – 4.34 (m, 2H), 3.80 – 3.70 (m, 2H), 3.36 (s, 3H). 13C NMR (150 MHz,
DMSO-d6) δ 160.19, 150.51, 144.96, 137.26, 129.74, 127.00, 125.42, 121.96, 119.57, 115.33,
114.59, 101.41, 69.99, 69.09, 58.43. ESI-HRMS calcd. for C17H16N5O2 [M + H]+ 322.1299,
found: 322.1306.
2-Isopropoxy-5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitrile (1g)
1
An off-white powder, yield 57.5%. H NMR (600 MHz, DMSO-d6) δ 9.50 (s, 1H), 8.73
(br s, 2H), 8.33 (d, J = 2.2 Hz, 1H), 8.21 (dd, J = 9.1, 2.2 Hz, 1H), 7.87 (s, 2H), 7.56 (d, J =
9.2 Hz, 1H), 4.91 (m, 1H), 1.37 (d, J = 5.9 Hz, 6H). 13C NMR (150 MHz, DMSO-d6) δ
159.40, 150.53, 144.98, 137.18, 129.45, 127.04, 125.56, 121.97, 119.97, 115.51, 115.47,
102.12, 72.27, 21.54. ESI-HRMS calcd. for C17H16N5O [M + H]+ 306.1349, found: 306.1355.
2-Isobutoxy-5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitrile (1h)
1
An off-white powder, yield 49.7%. H NMR (600 MHz, DMSO-d6) δ 9.51 (s, 1H), 8.70
(d, J = 4.6 Hz, 2H), 8.35 (d, J = 2.7 Hz, 1H), 8.28 – 8.14 (m, 1H), 7.86 (d, J = 6.0 Hz, 2H),
7.52 (d, J = 9.2 Hz, 1H), 4.03 (d, J = 6.5 Hz, 2H), 2.11 (m, 1H), 1.04 (d, J = 6.7 Hz, 6H). 13C
NMR (150 MHz, DMSO-d6) δ 160.36, 150.51, 144.95, 137.28, 129.60, 127.02, 125.28,
121.93, 119.55, 115.23, 114.49, 101.37, 75.27, 27.59, 18.72. ESI-HRMS calcd. for
C18H18N5O [M + H]+ 320.1506, found: 320.1509.
2-(sec-Butoxy)-5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitrile (1i)
A yellow powder, yield 38.5%. 1H NMR (600 MHz, DMSO-d6) δ 9.50 (s, 1H), 8.77 (br s,
2H), 8.33 (d, J = 2.8 Hz, 1H), 8.20 (dd, J = 9.2, 2.8 Hz, 1H), 7.89 (s, 2H), 7.55 (d, J = 9.3 Hz,
1H), 4.79 – 4.63 (m, 1H), 1.72 (m, 2H), 1.33 (d, J = 6.1 Hz, 3H), 0.97 (d, J = 7.4 Hz, 3H). 13C
NMR (150 MHz, DMSO-d6) δ 159.66, 150.44, 144.98, 137.13, 129.42, 126.95, 125.45,
121.87, 120.15, 115.44, 115.40, 102.10, 76.89, 28.33, 18.74, 9.17. ESI-HRMS calcd. for
This article is protected by copyright. All rights reserved.

C18H18N5O [M + H]+ 320.1506, found: 320.1519.
2-Isopentyloxy-5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitrile (1j)
1
A white powder, yield 29.7%. H NMR (600 MHz, DMSO-d6) δ 9.50 (s, 1H), 8.69 (d, J
= 4.4 Hz, 2H), 8.33 (d, J = 2.6 Hz, 1H), 8.22 (dd, J = 9.1, 2.6 Hz, 1H), 7.85 (d, J = 5.7 Hz,
2H), 7.53 (d, J = 9.2 Hz, 1H), 4.34 – 4.22 (m, 2H), 1.83 (m, 1H), 1.70 (q, J = 6.6 Hz, 2H),
13
0.97 (d, J = 6.6 Hz, 6H). C NMR (150 MHz, DMSO-d6) δ 160.30, 129.60, 127.07, 125.37,
121.54, 115.29, 114.47, 101.34, 68.07, 36.95, 24.62, 22.38. ESI-HRMS calcd. for C19H20N5O
[M + H]+ 334.1662, found: 334.1669.
2-Cyclopentyloxy-5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitrile（1k）
1
A white powder, yield 55.0%. H NMR (600 MHz, DMSO-d6) δ 9.50 (s, 1H), 8.70 (s,
2H), 8.33 (d, J = 2.7 Hz, 1H), 8.22 (dd, J = 9.1, 2.8 Hz, 1H), 7.86 (d, J = 5.4 Hz, 2H), 7.52 (d,
J = 9.2 Hz, 1H), 5.25 – 4.97 (m, 1H), 2.16 – 1.91 (m, 2H), 1.91 – 1.70 (m, 4H), 1.65 (dd, J =
7.7, 5.5 Hz, 2H). ESI-HRMS calcd. for C19H18N5O [M + H]+ 332.1506, found: 332.1512.
2-Cyclohexyloxy-5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitrile（1l）
An off-white powder, yield 50.8%. 1H NMR (600 MHz, DMSO-d6) δ 9.50 (s, 1H), 8.82
(br s, 2H), 8.33 (d, J = 2.7 Hz, 1H), 8.20 (dd, J = 9.2, 2.7 Hz, 1H), 7.91 (br s, 2H), 7.58 (d, J
= 9.3 Hz, 1H), 4.72 (m, 1H), 1.94 (m, 2H), 1.80 – 1.69 (m, 2H), 1.60 (m, 2H), 1.55 – 1.34 (m,
4H). 13C NMR (150 MHz, DMSO-d6) δ 159.24, 150.54, 144.99, 137.13, 129.48, 126.99,
125.52, 121.97, 119.82, 115.76, 115.43, 102.25, 76.37, 30.65, 24.88, 22.60. ESI-HRMS calcd.
for C20H20N5O [M + H]+ 346.1662, found: 346.1677.
2-Benzyloxy-5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitrile（1m）
1
An off-white powder, yield 37.8%. H NMR (600 MHz, DMSO-d6) δ 9.51 (s, 1H), 8.74
(br s, 2H), 8.38 (d, J = 2.7 Hz, 1H), 8.25 (dd, J = 9.1, 2.7 Hz, 1H), 7.87 (s, 2H), 7.63 (d, J =
9.2 Hz, 1H), 7.52 (d, J = 7.3 Hz, 2H), 7.45 (t, J = 7.6 Hz, 2H), 7.39 (t, J = 7.3 Hz, 1H), 5.41
13
(s, 2H). C NMR (150 MHz, DMSO-d6) δ 159.92, 150.54, 144.98, 137.23, 135.65, 129.90,
This article is protected by copyright. All rights reserved.

128.65, 128.33, 127.71, 127.09, 125.54, 122.05, 119.44, 115.32, 115.01, 101.71, 70.77.
ESI-HRMS calcd. for C21H16N5O [M + H]+ 354.1349, found: 354.1338.
2-((4-Methoxybenzyl)oxy)-5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitrile（1n）
1
An off-white powder, yield 58.2%. H NMR (600 MHz, DMSO-d6) δ 9.51 (s, 1H), 9.10
(br s, 2H), 8.36 (d, J = 2.7 Hz, 1H), 8.24 (dd, J = 9.1, 2.7 Hz, 1H), 8.04 (br s, 2H), 7.63 (d, J
= 9.2 Hz, 1H), 7.45 (d, J = 8.6 Hz, 2H), 7.00 (d, J = 8.6 Hz, 2H), 5.31 (s, 2H), 3.77 (s, 3H).
13C NMR (150 MHz, DMSO-d6) δ 160.00, 159.37, 150.18, 145.17, 129.79, 129.73, 127.43,
127.02, 125.49, 121.96, 120.15, 115.35, 115.06, 114.03, 109.52, 101.69, 70.69, 55.13.
ESI-HRMS calcd. for C22H18N5O2 [M + H]+ 384.1455, found: 384.1462.
2-((4-Chlorobenzyl)oxy)-5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitrile（1o）
1
An off-white powder, yield 49.2%. H NMR (600 MHz, DMSO-d6) δ 9.51 (s, 1H), 8.70
(br s, 2H), 8.38 (d, J = 2.7 Hz, 1H), 8.26 (dd, J = 9.1, 2.7 Hz, 1H), 7.86 (d, J = 5.0 Hz, 2H),
7.61 (d, J = 9.2 Hz, 1H), 7.53 (m, 4H), 5.40 (s, 2H). 13C NMR (150 MHz, DMSO-d6) δ
159.72, 150.53, 144.97, 137.25, 134.69, 132.96, 129.98, 129.56, 128.68, 127.05, 125.51,
122.02, 119.61, 115.25, 114.98, 101.73, 69.94. ESI-HRMS calcd. for C21H15ClN5O [M + H]+
388.0960, found: 388.0968.
2-((4-Cyanobenzyl)oxy)-5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitrile（1p）
1
An off-white powder, yield 31.0%. H NMR (600 MHz, DMSO-d6) δ 9.52 (s, 1H), 8.74
(br s, 2H), 8.41 (d, J = 2.7 Hz, 1H), 8.27 (dd, J = 9.1, 2.7 Hz, 1H), 7.94 (d, J = 8.2 Hz, 2H),
13
7.88 (br s, 2H), 7.71 (d, J = 8.2 Hz, 2H), 7.60 (d, J = 9.2 Hz, 1H), 5.53 (s, 2H). C NMR
(150 MHz, DMSO-d6) δ 159.52, 150.54, 145.04, 141.38, 132.64, 130.13, 128.00, 127.11,
125.56, 122.02, 121.28, 118.65, 115.20, 114.93, 110.95, 101.77, 69.73. ESI-HRMS calcd. for
C22H15N6O [M + H]+ 379.1302, found: 379.1312.
XO inhibitory activity
Bovine XO inhibitory potency in vitro was assayed spectrophotometrically by measuring
the production of uric acid at 294 nm using a UV-2100 spectrophotometer (UNICO). The
testing method was based on the procedure reported by Matsumoto et al.,[17] with
This article is protected by copyright. All rights reserved.

modification. The reactive mixture contained 0.1 M sodium pyrophosphate buffer (pH 8.3),
0.3 mM Na2EDTA, 100 μM xanthine, 25 U/L XO (Sigma, X4875) and the tested compound.
The inhibition of XO was evaluated by the reduction of the uric acid formation. The enzyme
was pre-incubated for 10 min with the tested compound at 25℃, and the reaction was started
by an addition of xanthine. All tests were performed in triplicate. Compounds presenting
inhibitory effects over 50% at a concentration of 50 μM were further tested at a wide range of
concentrations to calculate their IC50 values using SPSS 20.0 software.
Molecular modeling
Molecular modeling studies were performed with MOE software.[38] The crystal
structure of bovine XO in complex with topiroxostat (PDB code: 1V97) was adopted in
docking calculations.[39] The structure was protonated, polar hydrogens were added and
energy minimization was carried out (RMSD gradient = 0.1 kcal/mol, AMBER10: EHT
field).[40] Ligand structures were built and minimized with MOE software. The docking
procedure adopted the standard protocol implemented in MOE. The binding site was
designated by the original ligand atoms and other parameters were maintained as the
defaults.[41] The 3-D binding modes were analyzed by Surfaces and Maps tool of the MOE
software.
Acknowledgments
This work was supported by the Chinese National Science Foundation (Grant Nos.
81274182 and 81573687) and the project of the Liaoning distinguished professor.
Conflict of interest
The authors have declared no conflict of interest.
This article is protected by copyright. All rights reserved.

Legend for Fig.1, Fig. 2, Fig. 3 and Scheme 1.
Fig. 1 The chemical structures of some XO inhibitors and designed compounds 1a-p.
Fig. 2 (A) docking conformation of topiroxostat (green) overlaid with the crystal
conformation of topiroxostat (orange); (B) and (C) docking conformation of 1k (cyan)
overlaid with the crystal conformation of topiroxostat (orange); (D) docking conformation of
1k (cyan) overlaid with its energy minimized conformation (pink) calculated by MOE
software.
Fig. 3 Lineweaver-Burk plot analysis of XO inhibition by compound 1k.
Scheme 1. Reagents and conditions: (i) HNO3, AcOH, 50°C, 4 h; (ii) RCl or RBr, K2CO3, KI,
DMF, 60°C, overnight; (iii) Fe, NH4Cl, EtOH, H2O, reflux, 3 h; (iv) 1) NaNO2, AcOH, H2O,
-10°C, 30 min; 2) NaN3, 0°C, 3 h; (v) 4-ethynylpyridine hydrochloride, CuSO4, vitamin C,
50°C, microwave for 8 min.
References
[1]
[2]
[3]
[4]
[5]
M. Gliozzi, N. Malara, S. Muscoli, V. Mollace. Int J Cardiol, 2016, 213, 23.
C. Chen, J.-M. Lü, Q. Yao. Med Sci Monit, 2016, 22, 2501.
R. Edward, D. Michael. Clin Med (Northfield Il), 2013, 13, 400.
J.-M. Lü, Q. Yao, C. Chen. Biochem Pharmacol, 2013, 86, 1328.
J. Evenäs, F. Edfeldt, M. Lepistö, N. Svitacheva, A. Synnergren, B. Lundquist, M. Gränse, A. Rönnholm,
M. Varga, J. Wright et al. Biorg Med Chem Lett, 2014, 24, 1315.
[6]
[7]
D. Yasuda, K. Takahashi, T. Kakinoki, Y. Tanaka, T. Ohe, S. Nakamura, T. Mashino. MedChemComm, 2013,
4, 527.
Z. Smelcerovic, A. Veljkovic, G. Kocic, D. Yancheva, Z. Petronijevic, M. Anderluh, A. Smelcerovic.
Chem-Biol Interact, 2015, 229, 73.
[8]
[9]
H. Singh, S. Sharma, R. Ojha, M.K. Gupta, K. Nepali, P.M.S. Bedi. Biorg Med Chem Lett, 2014, 24, 4192.
A. Šmelcerović, K. Tomović, Ž. Šmelcerović, Ž. Petronijević, G. Kocić, T. Tomašič, Ž. Jakopin, M. Anderluh.
Eur J Med Chem, 2017, 135, 491.
[10]
[11]
K. Okamoto, B.T. Eger, T. Nishino, S. Kondo, E.F. Pai. J Biol Chem, 2002, 278, 1848.
S. Ishibuchi, H. Morimoto, T. Oe, T. Ikebe, H. Inoue, A. Fukunari, M. Kamezawa, I. Yamada, Y. Naka.
Biorg Med Chem Lett, 2001, 11, 879.
[12]
Q. Guan, Z. Cheng, X. Ma, L. Wang, D. Feng, Y. Cui, K. Bao, L. Wu, W. Zhang. Eur J Med Chem, 2014, 85,
This article is protected by copyright. All rights reserved.

508.
[13]
J.U. Song, S.P. Choi, T.H. Kim, C.-K. Jung, J.-Y. Lee, S.-H. Jung, G.T. Kim. Biorg Med Chem Lett, 2015, 25,
1254.
[14]
[15]
[16]
S. Wang, J. Yan, J. Wang, J. Chen, T. Zhang, Y. Zhao, M. Xue. Eur J Med Chem, 2010, 45, 2663.
S. Chen, T. Zhang, J. Wang, F. Wang, H. Niu, C. Wu, S. Wang. Eur J Med Chem, 2015, 103, 343.
T.-j. Zhang, Q.-x. Wu, S.-y. Li, L. Wang, Q. Sun, Y. Zhang, F.-h. Meng, H. Gao. Biorg Med Chem Lett, 2017,
27, 3812.
[17]
[18]
[19]
K. Matsumoto, K. Okamoto, N. Ashizawa, T. Nishino. J Pharmacol Exp Ther, 2010, 336, 95.
E. Cristofer, T.E. Bryan, O. Ken, N. Tomoko, N. Takeshi, F.P. Emil. PNAS, 2000, 97, 10723.
T. Sato, N. Ashizawa, T. Iwanaga, H. Nakamura, K. Matsumoto, T. Inoue, O. Nagata. Biorg Med Chem
Lett, 2009, 19, 184.
[20]
[21]
[22]
[23]
[24]
[25]
[26]
C. B-Rao, A. Kulkarni-Almeida, K.V. Katkar, S. Khanna, U. Ghosh, A. Keche, P. Shah, A. Srivastava, V.
Korde, K.V.S. Nemmani et al. Biorg Med Chem, 2012, 20, 2930.
S. Khanna, S. Burudkar, K. Bajaj, P. Shah, A. Keche, U. Ghosh, A. Desai, A. Srivastava, A.
Kulkarni-Almeida, N.J. Deshmukh et al. Biorg Med Chem Lett, 2012, 22, 7543.
K. Bajaj, S. Burudkar, P. Shah, A. Keche, U. Ghosh, P. Tannu, S. Khanna, A. Srivastava, N.J. Deshmukh, A.
Dixit et al. Biorg Med Chem Lett, 2013, 23, 834.
K. Nepali, A. Agarwal, S. Sapra, V. Mittal, R. Kumar, U.C. Banerjee, M.K. Gupta, N.K. Satti, O.P. Suri, K.L.
Dhar. Biorg Med Chem, 2011, 19, 5569.
K. Nepali, G. Singh, A. Turan, A. Agarwal, S. Sapra, R. Kumar, U.C. Banerjee, P.K. Verma, N.K. Satti, M.K.
Gupta et al. Biorg Med Chem, 2011, 19, 1950.
E. Hofmann, J. Webster, T. Do, R. Kline, L. Snider, Q. Hauser, G. Higginbottom, A. Campbell, L. Ma, S.
Paula. Biorg Med Chem, 2016, 24, 578.
M.V.N. Rodrigues, A.F. Barbosa, J.F. da Silva, D.A. dos Santos, K.L. Vanzolini, M.C. de Moraes, A.G.
Corrêa, Q.B. Cass. Biorg Med Chem, 2016, 24, 226.
[27]
[28]
S.H. Nile, B. Kumar, S.W. Park. Chem Biol Drug Des, 2013, 82, 290.
Y. Li, C.M. Frenz, Z. Li, M. Chen, Y. Wang, F. Li, C. Luo, J. Sun, L. bohlin, Z. Li et al. Chem Biol Drug Des,
2011, 537.
[29]
[30]
A. Nagao, M. Seki, H. Kobayashi. Biosci, Biotechnol, Biochem, 1999, 63, 1787.
R.M. Vitale, L. Antenucci, M. Gavagnin, G. Raimo, P. Amodeo. J Enzyme Inhib Med Chem, 2017, 32,
345.
[31]
M. Kaur, A. Kaur, S. Mankotia, H. Singh, A. Singh, J.V. Singh, M.K. Gupta, S. Sharma, K. Nepali, P.M. Bedi.
Eur J Med Chem, 2017, 131, 14.
[32]
[33]
H.J. Tang, X.W. Zhang, L. Yang, W. Li, J.H. Li, J.X. Wang, J. Chen. Eur J Med Chem, 2016, 124, 637.
T.-j. Zhang, S.-y. Li, W.-y. Yuan, Q.-x. Wu, L. Wang, S. Yang, Q. Sun, F.-h. Meng. Biorg Med Chem Lett,
2017, 27, 729.
[34]
[35]
[36]
[37]
[38]
[39]
P. Thirumurugan, D. Matosiuk, K. Jozwiak. Chem Rev, 2013, 113, 4905.
D. Dheer, V. Singh, R. Shankar. Bioorg Chem, 2017, 71, 30.
F. Meng, T. Zhang, Q. Wu, L. Wang, Q. Sun, J. Liang, X. He. CN105294584A. 2016.
L.D. Kong, Y. Zhang, X. Pan, R.X. Tan, C.H. Cheng. Cell Mol Life Sci, 2000, 57, 500.
S. Vilar, G. Cozza, S. Moro. Curr Top Med Chem, 2008, 8, 1555.
K. Okamoto, K. Matsumoto, R. Hille, B.T. Eger, E.F. Pai, T. Nishino. Proc Natl Acad Sci U S A, 2004, 101,
7931.
[40]
S.H. Abbas, G.E.-D.A.A. Abuo-Rahma, M. Abdel-Aziz, O.M. Aly, E.A. Beshr, A.M. Gamal-Eldeen. Bioorg
This article is protected by copyright. All rights reserved.

Chem, 2016, 66, 46.
[41]
D. Dal Ben, M. Buccioni, C. Lambertucci, A. Thomas, K.-N. Klotz, S. Federico, B. Cacciari, G. Spalluto, R.
Volpini. Eur J Med Chem, 2013, 70, 525.
This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.