Discovery and biological evaluation of some (1H-1,2,3-triazol-4-yl)methoxybenzaldehyde derivatives containing an anthraquinone moiety as potent xanthine oxidase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.01.049

A series of (1H-1,2,3-triazol-4-yl)methoxybenzaldehyde derivatives containing an anthraquinone moiety were synthesized and identified as novel xanthine oxidase inhibitors. Among them, the most promising compounds 1h and 1k were obtained with IC₅₀values of 0.6?μM and 0.8?μM, respectively, which were more than 10-fold potent compared with allopurinol. The Lineweaver-Burk plot revealed that compound 1h acted as a mixed-type xanthine oxidase inhibitor. SAR analysis showed that the benzaldehyde moiety played a more important role than the anthraquinone moiety for inhibition potency. The basis of significant inhibition of xanthine oxidase by 1h was rationalized by molecular modeling studies.

Full text of DOI:10.1016/j.bmcl.2017.01.049

Bioorganic & Medicinal Chemistry Letters 27 (2017) 729–732
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and biological evaluation of some (1H-1,2,3-triazol-4-yl)
methoxybenzaldehyde derivatives containing an anthraquinone moiety
as potent xanthine oxidase inhibitors
1
1
⇑
Ting-jian Zhang, Song-ye Li , Wei-yan Yuan , Qing-xia Wu, Lin Wang, Su Yang, Qi Sun, Fan-hao Meng
School of Pharmacy, China Medical University, 77 Puhe Road, North New Area, Shenyang 110122, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of (1H-1,2,3-triazol-4-yl)methoxybenzaldehyde derivatives containing an anthraquinone moiety
Received 14 September 2016
Revised 28 December 2016
Accepted 14 January 2017
Available online 16 January 2017
were synthesized and identified as novel xanthine oxidase inhibitors. Among them, the most promising
compounds 1h and 1k were obtained with IC50 values of 0.6 lM and 0.8 lM, respectively, which were
more than 10-fold potent compared with allopurinol. The Lineweaver-Burk plot revealed that compound
1h acted as a mixed-type xanthine oxidase inhibitor. SAR analysis showed that the benzaldehyde moiety
played a more important role than the anthraquinone moiety for inhibition potency. The basis of signif-
icant inhibition of xanthine oxidase by 1h was rationalized by molecular modeling studies.
Ó 2017 Elsevier Ltd. All rights reserved.
Keywords:
Anthraquinone
Xanthine oxidase
Hyperuricemia
Xanthine oxidase (XO) is a well-known target for the treatment
of hyperuricemia and gout. Inhibition of XO could block the
hydroxylation of both hypoxanthine and xanthine in the last two
steps of uric acid biosynthesis in humans.1 Indeed, the over-
production of uric acid is the key cause of hyperuricemia and gout.2
Therefore, inhibitors of XO could decrease the generation of uric
acid and benefit these pathological conditions.3 Reactive oxygen
species (ROS) are generated in concert with the oxidation process.
An excess of ROS could induce various pathological states such as
inflammation, metabolic disorders, atherosclerosis, cancer and
chronic obstructive pulmonary disease.4 Thus, inhibition of XO is
a potential treatment of diseases caused by the XO-derived ROS
as well.5
Reported XO inhibitors can be simply divided into two cate-
gories: purine analogs and non-purine XO inhibitors. Allopurinol
(Fig. 1), a prototypical inhibitor of XO with a recognizable purine
backbone, has been widely prescribed in the treatment of hyper-
uricemia and gout for several decades. However, in some cases,
severe life-threatening side effects of allopurinol have been
reported due to the purine backbone, such as fulminant hepatitis,
renal failure, and Stevens–Johnson syndrome.6 Given these limita-
tions, research has focused on the development of novel non-pur-
ine XO inhibitors with potent XO inhibitory potency, but with
fewer side effects. In recent decades, a great amount of non-purine
XO inhibitors of various chemotypes have been reported, such as
Febuxostat (approved in USA, 2009),7 Y-700,8 Topiroxostat
(approved in Japan, 2013),9 isoxazoles,10 schiff bases of benzalde-
hydes,11 N-(1,3-diaryl-3-oxo-propyl)amides,12 N-acetyl pyrazoli-
nes,13 isocytosines,14 selenazoles,15 imidazoles,16 2-(indol-5-yl)
thiazoles,17 chalcones,18 and 9-deazaguanines.19
In our previous studies on anthraquinone compounds as anti-
tumor agents, we unexpectedly found a compound (1a), which
contained a benzaldehyde moiety and an anthraquinone moiety
linked by a 1,2,3-triazole (Fig. 1), presented poor anti-tumor activ-
ity, but remarkable inhibitory potency in vitro against XO. Since
both benzaldehyde and anthraquinone have appeared as scaffolds
of XO inhibitors in recent studies,20 we investigated the activities
and the structure–activity relationship (SAR) around 1a.
The synthesis of compounds 1a-q is shown in the Scheme 1.
Commercially available phthalic anhydride reacted with toluene
via a Friedel-Craft reaction to provide 2-(4-methylbenzoyl)benzoic
acid 2, which underwent a intramolecular cyclization in acid-cat-
alyzed conditions, leading to 2-methylanthracene-9,10-dione 3.
The bromination of 3 with NBS obtained 2-(bromomethyl)an-
thracene-9,10-dione 4, which was then treated with sodium azide
to yield 2-(azidomethyl)anthracene-9,10-dione 5. The cyclization
of 5 with various alkyne analogs in the presence of copper sulfate
and vitamin C in a microwave condition resulted in compounds 1a-
o with good yields. Hydrolysis of compound 1o with sodium
hydroxide resulted in 1p, which was acetylated to yield 1q. The
⇑
Corresponding author.
E-mail address: fhmeng@cmu.edu.cn (F.-h. Meng).
These authors contributed equally.
1
http://dx.doi.org/10.1016/j.bmcl.2017.01.049
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.

730
T.-j. Zhang et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 729–732
testing method has been described in our previous study.16 Allop-
urinol was included as a reference compound. Compounds pre-
senting inhibitory effects higher than 60% at the concentration of
50 lM were further tested at a wide range of concentrations to cal-
culate associated IC50 values. The results are shown in Table 1.
COOH
CN
N
OH
N
S
N
N
HN
NC
O
N
N
N
N
H
N
Compound 1a was initially discovered as a weak XO inhibitor
Allopurinol
Febuxostat
Topiroxostat
(IC50 = 30.5
(IC50 = 9.8
l
M), which was 3-fold less potent than allopurinol
lM). Removing the phenyl group led to inactive com-
pounds 1p and 1q, which implied that the phenyl group may
strongly affect the XO inhibitory activity. Therefore, structure-
modification efforts to enhance the inhibitory potency were mainly
focused on this phenyl group.
The preliminary structure–activity relationship (SAR) around 1a
indicated that the formyl group played a significant role for XO
inhibition in this series, in spite of its lower drug-like features.
Removal of the formyl group resulted in 1b and its analog 1c,
accompanied by a decrease of potency. Furthermore, when the
para-formyl group was changed into a cyano (1e) or an acety-
lamino (1f) group, the activities of both were diminished, as well.
However, the introduction of two methoxy groups into the ortho-
O
O
R1
N
N
R2
R3
O
N
R4
1a: R1 = R3 = R4 = H, R2 = formyl.
Fig. 1. Chemical structures of Allopurinol, Febuxostat, Topiroxostat and compound
1a.
structures of the synthesized compounds were elucidated by 1H
NMR, 13C NMR, and MS. All spectral data were in accordance with
assumed structures.
position of phenyl ether (1d, IC50 = 16.8 lM) led to a 1.8-fold
increase in potency.
Bovine XO inhibitory potencies in vitro were spectrophotomet-
rically measured by determining the uric acid levels at 294 nm. The
As shown in Table 1, the position of the formyl group noticeably
affected the inhibitory potency. When the para-formyl group of 1a
O
O
O
COOH
ϸ
Ϲ
Ϻ
Br
O
O
O
O
O
2
3
4
O
O
R3
R4
ϼ
ϻ
N3
N
N
R2
O
N
R1
O
O
1a-n
5
ϼ
O
O
O
O
Ͻ
N
N
N
N
OR5
O
N
N
O
1o
1p, R5 = H
1q, R5 = acetyl
Ͼ
Scheme 1. Synthesis of compounds 1a-q. Reagents and conditions: (i) toluene, AlCl3, 50 °C, 4 h; (ii) H2SO4, 100 °C, 1 h; (iii) NBS, CCl4, reflux, 24 h; (iv) NaN3, THF, 40 °C,
overnight; (v) alkyne derivatives, CuSO4, vitamin C, EtOH, H2O, 80 °C, microwave 10 min; (vi) NaOH, MeOH, H2O, 50 °C, 1.5 h; (vii) acetyl chloride, Et3N, dichloromethane, 0 °C
then 25 °C, 1 h.
Table 1
XO in vitro inhibitory potencies of compounds 1a-q.
Compounds
R1
R2
R3
R4
Inhibition rate at 50
l
M
IC50 (lM)
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1p
1q
H
H
Cl
OCH3
H
H
H
Formyl
Carboxyl
Formyl
Formyl
Formyl
Formyl
Formyl
/
Formyl
H
H
Formyl
CN
Acetylamino
H
H
H
H
F
Cl
Br
H
/
H
H
H
H
H
H
H
H
Cl
OCH3
H
H
H
H
H
H
H
H
H
H
/
67.5
18.4
12.7
70.3
7.0
20.1
77.2
90.7
24.2
9.8
88.0
61.1
21.1
70.8
31.0
19.4
95.1
30.5
/
/
16.8
/
/
15.1
0.6
/
Formyl
H
H
N(C2H5)2
/
H
H
H
F
/
0.8
46.5
/
6.5
/
/
/
/
/
/
/
/
/
/
9.8
Allopurinol

T.-j. Zhang et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 729–732
731
was transferred to the meta-position (1g, IC50 = 15.1
potency was doubled. Moreover, the transition of the formyl group
to the ortho-position produced the most promising compound 1h
(IC50 = 0.6 lM), which was 51 times more potent than 1a.
The following modifications based on 1h were mainly carried
out by introduction of several substituents onto the phenyl unit,
such as halogens and N,N-diethylamino. Unfortunately, most of
the obtained compounds resulted either in a steep loss in potency
l
M), the
(IC50 = 9.0 lM), although there was nearly a 10-fold loss in potency
compared with 1h. For the R6 group, the broad structural tolerance
implied that the anthraquinone moiety may act as a supporting
role in inhibition of XO. Because the XO binding pocket presents
as a long, narrow cavity leading toward the molybdenum-pterin
center,7 it was concluded that the anthraquinone moiety may be
located at the outer region of active pocket, as in the case of the
isobutoxy group of Febuxostat, which possessed more tolerance
for the structure of the ligands. This hypothesis was confirmed
by molecular simulation studies.
(1l, IC50 = 46.5
(1j and 1m). The exception was 1k (IC50 = 0.8
l
M and 1n, IC50 = 6.5
l
M) or were entirely inactive
M), possessing a
l
fluorine atom for R2, which displayed comparable potency to com-
pound 1h. In addition, formyl oxidation to carboxyl (1i) also dimin-
ished the potency. Therefore, the only modification that could
maintain the potency was achieved by introducing a fluorine atom
for R2 group in this series, i.e., 1k.
To investigate the function of the anthraquinone moiety, 1h
analogs with the anthraquinone moiety replaced by a benzyl group
or a hexyl group were synthesized (Scheme 2) and evaluated for
potency against XO (Table 2). Surprisingly, some of these com-
To further understand the binding mode of the synthesized
compounds, molecular simulations of 1h into the binding pocket
of XO were performed. As the molybdenum-pterin sites of XO
and xanthine oxidoreductase are structurally equivalent,21 the
crystal structure of xanthine oxidoreductase/Febuxostat complex
(PDB code 1N5X) was employed as the protein template. A molec-
ular docking study was undertaken with AutoDock 422 with the
default settings. The Lamarckian genetic algorithm was used as
the search parameters. MOE (Molecular Operating Environment,
version 2015.1001) software was used for graphic display
(Fig. 2). According to this model, the benzaldehyde moiety inserted
into the long-narrow cavity of the binding pocket and partially
overlapped with phenyl moiety of Febuxostat. Additionally, the
formyl group accepted a hydrogen bond from the hydroxyl of
Ser876. Moreover, the anthraquinone moiety occupied a sub-
pocket formed by Phe1013, Leu1014, Ser1075, Lys771 and
Met770 outside the cavity, and an H-bond interaction between a
carbonyl of anthraquinone and the amino of Lys771 was observed.
These interactions may provide a reasonable explanation for the
SAR results in the study.
pounds were also active, such as 7b (IC50 = 21.5 lM) and 8b
R2
CHO
O
6a, R2 = H
6b, R2 = F
N
CHO
7a, R2 = H, R6 = benzyl
N
N
ϸ
2
6
R2
O
7b
, R = F, R = benzyl
R6
8a, R2 = H, R6 = hexyl
8b, R2 = F, R6 = hexyl
7a-b
8a-b
Scheme 2. Synthesis of compounds 7a-b and 8a-b. Reagents and conditions: (i)
(azidomethyl)benzene or azidohexane, CuSO4, vitamin C, EtOH, H2O, 80 °C,
microwave 10 min.
Table 2
XO in vitro inhibitory potencies of 1h analogs.
Compounds
R2
R6
Inhibition rate at 50
l
M
IC50 (lM)
7a
7b
8a
8b
H
F
H
F
Benzyl
Benzyl
Hexyl
Hexyl
46.7
83.4
43.5
93.0
/
21.5
/
9.0
Fig. 2. Docking pose of compound 1h (orange) within the protein binding pocket overlaid with the reference ligand Febuxostat (cyan).

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T.-j. Zhang et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 729–732
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Supplementary material
tight binding inhibitor. J Biol Chem. 1995;270:7816.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.01.
049. These data include MOL files and InChiKeys of the most
important compounds described in this article.