Development of benzoxazole deoxybenzoin oxime and acyloxylamine derivatives targeting innate immune sensors and xanthine oxidase for treatment of gout

Article abstract of DOI:10.1016/j.bmc.2018.02.013

Both the inhibition of inflammatory flares and the treatment of hyperuricemia itself are included in the management of gout. Extending our efforts to development of gout therapy, two series of benzoxazole deoxybenzoin oxime derivatives as inhibitors of innate immune sensors and xanthine oxidase (XOD) were discovered in improving hyperuricemia and acute gouty arthritis. In vitro studies revealed that most compounds not only suppressed XOD activity, but blocked activations of NOD-like receptor (NLRP3) inflammasome and Toll-like receptor 4 (TLR4) signaling pathway. More importantly, (E)-1-(6-methoxybenzo[d]oxazol-2-yl)-2-(4-methoxyphenyl)ethanone oxime (5d) exhibited anti-hyperuricemic and anti-acute gouty arthritis activities through regulating XOD, NLRP3 and TLR4. Compound 5d may serve as a tool compound for further design of anti-gout drugs targeting both innate immune sensors and XOD.

Full text of DOI:10.1016/j.bmc.2018.02.013

Accepted Manuscript
Development of benzoxazole deoxybenzoin oxime and acyloxylamine deriva-
tives targeting innate immune sensors and xanthine oxidase for treatment of gout
Jun Huang, Zehao Zhou, Mengze Zhou, Mingxing Miao, Huanqiu Li, Qinghua
Hu
PII:
S0968-0896(18)30156-1
https://doi.org/10.1016/j.bmc.2018.02.013
BMC 14199
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
24 January 2018
9 February 2018
11 February 2018
Please cite this article as: Huang, J., Zhou, Z., Zhou, M., Miao, M., Li, H., Hu, Q., Development of benzoxazole
deoxybenzoin oxime and acyloxylamine derivatives targeting innate immune sensors and xanthine oxidase for
treatment of gout, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.02.013
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Development of benzoxazole deoxybenzoin oxime and acyloxylamine derivatives
targeting innate immune sensors and xanthine oxidase for treatment of gout
Jun Huanga, #, Zehao Zhou a, #, Mengze Zhou b, #, Mingxing Miao b, Huanqiu Li a, *
Qinghua Hu b, *
,
a
Department of Medicinal Chemistry, College of Pharmaceutical Sciences, Soochow
University, Suzhou 215123, PR China
b Department of Pharmacology, School of Pharmacy, China Pharmaceutical University,
Nanjing 210009, PR China
* Corresponding author.
Email: huqh@cpu.edu.cn (Qing-Hua Hu); huanqiuli@suda.edu.cn (Huan-Qiu Li)
# These authors contributed equally to this study
Abstract
Both the inhibition of inflammatory flares and the treatment of hyperuricemia
itself are included in the management of gout. Extending our efforts to development
of gout therapy, two series of benzoxazole deoxybenzoin oxime derivatives as
inhibitors of innate immune sensors and xanthine oxidase (XOD) were discovered in
improving hyperuricemia and acute gouty arthritis. In vitro studies revealed that most
compounds not only suppressed XOD activity, but blocked activations of NOD-like
receptor (NLRP3) inflammasome and Toll-like receptor 4 (TLR4) signaling pathway.
More
importantly,
(E)-1-(6-methoxybenzo[d]oxazol-2-yl)-2-(4-methoxyphenyl)
ethanone oxime (5d) exhibited anti-hyperuricemic and anti-acute gouty arthritis
activities through regulating XOD, NLRP3 and TLR4. Compound 5d may serve as a
tool compound for further design of anti-gout drugs targeting both innate immune
sensors and XOD.
Keywords ： xanthine oxidase; NOD-like receptor 3; Toll-like receptor 4;
anti-hyperuricemia; gout
1. Introduction
1

Gout is an inflammatory arthritis characterized by abrupt self-limiting attacks of
inflammation triggered by deposition of monosodium urate crystals (MSU) in the
joint secondary to longstanding hyperuricemia, which has been associated with poor
quality of life [1]. Therefore, prevention and treatment of gout should be studied and
solved urgently.
Both the inhibition of inflammatory flares and the treatment of hyperuricemia
itself are included in the management of gout [2]. For anti-inflammation, colchicine,
corticosteroids and NSAIDs could effectively treated acute exacerbations of gout,
nevertheless, broad drug toxicities, particularly in subjects with significant
co-morbidities limited their application in clinic [3]. In addition, although blocking
therapies, such as anakinra, canakinumab and rilonacept, have an increasing role in
the management of difficult-to-treat gout, high cost and inconvenient route of
administration made this type of drug difficult to be promoted [4]. Recent studies
have demonstrated that innate immune sensors including Nod-like receptor 3 (NLRP3)
and Toll-like receptor 4 (TLR4) are involved in sensing MSU deposition and
subsequent activation of the downstream inflammatory response [5]. Assembly of
NLPR3 inflammasome composed by NLRP3, apoptosis-associated speck-like protein
(ASC) and caspase-1 led to cleavage of interleukin 1 beta (IL-1β) precursor to
produce active IL-1β, playing a critical role in the pathogenesis of acute gouty
arthritis [6]. On the other hand, TLR4 - Myeloid differentiation primary response gene
88 (MyD88) - NF-κB signaling also contributed to development of acute
inflammation in primary gout patients [7]. Consistently, several natural compounds or
traditional Chinese medicines exhibited beneficial effects on urate-related diseases
through inhibition of NLRP3 and TLR4 [8, 9]. Moreover, some NLRP3 inhibitors for
gout treatment have entered the phase of clinical research, such as bucillamine.
Nevertheless, there are no dual inhibitors targeting NLRP3 and TLR4 were
discovered for gout therapy.
More importantly, in spite of anti-inflammatory therapies successfully suppress
MSU-induced inflammatory cascade, the serum uric acid still maintains a high level,
which might lead to recurrence of gout attack [10]. Xanthine oxidase (XOD) involved
2

in uric acid production, catalyzes the oxidation of xanthine to uric acid, which has
been regarded as an effective target for treatment of hyperuricemia and gout.
Febuxostat and allopurinol have been developed as XOD-targeted anti-hyperuricemia
drugs commonly used in clinic [11]. Nevertheless, both febuxostat and allopurinol
exhibited serious side effects, while they seem to be not effective for acute gouty
arthritis [12, 13]. Whereas there are no dual functional drugs which have
anti-inflammatory and anti-hyperuricemic activities for gout therapy till now, the
development of novel compounds that act by dual inhibition of innate immune sensors
and XOD could be a promising approach for treatment of gout.
Interestingly, evidences from previous studies suggested that several kinds of
flavonoids, such as quercetin and morin, not only exhibited potential
anti-hyperuricemia effects in oxonate-treated mice by inhibiting XOD activities, but
attenuated MSU-induced acute gouty arthritis through regulating innate immune
sensors in rats [14, 15]. Isoliquiritigenin, another type of flavones derivatives, has
been regarded as a dual inhibitor of NLRP3 and TLR4, but high toxicity limited its
clinical application [16, 17]. Additionally, deoxybenzoins, intermediates in the
synthesis of flavones, their oximes derivatives also exhibited similar biological
activities including anti-hyperuricemic and immunosuppressive activities in our
previous reports [18, 19]. In view of the above, based on the deoxybenzoins oximes
skeleton, we designed two series of benzoxazole deoxybenzoin oxime derivatives as
dual inhibitors of innate immune sensors and XOD (Scheme 1). Notably, compound
5d is multi-targeting inhibitor of NLRP3, TLR4 and XOD with excellent potency in
treating hyperuricemia and acute gouty arthritis.
3

Scheme 1. Design of the target compounds.
2. Chemistry
The preparation of the benzoxazole deoxybenzoin oxime derivatives is shown in
Scheme 1. As summarized in Scheme 2, our chemical synthesis utilized a series of
commercially available 2-aminophenols 1a-1f. Compounds 1a-1f reacted with an
equal equivalent of lactic acid to yield 2a-2f. The intermediates 2a-2f were then
oxidized by an equal equivalent of chromium trioxide in acetic acid under reflux to
afford benzoxazole ketones 3a-3f. Compounds 3a-3f reacted with bromobenzene
under Pd(dba)2/DTPF-catalyzed R-arylation reaction to afford benzoxazole
deoxybenzoin 4a-4n. The treatment of selected deoxybenzoins derivatives 4a–4n with
hydroxylamine hydrochloride in ethanol afforded the benzoxazole deoxybenzoin
oxime derivatives 5a–5n with sodium acetate as catalyst and alkaline environment
provider.
As shown in Scheme 3, benzoxazole acyloxylamines 7a-7h with an oxime linking
moiety were synthesized by reaction of appropriate acid chloride with oxime 6, which
was itself prepared from the corresponding benzoxazole ketone 3a by condensation
with hydroxylamine hydrochloride (Scheme 3).
4

Compound
R1
H
R2
H
5a
5b
5c
5d
5e
5f
5-Br
6-Cl
6-OMe
6-Cl
6-Me
5-Cl
6-Cl
6-Me
5-Cl
5-Me
5-Me
6-Br
6-Br
H
4-F
4-OMe
4-OMe
4-F
H
5g
5h
5i
H
H
4-F
4-F
H
5j
5k
5l
H
5m
5n
H
Scheme 2. Synthesis and chemical structures of oxime derivatives 5a-5n.
5

Compound.
R
Ph
7a
7b
7c
7d
7e
7f
toluene
4-Br-Ph
2-Br-Ph
2-Me-Ph
4-OMe-Ph
4-NO2-Ph
ethylbenzene
7g
7h
Scheme 3. Synthesis and chemical structures of acyloxylamines 7a-7h.
3. Results and Discussion
3.1 In vitro XOD inhibitory activities screen and the Binding Modes
Since the xanthine oxidase (XOD) was the rate-limiting enzyme involved in uric
acid production, we examined XOD inhibitory activities of the synthesized
compounds. The results were summarized in Table 3. Preliminary SAR (Structure
Activity Relationship) studies were performed to deduce how the structure variation
and modification could affect the XOD inhibitory activities. As shown in Table 1,
several benzoxazole deoxybenzoin oxime derivatives (e.g., 5d, 5e, 5h, 5i, 5k and 5l)
showed good to excellent XOD inhibitory activity. As to compounds 5a-5n, the
electron-donating substituents on the phenyl ring at R1 and R2 position input
substantial effects on the XOD inhibitory capability of the compounds. 5a with no
substituent for R1 and R2 exhibited IC50 value of 39.1 μM, and 5d with methoxy
6

group for R1 and R2 showed much increased inhibitory activity (IC50 = 3.7 μM),
comparable to the positive control allopurinol (IC50 = 2.9 μM). Introduction of a
chloro group on the R1 led to the loss of XOD inhibitory activity. Compound 5e (R1 =
6-Cl) showed lower activity (IC50 = 17.8 μM) than 5d (R1 = 6-OMe), so the methoxy
group is beneficial for increasing XOD inhibitory activity.
Secondly, benzoxazole acyloxylamines 7a-7h showed moderate XOD inhibitory
activity (IC50 range: 15.2 μM-64.3 μM). Compounds with R2 substitution at the para
position (7c, 7f and 7g) showed better activities than others. Especially, compound 7f
with para-methoxy group on phenyl ring showed potent inhibitory activity and
similar to the tendency for benzoxazole deoxybenzoin oxime derivatives.
Enzyme kinetics studies were performed for the representative compound 5d. The
Lineweaver-Burk plot (Figure 1) revealed that compound 5d acted as a
competitive-type inhibitor of XOD with a Ki value of 2.89 μM. To investigate the
binding modes between benzoxazole deoxybenzoin oxime derivatives and XOD,
docking of the most potent compound 5d was performed using GLIDE
(2015, Schrodinger suite),
and
the
binding
model
was
illustrated
by Discovery Studio 3.5 client. Figure 2 shows the docking conformations of the hit
compound at the binding site of XOD. In the binding model, compound 5d is nicely
bound to the region of XOD, docking score (-6.62 kcal/mol) is less than score of
allopurinol (-7.24 kcal/mol) and the benzoxazole ring is inserted well inside the
pocket. Compound 5d shows important interactions between benzoxazole ring and the
residues known to be important for binding of endogeneous substrate of XOD, play an
important role in stabilizing the three-dimensional structure of a protein. The methoxy
group on benzoxazole ring interacts with Thr1010 residue via hydrogen bond
interaction, which is consistent with the fact that the methoxy group is beneficial for
increasing XOD inhibitory activity.
Table 1. In vitro inhibitory effects of the selected compounds on XOD activity.
Compound.
In Vitro IC50
Compound.
In Vitro IC50
(μM)
(μM)
7

39.1±4.2
> 100
44.3±8.4
3.7±0.5
11.6±1.1
26.9±3.7
> 100
5a
5b
5d
5f
5c
17.8±2.3
58.1±7.8
17.5±1.9
9.8±1.2
> 100
5e
5g
5h
5j
5i
15.2±2.1
> 100
5k
5l
5m
5n
7b
7d
7f
50.5±5.7
25.6±3.8
38.8±6.3
20.6±3.4
2.9±0.4
48.9±7.9
64.3±7.4
15.2±2.2
52.5±6.3
7a
7c
7e
7g
7h
Allopurinol
100
80
5d 0 M
5d 0.8 M
5d 4 M
5d 20 M
60
40
20
-0.01
0.01
0.02
1/s
0.03
0.04
0.05
-20
Figure 1. Lineweaver-Burk plot analysis of xanthine oxidase by compound 5d.
Figure 2. The 3D mode structure of compound 5d binding model with XOD complex structure
(1N5X.pdb), and ligand interaction diagram of compound 5d with XOD complex structure.
8

3.2 NLRP3/TLR4 inhibitory activity and Binding Modes
NLRP3 inflammasome and TLR4 signaling pathway activation play a causal role
in the course of acute gouty arthritis, inhibition of which could ameliorate
inflammatory reactions. Thus, we examined NLRP3 and TLR4 inhibitory activities of
benzoxazole derivatives 5d-5g, 5i, 5k and 7f, which exhibited their potent capacities
on XOD inhibitory activity. As shown in Table 2, compounds 5d displayed the most
potent NLRP3 inhibitory activity, comparable to the positive control Isoliquiritigenin.
This biological assay indicated that benzoxazole deoxybenzoin oxime derivative 5d is
potential small-molecule NLRP3 inhibitor as anti-hyperuricemia agent. In Table 3 is
show that compound 5d suppressed TLR4 signaling pathway characterized by
decreased secreted embryonic alkaline phosphatase (SEAP) concentrations with IC50
value of 0.54 μM), which seemed to be more effective than positive control TAK-242.
To investigate the binding mode between benzoxazole deoxybenzoin oxime
derivative 5d and NLRP3, this compound was subjected to molecular docking. As
depicted in Figure 3, 5d was observed to be well-fitted into the LRR domain of
NLRP3. The methoxy group on benzoxazole ring forms a hydrogen bond with
LYS540, the oxime hydroxyl group also forms a hydrogen bond with GLU541
residue of the LRR domain of NLRP3, which was consistent with the fact that the
benzoxazole deoxybenzoin oxime derivatives showed better activity than benzoxazole
acyloxylamine 7f.
Figure 3. The 3D mode structure of compound 5d binding model with LRR domain of NLRP3
and ligand interaction diagram of compound 5d with NLRP3 complex structure.
9

Table 2. Inhibitory effects of the selected compounds on IL-1β production induced by MSU in
THP-1 cells.
Compound.
In Vitro IC50
(μM)
Compd.
In Vitro IC50
(μM)
1.22
5.72
5d
5e
5i
7f
21.95
-
15.88
39.47
5h
5k
Isoliquiritigenin
1.54
Table 3. In vitro inhibitory effects of the selected compounds on SEAP production induced by
lipopolysaccharide (LPS) in HEK293-derived cells.
Compound
In Vitro IC50
Compound
In Vitro IC50
(μM)
(μM)
0.54
-
5.42
19.25
25.90
5d
5h
5e
5i
7f
45.65
0.68
5k
TAK242
3.3 In vivo anti-hyperuricemia effect of 5d through inhibition of XOD activity
As shown in Figure 4, potassium oxonate administration caused remarkable
elevation of serum uric acid levels (P < 0.001), which could be attenuated by
treatment with 5d at three tested concentrations in a dose-dependent manner, as well
as allopurinol at 10 mg/kg. Consistently with in vitro study,
compound 5d at concentrations of 2.5, 5, 10 mg/kg
dose-dependently
inhibited
hepatic XOD activity by 19.2, 35.8 and 41.9%. Allopurinol at 10 mg/kg also
significantly suppressed hepatic XOD activity by 47.1% in hyperuricemic mice.
10

In normal mice, allopurinol treatment resulted in hypouricemia (P < 0.05),
exhibiting potential side effects, while 5d had no effects on serum uric acid levels.
Moreover, compared with normal + vehicle group, 5d at 2.5, 5, 10 mg/kg reduced
hepatic XOD activity by 3.7%, 6.8% and 10.2% with no significance while
allopurinol remarkably decreased liver XOD activity by 27.9 % in normal mice.
Despite the less potent inhibitory effect on XOD activity, 5d exhibited higher safety
than allopurinol.
A
8
Normal
###
Oxonate-treated
*
**
***
***
4
0
#
Vehicle
2.5
5
10
10
Allopurinol
(mg/kg)
5d
B
20
15
10
5
47.1%
41.9%
35.8%
19.2%
Vehicle
2.5 mg/kg 5d
5 mg/kg 5d
27.9%
10.2%
6.8%
3.7%
###
10 mg/kg 5d
10 mg/kg Allopurinol
*
***
***
***
#
0
Normal
Oxonate-treated
Data were expressed as mean ± S.E.M (n = 8). #P<0.05, ###P<0.001, compared with normal +
vehicle group; *P<0.05, **P<0.01, ***P<0.001, compared with oxonate-treated + vehicle group.
Figure 4. Effects of 5d and allopurinol on serum uric acid levels (A) and hepatic XOD activity (B)
in normal and oxonate-treated mice.
11

3.4 In vivo anti-acute gouty arthritis effect of 5d through regulation of NLRP3
inflamasome and TLR4 signaling pathway
As shown in Table 4, MSU injection led to a significant increase in the ankle
diameter of the rats for 4, 8, 12, 24 h, compared with the normal rats. Of note,
treatment with 5d exhibited remarkable effects on MSU-induced ankle swelling in a
dose-dependent manner, which was comparable with that of the positive control drug,
colchicine. Moreover, MSU injection caused elevated IL-1β and TNF-α
concentrations in serum and synovial homogenates, which was attenuated by
treatment with 5d, as well as colchicine (Figure 5).
Western blot analysis revealed that protein expression of NLRP3, ASC and
caspase-1 (p20) were significantly up-regulated in synovium of MSU-induced rats.
Treatment with compound 5d could effectively down-regulated these protein
expressions, blocking synovial NLRP3 inflammasome activation. In addition,
colchicine at 10 mg/kg also inhibited synovial NLRP3 inflammasome activation less
potent than 5d (Figure 6). On the other hand, TLR4 signaling pathway was also
induced by MSU injection characterized by over-expressions of TLR4, MyD88, p-IκB
and nuclear p65 NF-κB in synovium of rats, which could be recovered by treatment
with 5d at various concentrations. Notably, colchicine at 10 mg/kg significantly
suppressed TLR4 signaling pathway, but failed to achieve the similar effect as 5d at
10 mg/kg (Figure 7).
Table 4. Effects of 5d and colchicine on the perimeter of the ankle (cm) at different intervals for
24 h in MSU-treated rats.
Group
Initial
2h
4h
8h
12h
24h
Normal + vehicle
MSU + vehicle
2.45±0.52
2.64±0.81
2.65±0.77
2.70±0.71
2.56±0.62
2.65±0.64
2.82±0.75
2.78±0.55
2.56±0.60
2.69±0.58
2.71±0.51
3.18±0.64##
2.95±0.45*
2.84±0.65*
2.79±0.35**
2.58±0.43
3.27±0.56###
2.96±0.44*
2.88±0.71*
2.72±0.42***
2.55±0.68
2.51±0.30
3.51±0.52###
3.14±0.48**
2.99±0.55***
2.70±0.38***
3.35±0.50###
2.97±0.73**
2.80±0.50***
2.73±0.46***
MSU + 5d 2.5 mg/kg
MSU + 5d 5 mg/kg
MSU + 5d 10 mg/kg
12

MSU + Colchicine 10 mg/kg
2.49±0.47
2.73±0.75
2.81±0.39**
2.77±0.59***
2.80±0.45***
2.79±0.57***
Data were expressed as mean ± S.E.M. (n = 8). ##P < 0.01, ###P < 0.001, compared with normal +
vehicle group; *P < 0.05, **P < 0.01, ***P < 0.001, compared with MSU + vehicle group.
MSU
25
IL-1
###
TNF-
20
**
**
***
15
10
###
5
**
***
***
0
Normal Vehicle 2.5
5
10
10 (mg/kg)
5d
Colchicine
MSU
800
IL-1
TNF-
###
*
*
***
***
###
400
**
**
***
0
Normal Vehicle 2.5
5
10
10 (mg/kg)
Colchicine
5d
Data were expressed as mean ± S.E.M. (n = 8). ###P < 0.001, compared with normal + Vehicle
group; *P < 0.05, **P < 0.01, ***P < 0.001, compared with MSU + vehicle group.
Figure 5. Effects of 5d and colchicine on cytokine levels in serum (A) and synovial homogenates
(B) in normal and MSU-treated rats.
MSU
5d
Colchicine
10 10 (mg/kg)
Normal Vehicle 2.5
5
NLRP3
ASC
120 kDa
22 kDa
20 kDa
55 kDa
Caspase1 (p20)
α-tubulin
13

A
B
MSU
MSU
3.0
3.0
###
##
*
**
**
1.5
0.0
**
1.5
0.0
**
**
(mg/kg)
Normal Vehicle
2.5
5
10
10
(mg/kg)
Normal Vehicle
2.5
5
10
10
5d
5d
Colchicine
Colchicine
C
MSU
3.0
###
*
**
***
***
1.5
0.0
(mg/kg)
Normal Vehicle
2.5
5
10
10
5d
Colchicine
Data were expressed as mean ± S.E.M. (n = 4). ##P < 0.05, ###P < 0.001, compared with normal
group; *P < 0.05, **P < 0.01, ***P < 0.001, compared with MSU + vehicle group.
Figure 6. Effects of 5d and colchicine on synovial protein expressions of NLRP3 (A), ASC (B)
and caspase-1 (p20) (C) in MSU-treated rats.
MSU
5d
5
Colchicine
10 10 (mg/kg)
Normal Vehicle 2.5
TLR4
90 kDa
34 kDa
55 kDa
40 kDa
39 kDa
65 kDa
MyD88
α-tubulin
p-IκBα
IκBα
\
p65 NF-κB
14

TBP
38 kDa
A
B
MSU
MSU
3.0
1.5
0.0
3.0
###
##
Normal Vehicle
###
*
*
*
**
1.5
0.0
**
**
***
(mg/kg)
(mg/kg)
2.5
5
10
10
Normal Vehicle
2.5
5
10
10
5d
5d
Colchicine
Colchicine
C
D
MSU
MSU
3.0
1.5
0.0
3.0
###
*
**
**
***
*
**
1.5
0.0
***
(mg/kg)
(mg/kg)
Normal Vehicle
2.5
5
10
10
Normal Vehicle
2.5
5
10
10
5d
5d
Colchicine
Colchicine
Data were expressed as mean ± S.E.M. (n = 4). ##P < 0.05, ###P < 0.001, compared with normal
group; *P < 0.05, **P < 0.01, ***P < 0.001, compared with MSU + vehicle group.
Figure 7. Effects of 5d and colchicine on synovial protein expression of TLR4 (A), MyD88 (B),
p-IκBα (C) and nuclear p65 NF-κB (D) in MSU-treated rats.
4. Conclusion
The current study prepared two series of benzoxazole deoxybenzoin oxime
derivatives as innate immune sensors and XOD inhibitors. The significance of this
work is firstly providing dual inhibitors of innate immune sensors and XOD for the
treatment of acute gouty arthritis. Notably, compound 5d is a multi-targeting
anti-hyperuricemic and anti-acute gouty arthritis candidate with excellent in vivo
15

potency. Further mechanism of anti-hyperuricemic activity and structural
optimization are in progress.
5. Experimental section
5.1. General chemistry
All chemicals (reagent grade) used were purchased from Sigma–Aldrich (USA) and
1
Sinopharm Chemical Reagent Co. Ltd. (China). H NMR spectra were measured on
Varian Unity Inova 300/400 MHz NMR Spectrometer at 25°C and referenced to TMS.
Chemical shifts are reported in ppm (δ) using the residual solvent line as internal
standard. Splitting patterns are designed as s, singlet; d, doublet; t, triplet; m, multiplet.
HRMS spectra were acquired on Bruker Esquire Liquid Chromatography-Ion Trap
Mass Spectrometer. Analytical thin-layer chromatography (TLC) was performed on
the glass-backed silica gel sheets (silica gel 60 Å GF254). All compounds were
detected using UV light (254 or 365 nm). All solvents and reagents were analytically
pure, and no further purification was needed. All starting materials were
commercially available.
5.2. General procedure for the preparation of compounds 5a-5n
Substituted 2-aminophenols 1a-1f (0.1 mol) and lactic acid (7.5 mL, 0.1mol)
were dissolved in 30 mL of HCl (4 M). The mixture was reflux for 0.5 h. After
cooling to room temperature, the reaction mixture was poured into water. Next, the
pH was adjusted to 10-11 with ammonia. The mixture was filtered, and the residue
was recrystallized from water to give 2a-2f. Next, compounds 2a-2f (0.04 mol) were
o
each dissolved in 20 mL of acetic acid. When the mixture was heated to 90 C,
o
chromium trioxide solution was added. The reaction mixture was stirred at 95 C for
3h. After cooling to room temperature, the reaction mixture was poured into water
and then extracted by ethyl acetate (EtOAc). The organic layers were combined, dried
with MgSO4, filtered and then concentrated under reduced pressure. The residue was
recrystallized from toluene to give the intermediates 3a-3f.
Substituted 2-bromotoluene (0.2 mmol) was dissolved in THF solvent (5 mL) in a
16

round bottom flask fitted with a condensor and an N2 inlet. KHMDS (0.080 g, 0.4
mmol), Pd(dba)2 (0.009 g, 0.013 mmol), and DTPF (0.012 g, 0.02 mmol) were added
to the reaction under an N2 atmosphere. Acetophenone 3a-3f (0.2 mmol) was added to
the orange homogenous reaction. The reaction was heated to reflux for 45 min. After
cooling the reaction to room temperature and diluting with Et2O (20 mL), it was
washed with 0.5 M citric acid (15 mL), H2O (15 mL), and brine (15 mL). The ether
solution wash dried over Na2SO4, filtered, and concentrated in vacuo. The crude
product was purified by flash chromatography using a gradient of 100% hexanes to 5%
Et2O in hexanes as the eluent to provided 4a-4n. A solution of 4a-4n (0.90 mmol) and
hydroxylamine hydrochloride (5.4 mmol) in ethanol (18 mL) was refluxed for 4-4.5 h
at 60oC, and sodium acetate (0.90 mol) was added. The reaction mixture was allowed
to cool to room temperature and evaporated to dryness in vacuo, and the residue was
partitioned between water (10 mL) and CH2Cl2 (10 mL). The organic extract was
washed with water (2-10 mL), and the aqueous layer was extracted with CH2Cl2 (2-10
mL). The combined organic extracts dried over anhydrous sodium sulfate and
evaporated in vacuo gave the corresponding oximes 5a-5n. All new oximes (Table 1)
were fully characterized by spectroscopic methods and the elemental analysis.
(E)-1-(benzo[d]oxazol-2-yl)-2-phenylethanone oxime (5a) Yield: 74%, Mp:
157-158 °C. 1H-NMR (400MHz, DMSO), δ (ppm): 9.31 (s, 1H), 7.71 (d, 1H, J =
8.0Hz), 7.22-7.34 (m, 4H), 6.79-6.91 (m, 4H), 3.71 (s, 2H). HR-MS: Calcd. For
C15H12N2O2[M+H]+: 253.2791, Found: 252.2778.
(E)-1-(5-bromobenzo[d]oxazol-2-yl)-2-phenylethanone oxime (5b) Yield: 82%,
1
Mp: 198-199 °C. H-NMR(400MHz, DMSO), δ ppm): 9.34 (s, 1H), 8.06 (s, 1H),
7.23-7.32 (m, 4H), 7.03-7.06 (m, 1H), 6.78 (d, J = 8.0Hz, 2H) 3.73 (s, 2H). HR-MS:
Calcd. For C15H11BrN2O2[M+H]+: 332.1632, Found: 332.1645.
(E)-1-(6-chlorobenzo[d]oxazol-2-yl)-2-(4-fluorophenyl)ethanone oxime (5c)
1
Yield: 81%, Mp: 138~140 °C. H-NMR (400MHz, DMSO), δ (ppm): 9.36 (s, 1H),
7.80 (d, J = 8.0Hz, 1H), 7.12-7.37 (m, 4H), 6.78 (d, J = 8.0Hz, 2H), 3.56 (s, 2H).
HR-MS: Calcd. For C15H10ClFN2O2[M+H]+: 305.7058, Found: 305.7074.
(E)-1-(6-methoxybenzo[d]oxazol-2-yl)-2-(4-methoxyphenyl)ethanone oxime (5d)
17

Yield: 86%, Mp: 151-152 °C. 1H-NMR (400MHz, DMSO), δ (ppm): 9.31(s, 1H), 7.80
(d, J = 8.0Hz, 1H), 7.36-7.39 (m, 2H), 7.12-7.16 (m, 2H), 6.66 (s, 1H), 6.55 (d, J =
4.0Hz, 1H), 3.72 (s, 2H), 3.53 (s, 6H),. HR-MS: Calcd. For C17H16N2O4[M+H]+:
313.1142, Found: 313.1154.
(E)-1-(6-chlorobenzo[d]oxazol-2-yl)-2-(4-methoxyphenyl)ethanone oxime (5e)
1
Yield: 91%, Mp: 182-183 °C. H-NMR (400MHz, DMSO), δ (ppm): 9.29 (s, 1H),
7.84 (d, J = 8.0Hz, 1H), 7.16-7.28 (m, 4H), 6.78-6.86 (m, 2H), 3.67 (s, 2H), 3.49 (s,
3H). HR-MS: Calcd. For C16H13ClN2O3[M+H]+: 317.0665, Found: 317.0694.
(E)-2-(4-fluorophenyl)-1-(6-methylbenzo[d]oxazol-2-yl)ethanone oxime (5f)
Yield: 77%, Mp: 174-175 °C. 1H-NMR (400MHz, DMSO), δ (ppm): 9.30 (s, 1H),
7.58 (d, J = 8.0Hz, 1H), 7.36-7.39 (m, 2H), 7.13-7.16 (m, 2H), 6.66 (s, 1H), 6.55 (d, J
= 4.0Hz, 1H), 3.73 (s, 2H), 2.21 (s, 3H). HR-MS: Calcd. For C16H13FN2O2[M+H]+:
285.1018, Found: 285.1065.
(E)-1-(5-chlorobenzo[d]oxazol-2-yl)-2-phenylethanone oxime (5g) Yield: 86%,
1
Mp: 177-178 °C. H-NMR (400MHz, DMSO), δ (ppm): 9.34 (s, 1H), 7.83 (d, J =
4.0Hz, 1H), 7.24-7.35 (m, 5H), 6.79-6.87 (m, 2H), 3.74 (s, 2H). HR-MS: Calcd. For
C15H11ClN2O2[M+H]+: 287.7123, Found: 287.7188.
(E)-1-(6-chlorobenzo[d]oxazol-2-yl)-2-phenylethanone oxime (5h) Yield: 87%,
1
Mp: 152-153 °C. H-NMR (400MHz, DMSO), δ (ppm): 9.35 (s, 1H), 7.99 (s, 1H),
7.23-7.35 (m, 5H), 6.93-6.95 (m, 1H), 6.84 (d, J = 4.0Hz, 1H), 3.76 (s, 2H). HR-MS:
Calcd. For C15H11ClN2O2[M+H]+: 287.0554, Found: 287.0527.
(E)-1-(6-methylbenzo[d]oxazol-2-yl)-2-phenylethanone oxime (5i) Yield: 75%,
1
Mp: 158-160 °C. H-NMR (400MHz, DMSO), δ (ppm): 9.37 (s, 1H), 7.60 (d, J =
4.0Hz, 1H), 7.28-7.39 (m, 5H), 6.71 (s, 1H), 6.60 (d, J = 8.0Hz, 1H), 3.75 (s, 2H),
2.13 (s, 3H). HR-MS: Calcd. For C16H14N2O2[M+H]+: 267.1154, Found: 267.1136.
(E)-1-(5-chlorobenzo[d]oxazol-2-yl)-2-(4-fluorophenyl)ethanone oxime (5j)
Yield: 72%, Mp: 153-154 °C. 1H-NMR (400MHz, DMSO), δ (ppm): 9.43 (s, 1H),
8.01 (s, 1H), 7.42-7.44 (m, 2H), 7.20 (d, J = 8.0Hz, 1H), 6.91-7.02 (m, 2H), 6.65 (d, J
= 8.0Hz, 1H), 3.81 (s, 2H). HR-MS: Calcd. For C16H14N2O2[M+H]+: 305.0462,
Found: 305.0438.
18

(E)-2-(4-fluorophenyl)-1-(5-methylbenzo[d]oxazol-2-yl)ethanone oxime (5k)
Yield: 82%, Mp: 155-156 °C. 1H-NMR (400MHz, DMSO), δ (ppm): 9.35 (s, 1H),
7.58 (s, 1H), 7.41 (t, J = 6.0Hz, 2H), 7.17-7.20 (m, 2H), 6.78 (s, 2H), 3.75 (s, 2H),
2.12 (s, 3H). HR-MS: Calcd. For C16H13FN2O2[M+H]+: 285.1016, Found: 285.1042.
(E)-1-(5-methylbenzo[d]oxazol-2-yl)-2-phenylethanone oxime (5l) Yield: 77%,
Mp: 149-150 °C. 1H-NMR (400MHz, DMSO), δ (ppm): 9.29 (s, 1H), 7.57 (s, 1H),
7.24-7.36 (m, 5H), 6.73 (s, 2H), 3.7 3(s, 2H), 2.16 (s, 3H). HR-MS: Calcd. For
C16H14N2O2[M+H]+: 267.1174, Found: 267.1152.
(E)-1-(6-bromobenzo[d]oxazol-2-yl)-2-(4-fluorophenyl)ethanone oxime (5m)
Yield: 82%, Mp: 159-161 °C. 1H-NMR (400MHz, DMSO), δ (ppm): 9.36 (s, 1H),
7.79 (d, J=8.0Hz, 1H), 7.36-7.39 (m, 2H), 7.14-7.17 (m, 2H), 7.02 (s, 1H), 6.92 (d, J =
8.0Hz, 1H), 3.75 (s, 2H). HR-MS: Calcd. For C15H10BrFN2O2[M+H]+: 349.0215,
Found: 349.0228.
(E)-1-(6-bromobenzo[d]oxazol-2-yl)-2-phenylethanone oxime (5n) Yield: 76%,
Mp: 162-163 °C. 1H-NMR (400MHz, DMSO), δ (ppm): 9.34 (s, 1H), 7.80 (d, J =
4.0Hz, 1H), 7.24-7.35 (m, 5H), 7.01 (s, 1H), 6.93 (d, J = 4.0Hz, 1H), 3.75 (s, 2H).
HR-MS: Calcd. For C15H10BrFN2O2[M+H]+: 331.1643, Found: 331.1664.
(E)-1-(benzo[d]oxazol-2-yl)ethanone O-benzoyl oxime (7a) Yield: 82%, Mp:
1
180-182 °C. H NMR (400 MHz, Chloroform-d) δ 7.85 – 7.78 (m, 1H), 7.63 (d, J =
8.0 Hz, 1H), 7.44 (dtd, J = 23.7, 7.5, 1.2 Hz, 2H), 4.36 (s, 2H), 2.62 (s, 3H). 13C NMR
δ (ppm) 164.32, 157.46, 154.74, 150.89, 140.87, 127.52, 125.24, 120.97, 111.55,
39.79, 13.65.
(E)-1-(benzo[d]oxazol-2-yl)ethanone O-(2-phenylacetyl) oxime (7b) Yield: 85%,
Mp: 112-114 °C. 1H NMR (600 MHz, Chloroform-d) δ 7.80 (dt, J = 7.9, 1.0 Hz, 1H),
7.62 (dt, J = 8.3, 0.9 Hz, 1H), 7.45 (ddd, J = 8.4, 7.4, 1.3 Hz, 1H), 7.41 – 7.34 (m, 5H),
13
7.31 (ddt, J = 8.5, 5.4, 2.7 Hz, 1H), 3.90 (s, 2H), 2.51 (s, 3H). C NMR δ (ppm)
167.70, 157.98, 153.67, 150.88, 140.93, 132.75, 129.29, 128.74, 127.47, 127.28,
125.09, 120.84, 111.56, 40.02, 13.48.
(E)-1-(benzo[d]oxazol-2-yl)ethanone O-(4-bromobenzoyl) oxime (7c) Yield:
1
78%, Mp: 205-207 °C. H NMR (400 MHz, Chloroform-d) δ 8.05 – 7.98 (m, 2H),
19

7.83 (ddd, J = 7.8, 1.4, 0.7 Hz, 1H), 7.71 – 7.62 (m, 3H), 7.44 (dtd, J = 23.2, 7.5, 1.3
13
Hz, 2H), 2.73 (s, 3H). C NMR δ(ppm) 162.13, 157.94, 154.28, 150.96, 140.97,
132.14, 131.27, 129.16, 127.40, 127.04, 125.16, 120.89, 111.64, 13.78.
(E)-1-(benzo[d]oxazol-2-yl)ethanone O-(2-bromobenzoyl) oxime (7d) Yield:
1
74%, Mp: 174-178 °C. H NMR (400 MHz, Chloroform-d) δ 7.89 – 7.81 (m, 2H),
7.73 (dd, J = 7.6, 1.6 Hz, 1H), 7.66 (dd, J = 7.9, 1.1 Hz, 1H), 7.50 – 7.39 (m, 4H),
2.70 (s, 3H). 13C NMR δ(ppm) 162.82, 157.93, 154.58, 150.96, 140.98, 134.41,
133.23, 131.61, 130.84, 127.39, 127.37, 125.15, 121.67, 120.90, 111.63, 14.18.
(E)-1-(benzo[d]oxazol-2-yl)ethanone O-(2-methylbenzoyl) oxime (7e) Yield:
88%, Mp: 140-142 °C. 1H NMR (600 MHz, Chloroform-d) δ 7.99 (dd, J = 7.8, 1.4 Hz,
1H), 7.83 (dd, J = 7.9, 1.1 Hz, 1H), 7.65 (d, J = 8.1 Hz, 1H), 7.48 (dtd, J = 15.4, 7.8,
1.4 Hz, 2H), 7.41 (td, J = 7.7, 1.1 Hz, 1H), 7.36 – 7.30 (m, 2H), 2.69 (s, 6H). 13C
NMR δ (ppm) 163.47, 158.22, 153.65, 150.96, 141.11, 141.00, 132.79, 131.97, 130.3 ,
127.46 , 127.27, 125.88, 125.09, 120.83, 111.60, 21.60, 13.88.
(E)-1-(benzo[d]oxazol-2-yl)ethanone O-(2-methoxybenzoyl) oxime (7f) Yield:
88%, Mp: 178-179 °C. 1H NMR (600 MHz, Chloroform-d) δ 7.99 (dd, J = 7.8, 1.4 Hz,
1H), 7.83 (dd, J = 7.9, 1.1 Hz, 1H), 7.65 (d, J = 8.1 Hz, 1H), 7.48 (dtd, J = 15.4, 7.8,
1.4 Hz, 2H), 7.41 (td, J = 7.7, 1.1 Hz, 1H), 7.36 – 7.30 (m, 2H), 361 (s, 3H), 2.71 (s,
3H). 13C NMR δ(ppm) 163.47, 158.22, 153.65, 150.96, 141.11, 141.00, 132.79,
131.97, 130.31, 127.46, 127.27, 125.88, 125.09, 120.83, 111.60, 21.60, 13.88.
(E)-1-(benzo[d]oxazol-2-yl)ethanone O-(4-nitrobenzoyl) oxime (7g) Yield: 79%,
1
Mp: 199-200 °C. H NMR (600 MHz, Chloroform-d) δ 8.42 – 8.32 (m, 4H), 7.87 –
7.82 (m, 1H), 7.71 – 7.63 (m, 1H), 7.49 (td, J = 8.3, 7.8, 1.3 Hz, 1H), 7.45 – 7.42 (m,
13
1H), 2.77 (s, 3H). C NMR δ(ppm) 161.67, 151.50, 151.08, 150.11, 140.37, 133.77,
131.5 , 128.59, 126.29, 124.72, 121.88, 112.25, 40.34, 19.31.
(E)-1-(benzo[d]oxazol-2-yl)ethanone O-(3-phenylpropanoyl) oxime (7h) Yield:
1
92%, Mp: 115-116 °C. H NMR (600 MHz, Chloroform-d) δ 7.83 – 7.78 (m, 1H),
7.62 (dt, J = 8.2, 0.9 Hz, 1H), 7.45 (ddd, J = 8.3, 7.4, 1.3 Hz, 1H), 7.39 (td, J = 7.7,
1.1 Hz, 1H), 7.31 (t, J = 7.6 Hz, 2H), 7.27 (d, J = 8.0 Hz, 2H), 7.24 – 7.21 (m, 1H),
13
3.10 (t, J = 7.7 Hz, 2H), 2.90 (dd, J = 8.3, 7.2 Hz, 2H), 2.50 (s, 3H). C NMR δ (ppm)
20

169.40, 158.07 , 153.35, 150.88, 140.93, 139.88, 128.61, 128.35, 127.25, 126.50,
125.09, 120.82, 111.55, 34.50, 30.79, 13.45.
5.3. Biological assay
Evaluation of XOD inhibitory effects of benzoxazole deoxybenzoin oxime derivatives
in vitro
Assay of XOD inhibitory activity was performed as our previous studies [19, 20].
The reaction mixture contained uric acid in 67 mM phosphate buffer (pH 7.4) and 20
nM XOD with an activity of 5 mU/mL, with or without test compounds at different
concentrations (0.16, 0.8, 4, 20, 100 μM). After pre-incubating the mixture for 1-5
min at 25 oC, 50 mM xanthine was added to the mixture, the absorption increments at
295 nm indicating the formation of uric acid were monitored. Three replicates were
made for each test sample. The percent inhibition ratio (%) was calculated according
to the following equation, thereby the agent concentration needed for 50% inhibition
was calculated as IC50 values. Test compounds and allopurinol were dissolved in
DMSO.
% inhibition = [(rate of control reaction – rate of sample reaction)/rate of control
reaction] × 100
Determination of NLRP3 inflammasome inhibitory effects of compounds in THP-1
cells
THP-1 cells obtained from American Type Culture Collection (Manassas, VA,
USA) were cultured in RPMI1640 medium supplemented with 10% fetal calf serum
o
and penicillin-streptomycin, which were set to temperatures of 37 C and
supplemented with 5% CO2. THP-1 cells were seeded at 5×105/mL in 96 well plates
and treated for 24 h with 100 ng/mL PMA before stimulation. After PMA treatment,
the medium was removed and replaced with serum free medium containing test
compounds and isoliquiritigenin at different concentrations (0.16, 0.8, 4, 20, 100 μM)
for 1 h before applying MSU (500 ug/mL). The supernatants were collected after
stimulation with MSU for 6 h in order to detect IL-1β concentration. The percent
21

inhibition ratio (%) was calculated according to the following equation, as well as
IC50 values.
% inhibition = [(IL-1β concentration in presence of MSU without test compounds
treatment – IL-1β concentration in presence of MSU with test compounds treatment)/
(IL-1β concentration in presence of MSU without test compounds treatment – IL-1β
concentration in absence of MSU without test compounds treatment] × 100
Determination of TLR4 signaling inhibitory effects of compounds in HEK293-derived
cells
HEK-BlueTM-hTLR4 obtained by co-transfection of the human TLR4, MD-2 and
CD14 co-receptor genes were purchased from Invivogen (CA, USA), activation of
which could induce the production of SEAP. The cells were cultured in DMEM
medium supplemented with 10% fetal calf serum and penicillin-streptomycin, as well
as 4.5 g/L glucose, 2-4 mM L-glutamine and 100 μg/mL Normocin™, which were set
o
to temperatures of 37 C and supplemented with 5% CO2. The cells were
pre-incubated with test compounds and TAK-242 for 2 h at various concentrations
(0.16, 0.8, 4, 20, 100 μM) before stimulated with LPS for 20 h. The next day, the
supernatants were sampled and analyzed colorimetrically for the presence of SEAP.
The IC50 values of these test compounds were calculated based on their inhibition of
SEAP expression using Secreted Alkaline Phosphatase Reporter Gene Assay Kit
(Luminescence) (Cayman Chemical, MA, USA). The percent inhibition ratio (%) was
calculated according to the following equation.
% inhibition = [(absorbance in presence of LPS without test compounds treatment
–absorbance concentration in presence of LPS with test compounds treatment)/
(absorbance in presence of LPS without test compounds treatment – absorbance in
absence of LPS without test compounds treatment] × 100
Animal models of hyperuricemia and acute gouty arthritis
Male Kun-Ming strain of mice (18-22 g) and Sprague-Dawley rats (180-200 g)
were purchased from the Central Institute for Experimental Animals of Zhejiang and
22

were allowed one week to adapt to the laboratory environment before used for
experiments. All animal studies were performed in accordance with the Animal Ethics
Committee of Nanjing Medical University.
Hyperuricemia was developed by uricase inhibitor potassium oxonate, as
described previously [19]. Mice were randomly divided into 10 groups: normal +
vehicle, oxonate + vehicle, oxonate + 5d (2.5 mg/kg, 5 mg/kg, 10 mg/kg), oxonate +
10 mg/kg allopurinol, normal + 5d (2.5 mg/kg, 5 mg/kg, 10 mg/kg), normal + 10
mg/kg allopurinol. Oxoanate, 5d and allopurinol at various concentrations were
dissolved or suspended in 0.5% CMC-Na, and the dosages of all the agents were
selected based on our preliminary experiments. Standard diets, but not water, were
withdrawn from the animals 1 h prior to the administration. Briefly, mice were
administered in a volume of 15 mL/kg by intraperitoneally injection with oxonate
(250 mg/kg) or 0.5% CMC-Na (vehicle) 1 h before oral administration of 5d or
allopurinol. Whole blood samples were collected 1 h after 5d or allopurinol
administration and centrifuged to obtain serum for uric acid assays. Liver tissues of
mice were rapidly separated on ice plate for assays of XOD activities.
MSU crystals (1.0 mg suspended in 50 μl sterile saline) were injected in the left
ankle joint of rats to establish acute gouty arthritis animal models. Rats were
randomly divided into 10 groups: normal + vehicle, MSU + vehicle, MSU + 5d (2.5
mg/kg, 5 mg/kg, 10 mg/kg), oxonate + 10 mg/kg colchicine, normal + 5d (2.5 mg/kg,
5 mg/kg, 10 mg/kg), normal + 10 mg/kg colchicine. 5d and colchicine at various
concentrations were dissolved or suspended in 0.5% CMC-Na, which were orally
gavaged once daily for 5 days. On the 5th day, the MSU crystal was injected 1 h later
after 5d or colchicine treatment. The perimeter of the joint at different intervals was
measured for 2 day at 2, 4, 8, 12, and 24 h. At the end of the experimental period,
blood from each rat was collected for serum separation. Synovial tissues were
separated on ice plate for next experiments.
XOD activity assay
For XOD activity assay, enzyme extraction of liver tissues has been performed as
23

described in our previous study [19]. XOD activity in extraction of mice livers was
determined by the XOD activity detection kit. The experimental process was
performed according to manufacturer’s instructions.
Uric acid assay
Phosphotungstic acid method was adopted to estimate serum uric acid
concentrations [21].
Il-1β and TNF-α concentrations assay
IL-1β concentration in culture supernatant and synovium tissues, as well as TNF-α
level in synovium tissues were measured by ELISA (R&D Systems) according to the
manufacturer’s protocol.
Western blot analysis
The protein extractions of rat synovial tissues were prepared for western blot
analyses of NLRP3, ASC, Caspase-1 (p20), TLR4, MyD88, p-IκB, IκB and α-tubulin,
while nuclear protein was extracted using kits purchased from Beyotime Biotech for
western blot analyses of p65 NF-κB and TBP. Western blot analysis was performed as
in our previous study [19]. The levels of target proteins were determined using a gel
imaging system (ChemiScope 2850, Clinx Science Instruments Co., Ltd., Shanghai,
China) and normalized to that of the reference band. Primary antibodies were listed in
Table 5.
Table 5. Antibodies used for western blot analysis.
Company
Description and titer
Catalog number
BIOSS Biotech
rabbit TLR4 antibody (1: 500)
rabbit MyD88 antibody (1: 500)
rabbit NLRP3 antibody (1: 500)
bs-1021R
bs-1047R
bs-6655R
(Beijing, P. R. China)
24

_______________________________________________________________________________
Cell Signaling Technology
(Boston, MA, USA)
NF-κB Pathway Sampler (1:1000)
rabbit α-tubulin antibody (1: 1000)
rabbit TBP antibody (1: 1000)
#9936
#2148
#8515
_________
Abcam
rabbit NLRP3 antibody (1: 1000)
rabbit ASC antibody (1: 1000)
rabbit Caspase-1 antibody (1: 1000)
ab214185
ab175449
ab179515
(Cambridge, MA, USA)
________
5.4. Enzyme kinetics
Compound 5d was further investigated for the type of inhibition and enzyme
kinetics study was carried out. Kinetic measurements were performed using three
different inhibitor concentrations (0.8, 4, 20 μM) and four different xanthine
concentrations (25-200 μM). According to the measured data, the Ki value of 5d for
XOD was calculated using the Lineweaver-Burk plot [22].
5.5. Protein structure prediction and molecular docking
The protein sequence of the NLRP3 protein (3QF2.pdb) was submitted, and the
tertiary structure of LRR domain of NLRP3 was predicted by RaptorX-ZY server [23],
which is one of the most top performing algorithm for difficult protein targets
(NLRP3.pdb). The potential ligand binding site of the NLRP3 protein located around
residue ASP750 was predicted by the sitemap module of the Schrodinger 2015 suite
[24]. In addition, the crystal structure of XOD complex (1N5X.pdb) was retrieved
from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/home.do).
Molecular docking was carried out using
the Glide 5.7. Van der Waals (VdW)
scaling of 0.8 and partial cut-off of 0.15 were set to soften the potential for non-polar
sites, and no constraints were specified. The ligand 5d was docked using the standard
precision (SP) mode. The best docked pose ranked by GlideScore value was recorded,
and saved for each ligand.
25

Acknowledgements
This work was supported by the National Natural Science Foundation of China (No.
81202573 and 81773745); the Suzhou Science & Technology Foundation (Grant
No.SYS201665) and PAPD (A Project Funded by the Priority Academic Program
Development of Jiangsu Higher Education Institutions).
Reference
[1]LK. Stamp, Major unanswered questions in the clinical gout field, Curr. Opin.
Rheumatol. 29 (2017) 171-177.
[2]K.Davies, MAS. Bukhari, Recent pharmacological advances in the management of
gout, Rheumatology. 14 (2017) .
[3]MD. Wechalekar, O. Vinik, JH. Moi, F. Sivera, IA. van Echteld ,C. van Durme, L.
Falzon, C. Bombardier, L. Carmona, D. Aletaha, RB. Landewé, DM. van der Heijde,
R. Buchbinder, The efficacy and safety of treatments for acute gout: results from a
series of systematic literature reviews including Cochrane reviews on intraarticular
glucocorticoids, colchicine, nonsteroidal antiinflammatory drugs, and interleukin-1
inhibitors, J. Rheumatol. Suppl. 92 (2014) 15-25.
[4]A. Dumusc, A. So, Interleukin-1 as a therapeutic target in gout, Curr. Opin.
Rheumatol. 27 (2015) 156-163.
[5]MC. Cleophas, TO. Crişan, LA. Joosten, Factors modulating the inflammatory
response in acute gouty arthritis, Curr. Opin. Rheumatol. 29 (2017) 163-170.
[6]SR. Kingsbury, PG. Conaghan, MF. McDermott, The role of the NLRP3
inflammasome in gout, J. Inflamm. Res. 4 (2011) 39-49.
[7]YF. Qing, QB. Zhang, JG. Zhou, L. Jiang, Changes in toll-like receptor
(TLR)4-NFκB-IL1β signaling in male gout patients might be involved in the
pathogenesis of primary gouty arthritis, Rheumatol. Int. 34 (2014) 213-220.
[8]Y. Yang, DM. Zhang, JH. Liu, LS. Hu, QC. Xue, XQ. Ding, LD. Kong, Wuling
San protects kidney dysfunction by inhibiting renal TLR4/MyD88 signaling and
NLRP3 inflammasome activation in high fructose-induced hyperuricemic mice, J.
26

Ethnopharmacol. 169(2017) 49-59.
[9]H. Wu, M. Zhou, G. Lu, Z. Yang, H. Ji, Q. Hu, Emodinol ameliorates urate
nephropathy by regulating renal organic ion transporters and inhibiting immune
inflammatory responses in rats, Biomed. Pharmacother. 96 (2017) 727-735.
[10]T. Pascart, P. Richette, Current and future therapies for gout, Expert. Opin.
Pharmacother. 18 (2017) 1201-1211.
[11]C. Chen, JM. Lü, Hyperuricemia-Related Diseases and Xanthine Oxidoreductase
(XOR) Inhibitors: An Overview, Med. Sci. Monit. 22 (2017) 2501-2512.
[12]BA. Hilmi, MI. Asmahan, A. Roseman, Use of newly available febuxostat in a
case of chronic tophaceous gout contraindicated to allopurinol and probenecid, Med. J.
Malaysia. 67 (2012) 125-126.
[13]SE. Sattui, AL. Gaffo, Treatment of hyperuricemia in gout: current therapeutic
options, latest developments and clinical implications. Ther. Adv. Musculoskelet. Dis.
8(2016) 145-159.
[14]KW. Ruiz-Miyazawa, L. Staurengo-Ferrari, SS. Mizokami, TP. Domiciano,
FTMC. Vicentini, D. Camilios-Neto, WR. Pavanelli, P. Pinge-Filho, FA. Amaral,
MM. Teixeira, R. Casagrande, WA. Jr. Verri, Quercetin inhibits gout arthritis in
mice:inductionof
an
opioid-dependent
regulation
of
inflammasome,
Inflammopharmacology. 15 (2017) [Epub ahead of print].
[15]C. Dhanasekar, M. Rasool, A dietary bioflavonol suppresses monosodium urate
crystal-induced inflammation in an animal model of acute gouty arthritis with
reference to NLRP3 inflammasome, hypo-xanthine phospho-ribosyl transferase, and
inflammatory mediators, Eur. J. Pharmacol. 768 (2017) 116-127.
[16]H. Honda, Y. Nagai, T. Matsunaga, N. Okamoto, Y. Watanabe, K. Tsuneyama, H.
Hayashi, I. Fujii, M. Ikutani, Y. Hirai, A. Muraguchi, K. Takatsu, Isoliquiritigenin is a
potent inhibitor of NLRP3 inflammasome activation and diet-induced adipose tissue
inflammation, J. Leukoc. Biol. 96 (2014) 1087-1100.
[17]SJ. Park, HS. Youn, Suppression of homodimerization of toll-like receptor 4 by
isoliquiritigenin, Phytochemistry. 71 (2010) 1736-1740.
[18]HQ. Li, Y. Luo, R. Song, ZL. Li, T. Yan, HL. Zhu, Design, synthesis, and
27

immunosuppressive activity of new deoxybenzoin derivatives, Chem. Med. Chem. 5
(2010) 1117-1122.
[19]Q. Hu, M. Zhou, H. Zhu, G. Lu, D. Zheng, H. Li, K. Hao,
(E)-2-(4-bromophenyl)-1-(2, 4-dihydroxyphenyl)ethanone oxime is a potential
therapeutic agent for treatment of hyperuricemia through its dual inhibitory effects on
XOD and URAT1, Biomed. Pharmacother. 86 (2017) 88-94.
[20]GZ. Ao, MZ Zhou, YY. Li, SN. Li, HN. Wang, QW. Wan, HQ. Li, QH. Hu,
Discovery of novel curcumin derivatives targeting xanthine oxidase and urate
transporter 1 as anti-hyperuricemic agents, Bioorg. Med. Chem. 25 (2017) 166-174.
[21]JJ. Carroll, H. Coburn, R. Douglass, AL. Babson, A simplified alkaline
phosphotungstate assay for uric acid in serum, Clin. Chem. 17 (1971) 158-160.
[22]M. Kaur, A. Kaur, S. Mankotia, H. Singh, A. Singh, JV. Singh, MK. Gupta, S.
Sharma, K. Nepali, PMS. Bedi, Synthesis, screening and docking of fused
pyrano[3,2-d]pyrimidine derivatives as xanthine oxidase inhibitor, Eur. J. Med. Chem.
131 (2017) 14-28.
[23]M. Källberg, H. Wang, S. Wang, J. Peng, Z. Wang, H. Lu, J. Xu. Template-based
protein structure modeling using the RaptorX web server. Nat. Protoc. 7 (2012)
1511–1522.
[24] Glide, Schrödinger, LLC, New York, NY, 2015.
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Development of benzoxazole deoxybenzoin oxime and acyloxylamines derivatives
targeting innate immune sensors and xanthine oxidase for treatment of gout
un Huanga, #, Zehao Zhou a, #, Mengze Zhou b, #, Mingxing Miao b, Huanqiu Li a, *
Qinghua Hu b, *
,
29