Design, synthesis and evaluation of 3-amide-5-aryl benzoic acid derivatives as novel P2Y14R antagonists with potential high efficiency against acute gouty arthritis

Article abstract of DOI:10.1016/j.ejmech.2021.113313

P2Y₁₄ nucleotide receptor plays important roles in series of physiological and pathologic events especially associated with immune and inflammation. Based on the 3-amide benzoic acid scaffold reported by our group previously, a series of 5-aryl-3-amide benzoic acid derivatives were designed as novel P2Y₁₄ antagonists with improved pharmacokinetic properties. Among which compound 11m showed most potent P2Y₁₄ antagonizing activity with an IC₅₀ value of 2.18 nM, furnishing greatly improved water solubility and bioavailability compared with PPTN. In MSU-induced acute gouty arthritis model in mice, 11m exerted promising in vivo efficacy in alleviating mice paw swelling and inflammatory infiltration. Mechanistically, compound 11m notably blocked pyroptosis of macrophages through inhibiting NLRP3 inflammasome activation. This work may contribute to the identification of potential therapeutic agents to intervene in acute gouty arthritis.

Full text of DOI:10.1016/j.ejmech.2021.113313

European Journal of Medicinal Chemistry 216 (2021) 113313
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design, synthesis and evaluation of 3-amide-5-aryl benzoic acid
derivatives as novel P2Y14R antagonists with potential high efficiency
against acute gouty arthritis
,
b
,
,
Ran Lu a 1, Yilin Wang a 1, Chunxiao Liu a, Zhenguo Zhang d, Baiyang Li a, Zibo Meng a,
Cheng Jiang a
*, Qinghua Hu a
,
b,
c
,
, c, **
a School of Pharmacy, China Pharmaceutical University, 639 Longmian Avenue, Nanjing, 211198, PR China
b Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, PR China
c State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, PR China
d Institute of Advanced Synthesis, School of Chemistry and Molecular Engineering, Nanjing Tech University, 30 Puzhu South Road, Nanjing, 211816, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
P2Y14 nucleotide receptor plays important roles in series of physiological and pathologic events espe-
cially associated with immune and inflammation. Based on the 3-amide benzoic acid scaffold reported by
our group previously, a series of 5-aryl-3-amide benzoic acid derivatives were designed as novel P2Y14
antagonists with improved pharmacokinetic properties. Among which compound 11m showed most
potent P2Y14 antagonizing activity with an IC50 value of 2.18 nM, furnishing greatly improved water
solubility and bioavailability compared with PPTN. In MSU-induced acute gouty arthritis model in mice,
11m exerted promising in vivo efficacy in alleviating mice paw swelling and inflammatory infiltration.
Mechanistically, compound 11m notably blocked pyroptosis of macrophages through inhibiting NLRP3
inflammasome activation. This work may contribute to the identification of potential therapeutic agents
to intervene in acute gouty arthritis.
Received 25 November 2020
Received in revised form
1 February 2021
Accepted 14 February 2021
Available online 24 February 2021
Keywords:
P2Y14 receptor
Antagonist
3-Amide-5-aryl benzoic acid derivatives
Anti-inflammation
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
suffered a significantly reduced glucose tolerance and changed
expression of components involved in insulin secretion, high-
P2 purinergic receptors are activated by purine or pyrimidine
nucleotides, including inotropic P2X1e7 receptors and metabotropic
P2Y1,2,4,6,11,12,13,14 receptors, which play key roles in physiology and
disease [1e4]. P2Y14 receptor is a member in P2Y receptors family,
which is activated by endogenic UDP-glucose (UDPG) and other
UDP sugars [5,6]. P2Y14 receptor has been reported broadly
expressed in astrocytes, microglia, immune cells and lung epithelial
cells [5,7,8].
lighting P2Y14R as a new modulator of maintaining energy ho-
meostasis by influencing insulin release [13,14]. Interestingly,
P2Y14R also plays a role in pro-inflammatory cytokines secretion to
mediate acute and chronic inflammatory conditions [15,16]. For
example, activation of P2Y14R in ischemic acute kidney injury
upregulated IRI-induced increase of chemokine expression in col-
lecting duct intercalated cells, and promoted neutrophil and
monocyte renal infiltration [17,18]. Another research illustrated
that P2Y14R knockout disrupted MSU-induced histopathologic
changes in rat synoviums, accompanying with a significant inhi-
bition of pyroptotic cell death and blockade of NRPL3 inflamma-
some activation in synovial tissues [19].
It has been considered that UDPG coupled P2Y14 receptors
promoted the hypersensitivity of microglia cells and the fluidity of
neutrophils [9e12]. Recent studies showed that knock-out mice
P2Y14R antagonists have been developed as a potential thera-
peutic for treating immune and inflammatory diseases. Well-
characterized potent and selective P2Y14R antagonists are invalu-
able tools for testing biological and therapeutic hypotheses. And
many efforts have been made to discover antagonists that can be
developed into a therapeutic drug (Fig. 1) [20]. The pyrido [4,3-d]
* Corresponding author. School of Pharmacy, China Pharmaceutical University,
639 Longmian Avenue, Nanjing, 211198, PR China.
** Corresponding author. School of Pharmacy, China Pharmaceutical University,
639 Longmian Avenue, Nanjing, 211198, PR China.
E-mail addresses: jc@cpu.edu.cn (C. Jiang), huqh@cpu.edu.cn (Q. Hu).
These authors contributed equally to this study.
1
https://doi.org/10.1016/j.ejmech.2021.113313
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

R. Lu, Y. Wang, C. Liu et al.
European Journal of Medicinal Chemistry 216 (2021) 113313
Fig. 1. Representative P2Y14R antagonists.
pyrimidines [21] was the first discovered scaffolds through HTS,
accompanying with hERG channel effects. Compound 1 (PPTN), one
of the reported 2-naphthalic acids derivatives, was tested for the
treatment of renal inflammatory with high activity and high
selectivity [22,23]. However, it suffered with poor physicochemical
properties and low oral bioavailability [7]. Some strategies have
been applied to overcomethis, such as esterifying the carboxyl
group into a pro-drug [23], or introduce a strong hydrophilic
polyethylene glycol chain on the amino group to increase water
solubility [24]. It has been reported several novel structures based
on the structure-guided modification and homogenous model [25],
including 3-alkynyl (compound 2) or triazole benzoic acids (com-
pound 3 and 4) [26,27], 2-amide benzoic acids (compound 5) [28]
and 3-amide benzoic acids (compound 6) [29]. The 3-amide ben-
zoic acids reported by our group showed potent P2Y14R antago-
nizing activity and excellent in vitro potency. However, the
optimized 6 suffered from poor bioavailability (F ¼ 6%) due to its
zwitterionic character. In this work, a series of 3-amide-5-aryl
benzoic acid derivatives were identified to overcome the limita-
tions through replacing the basic substitutions attached to the core
phenyl ring with substituted phenyl or aromatic groups.
Therefore, we introduced phenyl or heterocyclic group to replace
the phenylpiperidine in order to find new P2Y14R antagonists with
improved pharmacokinetic properties. A series of new 3-amide-5-
aryl benzoic acids were designed as shown in Table 1.
2.2. Chemistry
The synthetic route of target compounds was outlined in
Scheme 1. The commercially available 3-amino-5-bromobenzoic
acid (7) was methylated with SOCl2 in menthol to produce
methyl 3-amino-5-bromobenzoate (8). Subsequent coupling of the
obtained bromobenzoate ester with 4-methylbenzoic acid using
BOP reagent in the presence of DMAP as a base gave key interme-
diate (9). Then the resulted bromobenzoate esters were coupled
with appropriate aromatic boric acids or borates catalyzed with Pd
(PPh3)4 and Cs2CO3 in DMF under argon conditions to provide the
expected analogues of this series. Finally, the target compounds, 3-
(4-methylbenzamido) benzoic acids derivatives (11a-11q), were
obtained by hydrolyzing the corresponding methyl 3-bromo-5-(4-
methylbenzamido) benzoates in an aqueous solution of lithium
hydroxide with yield ranging from 74% to 89%.
2. Results and discussion
2.3. Biological evaluation
2.1. Molecule design
2.3.1. In vitro P2Y14R inhibition
All synthesized derivatives were tested for their in vitro inhibi-
tion based on the production of cAMP in a HEK293 cell line stably
expressing P2Y14R. The P2Y14R inhibitory activities of these de-
rivatives were screened at a relatively high single concentration of
inhibitor (100 nM) to identify the more potent antagonist. The IC50
values of the compounds with more than 80% inhibition at 100 nM
were further tested in the functional assay with full concentration-
response curves as shown in Table 1.
Firstly, piperidine group in 6 was removed and 11a was ob-
tained. This modification did not lose potency dramatically
(IC50 ¼ 4.85 nM). When phenyl was substituted with electron-
donating group (4-CH3O-), the P2Y14R inhibitory activities was
slightly decreased. Furthermore, the water solubility of 11a was
In our previous work, a series of 3-amide benzoic acids were
identified as novel and potent P2Y14R antagonists [28]. However,
compound 6 showed very poor bioavailability (F ¼ 6%) due to its
zwitterionic character. Based on the reported works, the carboxyl
group was necessary for maintaining activity, thus the design
strategy in this work was to retain the carboxyl group. As it has
been reported, the hydrophobic 4-(4-methyl)-benzamide moiety
attached to C-3 of the benzoic acid core was considered as the
optimal substitution at this position. Additionally, previous SAR
exploration of PPTN predicted that a charged amino group is not
essential, and diverse 5- and 6-membered ring linking the core
facing the extracellular loop side of the receptor were also efficient.
2

R. Lu, Y. Wang, C. Liu et al.
European Journal of Medicinal Chemistry 216 (2021) 113313
Table 1
In vitro P2Y14R affinity and cLogP values of compounds 11a-q.
Compound
R
P2Y14R affinity
cLog P
% inhibition at 100 nM
IC50 (nM)
PPTN (2)
6
e
96.31
85.33
1.98 0.006
1.77 0.26
6.18
4.05
11a
11b
11c
11d
11e
86.99
79.10
79.10
72.12
65.72
4.85 1.49
4.56
4.43
3.22
3.22
2.62
ND
ND
ND
ND
11f
68.57
72.50
ND
ND
2.33
3.01
11g
11h
11i
11j
82.96
68.76
60.19
9.74 0.19
4.48
4.54
4.88
ND
ND
11k
43.70
ND
3.91
11l
61.09
93.39
16.05
82.27
ND
3.17
3.12
3.51
3.77
11m
11n
11o
2.18 0.42
ND
2.95 0.47
11p
11q
46.01
86.79
ND
3.1
2.63 0.67
3.08
decreased compared with 6. Next several five or six-membered
heterocyclic rings as the bioisostere of phenyl ring were intro-
duced to replace the phenyl group in order to improve the aqueous
solubility. According to the results of P2Y14R affinities, compounds
(11c, 11d and 11e) replaced by nitrogen-containing aromatic het-
erocycles didn’t exhibit a good inhibition ratio (<80%). Compounds
that displayed inhibition of >80% on the P2Y14 R at 100 nM were
five-membered heterocyclic rings containing one heteroatom at
position 3 of these structure, such as S (11h, 82.96%), O (11m,
93.39%; 11o, 82.27%), NH (11q, 86.79%). A closer inspection of the
affinity data reveals that, in most cases, the introduction of
pentagonal heterocycles linked at position 2 (compound 11k-m,
11o-p and 11r), diminishes the affinity compared to the 3-
substituted ones. For example, the 3-furyl derivative 11m was
considered as the most appealing P2Y14R antagonist (93.39% inhi-
bition at 100 nM, IC50 ¼ 2.18 nM) identified in this work, while its
close analogue 11l exhibited a decreased P2Y14R inhibitory activity
(61.09% inhibition at 100 nM). Moreover, other compounds for
instance a thienyl or furyl group substituted by a methyl (11j, 11n)
or acetyl (11k) at position 3 showed decreased affinity. The IC50
3

R. Lu, Y. Wang, C. Liu et al.
European Journal of Medicinal Chemistry 216 (2021) 113313
Scheme 1. Reagents and conditions:(a) CH3OH, SOCl2, 0e25 ꢀC; (b) BOP reagent, DMAP, DCM; (c) Pd(PPh3)4, Cs2CO3, DMF, 100 ꢀC; (d) LiOH(1 M aq). MeOH- THF-DMSO 1/1/1 (v/v/
v). R are defined in Table 1.
values of the compounds with more than 80% inhibition to the
P2Y14 R at 100 nM were further tested in the functional assay with
full concentration-response curves as shown in Table 1. The IC50
values for 11a, 11h, 11m, 11o and 11q were 4.85, 9.74, 2.18, 2.95 and
2.63 nM, respectively.
After optimization, several highly potent P2Y14R antagonists
were obtained. Compounds 11o and 11q, two of the most potent
compounds, were chemically unstable, which were oxidized easily
at room temperature in DMSO. Thus, the stable and most potent
one, 11m, was selected for further evaluation.
2.3.4. Water solubility
An important aim in the development of new P2Y14R antago-
nists is to improve the aqueous solubility. PPTN has an aqueous
solubility less than 0.005 mg/mL, and the solubility of 11a is
0.0463 mg/mL (pure water). While 11m shows an aqueous solu-
bility of 0.0776 mg/mL (pure water), which is more than 10-fold
improvement compared with PPTN and more than 1.5-fold for
11a.The improved aqueous solubility will provide an opportunity to
evaluate their in vivo functional profiles.
2.3.5. Anti-inflammatory effects of compound 11m
As shown in Fig. 2, in MSU-induced animal model, control mice
2.3.2. Metabolic stability of 11m in human liver microsomes
11m was evaluated for its metabolic stability in human micro-
somes. Compound 11m demonstrated excellent microsomal sta-
bility (T1/2 > 60 min).
with MSU treatment showed
a continuous stability of paw
2.3.3. Pharmacokinetic (PK) study of compound 11m in SD mice
Compound 11m was evaluated for pharmacokinetics (PK) in
male SD mice at 5 mg/kg and 10 mg/kg for intravenous (iv) and oral
(po) administration. As shown in Table 2, 11m showed dramatically
improved oral bioavailability (48.32% for compound 11m vs 7% for
compound PPTN) and more favorable PK profile. The moderate
volume of distribution (Vss) of 0.78 L/kg suggested that compound
11m penetrated to tissues well, and high systemic exposure (oral
AUCall ¼ 436599 min*ng/mL) indicated that compound 11m could
be well absorbed after oral administration, while the oral AUCall
value of compound 1 was 182083 min*ng/mL. Compound 11m
exhibited reasonable plasma clearance level (CL ¼ 6.03 mL/min/kg)
and half-life (T1/2 ¼ 138.93 min and 153.54 min for iv and oral
administration, respectively). According to the results, bioavail-
ability of 11m was greatly improved compared with PPTN (F ¼ 7%)
or compound 6 (F ¼ 6%).
Fig. 2. The thickness of mice foot was increased after the injection of MSU. PPTN and
11m abolished the effective effect on the paw swelling. The injected foot thickness of
each mouse was determined at 0, 1, 2, 4, 6, 8, 12, 24 h after MSU stimulation (n ¼ 6).
Data are shown as means SD (n ¼ 3). #P < 0.05, ##P < 0.01 versus control group,
*P < 0.05, **P < 0.01 versus model group.
Table 2
Pharmacokinetic (PK) results of compound 11m.
compounds
admin (dosage)
sample
C
max (ng/mL)
T1/2(min)
AUCall (min*ng/mL)
Vss (mL/kg)
CL (mL/min/kg)
F(%)
11m
iv (5 mg/kg)a
plasma
plasma
19270b
1801
138.93
153.54
843849
436599.5
781.93
212.67
121.95
6.03
po (10 mg/kg)a
48.32%
a
n ¼ 5.
b
Cmax ¼ C5 (t ¼ 5 min) for iv administration.
4

R. Lu, Y. Wang, C. Liu et al.
European Journal of Medicinal Chemistry 216 (2021) 113313
thickness during experimental period. In contrast, an obvious
elevation in paw thickness could be observed in model mice
receiving MSU, suggesting the occurrence of exaggerated paw
swelling. Most importantly, 11m or PPTN treatment efficiently
reversed the paw swelling and infiltration of inflammatory cells in
foot tissues (Fig. 3). Besides, compound 11m was similar to PPTN in
the first 4 h, however, it recovered paw swelling more significantly
after 24 h.
compared with control group, which were reversed by treatment of
compound 11m and PPTN. Meanwhile, increased level of IL-1b, IL-8
and GSDMD-NT in model group were apparently inhibited by
compound 11m and PPTN (Fig. 4).
Consistently, MSU exposure in the model group exhibited an
obvious increment in the proportion of pyroptotic cells character-
ized by Caspase-1/PI double positive staining analyzed by flow
cytometry compared with control ones (Fig. 5), which was notably
ameliorated by 11m and PPTN intervention.
MSU stimulation contribute to the up-regulation of NLRP3, ASC
and Caspase-1 p20 protein expressions in mice foot tissues
Fig. 3. Representative photographs of histo-pathological changes in foot tissues are presented (200 ꢁ , 400 ꢁ ). The small boxed areas show higher magnification views of the large
boxes.
5

R. Lu, Y. Wang, C. Liu et al.
European Journal of Medicinal Chemistry 216 (2021) 113313
Fig. 4. (A) Relative protein expressions of NLRP3, ACS, Caspase-1 (p20), GSDMD, IL-1
b
and IL-8 in foot tissues were assayed by western blot. (B) Quantification of the protein level
was expressed as the ratio (in percentage) of control group. Data are shown as means SD (n ¼ 3). ###P < 0.001 versus control group, *P < 0.05, **P < 0.01 versus model group.
Fig. 5. Effects of compound 11m and PPTN on MSU-induced pyroptosis of macrophages in mice air pouch exudates. The rate of pyroptotic cell death was examined with PI and
active Caspase-1 double staining by flow cytometry.
3. Conclusion
identified as novel P2Y14R antagonists with improved pharmaco-
kinetic profiles by zwitterionic character. The optimized compound
11m showed potent P2Y14R antagonizing activity and the water
In summary, a series of 3-amide-5-aryl benzoic acids were
6

R. Lu, Y. Wang, C. Liu et al.
European Journal of Medicinal Chemistry 216 (2021) 113313
solubility of 11m was greatly improved compared with PPTN.
Compound 11m showed excellent microsomal stability in human
microsomes and moderate bioavailability (F ¼ 48%) in rats. In vivo
anti-inflammatory evaluation of 11m in MSU-induced mice paw
swelling model demonstrated that 11m can efficiently reversed the
paw swelling and infiltration of inflammatory cells in foot tissues.
Most importantly, in the MSU-induced mice paw swelling model,
11m exerted better therapeutic effects in lowering increments of
mice paw thickness (~0.13 mm) than PPTN (~0.40 mm) at 24 h,
exhibiting more potential P2Y14R inhibitory actions. On the other
hand, 11m significantly reversed NLRP3 inflammasome activation
induced by MSU-injection, contributing to decreased proportion of
pyroptosis macrophages. Taken together, the identification of the 3-
amide-5-aryl benzoic acids P2Y14 receptor antagonists with
preferred in vitro and in vivo potencies may provide an alternative
therapy for acute gouty arthritis diseases.
was washed with 1M hydrochloric acid (20 mL*3). The organic
layer were dried over MgSO4 and the solvent was eliminated under
reduced pressure to give crude product. The residue was purified by
column chromatography (petroleum ether/ethyl acetate ¼ 4:1) to
give pure 9 as a white solid. Yield: 6.38g, 84%. m. p. 146e147 ꢀC. 1H
NMR (400 MHz, CDCl3-d3)
d
(ppm) 8.34 (t, J ¼ 2.0 Hz, 1H), 8.07 (t,
J ¼ 1.7 Hz, 1H), 7.93 (t, J ¼ 1.6 Hz, 1H), 7.79 (d, J ¼ 8.2 Hz, 2H), 7.30 (d,
J ¼ 9.1 Hz, 2H), 3.92 (s, 3H), 2.44 (s, 3H). MS (ESI-TOF) m/z calc’d for
C
18H15N3O3 [MþH]þ 348.0234, found 426.1857.
4.1.3. General procedure for the synthesis of 11a-11q
(1) To a solution of methyl 3-bromo-5-(4-methylbenzamido)
benzoate (200 mg, 0.57 mmol) in DMF (3 mL) was added
phenylboronic acid (84 mg, 0.69 mmol), cesium carbonate
(281 mg, 0.86 mmol) and Pd(PPh3)4 (66 mg, 0.057 mmol).
The mixture was stirred under argon for 10 h at 100 ꢀC. Upon
completion, the mixture was filtered, diluted with ethyl ac-
etate (10 mL * 3), washed with brine, dried over anhydrous
MgSO4 and concentrated to give a crude resin. The crude
product was further purified using column chromatography
(petroleum ether/ethyl acetate ¼ 1:1) to give a white solid of
10a.
4. Experimental
4.1. Chemistry
All reagents and solvents were commercially available and were
used without further purification. The high-resolution mass spec-
troscopic (HRMS) data were obtained on a proteomics optimized Q-
TOF-2 (Micromass-Waters) using external calibration with poly-
alanine. 1H NMR and 13C NMR spectra were recorded on Bruker
400 MHz or Bruker 300 MHz instruments using the solvent indi-
cated in each case (300 MHz or 400 MHz for 1H and 75 or 101 MHz
for 13C). The tested compounds were purified to >95% purity as
determined by HPLC. All chemical reagents were purchased from
bide. All reactions were monitored by thin-layer chromatography
(TLC) with 254 nm and 365 nm fluorescent indicators. The purifi-
cation of compounds by flash chromatography and preparative TLC
was carried out using silica-gel 300*400 mm mesh and GF254
plates, respectively. C LogP was calculated using ALOGPS 2.1 pro-
gram (www.vcclab.org/lab/alogps/). PPTN (1) was prepared as the
reported [22]. Compound 5 were synthesized according to our
previous work [28].
(2) A mixture of methyl 5-(4-methylbenzamido)-[1,10-
biphenyl]-3-carboxylate (1 mmol) (10a), 1M LiOH aqueous
(5 mL), MeOH (2 mL), DMSO (2 mL) and THF (2 mL) was
stirred at room temperature for 3 h. The solvent was
concentrated in a vacuum, and the residue was acidified with
1M hydrochloric acid to pH 1. The resulting precipitate was
filtered
giving
rise
to
corresponding
5-(4-
methylbenzamido)-[1,10-biphenyl]-3-carboxylic acid (11a).
Purified by preparative TLC (ethyl acetate: methanol ¼ 10:1).
4.1.3.1. 5-(4-Methylbenzamido)-[1,10-biphenyl]-3- carboxylic acid
(11a). A white solid, yield: 87%; HPLC purity: 96.95%. m. p. 260 ꢀC
(decomposed). 1H NMR (400 MHz, DMSO‑d6)
d (ppm) 13.10 (s, 1H),
10.42 (s, 1H), 8.46 (t, J ¼ 1.8 Hz, 1H), 8.38 (t, J ¼ 2.0 Hz, 1H),
8.00e7.87 (m, 3H), 7.72e7.68 (m, 1H), 7.68 (s, 1H), 7.53 (dd, J ¼ 8.4,
6.9 Hz, 2H), 7.46e7.40 (m, 1H), 7.38 (s, 1H), 7.36 (s, 1H), 2.40 (s, 3H).
4.1.1. Synthesis of methyl 3-amino-5-bromobenzoate (8)
To a solution of 3-amino-5-bromobenzoic acid (7) (5.00g,
23.14 mmol) in methanol (25 mL) was added SOCl2 (15 mL) drop-
wise at 0 ꢀC. Then, the mixture was stirred at room temperature for
2 h and was monitored by TLC. After the reaction completed, the
reaction was added saturate sodium hydrogen carbonate aqueous
solution until pH 7e8, followed stirring for another 30 min. The
mixture was extracted with ethyl acetate (50 mL* 3). The organic
phase was collected and dried over MgSO4. After filtration, the
solvent was removed under reduced pressure and the residue was
purified by column chromatography (petroleum ether/ethyl
acetate ¼ 1:1) to provide the pure product as white solid. Yield:
4.62g, 86.4%. m. p. 94e95 ꢀC. 1H NMR (300 MHz, CDCl3-d3) (ppm)
7.55 (t, J ¼ 1.6 Hz, 1H), 7.28 (dd, J ¼ 2.4, 1.4 Hz, 1H), 7.01 (t, J ¼ 2.0 Hz,
1H), 3.91 (s, 3H). MS (ESI-TOF) m/z calc’d for C18H15N3O3 [MþH]þ
229.9817, found 229.9814.
13C NMR (75 MHz, DMSO‑d6)
d (ppm) 167.70, 166.06, 142.31, 141.38,
140.65, 139.89, 132.44, 132.15, 129.58, 129.41, 128.43, 128.22, 127.19,
123.04, 122.94, 120.51, 21.46.MS (ESI-TOF) m/z calc’d for C21H17NO3
[MþH]þ 332.1287, found 332.1281.
4.1.3.2. 40-Methoxy-5-(4-methylbenzamido)-[1,10-biphenyl]-3-
carboxylic acid (11b). A white solid, yield: 87%; HPLC purity:
99.78%. m. p. 247 ꢀC (decomposed). 1H NMR (300 MHz, DMSO‑d6)
d
(ppm) 13.05 (s, 1H), 10.41 (s, 1H), 8.43 (t, J ¼ 1.7 Hz, 1H), 8.34 (t,
J ¼ 1.9 Hz, 1H), 7.95 (d, J ¼ 8.2 Hz, 2H), 7.89 (t, J ¼ 1.6 Hz, 1H), 7.63 (d,
J ¼ 8.8 Hz, 2H), 7.36 (d, J ¼ 7.8 Hz, 2H), 7.08 (d, J ¼ 8.8 Hz, 2H), 3.81
(s, 3H), 2.39 (s, 3H). 13C NMR (75 MHz, DMSO‑d6)
d (ppm) 167.70,
165.99, 159.74, 142.31, 140.99, 140.55, 132.28, 132.16, 132.11, 129.43,
128.29, 128.21, 122.51, 122.40, 119.82, 115.01, 55.66, 21.49. MS (ESI-
TOF) m/z calc’d for C22H19NO4 [MþH]þ 362.1392, found 362.1390.
4.1.2. Synthesis of methyl 3-bromo-5-(4-methylbenzamido)
benzoate (9)
4.1.3.3. 3-(4-Methylbenzamido)-5-(pyridin-3-yl)benzoic acid (11c).
A white solid, yield: 75%; HPLC purity: 99.82%. m. p. 290 ꢀC
To a magnetically stirred suspension of 4-methylbenzoic acid
(4.44g, 32.60 mmol) in DCM (35 mL) were successively added N, N-
dimethylpyridin-4-amine (DMAP) (5.31g, 43.46 mmol) and BOP
reagent (14.42g, 32.60 mmol). The mixture was allowed to stir at
room temperature for 15 min. The methyl 3-amino-5-
bromobenzoate (8) (5.00g, 21.73 mmol) was then added, and the
solution was stirred at room temperature overnight. The mixture
(decomposed). 1H NMR (400 MHz, DMSO‑d6)
d (ppm) 13.19 (s, 1H),
10.48 (s, 1H), 8.94e8.86 (m, 1H), 8.64 (d, J ¼ 4.7 Hz, 1H), 8.53 (s, 1H),
8.41 (d, J ¼ 2.0 Hz, 1H), 8.11 (dt, J ¼ 8.1, 1.9 Hz, 1H), 7.94 (d, J ¼ 8.1 Hz,
3H), 7.55 (dd, J ¼ 8.0, 4.8 Hz,1H), 7.37 (d, J ¼ 7.8 Hz, 2H), 2.40 (s, 3H).
13C NMR (101 MHz, DMSO‑d6)
d (ppm) 167.45, 166.06, 149.50,
148.04, 142.43, 140.77, 138.33, 135.43, 134.81, 132.65, 132.03, 129.47,
128.22, 124.54, 123.10, 123.07, 121.05, 21.51. MS (ESI-TOF) m/z calc’d
7

R. Lu, Y. Wang, C. Liu et al.
European Journal of Medicinal Chemistry 216 (2021) 113313
for C20H16N2O3 [MþH]þ 333.1239, found 333.1238.
255 ꢀC (decomposed). 1H NMR (300 MHz, DMSO‑d6)
d (ppm) 10.43
(s, 1H), 8.40 (d, J ¼ 3.9 Hz, 2H), 7.97 (d, J ¼ 7.6 Hz, 3H), 7.34 (d,
4.1.3.4. 3-(4-Methylbenzamido)-5-(pyridin-4-yl)benzoic acid (11d).
A white solid, yield: 81%; HPLC purity: 99.04%. m. p. 215e217 ꢀC.1H
J ¼ 7.9 Hz, 2H), 6.89 (s, 1H), 6.24 (s, 1H), 2.38 (s, 6H). 13C NMR
(75 MHz, DMSO‑d6)
d (ppm) 167.62, 166.03, 152.74, 151.09, 142.33,
NMR (300 MHz, DMSO‑d6)
d
(ppm) 13.52 (s, 1H), 10.50 (s, 1H), 8.70
140.61, 132.47, 132.12, 131.59, 129.41, 128.25, 119.95, 119.35, 118.78,
108.92, 108.03, 21.46, 13.89. MS (ESI-TOF) m/z calc’d for C20H17NO3S
[MþH]þ 352.1007, found 352.0998.
(d, J ¼ 5.0 Hz, 2H), 8.55 (t, J ¼ 2.0 Hz,1H), 8.49 (t, J ¼ 2.0 Hz,1H), 8.02
(t, J ¼ 1.7 Hz, 1H), 7.95 (d, J ¼ 7.9 Hz, 2H), 7.72 (d, J ¼ 5.7 Hz, 2H), 7.38
(d, J ¼ 7.9 Hz, 2H), 2.41 (s, 3H). 13C NMR (101 MHz, DMSO‑d6)
d
(ppm) 167.34,166.10,150.90,146.80, 142.48, 140.87, 138.50, 132.73,
4.1.3.11. 3-(4-Methylbenzamido)-5-(5-acetylthiophen-2-yl)benzoic
131.98, 129.48, 128.22, 122.95, 122.92, 121.99, 121.79, 21.51. MS (ESI-
acid (11k). A white solid, yield: 85%; HPLC purity: 95.88%. m. p.
TOF) m/z calc’d for C20H16N2O3 [MþH]þ 333.1239, found 333.1238.
281 ꢀC (decomposed) 1H NMR (300 MHz, DMSO‑d6)
d (ppm) 13.29
(s, 1H), 10.47 (s, 1H), 8.49 (d, J ¼ 1.6 Hz, 2H), 8.00 (t, J ¼ 1.6 Hz, 1H),
7.98 (d, J ¼ 4.0 Hz, 1H), 7.94 (d, J ¼ 8.2 Hz, 2H), 7.69 (d, J ¼ 4.0 Hz,
1H), 7.37 (d, J ¼ 8.1 Hz, 2H), 2.57 (s, 3H), 2.40 (s, 3H). 13C NMR
4.1.3.5. 3-(4-Methylbenzamido)-5-(pyrimidin-5-yl)benzoic
(11e). A white solid, yield:84%; HPLC purity: 95.79%. m. p.
151e154 ꢀC.1H NMR (300 MHz, DMSO‑d6)
(ppm) 13.20 (s, 1H),
acid
d
(75 MHz, DMSO‑d6) d (ppm) 191.15, 167.15, 166.13, 150.55, 143.71,
10.50 (s, 1H), 9.25 (s, 1H), 9.14 (s, 2H), 8.55 (t, J ¼ 1.8 Hz, 1H), 8.41 (t,
142.52, 140.92, 135.65, 133.76, 132.79, 131.91, 129.47, 128.23, 126.09,
121.75, 121.66, 26.88, 21.51. MS (ESI-TOF) m/z calc’d for
C21H17N1O4S [MþH]þ 380.0957, found 380.0952.
J ¼ 1.9 Hz, 1H), 8.01 (t, J ¼ 1.6 Hz,1H), 7.94 (d, J ¼ 8.2 Hz, 2H), 7.36 (d,
J ¼ 8.3 Hz, 2H), 2.40 (s, 3H). 13C NMR (75 MHz, DMSO‑d6)
d (ppm)
167.31, 166.06, 158.15, 155.32, 142.47, 140.87, 135.11, 133.30, 132.88,
131.94, 129.47, 128.22, 123.20, 123.14, 121.72, 21.50. MS (ESI-TOF) m/
z calc’d for C19H15N3O3 [MþH]þ 334.1192, found 334.1190.
4.1.3.12. 3-(4-Methylbenzamido)-5-(furan-2-yl)benzoic acid (11l).
A white solid, yield: 81%; HPLC purity: 95.90%. m. p. 247 ꢀC
(decomposed). 1H NMR (300 MHz, DMSO‑d6)
d (ppm) 13.16 (s, 1H),
4.1.3.6. 3-(2-Aminopyrimidin-5-yl)-5-(4-methylbenzamido)benzoic
10.38 (s, 1H), 8.21 (d, J ¼ 10.8 Hz, 1H), 8.10 (t, J ¼ 6.3 Hz, 2H), 7.98 (t,
J ¼ 5.3 Hz, 2H), 7.90 (d, J ¼ 8.8 Hz, 1H), 7.80e7.70 (m, 1H), 7.33 (d,
J ¼ 7.9 Hz, 2H), 6.89 (d, J ¼ 2.5 Hz, 1H), 2.38 (s, 3H). 13C NMR
acid (11f). A white solid, yield: 89%; HPLC purity: 99.40%. m. p.
242e244 ꢀC. 1H NMR (300 MHz, DMSO‑d6)
d (ppm) 13.11 (s, 1H),
10.40 (s, 1H), 8.58 (s, 2H), 8.43 (t, J ¼ 1.7 Hz, 1H), 8.24 (t, J ¼ 1.9 Hz,
(75 MHz, DMSO‑d6) d (ppm) 167.78, 166.08, 144.89, 142.23, 140.54,
1H), 7.93 (d, J ¼ 8.2 Hz, 2H), 7.85 (t, J ¼ 1.6 Hz,1H), 7.37 (d, J ¼ 8.0 Hz,
140.11, 133.12, 132.39, 132.15, 129.33, 128.20, 125.84, 122.00, 120.40,
121.95,109.10, 21.35. MS (ESI-TOF) m/z calc’d for C15H19NO4 [MþH]þ
322.1079, found 322.1077.
2H), 6.91 (s, 2H), 2.40 (s, 3H). 13C NMR (75 MHz, DMSO‑d6)
d (ppm)
167.58, 165.98, 163.61, 156.46, 142.38, 140.64, 136.50, 132.52, 132.08,
129.46, 128.20, 121.86, 121.54, 119.99, 21.51. MS (ESI-TOF) m/z calc’d
for C19H16N4O3 [MþH]þ 349.1301, found 349.1290.
4.1.3.13. 3-(4-Methylbenzamido)-5-(furan-3-yl)benzoic acid (11m).
A white solid, yield: 87%; HPLC purity: 96.04%. m. p. 224 ꢀC
4.1.3.7. 3-(2-Methoxypyrimidin-5-yl)-5-(4-methylbenzamido)ben-
(decomposed). 1H NMR (300 MHz, DMSO‑d6)
d (ppm) 10.38 (s, 1H),
zoic acid (11g). A white solid, yield: 86%; HPLC purity: 99.62%. m. p.
8.25 (d, J ¼ 16.0 Hz, 2H), 7.97 (q, J ¼ 9.7, 6.8 Hz, 3H), 7.75 (s, 1H), 7.33
166e168 ꢀC. 1H NMR (300 MHz, DMSO‑d6)
d (ppm) 10.47 (s, 1H),
(d, J ¼ 7.7 Hz, 2H), 6.85 (d, J ¼ 3.2 Hz, 1H), 6.60 (t, J ¼ 2.8 Hz, 1H),
8.93 (s, 2H), 8.50 (t, J ¼ 1.7 Hz, 1H), 8.32 (t, J ¼ 1.9 Hz, 1H), 7.99e7.88
2.38 (s, 3H). 13C NMR (75 MHz, DMSO‑d6)
d (ppm) 167.39, 166.08,
(m, 3H), 7.37 (d, J ¼ 8.0 Hz, 2H), 3.99 (s, 3H), 2.40 (s, 3H). 13C NMR
142.82, 142.41, 140.76, 134.80, 132.55, 132.05, 129.44, 129.19, 128.24,
126.88, 124.76, 121.33, 121.26, 120.42, 21.50. MS (ESI-TOF) m/z calc’d
for C15H19NO4 [MþH]þ 322.1079, found 322.1079.
(101 MHz, DMSO‑d6)
d (ppm) 168.28, 166.91, 166.11, 158.73, 143.28,
141.60, 136.02, 133.54, 132.86, 130.31, 129.07, 128.29, 128.20, 123.48,
121.91, 56.15, 22.35. MS (ESI-TOF) m/z calc’d for C20H17N3O4
[MþH]þ 364.1297, found 364.1293.
4.1.3.14. 3-(4-Methylbenzamido)-5-(5-methylfuran-2-yl)benzoic
acid (11n). A white solid, yield: 89%; HPLC purity: 98.89%. m. p.
4.1.3.8. 3-(4-Methylbenzamido)-5-(thiophen-3-yl)benzoic
(11h). A white solid, yield: 83%; HPLC purity: 99.42%. m. p. 258 ꢀC
(decomposed). 1H NMR (300 MHz, DMSO‑d6)
(ppm) 13.10 (s, 1H),
acid
286 ꢀC (decomposed). 1H NMR (300 MHz, DMSO‑d6)
d (ppm) 10.42
(s, 1H), 8.40 (s, 2H), 7.95 (d, J ¼ 7.7 Hz, 3H), 7.33 (d, J ¼ 7.9 Hz, 2H),
d
6.88 (s, 1H), 6.23 (s, 1H), 2.37 (s, 6H). 13C NMR (75 MHz, DMSO‑d6)
10.38 (s, 1H), 8.45e8.33 (m, 2H), 7.95 (d, J ¼ 2.9 Hz, 2H), 7.93e7.88
d (ppm) 167.58, 166.00, 152.71, 151.06, 142.29, 140.57, 132.43, 132.08,
(m, 2H), 7.70 (dd, J ¼ 5.2, 2.8 Hz, 1H), 7.52 (d, J ¼ 5.0 Hz, 1H), 7.36 (d,
131.55, 129.37, 128.22, 119.92, 119.31, 118.75, 108.89, 108.00, 21.43,
13.86. MS (ESI-TOF) m/z calc’d for C20H17NO4 [MþH]þ 336.1236,
found 336.1234.
J ¼ 7.9 Hz, 2H), 2.40 (s, 3H). 13C NMR (75 MHz, DMSO‑d6)
d (ppm)
167.79, 166.04, 142.30, 141.11, 140.54, 136.26, 132.59, 132.15, 129.41,
128.22, 127.99, 126.46, 122.45, 122.38, 122.28, 120.38, 21.46. MS
(ESI-TOF) m/z calc’d for C19H15NO3S [MþH]þ 338.0851, found
338.0852.
4.1.3.15. 3-(4-Methylbenzamido)-5-(3,4-dimethylisoxazol-5-yl)ben-
zoic acid (11o). A white solid, yield: 89%; HPLC purity: 97.84%. m. p.
219 ꢀC (decomposed). 1H NMR (400 MHz, DMSO‑d6)
d (ppm) 13.15
4.1.3.9. 3-(4-Methylbenzamido)-5-(thiophen-2-yl)benzoic acid (11i).
(s, 1H), 10.43 (s, 1H), 8.45 (t, J ¼ 1.8 Hz, 1H), 8.05 (t, J ¼ 1.9 Hz, 1H),
A white solid, yield: 88%; HPLC purity: 96.35%. m. p. 260 ꢀC
7.92 (d, J ¼ 1.8 Hz, 1H), 7.90 (d, J ¼ 1.9 Hz, 1H), 7.62 (t, J ¼ 1.6 Hz, 1H),
(decomposed). 1H NMR (300 MHz, DMSO‑d6)
d
(ppm) 13.22 (s, 1H),
7.37 (s, 1H), 7.35 (s, 1H), 2.46 (s, 3H), 2.40 (s, 3H), 2.27 (s, 3H). 13
C
10.41 (s, 1H), 8.39 (d, J ¼ 3.1 Hz, 2H), 7.95 (s, 1H), 7.92 (d, J ¼ 2.3 Hz,
NMR (75 MHz, DMSO‑d6) d (ppm) 167.49, 166.15, 165.98, 158.40,
142.37, 140.51, 132.37, 132.14, 130.97, 129.42, 128.26, 124.87, 124.74,
2H), 7.69e7.46 (m, 2H), 7.36 (d, J ¼ 7.9 Hz, 2H), 7.18 (dt, J ¼ 6.3,
3.1 Hz, 1H), 2.40 (s, 3H). 13C NMR (75 MHz, DMSO‑d6)
d
(ppm)
120.50, 115.73, 21.47, 11.86, 11.01.MS (ESI-TOF) m/z calc’d for
167.39,166.08,142.82,142.40,140.76,134.79,132.54,132.05,129.44,
129.19, 128.23, 126.88, 124.76, 121.33, 121.26, 120.41, 21.51. MS (ESI-
TOF) m/z calc’d for C19H15NO3S [MþH]þ 338.0851, found 338.0851.
C
20H18N2O4 [MþH]þ 351.1345., found 351.1340.
4.1.3.16. 3-(4-Methylbenzamido)-5-(1H-pyrrol-2-yl)benzoic
(11p). A grey powder, yield: 74%; HPLC purity: 96.84%. m. p.
158e159 ꢀC. 1H NMR (300 MHz, DMSO‑d6)
(ppm) 11.50 (s, 1H),
10.33 (s, 1H), 8.24 (d, J ¼ 23.6 Hz, 2H), 8.00e7.89 (m, 3H), 7.36 (d,
acid
4.1.3.10. 3-(4-Methylbenzamido)-5-(5-methylthiophen-2-yl)benzoic
acid (11j). A white solid, yield: 85%; HPLC purity: 99.90%. m. p.
d
8

R. Lu, Y. Wang, C. Liu et al.
European Journal of Medicinal Chemistry 216 (2021) 113313
J ¼ 7.8 Hz, 2H), 6.87 (s, 1H), 6.50 (s, 1H), 6.16 (s, 1H), 2.40 (s, 3H). 13
C
4.2.4. Pyroptosis assay
NMR (101 MHz, DMSO‑d6)
d
(ppm) 167.81, 165.91, 142.27, 140.23,
Cell suspensions from the air pouch were centrifuged at
3000 rpm for 5 min. For the pyroptosis measurement, active
Caspase-1 and PI fluorescence of samples were evaluated using
flow cytometry. Active caspase-1 was measured by FLICA 660
Caspase-1 Detection Kit (Immunochemistry Technologies, U.S.A.)
according to the manufacturer’s instruction and propidium iodide
(PI) was used to assess cells with membrane pores (Beyotime
Biotechnology, Nanjing, CN).
134.13, 132.17, 130.79, 129.43, 129.17, 128.19, 120.49, 120.16, 119.80,
119.06,109.75,106.61, 21.50. MS (ESI-TOF) m/z calc’d for C19H16N2O3
[MþH]þ 321.1239, found 321.1233.
4.1.3.17. 3-(4-Methylbenzamido)-5-(1H-pyrazol-3-yl)benzoic
(11q). A pale yellow solid, yield: 81%; HPLC purity: 98.04%. m. p.
68 ꢀC (decomposed) 1H NMR (400 MHz, DMSO‑d6)
(ppm) 10.48 (s,
acid
d
1H), 8.41 (t, J ¼ 1.7 Hz, 1H), 8.38 (t, J ¼ 2.0 Hz, 1H), 7.91 (d, J ¼ 7.9 Hz,
2H), 7.76 (t, J ¼ 1.7 Hz, 1H), 7.67e7.58 (m, 2H), 7.35 (d, J ¼ 7.9 Hz,
4.2.5. Microsomal stability assay
2H), 2.39 (s, 3H). 13C NMR (75 MHz, DMSO‑d6)
d (ppm) 166.56,
Liver microsomal incubations were conducted in triplicate. In-
cubation mixtures consisted of 20 mM of each test compound,
0.2 mg/mL human liver microsomes, and 1 mM cofactor NADPH in
a total volume of 400 mL of 100 mM potassium phosphate buffer
(pH 7.4 containing 5 mM MgCl2).The mixture was shaken for 5 min
for preincubation in a shaking water bath at 37 ꢀC. For metabolic
166.27, 142.58, 141.45, 133.88, 133.42, 132.60, 131.99, 129.43, 129.29,
128.23,126.94,126.58,121.88,120.38, 21.46. MS (ESI-TOF) m/z calc’d
for C18H15N3O3 [MþNa]þ 322.1192, found 322.1188.
4.2. Biological evaluation
stability studies, aliquots of 50
were collected at 0, 5, 10, 15, 30, and 45 min. After collection of
samples, the reaction was terminated with 100 L of EtOAc con-
mL of the incubation sample mixture
4.2.1. P2Y14R inhibitory activity screening
HEK293 cell lines stably expressing P2Y14R were plated in 384-
well plates approximately 24h before assay at the density of
m
taining the internal standard. The mixture was then centrifuged at
10000g to remove the protein and the supernatant was subse-
quently applied to LC-MS/MS analysis. The natural log of the
amount of parent compound remaining was plotted against time to
calculate the rate of disappearance and the half-life of the tested
compounds.
10 000 cells per well. Cells were incubated with 7.5 mL induction
buffer containing Forskolin, UDPG and various concentrations of
test compounds for 30 min at 37 ꢀC. Then P2Y14R inhibitory ac-
tivities were evaluated by detecting the levels of cAMP in
HEK293 cell lysates with cAMP-Glo™ Assay Kits (Promega, WI,
USA) followed the manufacturer’s instructions and calculated IC50
values.
4.2.6. Statistical analysis
Statistical analysis was performed using an Unpaired Student t-
test, or one-way ANOVA, in GraphPad Prism version 8 (GraphPad
software Inc., CA, USA). Alpha value was 0.05 and the confidence
interval was 95%. Data are presented as the means standard de-
viation (SD) of at least three independent experiments, and was
considered statistically significant when p < 0.05.
4.2.2. In vivo Murine gout model (MSU-induced inflammation)
assay
ICR male mice were bred and housed in a conventional animal
facility. All animals used for the experiments were between 8 and
12 weeks old. Mice were injected with monosodium urate (MSU)
crystals (1.5 mg) suspended in 10
(PBS, pH 7.4) to simulate acute gouty arthritis. Test compounds
were administered by intravenous injection (25.6 M/kg) into the
footpads. The positive control, PPTN (10 M), and 11m (10 M) was
mL of phosphate-buffered saline
4.3. Water solubility
m
m
m
The compound powder and PBS buffer (pH 7.4) were mixed in
Eppendorf tubes, followed by vigorously vortexed for 1 min and
sonicated for another 10 min at room temperature, then the
mixture was incubated with shaking overnight to ensure com-
pound saturation at room temperature. Compound suspensions
were centrifuged at 10 000 RPM for 15 min, and supernatants were
filtered through 0.22 mm syringe filters. Concentrations of com-
pound in the supernatants were determined by UV-3000 absorp-
tion spectrometry with standard curves and absorption extinction
coefficient in the same buffer pre-determined.
administered subcutaneously (sc), immediately after subcutaneous
injection of MSU crystals on the paw plantar surface. PPTN and 11m
was dissolved in DMSO. Control mice received an equivalent vol-
ume of PBS (pH 7.4). After 24 h of MSU stimulation, foot tissues
were extracted and stored for further analysis. As for the mice air
pouch inflammation model, it was formed in the backs of ICR mice
by subcutaneous injection of sterile air as previously described. On
day 7, MSU crystals (2 mg suspended in 1 ml of PBS) and PPTN or
11m were injected into the pouch. 24 h later, the air pouch lavages
were collected and processed for pyroptosis assay.
Declaration of competing interest
4.2.3. Western blot
The mice foot tissues were lysed in a RIPA buffer, and the lysate
was harvested by centrifugation at 12 000 rpm for 10 min. Aliquots
of protein (40 mg/lane) were separated by 8e12% SDS-PAGE and
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
transferred to polyvinylidene fluoride membranes (Millipore Cor-
poration, Boston, MA, USA). Subsequently, the membranes were
blocked for 2 h in 5% nonfat dry milk-TBS-0.1% Tween 20. 2 h later,
the blots were incubated with primary antibodies at 4 ꢀC overnight
followed by a 2 h incubation with a horseradish peroxidase-
conjugated secondary anti-rabbit antibody at room temperature.
Finally, protein bands were visualized using enhanced chem-
iluminescence (ECL) detection solution (Beyotime, China) and
scanned with a Chemiluminescence imaging system (Gel Catcher
2850, China).
Acknowledgements
This work has been financially supported by National Natural
Science Foundation of China (No. 82073685, 81773745 and
81872867), “Double First-Class” University project (CPU2018GF02),
Open-end Funds of Jiangsu Key Laboratory of Marine Pharmaceu-
tical Compound Screening (HY201902) and the ‘‘Qinglan Project’’ of
Jiangsu Province.
9

R. Lu, Y. Wang, C. Liu et al.
European Journal of Medicinal Chemistry 216 (2021) 113313
P.G. Szekeres, et al., GPR105, a novel Gi/o-coupled UDP-glucose receptor
expressed on brain glia and peripheral immune cells, is regulated by immu-
nologic challenge: possible role in neuroimmune function, Mol. Brain Res. 118
(1e2) (2003) 10e23.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113313.
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