Development of FABP4/5 inhibitors with potential therapeutic effect on type 2 Diabetes Mellitus

Article abstract of DOI:10.1016/j.ejmech.2021.113720

Fatty acid-binding protein 4 (FABP4) and fatty acid-binding protein 5 (FABP5) are promising therapeutic targets for the treatment of various metabolic diseases. However, the weak potency, low selectivity over FABP3, or poor pharmacokinetic profiles of currently reported dual FABP4/5 inhibitors impeded further research. Here, we described the characterization of a series of dual FABP4/5 inhibitors with improved metabolic stabilities and physicochemical properties based on our previous studies. Among the compounds, D9 and E1 exhibited good inhibitory activities against FABP4/5 and favorable selectivity over FABP3 in vitro. In cell-based assays, D9 and E1 exerted a decrease of FABP4 secretion, a strong anti-lipolytic effect in mature adipocytes, and suppression of MCP-1 expression in THP-1 macrophages. Moreover, D9 and E1 possessed good metabolic stabilities in mouse hepatic microsomes and acceptable pharmacokinetics profiles in ICR mice. Further in vivo experiments showed that D9 and E1 could potently decrease serum FABP4 levels and ameliorate glucose metabolism disorders in obese diabetic db/db mice. These results demonstrated that D9 and E1 could serve as lead compounds for the development of novel anti-diabetic drugs.

Full text of DOI:10.1016/j.ejmech.2021.113720

European Journal of Medicinal Chemistry 224 (2021) 113720
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Development of FABP4/5 inhibitors with potential therapeutic effect
on type 2 Diabetes Mellitus
,
,
c
,
, **
Yu-Long He a 1, Meng-Ting Chen b 1, Ting Wang b, Ming-Ming Zhang a, Ying-Xia Li a
,
He-Yao Wang b ***, Ning Ding a
a School of Pharmacy, Fudan University, Shanghai, 201203, China
b State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China
c University of Chinese Academy of Sciences, Beijing, 100049, China
,
, *
a r t i c l e i n f o
a b s t r a c t
Article history:
Fatty acid-binding protein 4 (FABP4) and fatty acid-binding protein 5 (FABP5) are promising therapeutic
targets for the treatment of various metabolic diseases. However, the weak potency, low selectivity over
FABP3, or poor pharmacokinetic profiles of currently reported dual FABP4/5 inhibitors impeded further
research. Here, we described the characterization of a series of dual FABP4/5 inhibitors with improved
metabolic stabilities and physicochemical properties based on our previous studies. Among the com-
pounds, D9 and E1 exhibited good inhibitory activities against FABP4/5 and favorable selectivity over
FABP3 in vitro. In cell-based assays, D9 and E1 exerted a decrease of FABP4 secretion, a strong anti-
lipolytic effect in mature adipocytes, and suppression of MCP-1 expression in THP-1 macrophages.
Moreover, D9 and E1 possessed good metabolic stabilities in mouse hepatic microsomes and acceptable
pharmacokinetics profiles in ICR mice. Further in vivo experiments showed that D9 and E1 could potently
decrease serum FABP4 levels and ameliorate glucose metabolism disorders in obese diabetic db/db mice.
These results demonstrated that D9 and E1 could serve as lead compounds for the development of novel
anti-diabetic drugs.
Received 20 May 2021
Received in revised form
11 July 2021
Accepted 23 July 2021
Available online 24 July 2021
Keywords:
Type 2 diabetes mellitus
Dual FABP4/5 inhibitors
Anti-lipolytic and anti-inflammatory effects
Metabolic stability
Anti-diabetic activities
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
AFABP) is predominantly expressed in adipocytes and macrophages
[5], which functions as an intracellular lipid chaperone and is
Type 2 diabetes (T2D) is a chronic metabolic disease charac-
terized by persistent hyperglycemia associated with defects in in-
sulin secretion and insulin action [1], which is considered to be a
growing global health problem [2]. The progression of the disease
impairs insulin action, disrupts glucose homeostasis and produces
hyperglycemia, as well as aggravates insulin resistance of periph-
eral tissues [3]. Considering the limitations of the current hypo-
glycemic drugs [4], there is an imperative need for safe and efficient
therapeutic agents to develop personalized treatment for T2D
patients.
involved in modulating glucose and lipid metabolism and inflam-
mation responses [6]. It has been demonstrated that Fabp4-defi-
cient mice acquired modest therapeutic benefits for genetic or
dietary obesity-related hyperinsulinemia and insulin resistance
[7,8]. Additionally, deletion of Fabp4 decreased lipolysis both
in vitro and in vivo [9,10] and attenuated the inflammatory response
in macrophages [11]. It's worth noting that lack of Fabp4 in adi-
pocytes led to a compensatory upregulated expression of FABP5, a
minor isoform of FABP in adipocytes [12], which might be part of
the reason for the modest phenotype of Fabp4ꢀ/ꢀ mice. Similarly,
ablation of Fabp5 enhanced glucose transport in insulin-stimulated
adipocytes and increased systemic insulin sensitivity in dietary and
genetically obese mice [13]. Compared with Fabp4 or Fabp5 single-
knockout mice, Fabp4 and Fabp5 double-knockout mice (Fabp4ꢀ/ꢀ
Fabp5ꢀ/ꢀ) exhibited a stronger protection against T2D, non-
alcoholic fatty liver disease and atherosclerosis [14,15], indicating
that targeting FABP4 and FABP5 simultaneously might be a novel
therapeutic approach for the treatment of metabolic diseases. In
Fatty acid binding protein 4 (FABP4, also known as aP2 or
* Corresponding author.
** Corresponding author.
*** Corresponding author.
E-mail addresses: liyx417@fudan.edu.cn (Y.-X. Li), hywang@simm.ac.cn
(H.-Y. Wang), dingning@fudan.edu.cn (N. Ding).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2021.113720
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

Y.-L. He, M.-T. Chen, T. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113720
addition to performing intracellular biological functions, FABP4 can
be secreted from adipocytes in associated with lipolysis and act as
an adipokine to exert various effects on multiple peripheral tissues,
including hepatic glucose production, cardiomyocyte contraction
and insulin secretion [16e19]. Elevated serum FABP4 level is closely
correlated with the development of a variety of metabolic diseases
such as obesity, T2D and cardiovascular diseases [14,20]. Although
some agents have been reported to reduce serum concentration of
FABP4 [21,22], the modulation of plasma FABP4 level by dual
FABP4/5 inhibitors has not been reported.
metabolic stability, acceptable pharmacokinetic profiles, and clear
in vivo effects.
2. Results and discussion
2.1. Compounds design
Compound A16 was showed to be good FABP4/FABP5 inhibitor
in our previous work (IC50 ¼ 1.97
m
M for FABP4; IC50 ¼ 4.85
mM for
FABP5). However, further investigations indicated that A16 is
Some FABP inhibitors have been published over the past de-
cades. Several classes of representative FABP4 and FABP4/5 in-
hibitors were listed below (Fig. 1). BMS309403 (A) is a specific
FABP4 inhibitor that improved glucose homeostasis and insulin
sensitivity, reduced adipose tissue inflammation in diabetic mouse
models [23]. The 2-(phenylamino) benzoic acid FABP4 inhibitor B
displayed strong anti-inflammatory activity in vitro and in vivo [24].
Compounds C-G were effective FABP4/5 inhibitors synthesized for
the treatment or prophylaxis of T2D, atherosclerosis, chronic kid-
ney diseases, or nonalcoholic steatohepatitis. While these dual
FABP4/5 inhibitors were only profiled for inhibitory activity against
human FABP4/5 (Ki or IC50), results of the pharmacological evalu-
ation in animal models have not been revealed [25e29]. The dual
FABP4/5 inhibitor H, featured as a non-carboxylic-acid-containing
compound, was reported to show significant potency towards
FABP4 and FABP5, and inhibit lipolysis in 3T3-L1 adipocytes and
suppress MCP-1 release from THP-1 macrophages as well [30].
However, there is still no FABP inhibitor has entered the clinical
research phase in view of some ineligible issues, including poor
metabolic properties, toxicities, and potent drug resistances [31].
Therefore, it still needs to make more efforts to bring optimal
molecules into therapy.
In our previous study, two novel dual FABP4/5 inhibitors (A16
and B22) were disclosed based on a potent FABP4 inhibitor 16dk
[32]. Unfortunately, the poor hepatic microsomal stability of A16
and the low dynamic solubility of B22 hindered further in-
vestigations. In this work, to improve the metabolic stability of A16
and optimize the physicochemical properties of B22, two series of
novel dual FABP4/5 inhibitors (D and E, respectively.) basing on
these two compounds were designed, synthesized, and compre-
hensively evaluated in vitro and in vivo. As the results, compounds
D9 and E1 were discovered to show high inhibitory activity against
FABP4/5, strong anti-lipolytic effect on mature adipocytes, moder-
ate anti-inflammatory effect on THP-1 macrophages, good
metabolically labile in mouse liver microsomes (MLM), since the T1/
is only 23.5 min. The preliminary studies on the metabolism
2
products of A16 in MLM implied that the ether bond (CeO) might
be the major potential metabolic site (data not shown). Therefore,
the more stable CeC bond was introduced into the molecule to
replace the corresponding CeO ether bond (compound C1). To our
delight, the stability of compound C1 was greatly improved (T1/
> 500 h in MLM) as compared with that of A16. However, the
2
FABP4 inhibitory activity of C1 was significantly decreased
(IC50 ¼ 15.65
m
M for FABP4; IC50 ¼ 3.75
mM for FABP5) in contrast
with A16. Besides, the lipophilicity of C1 seems higher than ex-
pected (ClogP ¼ 7.8). Thus, to improve the FABP4/5 potency and the
druggability of C1, Series D compounds basing on the structure of
C1 were designed, which keeping the CeC bond linker in the side
chain between the naphthalene core and the aromatic sub-
stitutions (R1).
As for B22, another representative FABP4/5 inhibitor disclosed
in our previous work, displayed good potency against FABP4/5
(IC50 ¼ 2.63
m
M for FABP4; IC50 ¼ 8.97
mM for FABP5). However, B22
showed no lipolytic inhibition in 3T3-L1 adipocytes. Previous work
[32,33] indicated that the carboxylic acid is the key for B22 to keep
the FABP4/5 inhibitory activities. Interestingly, a slight modification
of B22 by inserting a CH2 group between the key carboxylic acid
and the phenyl group (E1) solved the above problems associated to
B22. Compound E1 not only exhibited excellent inhibitions against
both FABP4/5 (IC50 ¼ 3.78
m
M for FABP4; IC50 ¼ 5.72
mM for FABP5)
but also showed impressive activities on 3T3-L1 adipocytes and
THP-1 macrophages. Inspired by this work, series E compounds
with various R2 group were designed by keeping the phenylacetic
acid group in B22 to find more potent compounds (Fig. 2).
2.2. Chemistry
The preparation of D1-12 is depicted in Scheme 1. The synthesis
Fig. 1. Representative structures of FABP inhibitors.
2

Y.-L. He, M.-T. Chen, T. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113720
Fig. 2. The Structure optimization strategy based on A16 and B22.
Scheme 1. Synthesis of D1-12. Reagents and conditions: (i) HSO3Cl, CHCl3, 0 ꢁC, 1 h; (ii) NaCl solution; 61 % over 2 steps; (iii) SOCl2, DMF, 0 ꢁC, 1 h, 57 %; (iv) Py, acetone, 55 ꢁC, 1 h,
55 %; (v) PdCl2(dppf), CH2Cl2, DMF, K2CO3, H2O, 100 ꢁC, 12 h; (vi) NaOH, H2O, MeOH, 1 h.
commenced with the commercially available 1-bromonaphthalene
(1), which was converted to intermediate 2 by treated with chlor-
osulfonic acid and saturated sodium chloride solution. After
transformed to the corresponding sulfonyl chloride by reacted with
thionyl chloride in DMF, compound 3 coupled with methyl 5-
amino-2-fluorobenzoate hydrochloride (4) to yield key intermedi-
ate 5. Then 5 cross-coupled with a panel of aromatic boric acids or
borates via Suzuki reaction to provide the corresponding products.
After hydrolyzing the methyl esters, the designed compounds D1-
12 were prepared.
The preparation of compounds E1-14 was outlined in Scheme 2,
which adopted a similar strategy to that of series D (Scheme 2). The
commercially available 1-Naphthol (7) was converted to the cor-
responding sulfonyl chloride 10 through three steps [32]. After
condensed with phenylamine (11), the key intermediate 12 yielded
the corresponding cross-coupling products via Suzuki reaction.
After hydrolyzing the methyl esters, the designed compounds E1-
14 were furnished.
2.3. Biological evaluations
2.3.1. In vitro FABP4 and FABP5 inhibitory activity assays
The inhibitory activities of the compounds against FABP4/5 were
evaluated with the 8-anilino-1-naphthalene-sulfonic acid (1, 8-
ANS) displacement assay as previously reported [32]. The re-
ported FABP4 inhibitor BMS309403 and dual FABP4/5 inhibitor
compound H were used as the positive controls.
The results for D1-12 are summarized in Table 1. Compared with
A16 (IC50: 1.97
played a slight decline in FABP4 (IC50: 4.84
(IC50: 18.38 M). These results indicated that replacement of the
mM for FABP4; IC50: 4.85
m
M for FABP5), D1 dis-
mM) and FABP5 potency
m
ether side chain (CeOeC) of A16 to a CeC linked phenyl was well
tolerated. Further changing of the phenyl group of D1 to a 3, 5-
dimethylisoxazole or pyridyl group, compounds D2 and D3,
respectively, still had moderate inhibition against FABP4 but weak
inhibition against FABP5. Since the phenyl-containing compound
D1 seemed better than D2 and D3 at the current stage, we carried
3

Y.-L. He, M.-T. Chen, T. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113720
Scheme 2. Synthesis of compounds E1-E14. Reagents and conditions: (i) NBS, diethylamine, CH2Cl2, 40 ꢁC, overnight, 72 %; (ii) CH3I, KOH, CH3CN, 40 ꢁC, 12 h, 85 %; (iii) HSO3Cl,
CH3Cl, 0 ꢁC to rt.,82 %; (iv) Pyridine, acetone, 55 ꢁC, 1 h, 61 %; (v) ReB(OH)2, K2CO3, PdCl2(dppf). CH2Cl2, DMF, H2O; (vi) NaOH, H2O, MeOH, 1 h, rt.
Table 1
In vitro FABP4 and FABP5 inhibitory activities of Series D.
Compound
R1
FABP4
@25
FABP5
@25
m
M
IC50
(
m
M)
m
M
IC50 (mM)
Inhibition %
Inhibition %
D1
D2
82 %
4.84 0.19
N.d
62 %
18.38 0.13
N.d
61 %
19 %
D3
D4
D5
D6
D7
52 %
80 %
73 %
40 %
74 %
N.d
21 %
59 %
52 %
64 %
79 %
N.d
7.68 0.92
8.24 0.13
N.d
N.d
N.d
N.d
12.53 0.71
8.20 1.03
D8
74 %
86 %
85 %
85 %
78 %
11.91 0.31
4.68 0.13
4.70 0.19
5.00 0.19
7.84 0.17
67 %
75 %
69 %
74 %
84 %
16.84 1.90
10.72 0.33
14.03 1.03
11.16 0.33
6.33 0.83
D9
D10
D11
D12
A16
Compound H
BMS309403
92 %
97 %
99 %
1.97 0.15
6.34 0.01
2.36 0.02
66 %
52 %
N.d
4.85 1.03
23.42 0.32
N.d
N.d represent not determined.
4

Y.-L. He, M.-T. Chen, T. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113720
out the detailed SAR investigations focusing on the substitutions of
the phenyl of D1. Introduction of a para-substituent on the phenyl
group afforded compounds D4-6, which all demonstrated
decreased potency against FABP4 and comparative inhibitory rates
against FABP5. Compounds containing trifluoromethoxy (D7) and
bulky isopropyl group (D8) on the ortho position of the phenyl also
did not increase the FABP4 inhibition. Introducing halogens or/and
methyl to the phenyl group, D9-12 displayed similar inhibitory
activity against FABP4 and improved FABP5 potency to D1.
phenyl of E1, compound E2 kept the inhibitory potency on FABP4
but decreased the FABP5 inhibition around 3 folds. When a single
halogen (E3 or E4) or trifluoromethyl (E5) was incorporated on the
ortho-substitutions of the phenyl, there was a decline in FABP4
potency (Inhibition rate: 56 % ~ 87 %). Expanding the substituent
from methyl (E6) to ethyl (E7) and isopropyl (E8), the inhibitory
potency on FABP4 decreased. It should be noted that compound E8
bearing 2-isopropyl group was the most potent FABP5 inhibitor
that has been reported ever. Incorporation of a hydroxyl group (E9)
seemed well-tolerated in FABP4 and FABP5 activity. Further mod-
ifications on the ortho-substitutions of the phenyl group by the
introduction of two halogens (E10-E14) gave similar inhibitory
potency on FABP4 but a slight decreased FABP5 inhibition to E1.
The direct binding affinities of BMS309403, D9 and E1 to FABP4
The results for E1-14 are summarized in Table 2. Optimization of
B22 around the key carboxylic group led to the discovery of E1,
which showed very good inhibitory activities both on FABP4 (IC50
:
3.78 M) and FABP5 (IC50: 5.72 M). These results encouraged us to
m
m
explore the SAR around E1 deeply. Removing the methyl on the side
Table 2
In vitro FABP4 and FABP5 inhibitory activities of Series E.
Compound
R2
FABP4
@25
FABP5
m
M
IC50
(
mM)
@25
m
M
IC50 (mM)
Inhibition %
Inhibition %
E1
94 %
3.78 0.03
72 %
5.72 0.47
E2
E3
95 %
87 %
4.71 0.02
5.25 0.12
59 %
50 %
14.64 1.19
12.36 0.57
E4
E5
77 %
56 %
8.02 0.29
N.d
63 %
63 %
7.57 0.18
7.72 1.36
E6
E7
80 %
63 %
5.16 0.20
62 %
74 %
7.72 1.67
3.20 0.22
14.28 1.36
E8
E9
50 %
N.d
66 %
55 %
2.90 0.18
N.d
86 %.
8.03 1.24
E10
96 %
3.14 0.05
55 %
10.95 1.67
E11
E12
E13
90 %
88 %
84 %
4.77 0.11
5.00 0.40
6.45 0.07
62 %
64 %
63 %
6.08 1.21
12.82 0.99
11.18 2.81
E14
52 %
N.d
60 %
13.33 5.67
B22
Compound H
BMS309403
95 %
97 %
99 %
2.63 0.11
6.34 0.01
2.36 0.02
67 %
52 %
N.d
8.97 0.98
23.43 0.32
N.d
N.d represent not determined.
5

Y.-L. He, M.-T. Chen, T. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113720
and FABP5 were measured by using microscale thermophoresis
(MST). Results showed that the Kd values of BMS309403, D9 and E1
within the acceptable range. Besides, compound E4 and E8, which
displayed strong anti-lipolytic activities in mature adipocytes,
exhibited poor metabolic stability in MLM. Considering the above
results, D9 and E1 were selected to futher explore the anti-
inflammatory effects on THP-1 macrophages.
for FABP4 were 1.67
mM, 11.06 mM and 3.14 mM, respectively
(Fig. S3), indicating that D9 and E1 showed high affinity to FABP4 as
similar as BMS309403. The Kd values of BMS309403, D9 and E1 to
FABP5 were 2794.5
mM, 104.9 mM and 14.09 mM, respectively
(Fig. S4), which suggested E1 has a strong binding affinity to FABP5.
2.3.5. Anti-inflammatory effects of dual FABP4/5 inhibitors on THP-
1 macrophages
2.3.2. FABP3 selectivity evaluation
FABP4 deficiency in macrophages could suppress inflammatory
signaling responses by decreasing the production of pro-
inflammatory cytokines such as MCP-1/CCL2 [37]. It had also
been demonstrated that BMS309403 reduced the release of MCP-1
from THP-1 macrophages in a FABP4-dependent manner [23]. In
our study, based on the results of anti-lipolytic activity and liver
microsomal stability described above, D9 and E1 were selected to
investigate the anti-inflammatory effects on THP-1 macrophages,
BMS309403 was chosen as positive control. Firstly, the cytotoxicity
was detected by the CCK-8 assay, and the results showed that all the
compounds had no effects on the cell viability of THP-1 cells at the
It is worth noting that Fabp3-deficient mice suffered from
reduced tolerance to physical activity and regional cardiac hyper-
trophy [34]. Considering the potential cardiotoxicity caused by
FABP3 inhibition, it is of significant importance to consider the
FABP3 selectivity while developing dual FABP4/5 inhibitors. Basing
on the above screening, dual FABP4/5 inhibitors (D9, D12, E1) were
evaluated for their selectivity against FABP3/4/5 (Table 3).
BMS309403 and compound H were used as the positive controls.
The results showed all the selected compounds had much lower
inhibitory activities than those of the controls. Besides, the selec-
tivities of these compounds against FABP4 and FABP5 over FABP3
are excellent as compared with those of the controls.
concentration of 50
mM (Fig. 4A). The expression of pro-
inflammatory cytokines MCP-1 were robustly increased after the
stimulation of LPS, and compound D9, E1 and BMS309403
dramatically decreased the expression of MCP-1 in LPS-stimulated
THP-1 macrophages (Fig. 4B). The data suggested that dual FABP4/5
inhibitors may exert anti-inflammatory effect by inhibiting the
expression of MCP-1 in macrophages.
2.3.3. Effects of dual FABP4/5 inhibitors on lipolysis in mature
adipocytes
Pharmacological inhibition or deletion of FABP4 in adipocytes
exhibited reduced efficiency of lipolysis [35,36]. Therefore, we
selected several potent dual FABP4/5 inhibitors from series D (D9,
D10, D11, D12) and series E (E1, E4, E8, E11) to investigate their anti-
lipolytic effects on Forskolin (FSK)-stimulated mature adipocytes,
with the FABP4 inhibitor BMS309403 and dual FABP4/5 inhibitor
B22 as the positive controls. Firstly, the Cell Counting Kit (CCK-8)
assay was carried out to examine the cytotoxicity effects of the
selected compounds on 3T3-L1 preadipocytes at the concentration
2.4. In vivo pharmacokinetic properties
Pharmacokinetic studies of D9, E1 and BMS309403 was con-
ducted in ICR (CD1) mice at 1 mg/kg and 3 mg/kg doses for intra-
venous (iv) and oral (po) administration, respectively. As shown in
Table 5, compared with the FABP4 inhibitor BMS309403, D9 and E1
possessed more favorable PK profiles with a relative longer half-life
(T1/2 (poa): D9 ¼ 1.36 h; E1 ¼ 6.04 h vs. BMS309403 ¼ 0.63 h),
higher system exposure (AUC (poa): D9 ¼ 1093 (h*ng/mL);
E1 ¼ 1742 (h*ng/mL) vs. BMS309403 ¼ 90 (h*ng/mL)), lower
plasma clearance level (CL: D9 ¼ 8.73 (mL/min/kg); E1 ¼ 8.56 (mL/
min/kg) vs. BMS309403 ¼ 44.3 (mL/min/kg)) and better oral
bioavailability (F: 18.4 % for D9 and 29.9 for E1 vs. 7 % for
BMS309403). All these results indicated that D1 and E1 were
suitable for in vivo efficacy evaluation.
of 100
nificant effects on the cell viability of adipocytes. The lipolysis
experiment was then performed at the concentration of 80 M, and
mM. As shown in Fig. S1, all of the compounds had no sig-
m
the result was demonstrated in Fig. 3A and B. FSK significantly
increased the concentration of glycerol released from mature adi-
pocytes, and our compounds as well as BMS309403 robustly
inhibited the FSK-stimulated lipolysis in 3T3-L1 cells, while B22
exhibited no anti-lipolytic effect, validating the feasibility of series
E design strategy. Among the compounds, D9, D11, E4 showed
comparative potency with that of BMS309403, while E1 and E8
showed superior anti-lipolytic activity than that of BMS309403.
2.5. In vivo anti-diabetic efficacy of D9 and E1
2.3.4. In vitro liver microsomal stability
To gain insight into the potential therapeutic effects of D9 and
E1 in vivo, the studies in obese diabetic db/db mice were carried out.
The FABP4 inhibitor BMS309403 was used as the positive control.
Six-week-old C57BL/KsJ-db/db mice were orally administrated
with D9, E1 and BMS309403 at a dose of 50 mg/kg for 8 weeks.
First, all the three compounds D9, E1 and BMS309403 had no
obvious effects on body weight (Fig. S2A). As showed in Fig. 5A, D9
and E1 reduced the fasting blood glucose levels by 32.4 % and 28.1 %
at the 7th week as compared with that of the vehicle group, while
Based on the potency at the molecular- and cellular-level
screenings described above, dual FABP4/5 inhibitors D9, D11, E1,
E4 and E8 were subjected to evaluate the in vitro metabolic stability
in MLM. The results were summarized in Table 4. As compared with
A16, compounds D9 and D11 exhibited improved metabolic sta-
bility in MLM, reflected in the remarkable increase in the half-time
and a sharp decline in clearance ratio. In contrast with B22, com-
pound E1 exhibited a slight decrease of liver microsomal stability
Table 3
Selectivity of representative FABP4/5 inhibitor toward FABP3.
Compound
FABP3 IC50
(m
M)
FABP4 IC50
(
mM)
FABP5 IC50
(m
M)
FABP3/FABP4
FABP3/FABP5
D9
D12
E1
BMS309403
Compound H
103.70 3.54
103.05 0.49
84.36 1.91
35.78 2.12
1.95 0.01
4.68 0.13
7.84 0.17
3.78 0.03
2.36 0.02
6.34 0.01
10.72 0.33
6.33 0.83
5.72 0.47
N.d
22.15
13.14
22.31
15.16
0.31
9.67
16.27
14.74
N.d
23.42 0.32
0.08
N.d represent not determined.
6

Y.-L. He, M.-T. Chen, T. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113720
Fig. 3. (A) Effect of representative Series D compounds and BMS309403 on the release of glycerol from FSK-stimulated mature adipocytes at the concentration of 80
mM. (B) Effect of
representative Series E compounds, BMS309403 and B22 on the release of glycerol from FSK-stimulated mature adipocytes at the concentration of 80
****p < 0.0001vs. FSK group.
mM (n ¼ 6/group). **p < 0.01,
Table 5
In Vivo PK parameters for D9, E1, and BMS309403 in mice.
Table 4
Mouse liver microsomal stability of representative compounds.
Parameter
D9
E1
BMS309403
Compd.
T1/2 (min)
Clint (ml/min/gpot)
T1/2 (poa) (h)
T1/2 (iva) (h)
Cmax (ng/mL)
AUC (po) (h*ng/mL)
AUC (iv) (h*ng/mL)
Cl (mL/min/kg)
F (%)
1.36 0.13
2.88 2.16
782 463
1093 21
1981 484
8.73 2.52
18.4
6.04 7.37
1.78 0.10
478 195
1742 516
1941 383
8.56 1.69
29.9
0.63 0.23
1.53 0.17
151 58
90 29
427 185
44.3 20.1
7
A16
D9
D11
B22
E1
23.5
91
89.3
23.1
23.8
20.6
23.8
48.8
139.1
88.4
102.1
88.3
43.1
15.1
E4
E8
a
po (3 mg/kg); iv (1 mg/kg). Results are expressed as the mean SEM (n ¼ 3).
BMS309403 treatment exhibited the better reducing levels by
38.9 % at the same dose. Subsequently, the results of OGTT (Fig. 5B
and C) and ITT (Fig. 5D and E) along with Western blot of liver
(Fig. 5F and G) revealed a significant improvement of glucose
metabolism and insulin resistance in db/db mice treated with D9
and E1. In addition, D9, E1 and BMS309403 treatment significantly
elevated the serum level of high-density cholesterol (HDL-C)
(Fig. 5H), and compound D9 significantly reduced serum low-
density lipoprotein cholesterol (LDL-C) level (Fig. 5I). However,
the selected compounds had no effects on the serum level of tri-
glyceride (TG) and total cholesterol (TCH) (Figs. S2BeC). Further
investigations showed that the serum FABP4 level was dramatically
decreased by D9 and E1 (Fig. 5J). It had been reported that FABP4
was secreted from adipocytes and elevated circulating FABP4 was
associated with various metabolic diseases [14,38]. Reduced
circulating FABP4 levels was associated with improved metabolic
benefits [39].
We subsequently examined the effects of D9 and E1 on the
release of FABP4 from mature adipocytes. As shown in Fig. 6AeC,
compared with the control group, the FABP4 levels in the super-
natant were dramatically reduced by D9 and E1 at the concentra-
tion of 50
mM, which might explain the reduction of circulating
FABP4 levels.
Overall, the above investigations revealed that D9 and E1 acted
as effectively as BMS309403 on reducing the fasting blood glucose
Fig. 4. (A) Effects of compounds on the viability of THP-1 macrophages. Cell viability was determined by CCK-8 assay (n ¼ 6/group). (B) Effects of compounds on the gene expression
of MCP-1 in LPS-stimulated THP-1 macrophages. Gene expression level of cytokines was measured by RT-qPCR (n ¼ 4/group). ***p < 0.001, ****p < 0.0001 vs. LPS group.
7

Y.-L. He, M.-T. Chen, T. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113720
Fig. 5. Effects of D9, E1 and BMS309403 on glucose and lipid metabolism in db/db mice. (A) Six-hour fasting blood glucose level after 7-weeks of administration. (BeC) After 45-
days of administration, the mice were subjected to oral glucose tolerance test (OGTT). (DeE) After 51-days of administration, mice were subjected to insulin tolerance test (ITT).
Blood glucose level of indicated time points were measured and the AUC was calculated. (FeG) Representative blots of hepatic Akt and p-Akt (Ser473) and the statistical results of
bands analysis data. (HeJ) Serum HDL-C, LDL-C and FABP4 level of mice in the vehicle, compound-treated and lean groups. All serum parameters were measured using assay kits.
*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. vehicle group. Values are given as mean S.E.M (n ¼ 10/group).
levels, improving the oral glucose tolerance and insulin resistance
in the obese diabetic db/db mice, and exerted a certain degree of
amelioration on lipid metabolism disorders. It should be noted that
BMS309403 is not a neat FABP4 inhibitor and it could also activate
AMPK signaling pathway and thus promotes glucose uptake in
skeletal muscle cells [40]. The inhibition of FABP4 and simulta-
neous activation of AMPK signaling pathway made BMS309403
play strong effects on the anti-diabetic tests. On the other hand, our
compounds are potent dual FABP4/5 inhibitors. As mentioned
above, inhibition of Fabp4 in adipocytes led to the compensatory
upregulated expression of FABP5 in adipocytes [12]. FABP5 could
also cause the problems on glucose and lipid metabolism as FABP4
did [12]. Simultaneously targeting FABP4 and FABP5 should be a
novel therapeutic approach for the treatment of metabolic diseases.
Thus, our potent dual FABP4/5 inhibitors D9 and E1 could still be
regarded as useful lead compounds for personalized antidiabetic
treatment, even if they did not show strong advantages on glucose
and lipid metabolism in db/db mice as compared with BMS309403
from the data of Fig. 5.
8

Y.-L. He, M.-T. Chen, T. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113720
4.2. General method of synthesizing series D and series E
4.2.1. Preparation of intermediate 2
To a solution of 1-bromonaphthalene (2.0 g, 9.66 mmol) in
chloroform (15 ml) was added chlorosulfonic acid (1.69 g,
14.49 mmol) dropwise in ice bath. Then the mixture was moved to
room temperature and stirred for another 1 h. The mixture was
poured into cold water, extracted with ethyl acetate. Next, satu-
rated sodium chloride solution was added to the above ethyl ace-
tate solution under stirring conditions and a white solid was
precipitated, the suspension was filtered, and the residue was
washed with dichloromethane. The washed residue was dried to
obtain the desired derivatives of sodium 4-bromonaphthalene-1-
sulfonate as white solid (1.8 g, 61 %).
4.2.2. Preparation of intermediate 3
To a solution of intermediate 2 (1.5 g, 4.85 mmol) in DMF (8 mL)
was added thionyl chloride (2.89 g, 24.26 mmol) at 0 ꢁC and then
stirred at room temperature for another 1 h. The reaction system
was poured into the ice water, extracted with extracted with ethyl
acetate three times. The combined ethyl acetate layer was
sequentially washed with water, dried with anhydrous Na2SO4, and
concentrated to give the desired product as a brown oil (0.87 g,
57 %), which was used for the next step without further
purification.
Fig. 6. (A) Immunoblots of FABP4 and glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) using the cell lysate (CL) and conditioned medium (CM) for 24 h of mature
adipocytes at the concentration of 50
mM. (BeC) Densitometric analysis of the im-
munoblots of FABP4 in CL and CM. Data are expressed as mean
S.E.M and are
representative of three independent experiments. *p < 0.05, **p < 0.01 vs. Control
group.
3. Conclusion
4.2.3. Preparation of intermediate 5
To a solution of intermediate 3 (800 mg, 2.62 mmol) in acetone
(10 mL) was added methyl 5-amino-2-fluorobenzoate hydrochlo-
ride (538 mg, 2.62 mmol) and pyridine (0.63 ml, 7.85 mmol). The
mixture then was refluxed for 2 h and evaporated under vacuum.
The residue was diluted with water and extracted with ethyl ace-
tate (15 mL) three times. The combined ethyl acetate layer was
sequentially washed with brine, dried with anhydrous Na2SO4, and
concentrated to give the crude product. The crude was purified by
column chromatography with petroleum/ethyl acetate to yield in-
termediate 4 as a prey solid (0.63 g, 54.9 %).
In summary, we have prepared a series of potent and selective
dual FABP4/5 inhibitors with potential therapeutic effects in dia-
betic mouse model based on our previous study. Systematic SAR
explorations resulted in the identification of D9 and E1 with
favorable FABP4/FABP5 inhibitory activity for further investigation.
Compound D9 and E1 could not only potently inhibit the lipolysis
in FSK-stimulated mature adipocytes as well as the release of
FABP4, but also decreased the expression of MCP-1 in LPS-
stimulated THP-1 macrophages. Furthermore, compounds D9 and
E1 showed good metabolic stability in mouse liver microsomes and
decent PK properties in mice, and long-term oral administration of
D9 and E1 at the dose of 50 mg/kg could effectively ameliorate
glucose metabolic disorders in obese diabetic db/db mice. Addi-
tionally, the serum FABP4 level was prominent decreased by D9 and
E1. These data indicated that D9 and E1 could be regarded as useful
lead compounds to be developed into personalized oral antidia-
betic agents with acceptable efficacy and safety.
4.2.4. General procedure for the preparation of intermediates 6a-l
To a suspension of the intermediate 5 (1 equiv) in DMF (8 mL)
was sequentially added appropriate substituted aryl boric acid (1.3
equiv), K2CO3 (2 equiv), PdCl2 (dppf).CH2Cl2 (0.1 equiv) and water
(0.5 mL). After refluxing for 12 h under N2 atmosphere, the reaction
mixture was cooled and then poured into H2O. The mixture was
extracted with extracted with EtOAc (2x), washed with saturated
NaCl solution, dried over anhydrous Na2SO4, and concentrated in
vacuo. The residue was purified by silica gel column chromatog-
raphy to afford the target compounds as a white solid.
4. Experiment section
4.2.5. General procedure for the preparation of D1e12
4.1. General information
To a solution of intermediate 6a-6l (1 equiv) in methanol (8 mL)
was added 1 N NaOH solution (10 equiv) and stirred at room
temperature for 1 h. Then the solvent was removed under vacuum,
the residue was acidified to pH ¼ 2 with HCl (1 M) solution and
white solid precipitated. The mixture was extracted with ethyl
acetate twice and the combined organic layer was dried over
anhydrous sodium sulfate and concentrated to provide the target
compounds as white solid.
All the reagents and solvents were supplied by standard sup-
pliers without further purification. Reactions were monitored by
analytical thin layer chromatography (TLC) and visualized with a
UV lamp (254 nm). The column chromatography was carried out
using silica gel (200e300 mesh). 1H NMR and 13C NMR spectra were
recorded with a 400 MHz Varian or Bruker 600 MHz NMR spec-
trometer at ambient temperature, respectively. Coupling constants
(J) values are described as hertz and Chemical shifts (
d
) of NMR
4.2.5.1. 2-Fluoro-5-((4-phenylnaphthalene)-1-sulfonamido) benzoic
acid (D1). White solid, yield: 70 %. H NMR (400 MHz, DMSO‑d6)
1
spectra are expressed in parts per million (ppm) units downfield
from tetramethylsilane. Mass spectra (MS) of compounds were
measured on Agilent 6120 quadrupole LC/MS. High resolution mass
spectrometry (HRMS) determinations for all new compounds were
performed on AB SCIWX TRIPLETOF 5600þ.
d
13.34 (s, 1H), 10.96 (s, 1H), 8.78 (d, J ¼ 8.6 Hz, 1H), 8.23 (d,
J ¼ 7.5 Hz, 1H), 7.87 (d, J ¼ 8.5 Hz, 1H), 7.78 (t, J ¼ 7.7 Hz, 1H), 7.65 (t,
J ¼ 7.7 Hz, 1H), 7.55e7.51 (m, 5H), 7.48e7.43 (m, 2H), 7.32e7.28 (m,
1H), 7.18 (t, J ¼ 9.6 Hz, 1H). 13C NMR (150 MHz, DMSO‑d6)
d 164.20,
9

Y.-L. He, M.-T. Chen, T. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113720
156.54, 145.69, 138.53, 133.52, 133.33, 131.59, 129.46, 129.17, 128.42,
128.07, 127.90, 127.80, 127.20, 126.60, 125.24, 124.44, 122.26, 119.53,
117.84. ESI-HRMS [M ꢀ H]e calcd for C23H16FNO4S: 420.0711, found:
420.0713.
NMR (150 MHz, DMSO‑d6) d 164.09, 156.71, 145.64, 140.07, 133.96,
133.29, 132.04, 131.80, 130.49, 128.97, 128.01, 127.62, 127.37, 127.33,
126.17, 125.97, 125.65, 125.59, 124.39, 122.70, 121.14, 120.35, 119.44,
117.65. ESI-HRMS [M ꢀ H]e calcd for C24H15F4NO5S: 504.0534,
found: 504.0538.
4.2.5.2. 5-((4-(3,5-dimethylisoxazol-4-yl)naphthalene)-1-
sulfonamido)-2-fluorobenzoic acid (D2). White solid, yield: 51 %. 1H
4.2.5.8. 2-Fluoro-5-((4-(2-isopropylphenyl)naphthalene)-1-
NMR (400 MHz, DMSO‑d6)
d
10.92 (s, 1H), 8.79 (dd, J ¼ 8.8, 3.1 Hz,
sulfonamido)benzoic acid (D8). White solid, yield: 51 %. 1H NMR
1H), 8.19 (dd, J ¼ 7.6, 3.1 Hz, 1H), 7.83e7.78 (m, 1H), 7.73e7.65 (m,
2H), 7.58 (dd, J ¼ 7.7, 3.1 Hz, 1H), 7.44e7.41 (m, 1H), 7.30e7.27 (m,
1H), 7.22e7.13 (m, 1H), 2.20 (s, 3H), 1.97 (s, 3H). 13C NMR (150 MHz,
(400 MHz, DMSO‑d6)
d
13.16 (s, 1H), 10.85 (s, 1H), 8.77 (d, J ¼ 8.7 Hz,
1H), 8.16 (d, J ¼ 7.5 Hz, 1H), 7.75 (t, J ¼ 7.8 Hz, 1H), 7.57 (t, J ¼ 7.9 Hz,
1H), 7.51e7.37 (m, 5H), 7.31e7.23 (m, 2H), 7.15 (d, J ¼ 9.9 Hz, 1H),
7.09 (d, J ¼ 7.8 Hz, 1H), 2.18e2.12 (m, 1H), 0.92 (dd, J ¼ 6.6, 3.1 Hz,
DMSO‑d6)
d 166.49, 164.13, 158.64, 158.43, 134.34, 133.56, 133.34,
132.09, 129.02, 128.22, 127.71, 126.95, 126.11, 126.05, 124.71, 122.93,
6H). 13C NMR (150 MHz, DMSO‑d6)
d 164.08, 158.42, 146.38, 145.38,
119.49, 117.79, 113.42, 11.07, 9.94. ESI-HRMS [M ꢀ H]e calcd for
136.91, 133.41, 133.01, 132.55, 129.18, 128.60, 127.96, 127.35, 127.10,
126.60, 126.01, 125.47, 125.34, 125.16, 124.39, 122.89, 119.36, 117.52,
29.67, 24.07, 22.69. ESI-HRMS [M ꢀ H]e calcd for C26H22FNO4S:
462.1181, found: 462.1187.
C
22H17FN2O5S: 439.0769, found: 439.0773.
4.2.5.3. 2-Fluoro-5-((4-(pyridin-4-yl)naphthalene)-1-sulfonamido)
benzoic acid (D3). White solid, yield: 65 %. 1H NMR (400 MHz,
DMSO‑d6)
d
11.03 (s, 1H), 8.81 (d, J ¼ 8.8 Hz, 1H), 8.75e8.72 (m, 2H),
4.2.5.9. 5-((4-(2-chloro-4-fluorophenyl)naphthalene)-1-
sulfonamido)-2-fluorobenzoic acid (D9). White solid, yield: 58 %. 1H
8.26 (d, J ¼ 7.6 Hz,1H), 7.83e7.80 (m, 2H), 7.71e7.64 (m,1H), 7.59 (d,
J ¼ 7.5 Hz, 1H), 7.58e7.48 (m, 3H), 7.34e7.28 (m, 1H), 7.18 (t,
NMR (400 MHz, DMSO‑d6)
d
11.01 (s, 1H), 8.81 (d, J ¼ 8.6 Hz, 1H),
J ¼ 9.6 Hz, 1H). 13C NMR (150 MHz, DMSO‑d6)
d
164.20, 158.27,
8.26 (d, J ¼ 7.6 Hz, 1H), 7.90 (d, J ¼ 7.8 Hz, 2H), 7.81 (t, J ¼ 7.2 Hz, 2H),
7.71e7.64 (dd, J ¼ 18.1, 8.0 Hz, 3H), 7.60 (d, J ¼ 7.6 Hz, 1H), 7.56e7.53
(m, 1H), 7.32e7.29 (m, 1H), 7.18 (t, J ¼ 9.6 Hz, 1H). 13C NMR
156.59, 149.68, 146.20, 142.83, 134.43, 133.41, 130.92, 129.02, 128.18,
127.69, 126.11, 125.35, 125.31, 124.58, 124.43, 122.35, 119.61, 117.87.
ESI-HRMS [M ꢀ H]e calcd for C22H15FN2O4S: 421.0664, found:
421.0665.
(151 MHz, DMSO‑d6)
d 164.19, 158.26, 156.57, 144.01, 142.71, 134.11,
133.45, 131.31, 130.43, 129.03, 128.09, 127.73, 127.55, 126.31, 125.47,
125.30, 124.54, 122.32, 119.57, 117.70. ESI-HRMS [M ꢀ H]e calcd for
4.2.5.4. 2-Fluoro-5-((4-(4-fluorophenyl)naphthalene)-1-
sulfonamido)benzoic acid (D4). White solid, yield: 54 %. 1H NMR
C23H14ClF2NO4S: 472.0227, found: 472.0228.
(400 MHz, DMSO‑d6)
d
10.99 (s, 1H), 8.79 (d, J ¼ 8.7 Hz, 1H), 8.23 (d,
4.2.5.10. 5-((4-(2,4-difluorophenyl)naphthalene)-1-sulfonamido)-2-
fluorobenzoic acid (D10). White solid, yield: 60 %. 1H NMR
J ¼ 7.6 Hz, 1H), 7.84 (d, J ¼ 8.5 Hz, 1H), 7.79 (t, J ¼ 7.7 Hz, 1H), 7.65 (t,
J ¼ 7.7 Hz, 1H), 7.55e7.49 (m, 4H), 7.37 (t, J ¼ 8.6 Hz, 2H), 7.34e7.27
(m, 1H), 7.17 (t, J ¼ 9.6 Hz, 1H). 13C NMR (150 MHz, DMSO‑d6)
(400 MHz, DMSO‑d6)
d 11.02 (s, 1H), 8.80e8.77 (m, 1H), 8.26e8.23
(m, 1H), 7.82e7.77 (m, 1H), 7.69e7.58 (m, 3H), 7.57e7.44 (m, 3H),
d
164.20, 162.74, 156.54, 144.57, 134.82, 133.51, 131.63, 131.57, 129.11,
7.33e7.26 (m, 2H), 7.21e7.15 (m, 1H). 13C NMR (150 MHz, DMSO‑d6)
127.94, 127.78, 127.30, 126.48, 125.41, 125.25, 124.47, 122.26, 119.57,
117.83, 115.42. ESI-HRMS [M ꢀ H]e calcd for C23H15F2NO4S:
438.0617, found: 438.0621.
d 164.19, 161.63, 158.27, 156.58, 138.57, 134.47, 133.44, 132.92,
131.86, 128.93, 128.12, 127.56, 127.46, 126.38, 126.22, 125.29, 125.23,
124.50, 122.32, 119.60, 117.85, 112.00, 104.21. ESI-HRMS [M ꢀ H]e
calcd for C23H14F3NO4S: 456.0523, found: 456.0525.
4.2.5.5. 2-Fluoro-5-((4-(4-hydroxyphenyl)naphthalene)-1-
sulfonamido)benzoic acid(D5). White solid, yield: 66 %. 1H NMR
4.2.5.11. 2-Fluoro-5-((4-(4-fluoro-2-methylphenyl)naphthalene)-1-
sulfonamido)benzoic acid (D11). White solid, yield: 57 %. 1H NMR
(400 MHz, DMSO‑d6)
d
10.93 (s, 1H), 9.79 (s, 1H), 8.76 (d, J ¼ 8.6 Hz,
1H), 8.20 (d, J ¼ 7.6 Hz, 1H), 7.96 (d, J ¼ 8.5 Hz, 1H), 7.76 (t, J ¼ 7.7 Hz,
1H), 7.63 (t, J ¼ 7.7 Hz,1H), 7.54e7.52 (m,1H), 7.48 (d, J ¼ 7.6 Hz,1H),
7.28 (d, J ¼ 7.9 Hz, 3H), 7.16 (t, J ¼ 9.6 Hz, 1H), 6.92 (d, J ¼ 8.1 Hz, 2H).
(400 MHz, DMSO‑d6)
d
13.23 (s, 1H), 10.89 (s, 1H), 8.78 (d, J ¼ 8.3 Hz,
1H), 8.19 (d, J ¼ 7.4 Hz, 1H), 7.77 (t, J ¼ 7.7 Hz, 1H), 7.61 (t, J ¼ 7.6 Hz,
1H), 7.49e7.39 (m, 3H), 7.32e7.20 (m, 3H), 7.17e7.13 (m, 2H),1.83 (s,
13C NMR (150 MHz, DMSO‑d6)
d
164.21, 158.17, 157.45, 145.96,
3H). 13C NMR (150 MHz, DMSO‑d6)
d 164.12, 162.54, 156.68, 144.29,
133.57, 132.54, 131.74, 130.79, 129.29, 129.00, 127.91, 127.76, 126.93,
126.81, 125.15, 124.98, 124.37, 122.17, 119.55, 117.80, 115.27. ESI-
HRMS [M ꢀ H]e calcd for C23H16FNO5S: 436.0660, found: 436.0664.
138.51, 134.44, 133.48, 133.40, 132.05, 131.26, 129.19, 127.97, 127.50,
127.37, 126.28, 125.93, 125.38, 124.52, 122.82, 119.48, 117.59, 116.52,
112.61, 19.28. ESI-HRMS [M
452.0774, found: 452.0777.
ꢀ
H]e calcd for C24H17F2NO4S:
4.2.5.6. 2-Fluoro-5-((4-(4-(trifluoromethyl)phenyl)naphthalene)-1-
sulfonamido)benzoic acid (D6). White solid, yield: 62 %. 1H NMR
4.2.5.12. 5-((4-(3-chloro-4-fluorophenyl)naphthalene)-1-
sulfonamido)-2-fluorobenzoic acid (D12). White solid, yield: 72 %.
(400 MHz, DMSO‑d6)
d
11.01 (s, 1H), 8.81 (d, J ¼ 8.6 Hz, 1H), 8.26 (d,
J ¼ 7.6 Hz, 1H), 7.90 (d, J ¼ 7.8 Hz, 2H), 7.81 (t, J ¼ 7.2 Hz, 2H),
7.71e7.64 (dd, J ¼ 18.1, 8.0 Hz, 3H), 7.60 (d, J ¼ 7.6 Hz, 1H), 7.56e7.53
(m, 1H), 7.32e7.29 (m, 1H), 7.18 (t, J ¼ 9.6 Hz, 1H). 13C NMR
1H NMR (400 MHz, DMSO‑d6)
d
11.01 (s, 1H), 8.79 (d, J ¼ 8.6 Hz, 1H),
8.23 (d, J ¼ 7.6 Hz, 1H), 7.86e7.77 (m, 2H), 7.73 (d, J ¼ 7.1 Hz, 1H),
7.67 (t, J ¼ 7.7 Hz, 1H), 7.61e7.52 (m, 3H), 7.50e7.48 (m, 1H),
7.32e7.28 (m, 1H), 7.17 (t, J ¼ 9.6 Hz, 1H). 13C NMR (150 MHz,
(151 MHz, DMSO‑d6)
d 164.19, 158.26, 156.57, 144.01, 142.71, 134.11,
133.45, 131.31, 130.43, 129.03, 128.09, 127.73, 127.55, 126.31, 125.47,
DMSO‑d6) d 164.21, 158.23, 156.54, 143.13, 136.21, 134.00, 133.47,
125.30, 124.54, 122.32, 119.57, 117.70. ESI-HRMS [M ꢀ H]e calcd for
131.52, 130.39, 128.96, 128.04, 127.72, 127.51, 126.34, 125.62, 125.23,
125.17, 124.50, 122.26, 119.72, 119.61, 117.85, 116.80. ESI-HRMS
[M ꢀ H]e calcd for C23H14ClF2NO4S: 472.0227, found: 472.0231.
C
24H15F4NO4S: 488.0589, found: 488.0589.
4.2.5.7. 2-Fluoro-5-((4-(2-(trifluoromethoxy)phenyl)naphthalene)-
1-sulfonamido)benzoic acid (D7). White solid, yield: 42 %. 1H NMR
4.2.6. General procedure for the preparation of E1e14
(400 MHz, DMSO‑d6)
d
13.28 (s, 1H), 10.92 (s, 1H), 8.79 (d, J ¼ 8.7 Hz,
To a solution of intermediate 10 (3.7 g, 11.03 mmol) in acetone
(20 mL) was added methyl 2-(5-amino-2-fluorophenyl)acetate
hydrochloride (1.94 g, 8.82 mmol) and pyridine (2.62g,
1H), 8.21 (d, J ¼ 7.6 Hz, 1H), 7.77 (t, J ¼ 7.8 Hz, 1H), 7.68e7.60 (m,
2H), 7.57e7.46 (m, 6H), 7.25e7.21 (m, 1H), 7.11 (t, J ¼ 9.7 Hz, 1H). 13
C
10

Y.-L. He, M.-T. Chen, T. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113720
33.08 mmol). The mixture then was refluxed for 2 h and evaporated
under vacuum. The residue was diluted with water and extracted
with ethyl acetate (15 mL) three times. The combined ethyl acetate
layer was sequentially washed with brine, dried with anhydrous
Na2SO4, and concentrated to give the crude product. The crude was
purified by column chromatography with petroleum/ethyl acetate
to yield intermediate12 as a prey solid (3.25 g, 61 %)
7.02e6.94 (m, 2H), 6.90e6.86 (m, 1H), 3.53 (s, 3H), 3.46 (s, 2H). 13
NMR (150 MHz, DMSO‑d6) 171.08, 157.94, 157.26, 135.44, 133.25,
132.35, 131.80, 130.02, 129.40, 128.84, 128.67, 128.55, 128.17, 127.22,
124.48, 124.45, 123.28, 122.93, 122.90, 119.97, 115.43, 61.39, 34.08.
ESI-HRMS [M ꢀ H]e calcd for C25H19ClFNO5S: 498.0584, found:
498.0585.
C
d
To a suspension of the intermediate 12 (1 equiv) in DMF (8 mL)
was sequentially added appropriate substituted aryl boric acid (1.3
equiv), K2CO3 (2 equiv), PdCl2 (dppf).CH2Cl2 (0.1 equiv) and water
(0.5 mL). After refluxing for 12 h under N2 atmosphere, the reaction
mixture was cooled and then poured into H2O. The mixture was
extracted with extracted with EtOAc (2x), washed with saturated
NaCl solution, dried over anhydrous Na2SO4, and concentrated in
vacuo. The residue was purified by silica gel column chromatog-
raphy to afford the target compounds as a white solid.(13a-n).
To a solution of intermediate 13a-n (1 equiv) in methanol (8 mL)
was added 1 N NaOH solution (10 equiv) and stirred at room
temperature for 1 h. Then the solvent was removed under vacuum,
the residue was acidified to pH ¼ 2 with HCl (1 M) solution and
white solid precipitated. The mixture was extracted with ethyl
acetate twice and the combined organic layer was dried over
anhydrous sodium sulfate and concentrated to provide the target
compounds as white solids E1-14.
4.2.6.5. 2-(2-fluoro-5-((4-methoxy-3-(2-(trifluoromethyl)phenyl)
naphthalene)-1-sulfonamido) phenyl) acetic acid (E5). White solid,
33 %. 1H NMR (400 MHz, DMSO‑d6)
d
10.71 (s,1H), 8.74 (d, J ¼ 8.6 Hz,
1H), 8.26 (d, J ¼ 8.3 Hz, 1H), 7.95 (s, 1H), 7.91 (d, J ¼ 7.8 Hz, 1H),
7.84e7.69 (m, 4H), 7.54 (d, J ¼ 7.5 Hz, 1H), 6.97e6.92 (m, 2H),
6.88e6.81 (m, 1H), 3.55 (s, 3H), 3.44 (s, 2H). 13C NMR (150 MHz,
DMSO‑d6)
d 171.09, 157.91, 156.98, 135.07, 133.17, 132.77, 132.43,
132.18, 128.86, 128.68, 128.56, 127.88, 127.31, 126.24, 124.95, 124.54,
123.34, 122.89, 122.75, 119.81, 115.33, 61.50, 34.00. ESI-HRMS
[M ꢀ H]e calcd for C26H19F4NO5S: 532.0847, found: 532.0849.
4.2.6.6. 2-(2-fluoro-5-((4-methoxy-3-(o-tolyl)naphthalene)-1-
sulfonamido)phenyl)acetic acid (E6). White solid, 84 %. 1H NMR
(400 MHz, DMSO‑d6)
d
12.45 (s, 1H), 10.63 (s, 1H), 8.72 (d, J ¼ 8.7 Hz,
1H), 8.28 (d, J ¼ 8.4 Hz, 1H), 7.87 (s, 1H), 7.79 (t, J ¼ 7.9 Hz, 1H), 7.74
(d, J ¼ 8.0 Hz, 1H), 7.39e7.35 (m, 2H), 7.33e7.29 (m, 1H), 7.25 (d,
J ¼ 7.2 Hz, 1H), 7.02e6.95 (m, 2H), 6.91e6.84 (m, 1H), 3.45 (s, 5H),
1.98 (s, 3H). 13C NMR (150 MHz, DMSO‑d6)
d 171.07, 158.01, 156.78,
4.2.6.1. 2-(2-fluoro-5-((3-(4-fluoro-2-methylphenyl)-4-
methoxynaphthalene)-1sulfonamido)phenyl) acetic acid (E1).
136.54, 135.72, 133.42, 133.30, 129.93, 129.69, 128.69, 128.29,
128.26, 128.08, 127.11, 126.44, 125.77, 124.44, 123.17, 123.13, 120.21,
White solid, 71 %. 1H NMR (400 MHz, DMSO‑d6)
d
12.47 (s, 1H),
115.44, 61.06, 34.04, 19.31. ESI-HRMS [M
26H19F4NO5S: 478.1130, found:4 78.1126.
ꢀ
H]e calcd for
10.66 (s, 1H), 8.74 (d, J ¼ 8.4 Hz, 1H), 8.33e8.26 (m, 1H), 7.88 (s, 1H),
7.81 (d, J ¼ 7.9 Hz,1H), 7.75 (t, J ¼ 7.6 Hz,1H), 7.36e7.29 (m,1H), 7.25
(d, J ¼ 10.0 Hz, 1H), 7.20e7.12 (m, 1H), 7.02e6.97 (m, 2H), 6.90e6.86
(m, 1H), 3.52e3.45 (m, 5H), 1.99 (s, 3H). 13C NMR (150 MHz,
C
4.2.6.7. 2-(5-((3-(2-ethylphenyl)-4-methoxynaphthalene)-1-
sulfonamido)-2-fluorophenyl)acetic acid (E7). White solid, 58 %. 1H
DMSO‑d6)
d
171.08, 162.45, 157.01, 156.41, 138.80, 133.35, 133.27,
NMR (400 MHz, DMSO‑d6) d 12.46 (s, 1H), 10.64 (s, 1H), 8.75 (d,
132.75, 131.65, 128.82, 128.40, 128.24, 127.18, 125.61, 124.47, 123.18,
122.92, 120.26, 116.51, 115.44, 112.68, 61.16, 34.00, 19.35. ESI-HRMS
[M ꢀ H]e calcd for C26H21F2NO5S: 496.1036, found: 496.1040.
J ¼ 8.6 Hz, 1H), 8.28 (d, J ¼ 8.3 Hz, 1H), 7.87 (s, 1H), 7.81 (t, J ¼ 7.8 Hz,
1H), 7.74 (t, J ¼ 7.6 Hz, 1H), 7.40 (d, J ¼ 9.1 Hz, 2H), 7.31 (d, J ¼ 7.1 Hz,
1H), 7.25 (d, J ¼ 7.6 Hz, 1H), 6.98 (d, J ¼ 7.4 Hz, 2H), 6.86 (d,
J ¼ 8.9 Hz, 1H), 3.49 (s, 3H), 3.45 (s, 2H), 2.33e2.27 (m, 2H), 0.86 (t,
4.2.6.2. 2-(2-fluoro-5-((3-(4-fluorophenyl)-4-methoxynaphthalene)-
1-sulfonamido)phenyl)acetic acid (E2). White solid, 64 %. 1H NMR
J ¼ 7.8 Hz, 3H). 13C NMR (150 MHz, DMSO‑d6)
d 171.05, 158.05,
156.66, 141.67, 135.96, 133.53, 133.22, 129.84, 128.66, 128.30, 128.21,
127.14, 126.50, 125.71, 124.48, 123.41, 123.19, 122.93, 120.24, 115.40,
(400 MHz, DMSO‑d6)
d
8.69 (d, J ¼ 8.2 Hz, 1H), 8.28 (d, J ¼ 8.1 Hz,
1H), 8.11 (s, 1H), 7.74e7.70 (m, 2H), 7.65e7.61 (m, 2H), 7.35 (t,
J ¼ 8.6 Hz, 2H), 7.05e7.03 (m, 1H), 6.98 (t, J ¼ 9.2 Hz, 1H), 6.91e6.87
(m, 1H), 3.53 (s, 3H), 3.48 (s, 2H). 13C NMR (150 MHz, DMSO‑d6)
61.22, 34.04, 25.49, 14.51. ESI-HRMS [M
27H24FNO5S: 492.1286, found: 492.1284.
ꢀ
H]e calcd for
C
d
171.20, 162.48, 157.93, 156.82, 133.39, 132.70, 132.37, 131.00,
4.2.6.8. 2-(2-fluoro-5-((4-methoxy-3-(2-(trifluoromethyl)phenyl)
naphthalene)-1-sulfonamido) phenyl) acetic acid (E8). White solid,
130.95, 130.08, 128.56, 128.37, 128.32, 127.32, 126.25, 124.53, 123.14,
120.03,119.98,115.61,115.46, 61.32, 34.14. ESI-HRMS [M ꢀ H]e calcd
for C25H19F2NO5S: 482.0879, found: 482.0882.
61 %. 1H NMR (400 MHz, DMSO‑d6)
d 12.45 (s, 1H), 10.64 (s, 1H), 8.74
(d, J ¼ 8.5 Hz, 1H), 8.28 (d, J ¼ 8.3 Hz, 1H), 7.87 (s, 1H), 7.83e7.78 (m,
1H), 7.74 (t, J ¼ 7.5 Hz, 1H), 7.48e7.42 (m, 2H), 7.29 (t, J ¼ 7.0, 1H),
7.21 (d, J ¼ 7.6 Hz, 1H), 6.99e6.93 (m, 2H), 6.87e6.83 (m, 1H), 3.50
(s, 3H), 3.44 (s, 2H), 2.58e2.52 (m, 1H), 1.13e1.11 (m, 3H), 0.90e0.88
4.2.6.3. 2-(2-fluoro-5-((3-(2-fluorophenyl)-4-methoxynaphthalene)-
1-sulfonamido)phenyl)acetic acid (E3). White solid, 61 %. 1H NMR
(400 MHz, DMSO‑d6)
1H), 8.29 (d, J ¼ 8.3 Hz, 1H), 8.07 (s, 1H), 7.81 (t, J ¼ 7.7 Hz, 1H), 7.74
(t, J ¼ 7.6 Hz, 1H), 7.55e7.49 (m, 2H), 7.41e7.34 (m, 2H), 7.03e6.96
(m, 2H), 6.94e6.87 (m, 1H), 3.56 (s, 3H), 3.48 (s, 2H). 13C NMR
d
12.42 (s, 1H), 10.69 (s, 1H), 8.71 (d, J ¼ 8.5 Hz,
(m, 3H). 13C NMR (150 MHz, DMSO‑d6)
d 171.03, 158.06, 156.76,
146.31, 135.27, 133.53, 133.18, 129.80, 128.68, 128.54, 128.31, 128.18,
127.17, 126.60, 125.57, 125.40, 124.47, 123.52, 123.23, 122.90, 120.19,
115.38, 61.24, 34.01, 29.59, 24.21, 22.57. ESI-HRMS [M ꢀ H]e calcd
for C28H26FNO5S: 506.1443, found: 506.1439.
(150 MHz, DMSO‑d6) d 171.11, 159.81, 157.94, 156.34, 133.25, 133.03,
131.57, 130.51, 129.41, 128.68, 128.62, 128.22, 127.31, 124.61, 124.51,
124.07, 123.22, 122.98, 122.82, 121.28, 119.97, 115.76, 115.48, 61.58,
34.02. ESI-HRMS [M ꢀ H]e calcd for C25H19F2NO5S: 482.0879,
found: 482.0881.
4 . 2 . 6 . 9 . 2 - ( 2 - fl u o r o - 5 - ( ( 3 - ( 2 - h y d r o x y p h e n y l ) - 4 -
methoxynaphthalene)-1-sulfonamido)phenyl)acetic
acid
(E9).
White solid, 86 %. 1H NMR (400 MHz, DMSO‑d6)
d
12.42 (s,1H),10.64
(s, 1H), 9.65 (s, 1H), 8.65 (d, J ¼ 8.5 Hz, 1H), 8.26 (d, J ¼ 8.3 Hz, 1H),
8.10 (s, 1H), 7.76e7.67 (m, 2H), 7.31e7.22 (m, 2H), 7.02e6.90 (m,
4.2.6.4. 2-(5-((3-(2-chlorophenyl)-4-methoxynaphthalene)-1-
sulfonamido)-2-fluorophenyl)acetic acid (E4). White solid, 57 %. 1H
5H), 3.55 (s, 3H), 3.46 (s, 2H). 13C NMR (150 MHz, DMSO‑d6)
d 171.17,
NMR (400 MHz, DMSO‑d6)
d
12.44 (s, 1H), 10.69 (s, 1H), 8.72 (d,
157.76, 157.22, 154.41, 134.74, 133.43, 130.89, 129.35, 128.41, 128.31,
128.04,126.76, 124.29, 124.08, 123.59, 123.08, 122.86, 122.70,119.51,
118.99, 115.73, 115.41, 60.84, 34.11. ESI-HRMS [M ꢀ H]e calcd for
J ¼ 8.5 Hz, 1H), 8.29 (d, J ¼ 8.4 Hz, 1H), 7.97 (s, 1H), 7.81 (t, J ¼ 7.7 Hz,
1H), 7.74 (t, J ¼ 7.6 Hz, 1H), 7.69e7.63 (m, 1H), 7.55e7.47 (m, 3H),
11

Y.-L. He, M.-T. Chen, T. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113720
C25H20FNO6S: 480.0923, found: 480.0921.
4.3. In vitro FABPs inhibition assay
The methods for expression and purification of FABP4/FABP5/
FABP3 were as previously described [39]. The inhibitory activities of
the compounds against FABPs were evaluated using 1-
anilinonapthalene 8-sulfonic acid (1, 8-ANS) displacement assay
4.2.6.10. 2-(5-((3-(2,4-difluorophenyl)-4-methoxynaphthalene)-1-
sulfonamido)-2-fluorophenyl) acetic acid (E10). White solid, 72 %. 1H
NMR (400 MHz, DMSO‑d6)
d 12.43 (s, 1H), 10.70 (s, 1H), 8.71 (d,
J ¼ 8.5 Hz, 1H), 8.28 (d, J ¼ 8.4 Hz,1H), 8.06 (s, 1H), 7.81 (t, J ¼ 7.6 Hz,
1H), 7.75 (t, J ¼ 7.6 Hz, 1H), 7.61e7.55 (m, 1H), 7.45 (t, J ¼ 9.8 Hz, 1H),
7.28e7.24 (m, 1H), 7.05e6.96 (m, 2H), 6.91e6.87 (m, 1H), 3.56 (s,
[32,41]. Briefly, 1, 8-ANS (10
solution) was mixed with FABPs protein to a final concentration of
10 M. The compounds were then added and incubated for 3 min at
mM, diluted in phosphate buffered
m
3H), 3.48 (s, 2H). 13C NMR (150 MHz, DMSO‑d6)
d 171.13, 161.40,
room temperature. The fluorescence signal at 370 nm (excitation)/
470 nm (emission) was measured with a Flexstation III instrument
(Molecular Devices, CA, USA). Compounds with inhibition rate
157.93, 157.85, 133.23, 132.93, 132.78, 129.53, 128.75, 128.69, 128.18,
127.36, 124.54, 123.22, 122.96, 122.82, 120.59, 120.41, 119.99, 115.47,
111.91, 104.24, 61.66, 34.00. ESI-HRMS [M
25H18F3NO5S: 500.0785, found: 500.0781.
ꢀ
H]e calcd for
greater than 60% at the concentration of 25
IC50 values determination.
mM were selected for
C
4.4. Cell viability assay
4 . 2 . 6 . 11. 2 - ( 5 - ( ( 3 - ( 2 - c h l o r o - 4 - fl u o r o p h e n y l ) - 4 -
methoxynaphthalene)-1-sulfonamido)-2-fluoro phenyl) acetic acid
(E11). White solid, 76 %. 1H NMR (400 MHz, DMSO‑d6)
d 12.43 (s,
Mouse 3T3-L1 preadipocytes and human THP-1 cells were from
the American Type Culture Collection and were cultured as previ-
ously described [23,42]. For cell viability assay, 3T3-L1 cells were
seeded in 96-well plates at the density of 6000 cells/well and then
1H), 10.69 (s, 1H), 8.73 (d, J ¼ 8.6 Hz, 1H), 8.28 (d, J ¼ 8.4 Hz, 1H),
7.95 (d, J ¼ 1.3 Hz,1H), 7.85e7.79 (m,1H), 7.75 (t, J ¼ 7.7 Hz,1H), 7.67
(d, J ¼ 8.8 Hz, 1H), 7.57e7.53 m, 1H), 7.38 (t, J ¼ 8.5, Hz, 1H),
7.00e6.95 (t, J ¼ 8.5 Hz, 2H), 6.89e6.85 (m, 1H), 3.54 (s, 3H), 3.46 (s,
treated with fresh medium with or without 100 mM of compounds
2H). 13C NMR (150 MHz, DMSO‑d6)
d 171.10, 162.48, 157.94, 157.47,
for 24 h. THP-1 cells were seeded in 96-well plates at the density of
1*105 cells/well and simultaneously incubated with 100 nM of
phorbol myristic acid (PMA) for 24 h to facilitated the differentia-
tion to a macrophage phenotype. The cells were then treated with
133.41, 133.25, 131.88, 128.96, 128.75, 128.63, 128.14, 127.28, 124.50,
123.72, 123.29, 122.95, 122.78, 120.02, 116.81, 115.43, 114.65, 61.49,
34.05. ESI-HRMS [M ꢀ H]e calcd for C25H18ClF2NO5S: 516.0490,
found: 516.0486.
or without 50
mM of compounds for another 24 h. Appropriate
concentrations of DMSO in medium was used as control. After that,
the cell viability was assessed by using Cell Counting Kit-8 (CCK-8)
according to manufacturer's instructions. Briefly, the original me-
4 . 2 . 6 . 1 2 . 2 - ( 5 - ( ( 3 - ( 3 - c h l o r o - 4 - fl u o r o p h e n y l ) - 4 -
methoxynaphthalene)-1-sulfonamido)-2-fluoro phenyl) acetic acid
dium was discarded and 100 ml CCK-8 (10 %, v/v) reagent was added
(E12). White solid, 60 %.1H NMR (400 MHz, DMSO‑d6)
d 12.36 (s,
gently and incubated for 30 min at 37 ꢁC. The absorbance was then
read at 450 nm. Cell viability was calculated by (compound-treated
group absorbance value/control group absorbance value) ꢂ 100 %.
1H),10.69 (s,1H), 8.70 (d, J ¼ 8.3 Hz,1H), 8.29 (d, J ¼ 8.1 Hz,1H), 8.12
(s, 1H), 7.81e7.73 (m, 3H), 7.62e7.55 (m, 2H), 7.07e7.04 (m, 1H),
6.99 (t, J ¼ 9.0 Hz, 1H), 6.93e6.89 (m, 1H), 3.57 (s, 3H), 3.50 (s, 2H).
13C NMR (150 MHz, DMSO‑d6)
d 171.16, 157.96, 157.07, 156.35,
4.5. Lipolysis assay
134.02, 133.27, 132.13, 130.90, 130.12, 129.80, 128.64, 128.55, 128.46,
127.43, 125.16, 124.52, 123.23, 123.08, 122.98, 120.12, 119.69, 117.16,
115.54, 61.62, 33.98. ESI-HRMS [M ꢀ H]e calcd for C25H18ClF2NO5S:
516.0490, found: 516.0485.
3T3-L1 preadipocytes were seed in 150 mm * 25 mm Dish for
differentiation as previously described [42]. The mature adipocytes
were digested by trypsin and seeded into 96-well plates at a density
of 40000 cells/well and then incubated with compounds with
various concentrations or DMSO (final concentration 0.1 %, v/v) for
24 h. On the day of lipolysis measurement, the cells were incubated
4 . 2 . 6 . 1 3 . 2 - ( 5 - ( ( 3 - ( 3 - c h l o r o - 2 - fl u o r o p h e n y l ) - 4 -
methoxynaphthalene)-1-sulfonamido)-2-fluoro phenyl) acetic acid
in Krebs-Ringer-HEPES buffer with or without 20 mM forskolin for
(E13). White solid, 63 %. 1H NMR (400 MHz, DMSO‑d6)
d 12.43 (s,
1 h. Thereafter, the concentrations of glycerol in supernatants were
measured using a commercial glycerol assay kit (Applygen Tech-
nologies Inc, China).
1H), 10.70 (s, 1H), 8.72 (d, J ¼ 8.5 Hz, 1H), 8.29 (d, J ¼ 8.2 Hz, 1H),
8.09 (s, 1H), 7.82 (t, J ¼ 7.7 Hz, 1H), 7.78e7.71 (m, 2H), 7.49 (t,
J ¼ 7.2 Hz, 1H), 7.39 (t, J ¼ 8.0 Hz, 1H), 7.03e6.96 (m, 2H), 6.90e6.87
(m, 1H), 3.58 (s, 3H), 3.48 (s, 2H). 13C NMR (150 MHz, DMSO‑d6)
4.6. FABP4 secretion assay and western blotting
d
171.14,157.94,157.88,155.16,133.20,132.65,130.66,130.47,129.65,
128.92, 128.85, 128.12,127.45,125.81,125.49,124.58, 123.28, 122.95,
120.52, 120.13, 119.95, 115.48, 61.92, 33.98. ESI-HRMS [M ꢀ H]e
calcd for C25H18ClF2NO5S: 516.0490, found: 516.0484.
The mature adipocytes were starved in DMEM containing 0.5%
BSA for 6 h, and then treated with 50 mM compound for 24 h. Su-
pernatant was then collected for ultrafiltration while intracellular
proteins were collected. Protein concentration of cell lysate and
supernatant were determined by a bicinchoninic acid kit (Beyo-
time, China). Approximate 50 mg of liver tissues were lysed in RIPA
Lysis Buffer (Beyotime, China) and the supernatant was collected
after centrifugation (Eppendorf, Germany) as previously described
[43]. Samples were then subjected to 10% or 12% SDS-PAGE,
4.2.6.14. 2-(5-((3-(2,5-dichlorophenyl)-4-methoxynaphthalene)-1-
sulfonamido)-2-fluorophenyl) acetic acid (E14). White solid, 78 %. 1H
NMR (400 MHz, DMSO‑d6)
d 12.44 (s, 1H), 10.69 (s, 1H), 8.73 (d,
J ¼ 8.6 Hz, 1H), 8.29 (d, J ¼ 8.4 Hz, 1H), 7.96 (s, 1H), 7.83 (t, J ¼ 7.7 Hz,
1H), 7.75 (t, J ¼ 7.6 Hz, 1H), 7.69 (d, J ¼ 8.5 Hz, 1H), 7.62e7.57 (m,
2H), 6.99e6.95 (m, 2H), 6.89e6.85 (m, 1H), 3.57 (s, 3H), 3.46 (s, 2H).
transferred to a 0.45
mm PVDF membrane, incubated with 1 %
13C NMR (150 MHz, DMSO‑d6)
d
171.07,157.95,157.36, 137.15,133.19,
BSA and then probed with primary antibodies (FABP4 antibody,
2120, GAPDH, 5174, Akt antibody, 9272, phospho-Akt antibody,
9271, CST, USA, 1:1000 dilution) at 4 ꢁC overnight. The next day, the
PVDF membranes were washed with 1*TBST for 3 times and
incubated with peroxidase-conjugated AffiniPure goat anti-rabbit
132.81, 131.65, 131.30, 131.00, 129.87, 129.07, 128.87, 128.76, 128.08,
127.33, 124.52, 123.47, 123.33, 123.00, 122.78, 120.02, 115.42, 61.69,
34.04. ESI-HRMS [M ꢀ H]e calcd for C25H18Cl2FNO5S: 532.0194,
found: 532.0189.
12

Y.-L. He, M.-T. Chen, T. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113720
IgG (211-032-171, Jackson Laboratory, USA, 1:10000 dilution) for
90 min at room temperature. GAPDH was used as the loading
control. The bands were visualized using ECL detection reagent
(Thermo Fisher Scientific), and the intensity of the bands was
quantified by using Gel-Pro Analyzer software.
weight was recorded twice a week. The mice were fasted for 6 h,
and the blood glucose in the tail vain was monitored using a glucose
meter once a week (Accu-CHEK, Roche). The oral glucose tolerance
test (OGTT) and insulin tolerance test (ITT) were performed as
previously described [43]. After 45 days of administration, the mice
were fasted overnight and then were given 0.5 g/kg glucose via
intragastric administration for OGTT. After 51 days of administra-
tion, the mice were subjected to insulin (0.85 IU/kg) via intraperi-
toneal injection after 6-h fasting for ITT. The line chart of blood
glucose was plotted and the AUC were calculated. At the end of the
experiment, blood samples were collected after the mice were
fasted for 6 h and the serum parameters were determined using
assay kit according to the manufacturer's instructions (Jiancheng,
Nanjing, Chin; Biovendor, Brno, Czech Republic), including high
density lipoprotein cholesterol (HDL-C), low density lipoprotein
cholesterol (LDL-C), triglyceride (TG), total cholesterol (TCH) and
FABP4. All animal experiments were permitted by the Institutional
Animal Care and Use Committee of Shanghai Institute of Materia
Medica (number: 2020-04-WHY-17).
4.7. RNA isolation and quantitative RT-PCR
THP-1 cells were seeded into 48-well plates at the density of
3*105 cells/well and incubated with 100 nM PMA simultaneously
for 24 h. After differentiated into macrophages, THP-1 cells were
washed with PBS for three times gently. Compounds at the con-
centration of 25 mM or DMSO (final concentration 0.1 %, v/v) was
added and incubated for 18 h followed by 100 ng/ml LPS stimula-
tion for another 6 h. Total RNA was extracted from THP-1 cells using
the TRIzol reagent (Invitrogen, USA) according to the manufac-
turer's instructions. Reverse transcription was performed by using
All-in-One cDNA Synthesis SuperMix (Biomake, China) and the
expression level of MCP-1 was quantified by real-time PCR with
SYBR Green qPCR Master Mix (Biomake, China) and ABI VIIA7 Real-
Time PCR machine (Applied Biosystems, USA). b-Actin was used as
4.11. Statistical analysis
control. The relative expression level of MCP-1 was calculated using
DD
the 2ꢀ CT method [32]. The primers used for RT-qPCR were listed
The IC50 values of compounds for FABPs were calculated by
nonlinear regression using GraphPad Prism software (San Diego,
CA, USA) and expressed as mean standard deviation (SD) of three
independent experiments. Statistical significance of different
groups was performed by t-test or one-way analysis of variance
(ANOVA) followed by a post hoc Tukey's test and a p value less than
0.05 was considered significant.
as Table S1.
4.8. Metabolic stability in liver microsomes
Microsomes in 0.1 M TRIS buffer pH 7.4 (final concentration
0.33 mg/mL), co-factor MgCl2 (final concentration 5 mM) and
tested compound (final concentration 0.1
mM, co-solvent (0.01%
DMSO) and 0.005% Bovin serum albumin (BSA)) were incubated at
37 ꢁC for 10 min. The reaction was started by the addition of NADPH
(final concentration 1 mM). Aliquots were sampled at 0, 7, 17, 30
and 60 min respectively and methanol (cold in 4 ꢁC) was added to
terminate the reaction. After centrifugation (4000 rpm, 5 min),
samples were then analyzed by LC-MS/MS.
Declaration of competing interest
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.
Acknowledgements
4.9. In vivo PK study in ICR (CD-1) mice
The authors appreciate the financial support from the National
Natural Science Foundation of China (81973178, 81773576), Na-
tional Key R&D Program of China (2018YFC0310905); the
“Personalized Medicines-Molecular Signature-based Drug Discov-
ery and Development”, Strategic Priority Research Program of the
Chinese Academy of Sciences (XDA12040336).
Eighteen ICR male mice were randomly divided into six group,
weighting 20e25 g each. Compound D9, E1 and BMS309403 dis-
solved in phosphate-buffered saline containing 5% DMSO, and 0.5%
hydroxypropyl methylcellulose (HPMC) was administered orally at
a dose 3 mg/kg to three groups of mice. Another three groups of
mice were injected with a single dose (1 mg/kg) of compound E1,
D9 and BMS309403 which are dissolved in DMSO/ethanol (EtOH)/
PEG300/NaCl (5/5/40/50, v/v/v/v). Blood samples at seven time
points (0.25, 0.5, 1.0, 2.0, 4.0, 8.0, and 24 h) were collected. Plasma
concentrations of compound D9, E1 and BMS309403 were
analyzed by LC-MS/MS (Waters I-class LC system and Waters Xevo
TQ-S Mass Spectrometer System).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113720.
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