Design, synthesis and structure-activity relationship studies of novel phenoxyacetamide-based free fatty acid receptor 1 agonists for the treatment of type 2 diabetes

Article abstract of DOI:10.1016/j.bmc.2015.09.010

The free fatty acid receptor 1 (FFA1) has attracted extensive attention as a novel antidiabetic target in the last decade. Several FFA1 agonists reported in the literature have been suffered from relatively high molecular weight and lipophilicity. We have previously reported the FFA1 agonist 1. Based on the common amide structural characteristic of SAR1 and NIH screened compound, we here describe the continued structure-activity exploration to decrease the molecular weight and lipophilicity of the compound 1 series by converting various amide linkers. All of these efforts lead to the discovery of the preferable lead compound 18, a compound with considerable agonistic activity, high LE and LLE values, lower lipophilicity than previously reported agonists, and appreciable efficacy on glucose tolerance in both normal and type 2 diabetic mice.

Full text of DOI:10.1016/j.bmc.2015.09.010

Bioorganic & Medicinal Chemistry 23 (2015) 6666–6672
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and structure–activity relationship studies of novel
phenoxyacetamide-based free fatty acid receptor 1 agonists for the
treatment of type 2 diabetes
Zheng Li a, Xuekun Wang a, Xue Xu b, Jianyong Yang a, Qianqian Qiu a, Hao Qiang a, Wenlong Huang a,c,
,
⇑
Hai Qian a,c,
⇑
a Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
b Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
c Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 August 2015
Revised 3 September 2015
Accepted 4 September 2015
Available online 5 September 2015
Keywords:
FFA1 agonist
OGTT
Amide linkers
Ligand efficiency
Type 2 diabetes
The free fatty acid receptor 1 (FFA1) has attracted extensive attention as a novel antidiabetic target in
the last decade. Several FFA1 agonists reported in the literature have been suffered from relatively high
molecular weight and lipophilicity. We have previously reported the FFA1 agonist 1. Based on the com-
mon amide structural characteristic of SAR1 and NIH screened compound, we here describe the contin-
ued structure–activity exploration to decrease the molecular weight and lipophilicity of the compound 1
series by converting various amide linkers. All of these efforts lead to the discovery of the preferable lead
compound 18, a compound with considerable agonistic activity, high LE and LLE values, lower lipophilic-
ity than previously reported agonists, and appreciable efficacy on glucose tolerance in both normal and
type 2 diabetic mice.
Ó 2015 Published by Elsevier Ltd.
1. Introduction
last decade, play a key role in amplifying glucose-stimulated insulin
secretion (GSIS) on pancreatic b-cells but does not affect insulin
The increasing and alarming prevalence of type 2 diabetes
mellitus (T2DM) along with the undesirable side effects (such as
body weight gain, gastric symptoms, risk of hypoglycemia, etc.)
associated with many oral antidiabetic agents has promoted a great
development in evaluating novel targets to achieve preferable hypo-
glycemic drugs.1–4 The free fatty acid receptor 1 (FFA1, also known
as GPR40), the prominent ones of novel antidiabetic targets in the
secretion at low blood glucose levels.5–8 Therefore, this particular
mechanism of FFA1 provides the tremendous potential for boosting
insulin levels without the risk of hypoglycemia.
Recently, a number of synthetic FFA1 agonists contained acidic
moieties have been reported in the literature (Fig. 1),9–17 and the
compounds TAK-875, AMG-837 and LY2881835 were in clinical tri-
als for treatment of T2DM. However, many of these agonists have
relatively high molecular weight and lipophilicity (red mark in
Fig. 1), which most likely associated with poor water-solubility,
high promiscuity, strong metabolic toxicity, and correlated with a
high risk of attrition in clinical trials.18–22 Studies have suggested
⇑
Corresponding authors. Tel.: +86 25 83271302; fax: +86 25 83271480.
E-mail addresses: ydhuangwenlong@126.com (W. Huang), qianhai24@163.com
(H. Qian).
http://dx.doi.org/10.1016/j.bmc.2015.09.010
0968-0896/Ó 2015 Published by Elsevier Ltd.

Z. Li et al. / Bioorg. Med. Chem. 23 (2015) 6666–6672
6667
Figure 1. Selected examples of synthetic GPR40 agonists. clogP values are calculated with ChemDraw Ultra 12.0 using the ‘clogP’ option.
Figure 2. Our strategy to decrease the molecular weight and lipophilicity of compound 1.
that clogP values should not exceed 4–5,18,23 and concepts such as
ligand efficiency (LE) and ligand lipophilicity efficiency (LLE) have
been recommended to direct the optimization process.24,25 Inspired
by the relatively low molecular weight and lipophilicity of SAR1, we
first designed and synthesized compound 2, which was a hybrid
structure containing both the oxalamide moiety of SAR1 and phe-
noxyacetic acid structure of our previously reported compound 1
(Fig. 2). However, the compound 2 and its analogs appeared to
diminish the in vitro agonistic activity, indicating that the interac-
tion mode of compound 2 series was different from SAR1. Subse-
quently, a series of amide linkers were designed based on the
common amide structural characteristic of SAR1 and NIH screened
compound. Among them, the most potent amide linker phenoxyac-
etamide was selected to systematically explore the SAR, which lead
to the identification of lead compound 18, a compound with consid-
erable agonistic activity, high LE and LLE values, lower lipophilicity
than previously reported agonists, and appreciable antihyper-
glycemic effect in both normal and type 2 diabetic mice.
chloroacetate in the presence of K2CO3. The intermediate 3a was
treated with various substituted anilines and oxalyl chloride,
followed by basic hydrolysis, afforded the desired carboxylic acids
2–7. Acylation of the intermediate 3a with corresponding acyl
chloride, formed from commercially available carboxylic acid 4a
or 4b with oxalyl chloride catalyzed by DMF catalyze, generated
the desired esters, which were isolated pure from ethanol.
Hydrolysis of esters with lithium hydroxide provided the designed
compounds 8 and 9 in high yield. Mono-acylation of the
cyclopropane-1,1-dicarboxylic acid with aniline to afford compound
6a, which was subsequently converted to compounds 10 by
acylation with intermediate 3a and esterolysis. The desired
compound 12 was obtained from aniline and compound 3a in
the presence of triphosgene and Et3N, followed by hydrolysis.
The target compounds 11 and 13–36 were synthesized from the
starting material various substituted phenoxyacetic acid according
to the method for the synthesis of compound 8.
2.2. FFA1 agonistic activity and SAR study
2. Results and discussion
2.1. Chemistry
Inspired by the low molecular weight and lipophilicity of SAR1,
we first designed and synthesized compound 2, a hybrid structure
containing both the oxalamide moiety of SAR1 and phenoxyacetic
acid structure of compound 1. The compound 2, however,
appeared to diminish the FFA1 agonistic activity compared with
the parent compound 1 (Table 1). The introduction of various sub-
stituents in compound 2 to give compounds 3–7 did not appear to
The synthetic routes of target compounds 2–36 are summarized
in Scheme 1. The key intermediate 3a was prepared by the
reduction of nitrobenzene 2a, which was derived from the
substitution of commercially available phenol 1a with methyl

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Z. Li et al. / Bioorg. Med. Chem. 23 (2015) 6666–6672
Scheme 1. Synthesis of target compounds 2 to 36. Reagents and conditions: (a) methyl chloroacetate, K2CO3, acetonitrile, Ki, reflux, 6 h; (b) H2, Pd–C, rt, 18 h; (c) various
substituted anilines, oxalyl chloride, then 3a, acetonitrile; (d) LiOHÁH2O, THF/MeOH/H2O, rt, 4 h; (e) oxalyl chloride, dichloromethane, then 3a, Et3N; (f) aniline, SOCl2, Et3N,
THF, 0 °C to rt, 3 h; (g) aniline, triphosgene, Et3N, then 3a.
Table 1
concentration compared with the compound 2, which likely sug-
gesting that a planar template might be unfavorable. Subsequently,
In vitro activities and select physicochemical properties of target compounds
a methylene was attached in the amide linker of compound 8 to
reduce the planarity of molecule, and the resulting compound 9
indeed obtained an approximately 3-fold increase in potency over
the compound 8. The encouraging result prompted us to reevaluate
the compound 10, a more flexible analog of compound 2. The com-
pound 10 revealed a slight improvement on potency compared to
the parent compound 2 but still deviate the desired effect. Gratify-
ingly, the stretched compound 11 (phenoxyacetamide linker), with
relatively low molecular weight and lipophilicity, exhibited a
marked improvement on potency in comparison with the corre-
sponding compound 9. This result demonstrated that a more
appropriate conformation was induced by topological structure
of phenoxyacetamide linker. The urea derivative 12, designed as
a constrained planar analog of compound 9, turned out almost an
order of magnitude less active but still indicates that the receptor
binding pocket can accommodate the conformation with the ter-
minal phenyl extended in the plane of urea.
Based on these results above, we therefore selected this linker of
the most potent compound 11 as our starting point for further mod-
ification. Besides the agonistic activity, the LE and logD7.4, as well as
LLE were taken advantage of in the evaluation of derivatives. As
shown in Table 2, methyl substituent on the phenoxyacetamide lin-
ker (13) resulted in further improvement, likely attributed to the
hydrophobic interaction of methyl group. On the whole, the com-
pound 13 series shared almost the same SAR with the compound
11 series (11 vs 13, 23 vs 24, 30 vs 31 and 32 vs 33). Interestingly,
the introduction of gem-dimethyl group (14), however, showed a
drastic loss of activity despite increased lipophilicity, suggesting
that the limited space in the binding pocket around this area. Then,
an initial optimization of the terminal phenyl with methyl group
(15, 18, and 19) demonstrated that substitution is tolerated in all
positions and preferred in the meta-substituent. Further modifica-
tion in the meta-position suggested that the bulkier substitution
also tolerated in this position (21, 23 and 26), whereas the
Compd
R
Linker Act %a (300 nM) Act %b (100 nM) Mw
clogPc
TAK-875
1
2
3
4
5
6
7
8
76.01
67.32
26.54
29.86
36.27
27.89
33.67
19.85
12.78
39.57
34.17
58.68
23.69
65.32
54.94
9.78
11.35
17.65
10.23
13.28
6.78
524.63 4.697
516.58 4.511
332.29 0.761
346.31 1.259
346.31 1.259
366.73 1.474
366.73 1.474
362.31 0.679
289.26 2.154
303.29 2.203
372.35 1.824
319.29 2.182
304.28 2.569
H
A
A
A
A
A
A
B
C
D
E
4-Me
3-Me
2-Cl
3-Cl
2-OMe
H
H
H
H
H
3.56
9
19.87
13.56
29.75
8.47
10
11
12
F
a
Agonist activities mean values at a screening concentration of 300 nM were
obtained from three independent experiments.
Agonist activities mean values at a screening concentration of 100 nM were
obtained from three independent experiments.
b
c
clogP values were estimated with ChemDraw Ultra, version 12.0.
have a significant improvement on potency (though a slight
improvement was observed in meta-substituted compounds 4
and 6), indicating that the interaction mode of compound 2 series
was different from SAR1. Therefore, we focused our redesign
efforts on exploring optimal amide linker based on the common
amide structural characteristic of SAR1 and NIH screened
compound. The compound 8, directly structural analog of NIH
screened compound, led to 2-fold erosion of potency at 300 nM

Z. Li et al. / Bioorg. Med. Chem. 23 (2015) 6666–6672
6669
Table 2
In vitro activities and physicochemical properties of designed compounds
Act %a (100 nM)
EC50 (nM)
clogPc
LogD7.4
LE (LLE)e
b
d
Compd
R
R1
R2
TAK-875
1
65.32
54.94
29.75
35.67
3.56
29.6
62.3
ND
135.3
ND
4.697
4.511
2.182
2.491
2.801
2.681
2.759
2.574
2.681
2.681
3.210
3.609
3.609
4.008
4.317
4.008
2.271
2.271
3.109
3.380
3.310
3.619
3.708
2.43
2.31
À1.25
À0.18
ND
ND
ND
0.07
À0.99
ND
ND
0.15
ND
0.35
0.63
ND
ND
ND
ND
ND
0.27 (5.1)
0.27 (4.9)
11
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
H
H
H
2-Me
2-OEt
2-F
3-Me
4-Me
4-Et
3-Isopro
4-Isopro
3-tBu
3-tBu
4-tBu
3-OMe
4-OMe
4-OEt
4-OCF3
3-CF3
3-CF3
2-Me,
5-Isopro
2-Me,
5-Isopro
3,4-DiMe
3,5-DiMe
2,5-DiMe
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
H
Me
Me
H
H
H
H
H
H
Me
H
H
H
Me
H
H
Me
H
0.38 (7.0)
20.78
8.79
ND
ND
35.07
45.37
31.23
14.59
43.28
11.83
40.19
32.76
7.65
135.9
108.2
148.2
ND
115.6
ND
121.7
140.3
ND
147.6
ND
0.37 (6.8)
0.39 (8.0)
0.36 (6.7)
0.34 (6.5)
0.33 (6.2)
31.69
5.73
3.56
ND
ND
ND
ND
6.45
11.35
12.73
14.56
ND
ND
0.47
ND
33
Me
H
19.38
ND
4.017
ND
34
35
36
Me
Me
Me
H
H
H
36.75
25.67
18.65
132.9
ND
ND
3.439
3.489
3.489
0.33
ND
ND
0.37 (6.5)
ND = Not determined.
a
Agonist activities mean values at a screening concentration of 100 nM were obtained from three independent experiments.
EC50 values for FFA1 activities represent the mean of three independent determinations.
clogP values were estimated with ChemDraw Ultra, version 12.0.
b
c
d
e
LogD7.4 values were determined by shake-flask procedure.
LE values were calculated by À
D
g = RT lnKd, presuming EC50 ꢀ Kd, and the LLE values were calculated by the formula pEC50 À logD7.4
.
electron-withdrawing substituent such as 3-CF3 (30 and 31) indi-
cated that the electron-withdrawing effect in the area might reduce
agonistic activity of FFA1. To complete this SAR study, various sub-
stitutions in the ortho-position and para-position of terminal phenyl
were evaluated. For the ortho-substituted compounds, the agonistic
activity of 13 (2-H) > 17 (2-F) > 15 (2-Me) > 16 (2-OEt) suggested
that the steric effect in the ortho-position might influence agonistic
activity of FFA1. The para-substituted compounds, however,
revealed dual effects of the steric and electrical property. The ago-
nistic activity of 19 (4-Me) > 20 (4-Et) > 22 (4-isopro) > 25 (4-tBu),
showed a good correlation with the Van der Waals radius of substi-
tution. This result demonstrated that the large substitution in this
position may be undesirable. Meanwhile, strong electron-donating
group such as 4-OMe (27), 4-OEt (28) and 4-OCF3 (29) occupied in
the para-position turned out a significant decrease of activity,
implying that the electrical effect in the para-position was also cru-
cial to agonistic activity.
With this beneficial experience for meta-substituent resulted in
increased potency, a series of di-substituted compounds contain-
ing meta-substituents were synthesized and evaluated. Unfortu-
nately, all of the synthetically di-substituted compounds 32–36
showed a lower potency than the corresponding mono-substituted
analogs. Interestingly, although methyl substituent in the meta-po-
sition exhibited the best activity, the meta-disubstituted com-
pound 35 revealed a markedly lower agonistic activity than the
corresponding monomethyl analog 18. These results implied that
the polysubstituted compounds may introduce an unfavorable
steric interaction with the ligand-binding pocket of FFA1. Among
all of the tested compounds, the compound 18, a most potent
agonist in this series, had a significant advantage compared to
TAK-875 and compound 1 in terms of LE and LLE values. After
comprehensive investigation of SAR, a clear SAR picture of our
chemical scaffold was developed and summarized in Figure 3.
2.3. Effect of compound 18 on glucose tolerance
Based on these results above, the most potent compound 18
(20, 50 and 80 mg/kg) was selected to evaluate the in vivo
hypoglycemic effects in normal ICR mice by oral glucose tolerance
test (OGTT). As shown in Figure 4A, compound 18 exhibited a
dose-dependent response in blood glucose levels. Moreover, the
compound 18 showed
a significant improvement in glucose
tolerance at 80 mg/kg dose, similar to the hypoglycemic effect of
TAK-875 (20 mg/kg), the most advanced compound once in phase
III studies.
To further assess hypoglycemic effects in the diabetic state, STZ-
induced type 2 diabetic C57BL/6 mice were used to evaluate the
OGTT of compound 18, an orally bioavailable FFA1 agonist. As
shown in Figure 4B, the compound 18 was significantly improved
the hyperglycemia state of type 2 diabetic mice. These results
demonstrated that compound 18, with a significant advantage in
terms of physicochemical property, has a great potential for
improving the hyperglycemia state in both normal and type 2 dia-
betic mice.

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Z. Li et al. / Bioorg. Med. Chem. 23 (2015) 6666–6672
Figure 3. SAR summary.
Figure 4. (A) Effect of compound 18 on glucose tolerance in normal ICR mice. (B) OGTT in fasting type 2 diabetic C57BL/6 mice. Values are mean SEM (n = 6). *P 6 0.05 and
**P 6 0.01 compared to vehicle mice by Student’s t test. #P 6 0.05 compared to vehicle diabetic mice by Student’s t test.
the identification of lead compound 18, an excellent FFA1 agonist
with considerable agonistic activity, high LE and LLE values, lower
lipophilicity than previously reported agonists, and appreciable
hypoglycemic effect in both normal and type 2 diabetic mice.
Although the agonistic activity of compound 18 was inferior to
TAK-875, the information obtained from the SAR studies allowed
us to design more active FFA1 agonists with distinct advantage in
LE and LLE values.
3. Conclusion
With the aim of developing potent FFA1 agonists with reduced
molecular weight and lipophilicity, we have identified a new ser-
ies of phenoxyacetamide FFA1 agonists by comprehensive evalu-
ating 6 amide linkers based on the common amide structural
characteristic of SAR1 and NIH screened compound. Subse-
quently, systematic exploration of SAR in this series, leading to

Z. Li et al. / Bioorg. Med. Chem. 23 (2015) 6666–6672
6671
University (Jiangsu, China), acclimatized for 1 week before experi-
ments. The breeding room was keep on a constant 12 h light/black
cycle with temperature at 23 2 °C and relative humidity 50 10%
throughout the experimental period. Mice were allowed ad libitum
access to standard pellets and water unless otherwise stated, and
the vehicle used for drug administration was 0.5% Carboxy Methyl
Cellulose aqueous solution for all animal studies. All animal
experiments were performed in compliance with the relevant laws
and institutional guidelines, and our experiments have been
approved by the institutional committee of China Pharmaceutical
University.
4. Experimental section
4.1. Chemistry
All starting materials, solvents and reagents were obtained from
commercial sources and used without further purification unless
otherwise indicated. Column chromatography was carried out on
silica gel (200–300 mesh) and monitored by thin layer chromatog-
raphy performed on GF/UV 254 plates and were visualized by
using UV light at 254 and 365 nm. Melting points were measured
using a RY-1 melting-point apparatus, which was uncorrected.
All of the NMR spectra were recorded on a Bruker ACF-300Q instru-
ment (300 MHz for 1H NMR and 75 MHz for 13C NMR spectra),
chemical shifts are expressed as values relative to tetramethylsi-
lane as internal standard, and coupling constants (J values) were
given in hertz (Hz). LC/MS spectra were recorded on a Waters
liquid chromatography-mass spectrometer system (ESI). Elemental
analyses were performed by the Heraeus CHN-O-Rapid analyzer.
TAK-875 was synthesized via published procedures.10
Statistical analyses were performed using specific software
(GraphPad InStat version 5.00, GraphPad software, San Diego, CA,
USA). Unpaired comparisons were analyzed using the two-tailed
Student’s t-test, unless otherwise stated.
4.3.2.1. Effect of compound 18 on glucose tolerance explored in
male ICR mice.
Normal ICR mice 10 weeks old were fasted
overnight (12 h), weighted, bled via the tail tip, and randomized
into 5 groups (n = 6). Mice were administrated orally with a single
doses of vehicle, TAK-875 (10 mL kgÀ1; 20 mg kgÀ1), or compound
18 (20 mg kgÀ1, 50 mg kgÀ1, 80 mg kgÀ1) and subsequently dosed
orally with 30% glucose aqueous solution (3 g kgÀ1) after half an
hour. Blood samples were collected immediately before drug
administration (À30 min), before glucose challenge (0 min), and
at 15, 30, 45, 60 and 120 min post-dose. The blood glucose was
measured by blood glucose test strips (SanNuo ChangSha, China).
The physical characteristics, 1H NMR, 13C NMR, MS and elemen-
tal analysis data for all intermediates and target molecules, were
reported in the Supporting information.
4.2. Determination of logD7.4
In 10 mL glass vial, 40
added 1980 L phosphate buffer solution (0.01 M, pH = 7.4) and
1980 L 1-octanol (Sigma), obtaining 100 M final concentration
lL of 10 mM stock solution in DMSO was
l
l
l
of the test compounds. The glass vials were shaken at 700 rpm for
24 h and left for 1 h to allow the phases to separate. The 1-octanol
phase was pipetted out and diluted Â10 with a mixture of methanol
(containing 0.1% formic acid) and MilliQ H2O (4:1) prior to analysis
4.3.2.2. Hypoglycemic effects of compound 18 explored in type 2
diabetic mice.
Male C57BL/6 mice after 1 week adaptation
were fed with high-fat diet (45% calories from fat, from Medi-
science Ltd, Yangzhou, China) ad libitum for 4 weeks to induce
insulin resistance and then injected intraperitoneally (ip) with
low dose of STZ (10 mL kgÀ1; 80 mg kgÀ1). The mice were fed with
high-fat-diet for another 4 weeks, and the development of diabetes
was confirmed by measuring blood glucose levels. The mice with
fasting blood glucose level 11.1 mmol/L or higher were considered
to be diabetic and were used in the experiment as type 2 diabetic
mice model.26,27
on HPLC with 60
lL injections. The buffer phase was analyzed
directly in 120 L injections. Each HPLC analysis was performed in
l
duplicates by the method described above. The logD7.4 values were
calculated by dividing the peak area (mAU*min) at 254 nm of the
1-octanol phase by the corresponding peak area of the buffer
phase. Peak areas were corrected for systematic errors using two
calibration points per compound per solvent. All test compounds
were analyzed in three independent experiments.
Type 2 diabetic C57BL/6 mice were fasted overnight (12 h),
weighted, bled via the tail tip, and randomized into 3 groups
(n = 6), another group of normal fasting C57BL/6 mice was added
as negative control. Mice were administrated orally with a single
doses of vehicle, TAK-875 (10 mL kgÀ1; 20 mg kgÀ1), or compound
18 (10 mL kgÀ1; 50 mg kgÀ1) and subsequently dosed orally with
20% glucose aqueous solution (2 g kgÀ1) after half an hour. Blood
samples were collected immediately before drug administration
(À30 min), before glucose challenge (0 min), and at 15, 30, 45, 60
and 120 min post-dose. The blood glucose was measured by blood
glucose test strips (SanNuo ChangSha, China).
4.3. Biological methods
4.3.1. Ca2+ influx activity of CHO cells stably expressing human
FFA1 (FLIPR Assay)
CHO cells stably expressing human FFA1 (accession no.
NM_005303) were seeded into 96-well plates at a density of 15 K
cells/well and incubated 16 h in 5% CO2 at 37 °C. Then, the culture
medium was removed and washed with 100
Salt Solution. Subsequently, cells were incubated in loading buffer
(containing 2.5 g/mL fluorescent calcium indicator Fluo 4-AM,
2.5 mmol/L probenecid and 0.1% fatty acid-free BSA) for 1 h at
37 °C. Various concentrations of test compounds or -linolenic acid
lL of Hank’s Balanced
l
Acknowledgements
c
(Sigma) were added into the well and the intracellular calcium flux
signals were measured by FLIPR Tetra system (Molecular Devices).
The agonistic activities of test compounds on human FFA1 were
expressed as [(A À B)/(C À B)] Â 100 (increase of the intracellular
Ca2+ concentration (A) in the test compounds-treated cells and
This study was supported by Grants from the National Natural
Science Foundation of China (Grants 81172932 and 81273376),
the Natural Science Foundation of Jiangsu Province (Grant
BK2012356), and the Project Program of State Key Laboratory of
Natural Medicines, China Pharmaceutical University (Grant
JKGZ201103).
(B) in vehicle-treated cells, and (C) in 10 lM c-linolenic acid-trea-
ted cells). EC50 value of selected compound was obtained with
Prism 5 software (GraphPad).
Supplementary data
4.3.2. Animals and statistical analysis of the data
Male ICR mice (18–22 g) and male C57BL/6 mice (18–22 g)
were purchased from Comparative Medicine Centre of Yangzhou
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.09.010.

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Z. Li et al. / Bioorg. Med. Chem. 23 (2015) 6666–6672
13. Houze, J. B.; Zhu, L.; Sun, Y.; Akerman, M.; Qiu, W.; Zhang, A. J.; Sharma, R.;
References and notes
Schmitt, M.; Wang, Y.; Liu, J.; Liu, J.; Medina, J. C.; Reagan, J. D.; Luo, J.; Tonn, G.;
Zhang, J.; Lu, J. Y.-L.; Chen, M.; Lopez, E.; Nguyen, K.; Yang, L.; Tang, L.; Tian, H.;
Shuttleworth, S. J.; Lin, D. C. H. Bioorg. Med. Chem. Lett. 2012, 22, 1267.
14. Brown, S. P.; Dransfield, P. J.; Vimolratana, M.; Jiao, X.; Zhu, L.; Pattaropong, V.;
Sun, Y.; Liu, J.; Luo, J.; Zhang, J.; Wong, S.; Zhuang, R.; Guo, Q.; Li, F.; Medina, J.
C.; Swaminath, G.; Lin, D. C. H.; Houze, J. B. ACS Med. Chem. Lett. 2012, 3, 726.
15. Wang, Y.; Liu, J.; Dransfield, P. J.; Zhu, L.; Wang, Z.; Du, X.; Jiao, X.; Su, Y.; Li, A.-
R.; Brown, S. P.; Kasparian, A.; Vimolratana, M.; Yu, M.; Pattaropong, V.; Houze,
J. B.; Swaminath, G.; Thanhvien, T.; Khanh, N.; Guo, Q.; Zhang, J.; Zhuang, R.; Li,
F.; Miao, L.; Bartberger, M. D.; Correll, T. L.; Chow, D.; Wong, S.; Luo, J.; Lin, D. C.
H.; Medina, J. C. ACS Med. Chem. Lett. 2013, 4, 551.
16. Defossa, E.; Wagner, M. Bioorg. Med. Chem. Lett. 2014, 24, 2991.
17. Tikhonova, I. G.; Sum, C. S.; Neumann, S.; Engel, S.; Raaka, B. M.; Costanzi, S.;
Gershengorn, M. C. J. Med. Chem. 2008, 51, 625.
18. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Deliv. Rev.
2012, 64, 4.
1. Phung, O. J.; Scholle, J. M.; Talwar, M.; Coleman, C. I. JAMA 2010, 303, 1410.
2. Avery, M. A.; Mizuno, C. S.; Chittiboyina, A. G.; Kurtz, T. W.; Pershadsingh, H. A.
Curr. Med. Chem. 2008, 15, 61.
3. Stein, S. A.; Lamos, E. M.; Davis, S. N. Expert Opin. Drug Saf. 2013, 12, 153.
4. Lebovitz, H. E. Nat. Rev. Endocrinol. 2011, 7, 408.
5. Itoh, Y.; Kawamata, Y.; Harada, M.; Kobayashi, M.; Fujii, R.; Fukusumi, S.; Ogi,
K.; Hosoya, M.; Tanaka, Y.; Uejima, H.; Tanaka, H.; Maruyama, M.; Satoh, R.;
Okubo, S.; Kizawa, H.; Komatsu, H.; Matsumura, F.; Noguchi, Y.; Shinobara, T.;
Hinuma, S.; Fujisawa, Y.; Fujino, M. Nature 2003, 422, 173.
6. Briscoe, C. P.; Tadayyon, M.; Andrews, J. L.; Benson, W. G.; Chambers, J. K.;
Eilert, M. M.; Ellis, C.; Elshourbagy, N. A.; Goetz, A. S.; Minnick, D. T.; Murdock,
P. R.; Sauls, H. R.; Shabon, U.; Spinage, L. D.; Strum, J. C.; Szekeres, P. G.; Tan, K.
B.; Way, J. M.; Ignar, D. M.; Wilson, S.; Muir, A. I. J. Biol. Chem. 2003, 278, 11303.
7. Stoddart, L. A.; Smith, N. J.; Milligan, G. Pharmacol. Rev. 2008, 60, 405.
8. Wellendorph, P.; Johansen, L. D.; Bräuner-Osborne, H. Mol. Pharmacol. 2009, 76,
453.
9. Wang, X.; Zhao, T.; Yang, B.; Li, Z.; Cui, J.; Dai, Y.; Qiu, Q.; Qiang, H.; Huang, W.;
Qian, H. Bioorg. Med. Chem. 2015, 23, 132.
10. Negoro, N.; Sasaki, S.; Mikami, S.; Ito, M.; Suzuki, M.; Tsujihata, Y.; Ito, R.;
Harada, A.; Takeuchi, K.; Suzuki, N.; Miyazaki, J.; Santou, T.; Odani, T.; Kanzaki,
N.; Funami, M.; Tanaka, T.; Kogame, A.; Matsunaga, S.; Yasuma, T.; Momose, Y.
ACS Med. Chem. Lett. 2010, 1, 290.
11. Mikami, S.; Kitamura, S.; Negoro, N.; Sasaki, S.; Suzuki, M.; Tsujihata, Y.;
Miyazaki, T.; Ito, R.; Suzuki, N.; Miyazaki, J.; Santou, T.; Kanzaki, N.; Funami,
M.; Tanaka, T.; Yasuma, T.; Momose, Y. J. Med. Chem. 2012, 55, 3756.
12. Negoro, N.; Sasaki, S.; Ito, M.; Kitamura, S.; Tsujihata, Y.; Ito, R.; Suzuki, M.;
Takeuchi, K.; Suzuki, N.; Miyazaki, J.; Santou, T.; Odani, T.; Kanzaki, N.; Funami,
M.; Tanaka, T.; Yasuma, T.; Momose, Y. J. Med. Chem. 2012, 55, 1538.
19. Christiansen, E.; Urban, C.; Grundmann, M.; Due-Hansen, M. E.; Hagesaether,
E.; Schmidt, J.; Pardo, L.; Ullrich, S.; Kostenis, E.; Kassack, M.; Ulven, T. J. Med.
Chem. 2011, 54, 6691.
20. Waring, M. J. Expert Opin. Drug Discov. 2010, 5, 235.
21. Keserü, G. M.; Makara, G. M. Nat. Rev. Drug Disc. 2009, 8, 203.
22. Leeson, P. D.; Young, R. J. ACS Med. Chem. Lett. 2015, 6, 722.
23. Gleeson, M. P. J. Med. Chem. 2008, 51, 817.
24. Hopkins, A. L.; Groom, C. R.; Alex, A. Drug Discovery Today 2004, 9, 430.
25. Leeson, P. D.; Springthorpe, B. Nat. Rev. Drug Disc. 2007, 6, 881.
26. Winzell, M. S.; Ahrén, B. Diabetes 2004, 53, 215.
27. Koehler, J. A.; Baggio, L. L.; Lamont, B. J.; Ali, S.; Drucker, D. J. Diabetes 2009, 58,
2148.