Design, synthesis and biological activity of phenoxyacetic acid derivatives as novel free fatty acid receptor 1 agonists

Article abstract of DOI:10.1016/j.bmc.2015.10.011

The free fatty acid receptor 1 (FFA1) is a novel antidiabetic target for the treatment of type 2 diabetes based on particular mechanism in amplifying glucose-stimulated insulin secretion. We have previously identified a series of phenoxyacetic acid deriva

Full text of DOI:10.1016/j.bmc.2015.10.011

Bioorganic & Medicinal Chemistry 23 (2015) 7158–7164
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological activity of phenoxyacetic acid
derivatives as novel free fatty acid receptor 1 agonists
Zheng Li a, Xuekun Wang a,b, Xue Xu c, Jianyong Yang a, Wenting Xia a, Xianhao Zhou a,
Wenlong Huang a,d, , Hai Qian a,d,
⇑
⇑
a Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
b Department of Biomedical Engineering, College of Engineering and Applied Sciences, Nanjing University, Nanjing 210093, PR China
c Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
d 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:
The free fatty acid receptor 1 (FFA1) is a novel antidiabetic target for the treatment of type 2 diabetes
based on particular mechanism in amplifying glucose-stimulated insulin secretion. We have previously
identified a series of phenoxyacetic acid derivatives. Herein, we describe the further chemical modifica-
tion of this series directed by ligand efficiency and ligand lipophilicity efficiency. All of these efforts lead
to the discovery of the promising candidate 16, an excellent FFA1 agonist with robust agonistic activity
(43.6 nM), desired LE and LLE values. Moreover, compound 16 revealed a great potential for improving
the hyperglycemia levels in both normal and type 2 diabetic mice without the risk of hypoglycemia even
at the high dose of 40 mg/kg.
Received 21 September 2015
Revised 7 October 2015
Accepted 8 October 2015
Available online 8 October 2015
Keywords:
FFA1 agonist
Phenoxyacetic acid
Ligand efficiency
Type 2 diabetes
Hypoglycemia
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
treatment of T2DM. Owing to the structural similarity to fatty acids,
most of the reported agonists have relatively high lipophilicity that
Type 2 diabetes mellitus (T2DM) is becoming a common and
frequently occurring metabolic disease characterized by impaired
glucose homeostasis due to insulin deficiency and tissue resistance
to glucose uptake and utilization.1,2 Insulin secretagogues, such as
meglitinides and sulfonylureas, are commonly used for the treat-
ment of T2DM.3 However, these drugs enhance insulin secretion
independent of blood glucose levels, leading to the relatively high
risk of hypoglycemia.4 Novel insulin secretagogues with potent
antidiabetic effects and a low risk of hypoglycemia are thus desir-
able. The free fatty acid receptor 1 (FFA1, also known as GPR40) is
highly expressed in pancreatic b-cells and responds to medium- to
long-chain free fatty acids resulting in amplification of glucose-
stimulated insulin secretion.5–7 Therefore, this particular
mechanism of FFA1 provides the tremendous potential for boost-
ing insulin levels with decreased risk of hypoglycemia compared
to classic insulin secretagogues.
are found to negatively influence the ADMET properties and corre-
late with attrition.16–18 Therefore, the concepts such as ligand effi-
ciency (LE) and ligand lipophilicity efficiency (LLE) have been
extensively used to avoid undue increase in molecular size and
lipophilicity in the optimization process.19,20 We have previously
reported a series of phenoxyacetic acid derivatives which success-
fully improved b-oxidation of phenylpropanoic acid moiety.21,22
Herein, we report the further optimization of this compound series
aiming to expand the structure–activity relationship (SAR) studies
directed by LE and LLE (Fig. 2). After systematic exploration of SAR
and application of molecular modeling, the promising candidate
16 and its interaction mode were identified. In subsequent in vivo
pharmacological studies, compound 16 showed a robustly hypo-
glycemic effect in both normal and type 2 diabetic mice without
the risk of hypoglycemia even at the high dose of 40 mg/kg.
Recently, a variety of synthetic FFA1 agonists have been reported
in the literature (Fig. 1),8–15 of which the compounds TAK-875,
AMG-837 and LY2881835 have reached clinical trials for the
2. Results and discussion
2.1. Chemistry
⇑
The synthetic routes of target compounds 1–16 are detailed in
Scheme 1. Compound 34a was synthesized via our published
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.2015.10.011
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

Z. Li et al. / Bioorg. Med. Chem. 23 (2015) 7158–7164
7159
Figure 1. Selected examples of synthetic GPR40 agonists.
Figure 2. Optimization of phenoxyacetic acid derivatives.
Scheme 1. Synthesis of target compounds 1–16. Reagents and conditions: (a) (3-formylphenyl)boronic acid, Pd(PPh3)4, Na2CO3, toluene, ethanol, H2O, 80 °C, 12 h; (b) acetyl
chloride, NEt3, CH2Cl2, 0 °C, 2 h; (c) NaBH4, CH3OH, THF, 0 °C, 30 min; (d) SOCl2, CH2Cl2, DMF, 40 °C, 4 h; (e) K2CO3, acetone, KI, reflux, 12 h; (f) CH3ONa, MeOH, rt, 2 h; (g) R1Br
or R1OTs, K2CO3, acetone, reflux, 12 h; (h) LiOHÁH2O, THF/MeOH/H2O, rt, 4 h.
procedures.21 The key intermediate 32 was prepared by the
acetylation of intermediate 31, which was derived from the Suzuki
coupling of commercially available bromobenzene 30 with
(3-formylphenyl)boronic acid in the presence of Pd(PPh3)4. The
intermediate 32 was reduced with NaBH4 in the presence of
methanol, followed by treating with thionyl chloride catalyzed
by DMF catalyzer, generated the chlorinated intermediate 33.
Condensation of the obtained chlorinated intermediate 33 with
34a by using Williamson ether synthesis to afford the common
intermediate 35. The basic hydrolysis of intermediate 35 gives
the phenol intermediate 36 and the desired target molecular 8. After
stirring at reflux temperature for 12 h, the phenol intermediate 36
Scheme 2. Synthesis of target compounds 17–21. Reagents and conditions: (a) ethyl chloroacetate, NaHCO3, ethanol, rt, 24 h; (b) R3COCl, NEt3, CH2Cl2, 0 °C–rt, 16 h; (c) H2,
Pd–C, rt, 24 h; (d) NaBH4, CH3OH, THF, 0 °C, 30 min; (e) SOCl2, CH2Cl2, DMF, 40 °C, 4 h; (f) K2CO3, acetone, KI, reflux, 12 h; (g) LiOHÁH2O, THF/MeOH/H2O, rt, 4 h.

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Z. Li et al. / Bioorg. Med. Chem. 23 (2015) 7158–7164
Table 1
In vitro activities and physicochemical properties of target compounds
O
R3
X
R1
O
R2
COOH
Act%a (100 nM)
EC50 (nM)
LogD7.4
LE (LLE)d
b
c
Compd
R1
R2
R3
X
TAK-875
65.32
58.37
53.12
47.85
36.92
41.57
35.17
26.38
44.26
29.6
49.7
65.9
105.8
ND
ND
ND
ND
110.3
2.43
3.20
3.48
3.64
ND
ND
ND
ND
2.37
0.27 (5.1)
0.33 (4.1)
0.31 (3.7)
0.29 (3.3)
1
2
3
4
5
6
7
8
Me
Et
Pro
iso-Pro
Bu
iso-Bu
Bn
H
Me
Me
Me
Me
Me
Me
Me
Me
F
F
F
F
F
F
F
F
O
O
O
O
O
O
O
O
0.32 (4.5)
0.29 (3.8)
O
9
Me
Me
F
F
O
O
48.73
23.62
103.6
ND
3.15
ND
BnO
10
O
11
12
Me
Me
F
F
O
O
37.95
44.37
ND
ND
HO
109.5
2.73
0.28 (4.2)
N
O
13
14
Me
Me
F
F
O
O
13.49
18.35
ND
ND
ND
ND
O
N
O
O
O
O
S
S
S
15
16
17
Me
H
F
O
O
54.94
61.28
36.29
62.3
43.6
ND
1.81
1.63
ND
0.28 (5.3)
0.29 (5.6)
O
O
O
F
N
Me
H
O
O
N
N
S
S
18
19
Me
Me
Me
H
18.37
23.46
ND
ND
ND
ND
O
O
O
O
O
O
N
N
S
S
20
21
Me
Me
Me
H
2.57
ND
ND
ND
ND
O
O
O
O
O
28.85
O
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.
LogD7.4 values were determined by shake-flask procedure.
b
c
d
LE values were calculated by—
D
g = RT lnKD, presuming KD ꢀ EC50, and the LLE values were calculated by the formula pEC50 À LogD7.4
.
was alkylated by various alkylation reagents in acetone under the
action of K2CO3, followed by basic hydrolysis, afforded the target
compounds 1–7 and 9–16.
basic hydrolysis with lithium hydroxide, afforded the target com-
pounds 17–21.
The synthesis of the designed target compounds 17–21 are
depicted in Scheme 2. The compound 41a was synthesized via
published procedures.8 The compound 41a was reduced with
NaBH4, followed by treating with thionyl chloride catalyzed by
DMF catalyzer, generated the chlorinated intermediate 42a.
Mono-alkylation of the aniline 37 with ethyl chloroacetate to afford
compound 38, which was subsequently converted to intermediate
39 by acylation with various acyl chloride. After stirring at ambient
temperature for 24 h, the obtained intermediate 39 was debenzy-
lated by Pd–C under the hydrogen atmosphere to afford intermedi-
ate 40. The intermediate 40 was connected with 42a by using
Williamson ether synthesis in the presence of K2CO3, followed by
2.2. FFA1 agonistic activity and SAR study
The synthetic compounds were investigated by a fluorometric
imaging plate reader (FLIPR) assay in Chinese hamster ovary
(CHO) cells expressing human FFA1. Besides overall activity, the
LogD7.4 and LE, as well as LLE were taken advantage of in the
assessment of optimized analogs. From previous SAR studies on
other chemical series, it was revealed that incorporation of the var-
ious substituents into the para-position of the biphenyl ring was
tolerated.9,10 With these observations in mind, we firstly explored
the tolerability of the functionalities in our series by using
various alkyl groups (compounds 1–6). As shown in Table 1,

Z. Li et al. / Bioorg. Med. Chem. 23 (2015) 7158–7164
7161
Figure 3. Overlay of TAK-875 (green) and compound 16 (blue) bound to FFA1. Key residues are labeled in red, and hydrogen bonding interactions are represented by yellow
dashed lines.
methyl substituted derivative 1, with relatively high lipophicity
(LogD7.4 = 3.20), exhibited the strongest agonistic activity in alkyl
substituted derivatives. Moreover, more bulky isopropyl (4) and
isobutyl (6) derivative was still tolerated in this area. These results
imply that the FFA1 receptors would have a large cavity in the
ligand-binding pocket around the tail substituent. Interestingly,
the agonistic activity of compound 1 (4-Me) > 2 (4-Et) > 3 (4-Pro)
> 5 (4-Bu) > 4 (4-isoPro) > 6 (4-isoBu) > 7 (4-Bn), which revealed a
good correlation with Van der Waals radius of corresponding sub-
stituents, and this result suggested that the steric effect in the
para-position of the biphenyl moiety might influence agonistic
activity of FFA1. Gratifyingly, the compound 8, derived from com-
mon intermediate 35b, exhibited a potent agonistic activity and
considerable LLE value owing to significantly decreased lipophic-
ity. This result demonstrated that the polar functionality was also
tolerated in this region. Therefore, this position was thought to be
suitable for modulating the lipophilicity of ligands by introducing
hydrophilic substituents. A slight improvement in potency over
the compound 5 was obtained only by replacing a carbon atom
of compound 5 with oxygen atom in compound 9. We speculated
that the improvement of the activity was associated with solvent
effect of oxygen atom which stabilized the lowest energy confor-
mation of ligand. Moreover, the terminal hydroxyl group derivative
12, with lower lipophilicity, exhibited a significant improvement
on potency in comparison with the parent compound 11, and this
result further confirmed the above speculation. The introduction of
amide (compounds 13 and 14), however, showed a drastic drop of
activity despite increased solvent effect, likely attributing to the
steric effects of dimethylamino and morpholine. Consistent with
before-mentioned findings, replacement of the methyl group in
compound 9 with benzyl group gave compound 10, which turned
out an approximately 1-fold decrease in potency compared to the
compound 9 but still indicates that the receptor binding pocket can
accommodate the bulky group. Inspired by the terminal hydrophi-
lic methylsulfonyl fragment of TAK-875, we designed and synthe-
sized compound 15, a superior agonist revealed a small decrease in
agonistic activity relative to compound 1 but more than an order of
Based on these results above, we therefore selected this scaffold
of 15 as our starting point for further modification. The mono-
methyl analog 16, with relatively lower molecular weight and
lipophilicity, exhibited a marked improvement on potency and
LLE value in comparison with the parent compound 15. With these
beneficially optimized experiences for phenoxyacetic acid series,
our optimized efforts were directed to replace the oxygen atom
of phenoxyacetic acid with bioisostere nitrogen atom. Concerns
about relatively high dissociation energy between alkaline amino
and adjacent carboxylic group led us to introduce amide rather
than amino. Among them, the acetamide analog 17 revealed a
strongest potency in this series but still deviate the desired
potency. Furthermore, as the size of amide substituent increases
the potency decrease (compounds 17 > 21 > 19), suggesting that
the limited space in the binding pocket around this area. Incorpo-
ration of methyl group in ortho-position of acidic head to afford
compounds 18 and 20 revealed a markedly lower agonistic activity
than the corresponding unsubstituted compounds 17 and 19. One
possible explanation is that the incorporation of bulkier methyl
substituent moves the carboxylic acid moiety into an unfavorable
conformation. Among all of the tested compounds, the most potent
agonist 16, with excellent lipophilicity and desired LLE value, was
selected for further investigation.
2.3. Docking study of compound 16 with hGPR40
To better understand the SAR and interaction mode of com-
pound 16, a molecular modeling study based on the recently
reported X-ray structure of FFA1 (PDB accession code: 4PHU)
was performed by using the Molecular Operating Environment
(MOE) (Fig. 3).23 As shown in Figure 3, the interaction mode of
compound 16 was nearly perfect overlap with TAK-875, and the
carboxylic acid moiety was highly coordinated by Arg183,
Arg258 and Tyr91 forming anchor point. Moreover, Trp174 is ori-
ented nearly perpendicularly to the plane of the fluorobenzene ring
of compound 16 where it forms an edge-on interaction. These cru-
cial interactions render the acidic head is very sensitive to the
structural modifications such as the bioisostere amide derivatives
(compounds 17–21). Meanwhile, our model aligns with the SAR
magnitude reduced lipophilicity, and thereby
a significantly
increased LLE value compared to compounds 1 and 2.

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Z. Li et al. / Bioorg. Med. Chem. 23 (2015) 7158–7164
Figure 4. (A) Effect of compound 16 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.
ICR mice by oral administrating a high dose of compound 16 in
comparison with glibenclamide. As shown in Figure 5, the gliben-
clamide (15 mg/kg) treated group lowered the blood glucose levels
far below fasting normal mice. In contrast, compound 16, even at
the high dose of 40 mg/kg, only slightly reduced plasma sugar
levels in fasting ICR mice, and the change of blood glucose levels
was much smaller than that of glibenclamide. Therefore, these
results demonstrated that compound 16 revealed a low risk of
hypoglycemia, a serious side effect to sulfonylureas, which are
widely used in the treatment of T2DM as one of the first-line drugs.
3. Conclusion
In conclusion, starting from previously identified phenoxyacetic
acid derivatives, we have systematically explored the SAR of this
Figure 5. Effects of compound 16 on fasting plasma glucose in normal mice. Values
series directed by LE and LLE. Among them, the ideal lipophilic
are expressed as mean SEM for six animals in each group. *P 6 0.05, **P 6 0.01
compound 16, with robust in vitro agonistic activity, revealed a
compared to compound 16 treated mice by Student’s t-test.
great potential for improving the hyperglycemia levels in both nor-
mal and type 2 diabetic mice without the risk of hypoglycemia
even at the high dose of 40 mg/kg. According to these results, com-
pound 16 will be a promising candidate as a novel FFA1 agonist
with a low risk of hypoglycemia, and the information derived from
the SAR studies allowed us to design more competitive FFA1 ago-
nists that are structurally related.
determined above, which identified that the para-position of the
terminal biphenyl moiety could tolerate a variety of substituents
with different polarity and length while minimal effect on FFA1
agonistic activity.
2.4. Effect of compound 16 on glucose tolerance
4. Experimental section
4.1. Chemistry
Based on these results above, the most potent compound 16
(10, 20 and 40 mg/kg) was selected to further investigate the oral
glucose tolerance test (OGTT) in normal ICR mice. The time-
dependent changes of the plasma glucose levels are shown in Fig-
ure 4A. The compound 16 revealed a dose-dependent decrease in
blood glucose levels with stronger efficacy at 40 mg/kg compared
to TAK-875 at 20 mg/kg. To further evaluate the hypoglycemic
effects in the diabetic state, STZ-induced type 2 diabetic C57BL/6
mice were used to assess the efficacy of compound 16, an orally
bioavailable potent agonist. As shown in Figure 4B, the hyper-
glycemia state was significantly improved in compound 16 treated
mice, and the hypoglycemic effect was in close proximity to the
positive control TAK-875, the most advanced compound once in
phase III studies. These results demonstrated that compound 16,
the most potent agonist among our synthetic compounds, has a
great potential for improving the hyperglycemia levels in both nor-
mal and type 2 diabetic mice.
All starting materials, reagents and solvents 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
chromatography performed on GF/UV 254 plates and were visual-
ized by using UV light at 365 and 254 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 instrument (300 MHz for 1H NMR and 75 MHz for 13C
NMR spectra), chemical shifts are expressed as values relative
to tetramethylsilane 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.8
2.5. Effects of compound 16 on the risk of hypoglycemia
The physical characteristics, 1H NMR, 13C NMR, MS and elemen-
tal analysis data for all intermediates and target compounds, were
reported in the Supporting information.
Obtaining these positive results in pharmacological study, we
subsequently assessed the risk of hypoglycemia in fasting normal

Z. Li et al. / Bioorg. Med. Chem. 23 (2015) 7158–7164
7163
4.2. Molecular modeling
institutional guidelines, and our experiments have been approved
by the institutional committee of China Pharmaceutical University.
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.
Docking simulations were performed using MOE (version
2008.10, The Chemical Computing Group, Montreal, Canada). The
crystal structure of FFA1 (PDB ID: 4PHU) was downloaded from
the Protein Data Bank (PDB). Prior to ligand docking, the structure
was prepared with Protonate 3D and a Gaussian Contact surface
was draw around the binding site. Subsequently, the active site
was isolated and the backbone was removed. The ligand poses
was filtered using Pharmacophore Query Editor. The compound
structures were placed in the site with Pharmacophore method
and then ranked with the London dG scoring function. For the
energy minimization in the pocket, MOE Forcefield Refinement
was used and ranked with the London dG scoring function.
4.4.2.1. Effect of compound 16 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
16 (10 mg kgÀ1, 20 mg kgÀ1, 40 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).
4.3. 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
4.4.2.2. Hypoglycemic effects of compound 16 explored in type 2
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-octa-
nol phase was pipetted out and diluted Â10 with a mixture of
methanol (containing 0.1% formic acid) and MilliQ H2O (4:1) prior
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.24,25
to analysis on HPLC with 60
lL injections. The buffer phase was
analyzed directly in 120 L injections. Each HPLC analysis was per-
l
formed in 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
4.4. Biological methods
as negative control. Mice were administrated orally with
single doses of vehicle, TAK-875 (10 mL kgÀ1; 20 mg kgÀ1), or
compound 16 (10 mL kgÀ1 20 mg kgÀ1
and subsequently
a
4.4.1. 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
;
)
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).
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
4.4.2.3. Effects of compound 16 on the risk of hypo-
c
glycemia.
10 weeks old male normal ICR mice were fasted
(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
overnight and randomized into 3 groups (n = 6). Compound 16
(40 mg kgÀ1), glibenclamide (15 mg kgÀ1), or vehicle was orally
administered, and blood was collected from tail vein immediately
before administration (0 min) and at 30, 60, 90, 120 and 180 min
after administration and measure blood glucose as described
above.
(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).
Acknowledgements
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).
4.4.2. 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
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 exper-
iments were performed in compliance with the relevant laws and
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.10.011.

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11. McKeown, S. C.; Corbett, D. F.; Goetz, A. S.; Littleton, T. R.; Bigham, E.; Briscoe,
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