Discovery of a novel oxime ether scaffold as potent and orally bioavailable free fatty acid receptor 1 agonists

Article abstract of DOI:10.1039/c6ra07356e

The free fatty acid receptor 1 (FFA1) plays a key role in amplifying glucose-stimulated insulin secretion in pancreatic β-cells. Most of the reported FFA1 agonists contain a biphenyl scaffold, which is associated with toxicity as verified by Daiichi Sankyo. Herein, we describe the systematic exploration of a non-biphenyl scaffold to improve the druggability of GW9508 (β-oxidation, Fsp³ = 0.13, tPSA = 58.5 ?²) directed by Fsp³ and tPSA values. All these optimizations ultimately led to the identification of compound 21, an unconventional agonist (EC₅₀ = 72.5 nM) bearing a methyl oxime ether scaffold. Moreover, compound 21 revealed improved drug-like properties (Fsp³ = 0.23, tPSA = 86.6 ?²) when compared to GW9508 (Fsp³ = 0.13, tPSA = 58.5 ?²) and an even higher binding efficiency index (BEI = 15.3) than TAK-875 (BEI = 14.3). Further pharmacological studies suggested that compound 21 has a considerable hypoglycemic effect in both normal and type 2 diabetic mice with a low risk of hypoglycemia. In addition, the docking study promoted our understanding of the ligand-binding pocket. This information might help towards the design of more promising new molecular entities.

Full text of DOI:10.1039/c6ra07356e

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PAPER
Discovery of a novel oxime ether scaffold as potent
and orally bioavailable free fatty acid receptor 1
agonists†
Cite this: RSC Adv., 2016, 6, 46356
Zheng Li,a Jianyong Yang,a Weijie Gu,a Guoshen Cao,a Xiaoting Fu,a Xuedan Sun,a
a
a
ab
ab
*
*
Yu Zhang, Hui Jin, Wenlong Huang
and Hai Qian
The free fatty acid receptor 1 (FFA1) plays a key role in amplifying glucose-stimulated insulin secretion in
pancreatic b-cells. Most of the reported FFA1 agonists contain a biphenyl scaffold, which is associated
with toxicity as verified by Daiichi Sankyo. Herein, we describe the systematic exploration of a non-
biphenyl scaffold to improve the druggability of GW9508 (b-oxidation, Fsp ¼ 0.13, tPSA ¼ 58.5 A˚2)
3
3
directed by Fsp and tPSA values. All these optimizations ultimately led to the identification of compound
21, an unconventional agonist (EC50 ¼ 72.5 nM) bearing a methyl oxime ether scaffold. Moreover,
compound 21 revealed improved drug-like properties (Fsp ¼ 0.23, tPSA ¼ 86.6 A˚2) when compared to
3
GW9508 (Fsp ¼ 0.13, tPSA ¼ 58.5 A˚2) and an even higher binding efficiency index (BEI ¼ 15.3) than TAK-
3
Received 21st March 2016
Accepted 25th April 2016
875 (BEI ¼ 14.3). Further pharmacological studies suggested that compound 21 has a considerable
hypoglycemic effect in both normal and type 2 diabetic mice with a low risk of hypoglycemia. In
addition, the docking study promoted our understanding of the ligand-binding pocket. This information
might help towards the design of more promising new molecular entities.
DOI: 10.1039/c6ra07356e
www.rsc.org/advances
Recently, a variety of synthetic FFA1 agonists that contain
acidic moieties have been reported in the literature (Fig. 1),13–21
1. Introduction
of which compounds TAK-875 and AMG-837 are both in clinical
trials. However, in December 2013, Takeda decided to terminate
the phase III studies of TAK-875 due to concerns about liver
toxicity.22,23 Subsequently, the researchers at Daiichi Sankyo
have shown that the common biphenyl scaffold (red mark in
Fig. 1) of the FFA1 agonists was crucial for toxicity.24 Therefore,
it is extremely urgent to hunt for a new non-biphenyl scaffold.
We previously reported a series of phenoxyacetic acid deriva-
tives, which successfully removed the biphenyl moiety by
introducing a phenoxyacetamide linker.25 Herein, we chose
GW9508 (Fig. 1) as the non-biphenyl lead compound to explore
novel FFA1 agonists because it has outstanding efficacy and
a simple structure to avoid undue increase in molecular size
and lipophilicity during the modication process. On the other
hand, GW9508 was susceptible to b-oxidation at the phenyl-
propanoic acid moiety and is a rather at molecule with a frac-
Type 2 diabetes mellitus (T2DM) is a worldwide epidemic
characterized by impaired glucose homeostasis due to tissue
resistance and insulin deciency.1,2 Despite an inspiring array
of pharmacotherapies for T2DM, most of the oral antidiabetic
agents are associated with undesirable side effects such as
a high risk of hypoglycemia, body weight gain and gastric
symptoms.3–6 Hence, a novel drug with increased durability and
safety in achieving preferable glycemic control is still an urgent
need for T2DM.7,8 The free fatty acid receptor 1 (FFA1, also
known as GPR40), a new anti-diabetic target, has attracted
considerable attention over the last decade and plays a key role
in amplifying glucose-stimulated insulin secretion on pancre-
atic b-cells without the risk of hypoglycemia.9–12 Therefore, the
glucose concentration-dependent mechanism of FFA1 provides
enormous potential for improving insulin levels with
a decreased risk of hypoglycemia.
3
3
tion of saturated carbons (Fsp ) of 0.13. The Fsp (the number of
sp3 hybridized carbons/total carbon count) is a new index rep-
resenting drug-likeness and is extensively used in optimization
processes.26–29 Lovering et al. pointed out that an increase in the
aCenter of Drug Discovery, State Key Laboratory of Natural Medicines, China
Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China. E-mail:
ydhuangwenlong@126.com; qianhai24@163.com; Fax: +86-25-83271302; Tel: +86-
25-83271302
Fsp value can result in improved clinical success.30,31 Moreover,
3
FFA1 is expressed in the brain and its function is unclear.10
However, a structurally related agonist with low polar surface
area revealed signicant central nervous system (CNS) expo-
bJiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China
Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
† Electronic supplementary information (ESI) available: The physical
characteristics, 1H NMR, 13C NMR, MS and elemental analysis data for all
intermediates and target molecules. See DOI: 10.1039/c6ra07356e
sure.32 Thus, we considered that improvement of the Fsp and
3
tPSA values would be a rational approach to hunting for a new
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Fig. 1 Selected examples of synthetic GPR40 agonists.
Fig. 2 Design strategy starting from GW9508 as an initial lead.
non-biphenyl scaffold with improved drug-like properties hydrochloride. The dihydrobenzofuran intermediate 14a was
(Fig. 2). Aer comprehensive exploration of SAR information synthesized via literature procedures.15 Condensation of the
and utilization of molecular modeling, the potent lead obtained chlorinated intermediates 3a–b, 6a, 9a or 13a–c with
compound 21 bearing an oxime ether scaffold and its binding 14a using the Williamson ether synthesis followed by basic
mode were identied. In further pharmacological studies, hydrolysis, afforded the desired carboxylic acids 1–4 and 6–8.
2
˚
3
compound 21 with improved Fsp (0.23) and tPSA (86.6 A )
The syntheses of target compounds 5 and 9–14 are depicted
values compared to GW9508, revealed a low risk of hypogly- in Scheme 2. The commercial starting material 2-bromoaceto-
cemia and outstanding hypoglycemic effects in both normal phenone 4a was treated with 4-hydroxybenzyl alcohol to afford
and type 2 diabetic mice.
the common intermediate 15a, which was further converted to
the chlorinated intermediate 16a. Intermediate 17a was ach-
ieved from 15a via condensation with hydroxylammonium
chloride. Alkylation of intermediate 17a with the corresponding
alkylating reagent obtained intermediates 18a–f and was fol-
lowed by chlorination using thionyl chloride to yield the desired
intermediates 19a–f. Subsequently, connecting the obtained
chlorinated intermediates 16a or 19a–f with 14a in the presence
of K2CO3, followed by basic hydrolysis, afforded the desired
carboxylic acids 5 and 9–14.
The syntheses of the target compounds 15–30 are detailed in
Scheme 3. The preparation of intermediates 21a–c began with
the conversion of 7a to 20a–c via condensation with substituted
4-hydroxybenzyl alcohol. Similar conditions to 21a–c provided
intermediates 25a–m from intermediates 23a–m, formed from
commercially available 22a–m upon treatment with methoxy-
amine hydrochloride. Condensation of the obtained chlori-
nated intermediates 21a–c or 25a–m with 14a using the
Williamson ether synthesis followed by basic hydrolysis, affor-
ded the desired carboxylic acids 15–30.
2. Results and discussion
2.1. Chemistry
The synthetic routes used to prepare target compounds 1–4 and
6–8 are summarized in Scheme 1. The intermediates 3a–b were
prepared by chlorination of 2a–b, which were derived from the
Williamson ether synthesis of 3-hydroxybenzyl alcohol and
benzyl bromide or (2-bromoethyl)benzene in the presence of
K2CO3. The commercial starting material 2-bromoacetophe-
none 4a was treated with 3-hydroxybenzyl alcohol to afford the
desired product 5a, which was further converted to interme-
diate 6a via chlorination with thionyl chloride catalyzed by
DMF. The intermediate 7a was synthesized upon mixing 4a and
methoxyamine hydrochloride in DMSO. Condensation of 7a
with 3-hydroxybenzyl alcohol and then chlorination provided
intermediate 9a. Similar conditions to 9a provided intermedi-
ates 13a–c from intermediates 11a–c, formed from commer-
cially available 10a–c upon treatment with methoxyamine
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Scheme 1 Syntheses of the target compounds 1–4 and 6–8. Reagents and conditions: (a) BnBr or (2-bromoethyl)benzene, K2CO3, acetone, 45
ꢀC, 12 h; (b) SOCl2, CH2Cl2, DMF, 40 ꢀC, 4 h; (c) methoxyamine hydrochloride, DMSO, rt, 8 h; (d) K2CO3, acetone, KI, 60 ꢀC, 8 h and (e) LiOH$H2O,
THF/MeOH/H2O, rt, 4 h.
the nature of the binding pocket, the conformationally con-
2.2. FFA1 agonistic activity and SAR study
strained analog 3 was designed and synthesized. Compound 3,
The synthetic compounds were investigated in Chinese hamster
ovary (CHO) cells expressing human FFA1 using a uorometric
imaging plate reader (FLIPR) assay. As shown in Table 1,
2
˚
with an increased tPSA value (82.0 A ), revealed a signicant
improvement in potency when compared to the parent
3
compound 2, though a dramatically decreased Fsp value was
3
compound 1 was initially designed to increase the Fsp value
observed. Concerns about the potential metabolic instability
associated with the ketone moiety led us to introduce an oxime
ether scaffold based on Connexios' patent,33 whereas the affor-
3
(Fsp ¼ 0.21) and appeared to diminish the FFA1 agonistic
3
activity when compared with GW9508 (Fsp ¼ 0.13). An approx-
imately 2-fold increase in potency over compound 1 was ob-
tained by attaching an additional methylene in compound 1 to
2
˚
ded compound 4 (tPSA ¼ 86.6 A ) appeared to diminish the
agonistic activity. Interestingly, re-positioning the site of the
substituent from the 3-position to 4-position obtained
compounds 5 and 6, which exhibited a signicant improvement
in potency in comparison with the parent compounds 3 and 4,
respectively. Unlike compound 4, the oxime ether analog 6
revealed better activity than the ketone analog 5, which sug-
gested that the oxime ether occupied an additional binding site
3
3
achieve compound 2, which further improved the Fsp value (Fsp
¼ 0.24). We speculated that the improvement of activity was
associated with the larger rotational freedom of conformation
in compound 2 to adopt the required conformation. With the
dual purpose of improving the potency of compounds as well as
dening the active conformation of the ligand to understand
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Scheme 2 Syntheses of target compounds 5 and 9–14. Reagents and conditions: (a) 4-hydroxybenzyl alcohol, K2CO3, acetone, 45 ꢀC, 12 h; (b)
SOCl2, CH2Cl2, DMF, 40 ꢀC, 4 h; (c) hydroxylammonium chloride, DMSO, rt, 8 h; (d) RBr, K2CO3, acetone, 45 ꢀC, 12 h; (e) K2CO3, acetone, KI, 60
ꢀC, 8 h and (f) LiOH$H2O, THF/MeOH/H2O, rt, 4 h.
Scheme 3 Syntheses of target compounds 15–30. Reagents and conditions: (a) K2CO3, acetone, 45 ꢀC, 12 h; (b) SOCl2, CH2Cl2, DMF, 40 ꢀC, 4 h;
(c) methoxyamine hydrochloride, DMSO, rt, 8 h; (d) K2CO3, acetone, KI, 60 ꢀC, 8 h and (e) LiOH$H2O, THF/MeOH/H2O, rt, 4 h.
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Table 1 In vitro agonistic activities and selected parameters of the target compounds
3c
2
Acta% (300 nM)
Actb% (100 nM)
Fsp
tPSAd (A )
˚
Compd
R
Scaffold
TAK-875
GW9508
76.01
73.02
65.32
57.16
0.35
0.13
99.1
58.5
1
A
A
18.31
3.96
0.21
65.0
2
35.57
14.74
0.24
65.0
3
4
5
6
A
A
B
B
43.85
32.26
54.83
67.45
20.36
13.64
38.24
53.76
0.20
0.23
0.20
0.23
82.1
86.6
82.1
86.6
7
B
B
36.75
20.85
15.62
4.18
0.25
0.33
114.8
86.6
8
a
The mean value of agonist activity at a screening concentration of 300 nM was obtained from three independent experiments. b The mean value of
agonist activity at a screening concentration of 100 nM was obtained from three independent experiments. Fsp ¼ number of sp3 hybridized
c
3
carbons/total carbon count. d The topological polar surface area was calculated using ACD/I-Lab: https://ilab.acdlabs.com/iLab2/index.php.
or induced a more appropriate conformation. Moreover, the However, a bulky hydrophilic substituent (compound 14) was
bioisosteres, thiophene (7) and the non-aromatic substituent (8) well tolerated despite the increased steric effects.
were introduced to explore the importance of the phenyl ring in
compound 6. The obtained compounds 7 and 8 displayed with improved Fsp (0.23) and tPSA (86.6 A ) values, we then
a drastic loss of activity, indicating that the phenyl ring of shied our focus on evaluating the various substituents on the
Having identied a favorable methyl oxime ether scaffold
2
˚
3
compound 6 was crucial for FFA1 agonistic activity.
terminal and middle benzene rings. We had concerns about the
Based on the results described above, we therefore selected relatively high molecular weights that exist in most of the re-
the oxime ether scaffold of compound 6 as our starting point for ported agonists such as TAK-875 (524.63) and AM-1638 (514.64).
further modication. Our optimized efforts were directed to Therefore, besides the agonistic activity, the binding efficiency
probe the best substituent for oxime ether (Table 2). For index (BEI, pEC50/Mw),34,35 a metric incorporating the affinity
hydrophobic substituents, as the size of the substituent and molecular weight, was taken advantage of in the drug-like
increased, the potency decreased (compound 6 > 9 > 10 > 12 > 11 evaluation of the ligands (Table 3). From the results of the
> 13), implying that the steric effects of the substituent in this uorine screening in the middle benzene ring (compound 15
area might interrupt the desired interactions with the receptor. and 16), the 30-position seemed to be better than the 20-position
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Table 2 Agonistic activities and selected index of the designed compounds
3c
2
Acta% (300 nM)
Actb% (100 nM)
Fsp
tPSAd (A )
˚
Compd
R
TAK-875
GW9508
6
9
76.01
73.02
67.45
45.63
36.57
65.32
57.16
53.76
26.87
17.38
0.35
0.13
0.23
0.26
0.28
99.1
58.5
86.6
86.6
86.6
Me
Et
10
11
12
25.67
29.73
5.35
7.79
0.31
0.31
86.6
86.6
13
20.75
40.29
4.03
0.19
0.22
86.6
14
21.36
129.6
a
The mean value of agonist activity at a screening concentration of 300 nM was obtained from three independent experiments. b The mean value of
agonist activity at a screening concentration of 100 nM was obtained from three independent experiments. Fsp ¼ number of sp3 hybridized
c
3
carbons/total carbon count. d The topological polar surface area was calculated using ACD/I-Lab: https://ilab.acdlabs.com/iLab2/index.php.
Table 3 In vitro agonistic activities and index of the designed compoundsa
Actb% (100 nM)
EC50 (nM)
BEId
Fsp
tPSA (A )
c
3e
f
2
˚
Compd
R1
R2
TAK-875
GW9508
6
15
16
17
18
19
20
21
22
23
24
25
26
27
28
65.32
57.16
53.76
31.25
46.73
21.65
10.56
40.18
27.56
54.83
51.45
45.76
43.57
30.78
32.38
20.78
51.67
25.78
20.63
29.6
63.2
76.9
ND
ND
ND
ND
ND
ND
72.5
89.7
ND
ND
ND
ND
ND
88.9
ND
ND
14.3
20.6
15.9
0.35
0.13
0.23
0.23
0.23
0.26
0.26
0.26
0.26
0.23
0.23
0.23
0.23
0.23
0.26
0.26
0.26
0.23
0.23
99.1
58.5
86.6
86.6
86.6
95.8
86.6
86.6
86.6
86.6
86.6
86.6
86.6
86.6
86.6
86.6
95.8
86.6
86.6
H
H
H
H
2-Me
3-Me
4-Me
2-F
3-F
4-F
3-Cl
4-Cl
3-CF3
4-CF3
4-MeO
2,4-Di F
3,4-Di Cl
H
20-F
30-F
30-MeO
H
H
H
H
H
H
H
H
H
H
H
H
H
15.3
15.1
14.8
29
30
a
ND ¼ not determined. b The mean value of agonist activity at a screening concentration of 100 nM was obtained from three independent experiments.
c
3
The EC50 values for FFA1 activity represent the mean of three determinations. d BEI ¼ pEC50/Mw in kDa. e Fsp ¼ number of sp hybridized carbons/total
3
carbon count. f The topological polar surface area was calculated using ACD/I-Lab: https://ilab.acdlabs.com/iLab2/index.php.
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group has an additional interaction with the receptor. Inter-
estingly, although uorine in the ortho-position and para-posi-
tion exhibited excellent activity, the disubstituted compound 29
revealed a markedly lower agonistic activity than its corre-
sponding mono-substituted analogs 21 and 23. Consistent with
the abovementioned nding, the 3,4-di-chlorinated compound
30 was also inferior to its mono-substituted analogs, suggesting
that the polysubstituted groups may introduce an unfavorable
electrical or steric interactions with FFA1. Among all of the
tested compounds, compound 21, a most potent agonist with
considerable BEI value (15.3) in this series, had a signicant
3
advantage when compared to GW9508 in terms of the Fsp and
tPSA values despite a lower BEI value.
Fig. 3 Overlay of compound 6 and TAK-875 bound to FFA1. The key
residues are labeled in red and the hydrogen bonding interactions are
represented by yellow dashed lines.
2.3. Molecular modeling study
To comprehensively understand the interaction mode and SAR
of our oxime ether series, we performed a molecular docking
study of compound 6 using the X-ray structure of FFA1 (PDB
accession code: 4PHU).37 As shown in Fig. 3, compound 6 was
nearly a perfect dock to the same binding pocket of TAK-875 and
the docking study aligns with the SAR determined above. The
residues Tyr91, Arg183 and Arg2258 were highly coordinated
with head acid moiety forming three stable hydrogen bonds.
Moreover, Trp174 was oriented nearly orthogonal to the plane
of the dihydroisobenzofuran ring of compound 6 where it forms
an additional edge-on interaction. Notably, the terminal
benzene ring of compound 6 has a hydrophobic interaction
with the Leu135 residue, which had no interactions with TAK-
875. It was suggested that this hydrophobic interaction was
crucial for the robust agonistic activity of compound 6 rather
than the other non-phenyl compounds (such as 7 and 8).
Moreover, the methyl group in the oxime ether moiety extends
into hydrophobic portion of the lipid bilayer, which further
stabilized the binding conformation of the ligand. Therefore, it
showed better agonistic activity than the other types of
substituent.
to retain FFA1 agonistic activity. However, a bulkier methoxy
group at the 30-position (17) showed a markedly reduced
agonistic activity, indicating that the introduction of a substit-
uent with steric effects in the 30-position was unfavorable. Then,
an initial modication of the terminal benzene ring with
a methyl group (18, 19 and 20) demonstrated that substitution
was tolerated in the para-position and was preferred in the meta-
position. Gratifyingly, compound 21 (2-F) exhibited a marked
improvement in potency when compared with compound 18 (2-
Me). These activity differences could be attributed to the steric
hindrance of the methyl group, which moves the tail moiety of
the ligand into a different and less favorable conformation.
Further modication at the meta-position suggested that the
van der Waals radius of the substituent revealed a good corre-
lation with the agonistic activity: the agonistic activity of
˚
˚
˚
compound 22 (3-F, 1.47 A) > 24 (3-Cl, 1.75 A) > 19 (3-Me, 1.80 A) >
36
˚
26 (3-CF3, 2.20 A). To complete the SAR study, a variety of
substitutions in the para-position of the terminal phenyl ring
were evaluated. For the para-substituted compounds, the
agonistic activity of 6 (4-H) > 23 (4-F) > 25 (4-Cl) > 20 (4-Me) > 27
(4-CF3) demonstrated that steric effects in the para-position
might inuence the FFA1 agonistic activity. In contrast,
a methoxy group in the para-position (compound 28) was shown
to improve the affinity for FFA1, implying that the methoxy
2.4. Effect of compound 21 on the OGTT
Based on the favorable in vitro activity, the most potent
compound 21 (10, 20 and 40 mg kgꢁ1) was selected for further
Fig. 4 (A) Effect of compound 21 on the OGTT in normal ICR mice and glucose load (3 g kgꢁ1) at 0 min. (B) The OGTT of compound 21 in fasting
type 2 diabetic C57BL/6 mice and glucose load (2 g kgꢁ1) at 0 min. The values are reported as the mean ꢂ SEM (n ¼ 6). *P # 0.05 and **P # 0.01
compared to vehicle mice using the Student's t test. #P # 0.05 compared to vehicle diabetic mice using the Student's t test.
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the SAR information in the optimal methyl oxime ether scaffold
lead to the discovery of lead compound 21, a potent FFA1
agonist with robustly agonistic activity (EC50 ¼ 72.5 nM),
2
˚
3
improved drug-like properties (Fsp ¼ 0.23, tPSA ¼ 86.6 A ),
higher BEI value (15.3) than TAK-875 (BEI ¼ 14.3) and
outstanding antihyperglycemic effects in both normal and type
2 diabetic mice without the risk of hypoglycemia. Moreover, our
docking study expanded the comprehensive understanding of
the binding site and may help to design more active FFA1
agonists with non-biphenyl scaffolds.
4. Experimental section
4.1. Chemistry
Fig. 5 Hypoglycemic effects of compound 21 in fasted normal ICR
mice at a high dose of 100 mg kgꢁ1. The values are reported as the
mean ꢂ SEM (n ¼ 6). *P # 0.05 and **P # 0.01 compared to vehicle
mice using Student's t test.
Chromatographic purication was performed on a silica gel
(200–300 mesh) and monitored using thin layer chromatog-
raphy carried out on GF/UV 254 plates using UV light (254 and
365 nm). Melting points were determined on a RY-1 melting-
point apparatus and were not corrected. NMR (nuclear
magnetic resonance) spectra were obtained on a Bruker ACF-
300Q instrument (300 MHz for 1H NMR and 75 MHz for 13C
NMR spectra) with tetramethylsilane as an internal standard.
Chemical shis are reported in parts per million (ppm) and
coupling constants (J values) given in hertz (Hz). Elemental
analyses were carried out on the Heraeus CHN-O-Rapid analyzer
and have errors within ꢂ0.4% for CHN elements. The LC/MS
spectra were obtained on a Waters liquid chromatography-
mass spectrometer system (ESI†). All starting materials and
reagents were obtained from commercial suppliers and used
without further purication. TAK-875 and GW9508 were
synthesized via previous reported procedures.15,21
pharmacological evaluation using an oral glucose tolerance test
(OGTT) in normal ICR mice. Single oral doses of compound 21
signicantly improved the glucose tolerance in
a dose-
proportional manner from 10 to 40 mg kgꢁ1 (Fig. 4A).
Furthermore, compound 21 revealed a robust hypoglycemic
effect at a dose of 40 mg kgꢁ1, a potency that was similar with
that found with TAK-875 (20 mg kgꢁ1).
To assess the pharmacological effects of compound 21 in the
diabetic state, we performed an OGTT on STZ-induced type 2
diabetic C57BL/6 mice, a disease model with impaired glucose
tolerance.38,39 As shown in Fig. 4B, the hyperglycemia state was
signicantly controlled in compound 21 (40 mg kgꢁ1) treated
mice. These results suggested that compound 21, with
2
˚
3
improved drug-like properties (Fsp ¼ 0.23 and tPSA ¼ 86.6 A ),
The physical characteristics, 1H NMR, 13C NMR, MS and
elemental analysis data for all intermediates and target
compounds are reported in the ESI.†
has great potential for reducing the plasma glucose excursion in
both normal and type 2 diabetic mice.
2.5. Effects of compound 21 on the risk of hypoglycemia
4.2. Molecular modeling
Based on the positive result of the hypoglycemic effect, the risk
of hypoglycemia in fasted normal ICR mice was subsequently
evaluated using a high oral dose of compound 21 in contrast
with the positive control glibenclamide (a sulfonylurea insulin
The molecular docking study of compound 6 was performed
using MOE (version 2008.10, the Chemical Computing Group,
Montreal, Canada). The X-ray crystal structure of FFA1 (PDB
accession code: 4PHU) was retrieved from the Protein Data
Bank. Prior to docking, water molecules and ligands were
deleted except TAK-875. Then, the X-ray crystal structure was
prepared with Protonate 3D and a Gaussian contact surface was
drawn around the binding site of TAK-875. Then, the active site
was isolated and the backbone was removed. The ligand poses
were ltered using Pharmacophore Query Editor. The structure
of compound 6 was docked into the binding pocket using the
Pharmacophore method and then ranked with the London dG
scoring function. For energy minimization of the ligand in the
active site, MOE Forceeld Renement was used and ranked
with London dG scoring function.
secretagogue). As shown in Fig. 5, glibenclamide (15 mg kgꢁ1
)
treated mice displayed lowered blood glucose levels far below
that observed for normal fasted ICR mice. On the contrary,
compound 21 treated mice, even at an oral dose of 100 mg kgꢁ1
,
displayed only mildly decreased fasting glucose levels and the
reduction was far less than that found with glibenclamide.
Therefore, the research results show that compound 21
possesses a relatively low risk of hypoglycemia, a common
adverse effect to a sulfonylurea insulin secretagogue.
3. Conclusions
In conclusion, starting from the previously reported non-
biphenyl FFA1 agonist GW9508, we systematically explored
4.3. Biological methods
a series of non-biphenyl scaffolds directed by the Fsp and tPSA
4.3.1. Ca2+ inux activity of CHO cells stably expressing
human FFA1 (FLIPR assay). CHO cells stably expressing human
3
3
values to improve the drug-like properties of GW9508 (Fsp
¼
2
˚
0.13, tPSA ¼ 58.5 A ). Subsequently, a systematic exploration of FFA1 (accession no. NM_005303) were plated at a density of 15k
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cells per well and incubated for 12 h in 5% CO2 at 37 ꢀC. mg kgꢁ1) or compound 21 (40 mg kgꢁ1) was orally administered
Subsequently, the culture medium was removed and washed 30 min before the oral glucose load (2 g kgꢁ1). Blood samples
with Hank's balanced salt solution (100 mL). Then, the cells were were collected from the tail vein immediately before drug
incubated in loading buffer (recording medium containing 2.5 administration (ꢁ30 min), 0 min (just before the glucose load)
mg mLꢁ1 uorescent calcium indicator Fluo 4-AM, 0.1% fatty and at 15, 30, 45, 60 and 120 min post-glucose load. The blood
acid-free BSA and 2.5 mmol Lꢁ1 probenecid) for 1 h at 37 C. glucose levels were measured using blood glucose test strips
ꢀ
Various concentrations of test compounds or g-linolenic acid (SanNuo ChangSha, China).
(Sigma) were added to the cells and the intracellular Ca2+ ux
4.3.2.3. Effects of compound 21 on the risk of hypoglycemia. 10
signals aer the addition were monitored by FLIPR Tetra system weeks old male normal ICR mice were fasted overnight and
(Molecular Devices) for 90 s. The agonistic activities of the test randomized into 3 groups (n ¼ 6). Compound 21 (100 mg kgꢁ1),
compounds on human FFA1 were expressed as [(A ꢁ B)/(C ꢁ B)] glibenclamide (15 mg kgꢁ1) or vehicle was orally administered
ꢃ 100 (increase of the intracellular calcium concentration (A) in and blood was collected from the tail vein immediately before
the test compounds-treated cells and (B) in vehicle-treated cells administration (0 min) and at 30, 60, 90, 120 and 180 min post-
and (C) in 10 mM g-linolenic acid-treated cells). The EC50 values administration and the blood glucose levels were measured as
of selected compounds were obtained with Prism 5 soware described above.
(GraphPad).
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 obtained
Conflict of interest
from the Comparative Medicine Centre of Yangzhou University The authors have no conicts of interest to declare.
(Jiangsu, China). Mice were acclimatized for 1 week before the
experiments were performed and allowed ad libitum access to
standard pellets and water unless otherwise stated. The feeding
Acknowledgements
room was maintained on a constant 12 h light/dark cycle with This study was supported by grants from the National Natural
ꢀ
controlled temperature (23 ꢂ 1 C) and humidity (55% ꢂ 5%). Science Foundation of China (Grants 81172932 and 81273376),
The vehicle was orally administered 0.5% carboxymethyl cellu- the Natural Science Foundation of Jiangsu Province (Grant
lose aqueous solution for all the animal studies. All animal BK2012356), and the Fundamental Research Funds for the
experiments were performed in compliance with the relevant Central Universities, China Pharmaceutical University (Grant
laws and institutional guidelines, and our experiments have JKZD2013001).
been approved by the Institutional Committee of China Phar-
maceutical University.
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