Discovery of novel pyrrole-based scaffold as potent and orally bioavailable free fatty acid receptor 1 agonists for the treatment of type 2 diabetes

Article abstract of DOI:10.1016/j.bmc.2016.03.014

The free fatty acid receptor 1 (FFA1) has gained significant interest as a novel antidiabetic target. Most of FFA1 agonists reported in the literature bearing a common biphenyl scaffold, which was crucial for toxicity verified by the researchers of Daiichi Sankyo. Herein, we describe the systematic exploration of non-biphenyl scaffold and further chemical modification of the optimal pyrrole scaffold. All of these efforts led to the identification of compound 11 as a potent and orally bioavailable FFA1 agonist without the risk of hypoglycemia. Further molecular modeling studies promoted the understanding of ligand-binding pocket and might help to design more promising FFA1 agonists.

Full text of DOI:10.1016/j.bmc.2016.03.014

Accepted Manuscript
Discovery of Novel Pyrrole-Based Scaffold as Potent and Orally Bioavailable
Free Fatty Acid Receptor 1 Agonists for the Treatment of Type 2 Diabetes
Zheng Li, Miaobo Pan, Xin Su, Yuxuan Dai, Mian Fu, Xingguang Cai, Wei Shi,
Wenlong Huang, Hai Qian
PII:
S0968-0896(16)30163-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.03.014
BMC 12863
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
28 January 2016
4 March 2016
5 March 2016
Please cite this article as: Li, Z., Pan, M., Su, X., Dai, Y., Fu, M., Cai, X., Shi, W., Huang, W., Qian, H., Discovery
of Novel Pyrrole-Based Scaffold as Potent and Orally Bioavailable Free Fatty Acid Receptor 1 Agonists for the
Treatment of Type 2 Diabetes, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.
2
016.03.014
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Discovery of Novel Pyrrole-Based Scaffold as Potent and Orally Bioavailable Free Fatty
Acid Receptor 1 Agonists for the Treatment of Type 2 Diabetes
a
a
a
a
a
a
a
Zheng Li , Miaobo Pan , Xin Su , Yuxuan Dai , Mian Fu , Xingguang Cai , Wei Shi , Wenlong
a, b*
a, b*
Huang , Hai Qian
a
Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, PR China.
b
Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, PR China.
Abstract
The free fatty acid receptor 1 (FFA1) has gained significant interest as a novel antidiabetic target.
Most of FFA1 agonists reported in the literature bearing a common biphenyl scaffold, which was
crucial for toxicity verified by the researchers of Daiichi Sankyo. Herein, we describe the
systematic exploration of non-biphenyl scaffold and further chemical modification of the optimal
pyrrole scaffold. All of these efforts led to the identification of compound 11 as a potent and orally
bioavailable FFA1 agonist without the risk of hypoglycemia. Further molecular modeling studies
promoted the understanding of ligand-binding pocket and might help to design more promising
FFA1 agonists.
Keywords: FFA1 agonist, GPR40, ligand efficiency, type 2 diabetes, pyrrole.

Co-corresponding authors: Wenlong Huang and Hai Qian, Centre of Drug Discovery, State Key Laboratory of
Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China.
Tel: +86-25-83271302; Fax: +86-25-83271302.
E-mail: ydhuangwenlong@126.com (W.l. Huang), qianhai24@163.com (H. Qian).
1

1. Introduction
The increasing and alarming prevalence of type 2 diabetes mellitus (T2DM) along with the
undesirable side effects associated with many oral antidiabetic agents (most notably high risk of
hypoglycemia, body weight gain and gastric symptoms) has stimulated an intense effort to
1
-4
evaluate novel targets to achieve preferable glycemic control. The free fatty acid receptor 1
FFA1, also known as GPR40), a new drug target attracted considerable attention for the treatment
of T2DM, play a key role in amplifying insulin secretion in a glucose concentration-dependent
(
5-8
manner. Therefore, this glucose-stimulated insulin secretion mechanism of FFA1 provides the
enormous potentiality for improving insulin levels without the risk of hypoglycemia.
Recently, a number of synthetic FFA1 agonists have been reported in the literature (Figure
9-16
1
),
and the candidates AMG-837 and TAK-875 were both in clinical trials for treatment of
T2DM. However, many of these agonists bearing a common biphenyl scaffold (red mark in
Figure 1) have high lipophilicity, which likely correlated with poor water-solubility, metabolic
17-21
toxicity, and associated with a higher risk of attrition.
Furthermore, some studies derived from
2
2
Daiichi Sankyo have shown that the biphenyl moiety of FFA1 agonists was crucial for toxicity.
Therefore, it is of profound significance to hunt a novel scaffold by modifying the biphenyl moiety.
We have previously reported a series of phenoxyacetic acid derivatives which successfully
23
removed the biphenyl structure by introducing a phenoxyacetamide linker. Herein, we chose
compound 1 (Figure 1) as a starting structure to explore novel FFA1 agonists, because it has
robustly FFA1 agonistic activity and a simple structure to avoid undue increase in molecular size
and lipophilicity in the optimization process. Fortunately, the recently reported crystal structure of
FFA1 indicates that the terminal benzene ring of biphenyl moiety had no direct interactions with
2
4
the binding site of FFA1. Based on these results, we envisioned that replacement of the terminal
benzene ring of compound 1 with a polar heterocycle was a worthy approach to hunt a new
scaffold with desired drug-like properties (Figure 2). After systematic exploration of scaffold and
application of molecular docking, the promising FFA1 agonist 11 (EC50 = 34.7 nM, LE = 0.34)
and its binding mode (fit quality = 1.0) were identified. Further pharmacological studies
demonstrated that compound 11 has a robustly hypoglycemic effect in both normal and type 2
diabetic mice without hypoglycemia even at the oral dose of 80 mg/kg.
2

Figure 1: Selected examples of synthetic FFA1 agonists.
Figure 2: Our strategy to hunt a novel scaffold with decreased lipophilicity by modifying the biphenyl moiety of
compound 1.
2
2
. Results and Discussion
.1. Chemistry
The synthetic routes of target compounds 2-13 are summarized in Scheme 1. The key
intermediates 3a-e were prepared by the reduction of 2a-e, which were derived from Ullmann
2
5
coupling reaction of corresponding amine and methyl 3-iodobenzoate in the presence of CuI.
Next, the compounds 3a-e treated with thionyl chloride catalyzed by DMF catalyzer to generate
the chlorinated intermediates 4a-e. The intermediate 3f was synthesized upon simple mixing of
3
-aminobenzylalcohol and 2, 5-dimethoxytetrahydrofuran in the presence of FeCl
3
·7H
at 60 °C. It's worth mentioning that our initial effort was to afford the chlorinated intermediate of
f by treating with thionyl chloride. However, the 3f tend to produce a coke tar due to the
polymerization reaction of pyrrole in the strongly acidic system of thionyl chloride. Thus, the
2
O (2 mol%)
2
6
3
3
brominated intermediate 4f was successfully synthesized by treating PBr with pyridine in a
weakly basic system.
The common intermediates 2f and 2k were obtained from the intermolecular cyclization
between corresponding amine and acetonylacetone by using Paal-Knorr pyrrole synthesis.
3

Subsequently, the intermediate 2g was achieved from 2f by Vilsmeier-Haack-Arnold reaction.
Reduction of the 2g with NaBH and then methylation provided the intermediate 2h. Oxidation of
the 2g with NaClO in the presence of NaH PO generated the desired carboxylic acid 2i, which
4
2
2
4
was followed by amidation using DMAP and EDCI to yield the desired intermediate 2j.
Alkylation of 2k with corresponding haloalkane afforded the intermediates 2l-n. Reduction and
bromination of the intermediates 2h, 2j and 2l-n provide the brominated intermediates 4h-i and
11
4
l-n. The dihydrobenzofuran intermediate 1e was synthesized via published procedures.
Condensation of the obtained intermediates 4a-i or 4l-n with 1e by using Williamson ether
synthesis, followed by basic hydrolysis, afforded the target compounds 2-13.
Scheme 1. Synthesis of target compounds 2 to 13. Reagents and conditions: (a) corresponding amine, K
L-proline, 60 °C, 8 h; (b) NaBH , MeOH, THF, reflux, 2 h; (c) SOCl , CH Cl , DMF, 40 °C, 4 h; (d)
,5-dimethoxytetrahydrofuran, FeCl ·7H O (2 mol%), H O, 60 °C, 3 h; (e) acetonylacetone, p-toluenesulfonic acid,
toluene, reflux, 3 h; (f) PBr , pyridine, CH Cl , 0 °C, 1 h; (g) POCl , DMF, toluene, 70 °C, 4 h; (h) NaBH , THF, rt,
h; (i) NaH, MeI, THF, 0 °C to rt, 8 h; (j) NaH PO , NaClO , 2-methylbut-2-ene, t-BuOH, THF, rt, 3 h; (k)
2 3
CO , CuI,
4
2
2
2
2
3
2
2
3
2
2
3
4
2
2
4
2
2 3
dimethylamine in THF, DMAP, EDCI, DMF, 0 °C to rt, 8 h; (l) RBr or MeI, K CO , acetone, KI, 45 °C, 12 h; (m)
4

2 3 2 2
K CO , acetone, KI, 60 °C, 8 h; (n) LiOH·H O, THF/MeOH/H O, rt, 4 h.
2.2. FFA1 agonistic activity and SAR study
Lovering et al. pointed out that an increase of saturation is a practical strategy to improve the
2
7, 28
drug-like properties of compound.
Moreover, Ishikawa and Hashimoto were profoundly
reviewed an alternative tactic for improving physicochemical property by means of disruption of
2
9
molecular planarity. Inspired by these new perspectives, we first designed and synthesized
piperidine derivative 2, a more saturated analogue of compound 1 (Table 1). The compound 2
revealed a significant improvement on lipophilicity (clogP = 3.993) compared to the parent
compound 1 (clogP = 5.693) but unfortunately also markedly reduced potency. A slight
improvement in potency over the compound 2 was achieved only by replacing piperidine of
compound 2 with bioisosteres in compound 3 and 4. We speculated that the increased affinity was
attributed to the solvent effect of heteroatom which stabilized the binding conformation of ligand.
However, the compound 5 (clogP = 1.645), a sulphone analogue of compound 4 designed to
further improve the lipophilicity as TAK-875, showed a drastic loss of activity despite increased
solvent effect, likely suggesting that the limited space in the binding pocket around this area. On
the basis of the above analysis, we removed a carbon atom in compound 2 to reduce the steric
effect with ligand-binding pocket, and the obtained compound 6 showed a significant
improvement in potency compared with the compound 2. The pyrrole derivative 7, designed as a
constrained planar analogue of compound 6, appeared to diminish the in vitro agonistic activity.
Gratifyingly, borrowing elements of known FFA1 agonists such as the ortho-dimethyl moiety of
TAK-875, the obtained compound 8 exhibited a marked improvement on potency in comparison
with the corresponding compound 7. This result demonstrated that a more appropriate twisted
conformation was induced by 2, 5-dimethyl pyrrole.
Table 1: In vitro activities and select physicochemical properties of target compounds
a
b
c
Compd.
Het
Act%(300nM)
Act%(100nM)
Mw
clogP
5

TAK-875
76.01
74.37
65.32
62.58
524.63
388.17
4.697
5.693
1
2
3
4
5
6
7
8
50.32
55.73
52.18
25.86
56.26
41.87
71.34
34.65
41.17
38.25
8.28
367.45
369.42
385.48
417.48
353.42
349.39
377.44
3.993
2.611
3.444
1.645
3.434
4.306
5.556
43.68
28.56
59.86
a
Agonist activities mean values at screening concentration of 300 nM were obtained from three independent
b
experiments. Agonist activities mean values at screening concentration of 100 nM were obtained from three
c
independent experiments. clogP values were estimated with ChemDraw Ultra, version 12.0.
Based on these results above, we therefore selected 2, 5-dimethyl pyrrole scaffold of the most
potent compound 8 as our starting point for further modification. Besides the agonistic activity, the
30, 31
ligand efficiency dependent lipophilicity (clogP / LE, LELP),
a metric incorporated affinity
and lipophilicity, was taken advantage of in the drug-like evaluation of derivatives. Moreover, the
32, 33
fit quality (LE / LE_Scale, FQ),
with score close to 1 indicative of near perfect binding, was
also introduced to assess the binding characteristics with FFA1. As shown in Table 2,
incorporation of substituent at the 3-position of pyrrole (9 and 10) turned out a drastic loss of
activity, indicating that the introduction of a substituent at 3-pyrrole is unfavorable for FFA1
2
agonistic activity. Next, our optimized efforts were directed to evaluate the R group, the
substituent at the para-position of the central phenyl ring. Among them, the methoxy analogue 11
revealed a strongest potency (34.7 nM), desired LELP value (16.5) and perfect FQ score (1.0)
despite slightly increased lipophilicity. Furthermore, as the Van der Waals radius of substituent
increases the activity decrease (compound 11 > 12 > 13), implying that the steric bulkiness of
substituent might interrupt the proper interaction with the receptor. Meanwhile, the order of
6

13
potency is reversed relative to the TAK-875 series, suggesting that the previous SAR
information can not directly convert to our pyrrole series. Among all of the tested compounds, the
most potent agonist 11, with excellent LE (LELP) value and perfect FQ score, was selected for
further investigation.
Table 2: In vitro activities and physicochemical properties of designed compounds
Act%
EC50
c
d
e
Compd.
R
1
R
2
clogP
LE (LELP)
FQ
a
b
(
100nM)
(nM)
TAK-875
65.32
62.58
59.86
31.69
25.72
29.6
38.4
46.9
ND
4.697
5.693
5.556
5.304
4.207
0.27 (17.3)
0.34 (16.7)
0.35 (15.8)
0.94
0.97
0.98
1
8
9
H
H
H
H
1
0
1
ND
1
H
H
H
MeO
EtO
63.76
54.62
40.75
34.7
61.2
ND
5.662
6.191
7.430
0.34 (16.5)
0.31 (19.9)
1.0
1
1
2
3
0.94
BnO
a
ND = Not determined. Agonist activities mean values at screening concentration of 100 nM were obtained from
b
three independent experiments. EC50 values for FFA1 activities represent the mean of three independent
c
d
determinations. clogP values were estimated with ChemDraw Ultra, version 12.0. LE values were calculated by
e
–
Δg = RT lnKD, presuming EC50≈KD, and the LELP values were calculated by the formula clogP / LE. FQ (fit
2
3
quality) = LE / LE_Scale, and LE_Scale = 0.0715 + 7.5328/HAC + 25.7079/HAC – 361.4722/HAC , HAC:
heavy atom count.
2.3. Docking study of compound 11 with FFA1
To understand the interaction mode and SAR of pyrrole series in-depth, a molecular docking study
based on the crystal structure of FFA1 (PDB accession code: 4PHU) was performed by using the
24
Molecular Operating Environment (MOE). As shown in Figure 3, the compound 11 docked very
well to the same binding site for TAK-875, and the modeling study can explain the SAR
7

reasonably. The head acid moiety was highly coordinated by Tyr91, Arg183 and Arg2258 forming
anchor point. Meanwhile, the residue Trp174 is oriented nearly orthogonal to the plane of the
dihydroisobenzofuran ring of compound 11 where it forms an edge-on interaction. Moreover, the 2,
5
-dimethyl pyrrole scaffold is roughly perpendicular to the central phenyl ring, which fully
explained the activity difference between 2, 5-dimethyl pyrrole (compound 8) and non-substituted
pyrrole (compound 7). Interestingly, the methoxy group on the central phenyl moiety has
hydrophobic interaction with receptor, and the additional interaction render compound 11 showed
best agonistic activities in this series. As mentioned above, compound 11 was occupied the
binding pockets effectively, and it was confirmed as the values of LE and FQ (compound 11: 0.34
and 1.0, TAK-875: 0.27 and 0.94, respectively).
Figure 3: Overlay of TAK-875 and compound 11 in crystal structure of FFA1. Key residues are labeled in red, and
hydrogen bonding interactions are represented by yellow dashed lines.
2.4. Effect of compound 11 on glucose tolerance
On the basis of its excellent in vitro potency, compound 11 (10, 20 and 40 mg/kg) was selected for
further in vivo pharmacological evaluation in ICR mice by oral glucose tolerance test (OGTT). As
shown in Figure 4A, single oral doses of compound 11 robustly reduced the plasma glucose
excursion during an OGTT in a dose-dependent manner from 10 to 40 mg/kg. Notably, the
compound 11 revealed a significant blood glucose-lowering effect at 40 mg/kg dose, similar to the
8

hypoglycemic effect of positive control TAK-875 (20 mg/kg), the most advanced candidate once
in phase III studies. Furthermore, the blood glucose curve of compound 11 tended to flat after 60
min at the dose of 40 mg/kg. This phenomenon could be at least in part rationalized by the low
risk of hypoglycemia showed in compound 11.
Figure 4: (A) Effect of compound 11 on plasma glucose levels during an OGTT in ICR mice, and glucose load (3
g/kg) at 0 min. (B) Effects of compound 11 and glibenclamide on fasting blood glucose level in normal mice. Data
#
are mean ± SEM for six animals. *P≤0.05 and **P≤0.01 compared to vehicle mice by Student’s t test. P≤0.05 and
#
#
P≤0.01 compared to compound 11 treated mice by Student’s t test.
2.5. Effects of compound 11 on the risk of hypoglycemia
Obtaining a positive result in in vivo pharmacological study, the risk of hypoglycemia was
subsequently assessed in fasting normal mice by oral administrating a high dose of compound 11
in comparison with glibenclamide (a well-known sulfonylurea) to further confirm the above
speculation. As shown in Figure 4B, the glibenclamide (15 mg/kg) treated group reduced the
plasma sugar levels far below the normal fasting glucose levels. In contrast, compound 11, even at
an oral dose of 80 mg/kg, only slightly alter blood glucose levels in fasting mice with normal
glucose homeostasis, and the change of plasma glucose levels was much smaller compared to that
of glibenclamide. Thus, our results demonstrated that compound 11 may pose a low risk of
hypoglycemia, a serious adverse effect common to sulfonylureas.
2.6. Hypoglycemic effects of compound 11 explored in type 2 diabetic mice
9

To assess antihyperglycemic effects in the diabetic state, the orally bioavailable agonist 11 was
evaluated by the OGTT in STZ-induced type 2 diabetic C57BL/6 mice, a model with impaired
34, 35
insulin secretion.
As shown in Figure 5, the plasma glucose levels was significantly decreased
in compound 11 (20 mg/kg) treated group, which presented potency-similarity with TAK-875 (20
mg/kg) treated mice. This result indicated that compound 11 has an outstanding efficacy for
improving the diabetic state in type 2 diabetic mice.
Figure 5: Effect of compound 11 on plasma glucose levels during an OGTT in fasting type 2 diabetic C57BL/6
mice, and glucose load (2 g/kg) at 0 min. Data are mean ± SEM for six animals. *P≤0.05 compared to vehicle
diabetic mice by Student’s t test.
3. Conclusion
With the aim of developing potent FFA1 agonists with novel drug-like scaffold, we have identified
a series of non-biphenyl scaffold agonists by comprehensive evaluating 7 polar heterocycle.
Subsequently, systematic exploration of SAR in the optimal pyrrole scaffold, leading to the
discovery of compound 11, an excellent FFA1 agonist with robustly agonistic activity (EC50
=
34.7 nM), desired LE (LELP) value and perfect FQ score (FQ = 1). Moreover, compound 11
revealed a great potential for reducing the blood glucose levels in ICR mice and type 2 diabetic
C57BL/6 mice without the risk of hypoglycemia even at a high dose of 80 mg/kg. All of these
researches manifested that compound 11 was meaningful for further investigation, and the
information obtained from this study might help to design more competitive new chemical entities
that are structurally related.
10

4
4
. Experimental section
.1. Chemistry
Column chromatographic purification was carried out on silica gel (200-300 mesh) and monitored
by thin layer chromatography performed on GF/UV 254 plates by using UV light at 254 and 365
nm. Melting points were determined on a RY-1 melting-point apparatus, which was not calibrated.
The nuclear magnetic resonance (NMR) spectra were recorded at room temperature on a Bruker
1
13
ACF-300Q instrument (300 MHz for H NMR and 75 MHz for C NMR spectra). Chemical shifts
are given in parts per million (ppm) with tetramethylsilane as internal standard, and coupling
constants (J values) were given in hertz (Hz). Elemental analyses were carried out by the Heraeus
CHN-O-Rapid analyzer. The LC/MS spectra were determined on a Waters liquid chromatography
-
mass spectrometer system (ESI). All starting materials, reagents and solvents were purchased
from commercial sources and used without further purification. TAK-875 and compound 1 were
11
synthesized via published procedures.
1
13
The physical characteristics, H NMR, C NMR, MS and elemental analysis data for all
intermediates and target molecules, were reported in the supporting information.
4.2. Molecular modeling
The docking simulation of compound 11 was performed by using MOE (version 2008.10, The
Chemical Computing Group, Montreal, Canada). The crystal structure of FFA1 (PDB code: 4PHU)
was obtained from the Protein Data Bank. Prior to the procedure of docking, the crystal structure
of FFA1 was prepared with Protonate 3D and a Gaussian Contact surface was draw around the
binding pocket of ligand. Then, the active site was isolated and the backbone was removed. The
ligand poses were filtered using Pharmacophore Query Editor. The structures of compound 11 was
docked in the active site with Pharmacophore method and then ranked with the London dG
scoring function. For the energy minimization of ligand in the pocket, MOE Forcefield
Refinement was used and ranked with the London dG scoring function.
4
4
.3. Biological methods
2
+
.3.1. Ca influx activity of CHO cells stably expressing human FFA1 (FLIPR Assay)
CHO cells stably expressing human FFA1 (accession no. NM_005303) were plated into 96-well
11

2
plates at a density of 15K cells/well and incubated overnight in 5% CO at 37 °C. Then, the
medium was removed and washed with Hank’s Balanced Salt Solution (100 μL). Subsequently,
cells were incubated in loading buffer (medium containing 2.5 μg/mL fluorescent calcium
indicator Fluo 4-AM, 0.1% fatty acid-free BSA and 2.5 mmol/L probenecid) for 60 min at 37 °C.
Various concentrations of test compounds or γ-linolenic acid (Sigma) were added into the cells
2+
and the intracellular Ca flux signals were monitored by FLIPR Tetra system (Molecular Devices)
for 90 s. The agonistic activities of test compounds on human FFA1 were expressed as
[
(A−B)/(C−B)]×100 (increase of the intracellular calcium concentration (A) in the test
compounds-treated cells and (B) in vehicle-treated cells, and (C) in 10 μM γ-linolenic acid-treated
cells). EC50 values of selected compounds were obtained with Prism 5 software (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 from Comparative
Medicine Centre of Yangzhou University (Jiangsu, China). Mice were acclimatized for 7 days
before experiments. The feeding room was keep on a constant 12 h light/black cycle (lights on
from 7:30 to 19:30) with controlled temperature (23 ± 1 °C) and humidity (55 ± 5%) throughout
the experimental period. Mice were allowed ad libitum access to standard pellets and water unless
otherwise stated, and the vehicle was oral administrate 0.5% Caboxy 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.
Statistical analyses were performed using GraphPad software (GraphPad InStat version 5.00,
San Diego, CA, USA). Unpaired comparisons were analyzed using the two-tailed Student’s t-test.
4.3.2.1. Effect of compound 11 on OGTT explored in male ICR mice
Ten-week-old normal male ICR mice were fasted overnight (12 h), weighted, bled via the tail vein,
-
1
-1
and randomized into 5 groups (n = 6). A single doses of TAK-875 (10 mL·kg ; 20 mg·kg ),
-
1
-1
-1
vehicle or compound 11 (10 mg·kg , 20 mg·kg , 40 mg·kg ) were orally administered 30 min
-1
before dosed orally with 30% glucose aqueous solution (3 g·kg ). Blood samples were collected
immediately before drug administration (-30 min), 0 min (just before glucose load), and at 15, 30,
12

45, 60 and 120 min after glucose load. The plasma glucose levels were measured by blood glucose
test strips (SanNuo ChangSha, China).
4.3.2.2. Effects of compound 11 on the risk of hypoglycemia
Normal male ICR mice 10 weeks old were fasted overnight (12 h) and randomized into 3 groups
-
1
-1
(
n = 6). A single doses of compound 11 (80 mg·kg ), glibenclamide (15 mg·kg ), or vehicle was
oral administered, and blood samples were collected from tail tip immediately before drug
administration (0 min) and at 30, 60, 90, 120 and 180 min after administration. The blood glucose
levels were measured as described above.
4.3.2.3. Hypoglycemic effects of compound 11 explored in type 2 diabetic mice
Male C57BL/6 mice after 7 days adaptation were fed with high-fat diet (45% calories from fat,
from Mediscience Ltd., Yangzhou, China) ad libitum for further 4 weeks to induce insulin
-1
-1
resistance and then injected intraperitoneally (i.p.) with low dose of STZ (10 mL·kg ; 80 mg·kg ).
The C57BL/6 mice were fed with high-fat diet for another 4 weeks, and mice with fasting blood
34, 35
glucose level ≥ 11.1 mmol/L were used in the experiment as type 2 diabetic mice model.
Type 2 diabetic C57BL/6 mice were fasted overnight (12 h), weighted, bled via the tail vein,
and randomized into 3 groups (n = 6), another group of normal fasting C57BL/6 mice was added
-1
-1
as negative control. A single doses of TAK-875 (10 mL·kg ; 20 mg·kg ), vehicle or compound 11
-
1
-1
(
10 mL·kg ; 20 mg·kg ) were orally administered 30 min before dosed orally with 20% glucose
-1
aqueous solution (2 g·kg ). Blood samples were collected immediately before drug administration
(
-30 min), 0 min (just before glucose load), and at 15, 30, 45, 60 and 120 min after glucose load.
The blood glucose levels were measured by blood glucose test strips (SanNuo ChangSha, China).
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 Fundamental Research Funds for the Central Universities, China
Pharmaceutical University (Grant JKZD2013001).
13

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