Design, synthesis and structure–activity relationship studies of novel free fatty acid receptor 1 agonists bearing amide linker

Article abstract of DOI:10.1016/j.bmc.2017.03.001

The free fatty acid receptor 1 (FFA1/GPR40) has attracted extensive attention as a novel antidiabetic target. Aiming to explore the chemical space of FFA1 agonists, a new series of lead compounds with amide linker were designed and synthesized by combining the scaffolds of NIH screened lead compound 1 and GW9508. Among them, the optimal lead compound 17 exhibited a considerable agonistic activity (45.78%) compared to the NIH screened compound 1 (15.32%). During OGTT in normal mice, the compound 17 revealed a significant glucose-lowering effect (?23.7%) at the dose of 50?mg/kg, proximity to the hypoglycemic effect (?27.8%) of Metformin (200?mg/kg). In addition, the compound 17 (100?mg/kg) also exhibited a significant improvement in glucose tolerance with a 29.1% reduction of glucose AUC_0–2?h in type 2 diabetic mice. All of these results indicated that compound 17 was considered to be a promising lead structure suitable for further optimization.

Full text of DOI:10.1016/j.bmc.2017.03.001

Accepted Manuscript
Design, Synthesis and Structure−Activity Relationship Studies of Novel Free
Fatty Acid Receptor 1 Agonists Bearing Amide Linker
Jianyong Yang, Zheng Li, Huilan Li, Chunxia Liu, Nasi Wang, Wei Shi, Chen
Liao, Xingguang Cai, Wenlong Huang, Hai Qian
PII:
S0968-0896(17)30132-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.03.001
BMC 13596
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 January 2017
9 February 2017
1 March 2017
Please cite this article as: Yang, J., Li, Z., Li, H., Liu, C., Wang, N., Shi, W., Liao, C., Cai, X., Huang, W., Qian,
H., Design, Synthesis and Structure−Activity Relationship Studies of Novel Free Fatty Acid Receptor 1 Agonists
Bearing Amide Linker, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.
2017.03.001
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Design, Synthesis and Structure−Activity Relationship Studies of Novel Free Fatty Acid
Receptor 1 Agonists Bearing Amide Linker
Jianyong Yanga,1, Zheng Lia,1, Huilan Lia, Chunxia Liua, Nasi Wanga, Wei Shia, Chen Liaoa,
Xingguang Caia, Wenlong Huanga, b*, Hai Qiana, b*
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/GPR40) has attracted extensive attention as a novel
antidiabetic target. Aiming to explore the chemical space of FFA1 agonists, a new series of lead
compounds with amide linker were designed and synthesized by combining the scaffolds of NIH
screened lead compound 1 and GW9508. Among them, the optimal lead compound 17 exhibited a
considerable agonistic activity (45.78 %) compared to the NIH screened compound 1 (15.32 %).
During OGTT in normal mice, the compound 17 revealed a significant glucose-lowering effect
(-23.7 %) at the dose of 50 mg/kg, proximity to the hypoglycemic effect (-27.8 %) of Metformin
(200 mg/kg). In addition, the compound 17 (100 mg/kg) also exhibited a significant improvement
in glucose tolerance with a 29.1 % reduction of glucose AUC0-2h in type 2 diabetic mice. All of
these results indicated that compound 17 was considered to be a promising lead structure suitable
for further optimization.
Keywords: Amide, FFA1 agonist, GPR40, Hybrid, Type 2 diabetes.
1. Introduction
∗
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: +86-25-83271050; Fax: +86-25-83271050.
E-mail: ydhuangwenlong@126.com (W.l. Huang), qianhai24@163.com (H. Qian).
1 These authors contributed equally to this work.
1

The type 2 diabetes mellitus (T2DM), a worldwide epidemic syndrome characterized by impaired
insulin secretion and/or tissue resistance, has become a serious health problem.1, 2 Besides the
intervention of lifestyle, the common treatments are associated with series of side effects such as
the high risk of hypoglycemia, weight gain and gastric symptoms.3-6 Therefore, there is an urgent
need for preferable hypoglycemic drugs with increased safety and durability.7, 8 The free fatty acid
receptor 1 (FFA1, also known as GPR40), the prominent ones of antidiabetic targets, is
predominantly expressed in pancreatic β-cells and amplify glucose-stimulated insulin secretion.9-12
This glucose concentration-dependent mechanism provides the tremendous potential for reducing
the levels of plasma glucose without the risk of hypoglycemia.
Figure 1: Representative examples of FFA1 agonists.
Recently, a variety of FFA1 agonists have been published in the literature (Figure 1),13-20 and
the TAK-875 and AMG-837 were both in clinical trials. However, Takeda decided to terminate the
development of TAK-875 due to concerns about liver toxicity.21, 22 It is widely believed that the
hepatotoxicity is compound-related since the expression of FFA1 is quite low in the human liver.10,
23, 24
Therefore, it is urgent mission for medicinal chemists to extend the chemical space and
thereby obtaining novel FFA1 agonists suitable for further development. To explore the chemical
space of FFA1 agonists, our previous study have identified a series of potential agonists with
different building blocks such as thiazole,25 pyrrole,26 triazole,27 oxime ether,28 phenoxyacetic
acid29, 30 and phenoxyacetamide linker31. Inspired by the novel amide linker of NIH screened lead
compound 1,13 we first designed and synthesized the compound 2, which was a hybrid structure
containing both the amide linker of compound 1 and the phenylpropionic acid moiety of GW9508
2

(Figure 2). As expected, the compound 2 revealed a better activity than the parent compound 1,
which likely attributing to the incorporation of phenylpropionic acid. After systematic exploration
of structure-activity relationship (SAR) and the application of docking study, the promising lead
compound 17 and its interaction mode were identified. In subsequent pharmacological studies,
compound 17 showed a great potential for improving the glucose tolerance both in normal mice
and type 2 diabetic mice.
Figure 2: Our strategy to explore the chemical space of FFA1 agonists.
2. Results and Discussion
2.1. Chemistry
The synthetic routes of target compounds 2-17 are summarized in Scheme 1. The key intermediate
3a was prepared by the esterification of 2a, which was derived from the nitration reaction of
commercially available phenylpropionic acid. The resultant nitrobenzene 3a was reduced under
catalytic hydrogenation conditions, followed by amidation reaction with substituted benzoic acid
and further ester hydrolysis to provide the desired compounds 2-16. The key intermediate 7a was
synthesized by the classical Suzuki coupling of commercially available 2-bromo-m-xylene (5a)
with (3-formylphenyl)boronic acid 6a in the presence of Pd(PPh3)4. Oxidation of the 7a with
NaClO2 in the presence of NaH2PO4 generated the desired carboxylic acid 8a, which was followed
by amidation with intermediate 4a using DMAP and EDCI, then further convert to the desired
compound 17 by basic hydrolysis with lithium hydroxide.
Scheme 1. Synthesis of target compounds 2-17. Reagents and conditions: (a) HNO3, H2SO4, 0 °C, 1 h; (b) MeOH,
H2SO4, reflux, 3 h; (c) H2, Pd-C, CH3OH, r.t., 24 h; (d) DMAP, EDCI, CH2Cl2, r.t., 8 h; (e) LiOH·H2O,
3

THF/MeOH/H2O, r.t., 4 h; (f) Pd(PPh3)4, Na2CO3, toluene, ethanol, H2O, 80 °C, 12 h; (g) NaH2PO4, NaClO2,
2-methylbut-2-ene, t-BuOH, THF, r.t., 3 h.
2.2. FFA1 agonistic activity and SAR study
The target compounds were screened on the Chinese hamster ovary cells expressing human FFA1
by the fluorometric imaging plate reader (FLIPR) assay. As shown in Table 1, the compound 2, a
hybrid structure of NIH screened compound 1 and GW9508, revealed a better activity than the
parent compound 1, which likely attributing to the introduction of phenylpropionic acid moiety.
However, incorporation of a nitrogen atom into the left benzene ring, as with the pyridine
derivative 3, attenuated the agonistic activity compared to the compound 2. Generally, the pyridine
−electron
-deficient heterocycle, so we speculated that the loss
ring is known as hydrophilic and π
of activity on compound 3 might be attributed to the reduced capability of hydrophobic and/or
−electron interaction with the
π
binding pocket of FFA1. Indeed, the hydrophobic and
π−electron-rich heterocyclic derivatives 3-thiophene (4) and 2-thiophene (5) exhibited a marked
improvement on potency compared to the pyridine derivative 3, but it is still inferior to the
bioisostere benzene derivatives 2. Based on these encouraging results above, we therefore selected
the lead compound 2 as our starting point for further modification. The results of methyl scan in
left benzene ring (compound 6-8) demonstrated that the substitution is tolerated in para-position
and preferred in the meta-position. Similar results were also observed in the chlorine substituted
(9-11) and methoxy substituted derivatives (12 and 13). For the ortho-substituted derivatives, the
agonistic activity of 2 (2-H) > 9 (2-Cl) > 6 (2-Me) > 12 (2-MeO) > 14 (2-NO2) suggested that the
steric effect in the ortho-position might influence agonistic activity of FFA1. These activity
differences could be attributed to the steric hindrance of bulkier substituent moves the ligand into
a different and less favorable conformation. The para-substituted derivatives, however, seemed to
be insensitive for structural modification with different substituents (compound 2 vs 8, 11 and 15).
Borrowing elements of previleged scaffolds reported in FFA1 agonists, the obtained
meta-substituted derivatives 16 and 17 exhibited a significantly improved activity. Gratifyingly,
the compound 17, a potent agonist with biphenyl moiety of TAK-875, revealed even greater
potency than the direct analogue of GW9508 (compound 16).
4

Table 1: In vitro FFA1 agonistic activities of target compounds
Compd.
Ar
R
Act%(1 µM) a Compd.
Ar
R
Act%(1 µM) a
17.85
15.32
18.76
4.29
phenyl
phenyl
phenyl
4-Cl
1
2
3
11
12
13
phenyl
H
H
2-MeO
3-MeO
1.26
19.79
H
15.37
13.85
5.67
phenyl
phenyl
2-NO2
0.19
21.35
34.26
45.78
4
5
6
7
14
15
16
17
H
4-tBu
phenyl
phenyl
2-Me
3-Me
29.35
TAK-875
GW9508
phenyl
phenyl
phenyl
4-Me
2-Cl
3-Cl
19.42
7.73
96.35
83.76
8
9
25.36
10
a Agonist activities mean values at a screening concentration of 1 µM were obtained from three independent
experiments.
2.3. Molecular modeling study
To comprehensively understand the interaction mode and SAR of the present series, a docking
study of optimal compound 17 based on the X-ray structure of FFA1 (PDB code: 4PHU) was
performed.32 As shown in Figure 3, the compound 17 docked well to the same binding pocket for
TAK-875, and the docking study aligns with the SAR determined above. Just as TAK-875, an
edge-on interaction was also formed between the residue Trp174 and the right phenyl of
compound 17. The acid head, however, just form two hydrogen bonds with Tyr91 and Arg258
(Figure 3B), which explained the reason why the potency of compound 17 is inferior to TAK-875
(three hydrogen bonds). Moreover, the para-position of middle phenyl point to the outside of
binding pocket, which was consistent with the insensitive SAR for the para-substituted derivatives
(compounds 2, 8, 11 and 15).
5

Figure 3: The interaction mode of compound 17 bound to FFA1. Key residues are labeled in white, and hydrogen
bonding interactions are represented by yellow dashed lines. (A) Overlay of TAK-875 and 17 (silver white) bound
to FFA1. (B) Compound 17 bound to FFA1.
2.4. Effect of optimized compounds on OGTT
In order to screen orally bioavailable compound, the selected compounds 7, 10, 15, 16 and 17 (50
mg/kg) were evaluated for glucose-lowering effects by oral glucose tolerance test (OGTT) in
normal ICR mice. As shown in Figure 4, the time-dependent changes of glucose levels and the
corresponding area under the curve (AUC0-2h) indicated that the in vivo activities of the selected
compounds were in accordance with the in vitro activities. Exceptionally, the compound 7 exerts
stronger hypoglycemic effect than that of compound 16 despite the lower in vitro potency than
compound 16. One possible reasonable explanation is that the lower molecular weight for the
compound 7. Among these lead compounds, the compounds 7, 16 and 17 revealed marked
6

improvements in glucose tolerance with a 20.3 %, 12.8 % and 23.7 % decrease in glucose AUC0-2h
respectively. Notably, the compound 17 revealed a significant glucose-lowering effect (-23.7 %) at
the dose of 50 mg/kg, proximity to the hypoglycemic effect (-27.8 %) of Metformin (200 mg/kg),
a widely used first-line antidiabetic drug.
,
Figure 4: Effects of compounds on blood glucose levels during an OGTT in normal ICR mice. (A) Time
dependent changes of plasma glucose levels. (B) The AUC0-2h of blood glucose levels. Values are mean SEM (n
= 6). *p≤0.05, **p≤0.01 compared to vehicle mice by Student’s t-test.
2.5. Hypoglycemic effects of 17 explored in type 2 diabetic mice
To evaluate the glucose-lowering effect of compound 17 in diabetic state, the OGTT in
STZ-induced type 2 diabetic C57BL/6 mice was conducted.33, 34 As shown in Figure 5, single oral
doses of compound 17 distinctly decreased the glucose excursion in a dose-dependent manner
from 25 to 100 mg/kg. Moreover, the blood glucose AUC
suggested that the minimum
−
0 2h
effective dose of compound 17 was 50 mg/kg. During the OGTT, the hyperglycemia was
significantly controlled with a 29.1 % reduction of glucose AUC0-2h in compound 17 (100 mg/kg)
treated group despite the decrease was less than that of TAK-875 (20 mg/kg, -39.1 % glucose
AUC0-2h) treated group. These promising results illustrated the compound 17 possesses potential
for further research as an orally bioavailable lead structure.
7

Figure 5: Effects of 17 on blood glucose levels during an OGTT in fasting type 2 diabetic C57BL/6 mice. (A)
Time dependent changes of plasma glucose levels. (B) The AUC0-2h of blood glucose levels. Values are mean
SEM (n = 6). *P≤0.05 and **P≤0.01 compared to vehicle diabetic mice by Student’s t test.
3. Conclusion
With the purpose of exploring the chemical space of FFA1 agonists, we have identified a new
series of lead compounds with amide linker by combining the scaffolds of NIH screened lead
compound 1 and GW9508. Systematic exploration of SAR in present series result in the discovery
of lead compound 17, a potent FFA1 agonist revealed a stronger agonistic activity (45.78 %) than
the NIH screened compound 1 (15.32 %). Moreover, compound 17 showed a great potential for
improving the glucose tolerance both in normal mice and type 2 diabetic mice. Although the
activity of lead compound 17 was inferior to the most advanced candidate TAK-875, the
information achieved from our SAR studies might allow us to explore more extensive chemical
space of FFA1 agonists.
4. Experimental section
4.1. Chemistry
Chromatographic purification was performed on silica gel (200-300 mesh) and monitored by thin
layer chromatography carried out on GF/UV 254 plates by using UV light (254 and 365 nm).
Melting points were determined on a RY-1 melting-point apparatus and were not corrected. The
NMR (nuclear magnetic resonance) spectra were recorded on a Bruker ACF-300Q instrument
8

(300 MHz for 1H NMR and 75 MHz for 13C NMR spectra) with tetramethylsilane as an internal
standard. Chemical shifts are given in parts per million (ppm), and coupling constants (J values)
were given in hertz (Hz). Elemental analyses were carried out on the Heraeus CHN-O-Rapid
analyzer and have errors within 0.4% for CHN elements. The LC/MS spectra were run on the
Waters liquid chromatography-mass spectrometer system (ESI). All starting materials and reagents
were obtained from commercial suppliers and used without further purification. TAK-875 was
synthesized via previous reported procedures.19
1
The physical characteristics, H NMR, 13C NMR, MS and elemental analysis data for all
intermediates and target compounds, were reported in the supporting information.
4.2. Molecular modeling
The molecular docking study of compound 17 was performed by using MOE (version 2008.10,
The Chemical Computing Group, Montreal, Canada). The crystal structure of FFA1 (PDB
accession code: 4PHU) was retrieved from 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 draw around the binding site of TAK-875. Then,
the active site was isolated and the backbone was removed. The ligand poses were filtered by
using Pharmacophore Query Editor. The structure of compound 17 was docked into the binding
pocket with Pharmacophore method and then ranked with the London dG scoring function. For
the energy minimization of ligand in the active site, MOE Forcefield Refinement was used and
ranked with London dG scoring function.
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 were plated at a density of 15K cells/well and incubated
12 h in 5% CO2 at 37 °C. Subsequently, culture medium was removed and washed with Hank’s
Balanced Salt Solution (100 µL). Then, cells were incubated in loading buffer (recording 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 1 h at 37 °C. Various concentrations of test compounds or γ-linolenic acid
(Sigma) were added into the cells, and the intracellular Ca2+ flux signals after addition were
9

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). All test compounds were measured in three
independent experiments.
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 obtained from Comparative
Medicine Centre of Yangzhou University (Jiangsu, China). Mice were acclimatized for 1 week
before experiments, and allowed ad libitum access to standard pellets and water unless otherwise
stated. The feeding room was maintained on a constant 12 h light/black cycle with controlled
temperature (23 1 °C) and humidity (55
5%). The vehicle was oral administrated 0.5%
Caboxy Methyl Cellulose aqueous solution for all animal studies. All animal experimental
protocols were approved by the ethical committee at China Pharmaceutical University and
conducted according to the Laboratory Animal Management Regulations in China and adhered to
the Guide for the Care and Use of Laboratory Animals published by the National Institutes of
Health (NIH Publication NO. 85-23, revised 2011).
Statistical analyses were obtained with GraphPad software (GraphPad InStat version 5.00,
San Diego, CA, USA). Unpaired comparisons were analyzed using the two-tailed Student’s t-test.
4.4.2.1. Effect of compounds on OGTT explored in ICR mice
Ten-week-old male ICR mice were fasted 12 h, weighted, bled via the tail vein, and randomized
into eight groups (n = 6). A single doses of vehicle, Metformin (200 mg·kg-1), TAK-875 (20
mg·kg-1) or selected compounds (50 mg·kg-1) were orally administered 60 min before oral glucose
load (3 g·kg-1). Blood samples were collected from the tail vein immediately before drug
administration (-60 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 using blood glucose test strips (SanNuo
ChangSha, China).
4.4.2.2. Hypoglycemic effects of compound 17 explored in type 2 diabetic mice
10

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
resistance and then injected intraperitoneally (i.p.) with low dose of STZ (10 mL·kg-1; 80 mg·kg-1).
The C57BL/6 mice were fed with high-fat diet for another 4 weeks, and the development of
diabetes was confirmed by measuring blood glucose levels and glucose tolerance.33, 34
Type 2 diabetic C57BL/6 mice were fasted 12 h, weighted, bled via the tail vein, and
randomized into 5 groups (n = 6). A single doses of TAK-875 (20 mg·kg-1), vehicle or compound
17 (25, 50 and 100 mg·kg-1) were orally administered 60 min before oral glucose load (2 g·kg-1).
Blood samples were collected from the tail vein immediately before drug administration (-60 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 using blood glucose test strips (SanNuo ChangSha, China).
Conflict of interest
The authors have no conflicts of interest to declare.
Acknowledgements
This study was supported by grants from the National Natural Science Foundation of China
(Grants 81673299 and 81273376), and the innovation project of Jiangsu Province (Grant
KYLX16_1175).
11

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