Discovery of novel free fatty acid receptor 1 agonists bearing triazole core via click chemistry

Article abstract of DOI:10.1016/j.bmc.2016.08.068

The free fatty acid receptor 1 (FFA1/GPR40) is a novel antidiabetic target based on particular mechanism in enhancing glucose-stimulated insulin secretion. Most of reported FFA1 agonists, however, have been suffered from relatively high lipophilicity and

Full text of DOI:10.1016/j.bmc.2016.08.068

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of novel free fatty acid receptor 1 agonists bearing triazole
core via click chemistry
Zheng Li a, Jianyong Yang a, Xuekun Wang a, Huilan Li a, Chunxia Liu a, Nasi Wang a, Wenlong Huang a,b,
,
⇑
Hai Qian a,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
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/GPR40) is a novel antidiabetic target based on particular mech-
anism in enhancing glucose-stimulated insulin secretion. Most of reported FFA1 agonists, however,
have been suffered from relatively high lipophilicity and molecular weight. Aiming to develop potent
agonists with improved physicochemical property, 25 compounds containing triazole scaffold and
various carboxylic acid fragments were synthesized via the click chemistry. Among them, the optimal
lead compound 26 with relatively low lipophicity (LogD7.4 = 1.95) and molecular weight
(Mw = 391.78) exhibited a considerable FFA1 agonistic activity (36.15%). In addition, compound 26
revealed a significant improvement in the glucose tolerance with a 21.4% and 14.2% reduction of glu-
cose AUC0–2h in normal ICR mice and type 2 diabetic C57BL/6 mice, respectively. All of these results
demonstrated that compound 26 was considered to be a promising lead compound suitable for fur-
ther optimization.
Received 2 August 2016
Revised 29 August 2016
Accepted 31 August 2016
Available online xxxx
Keywords:
FFA1 agonist
GPR40
Triazole core
Click chemistry
Type 2 diabetes
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
It is widely believed that the hepatotoxicity is compound related
since the expression of FFA1 is not found in the human
The worldwide epidemic of type 2 diabetes mellitus (T2DM), a
metabolic syndrome characterized by impaired glucose homeosta-
sis, has become a serious health problem.1,2 Besides the lifestyle
intervention, the most common treatments are associated with
side effects such as risk of hypoglycemia, gastric symptoms and
weight gain.3–6 Hence, there is an urgent need for improved treat-
ment with increased safety and durability.7,8 The free fatty acid
receptor 1 (FFA1, previously known as GPR40), the prominent ones
of antidiabetic targets in the last decade, is predominantly
expressed in pancreatic b-cells and amplify glucose-stimulated
insulin secretion.9–12 This glucose concentration-dependent mech-
anism provides the potential for improving the insulin levels with-
out the risk of hypoglycemia.
liver.10,24–26 Most current FFA1 agonists have somewhat high
lipophilicity and molecular weight (red mark in Fig. 1), likely
correlated with poor water-solubility, high toxicity, metabolic
instability and associated with high risk of attrition in clinical
test.27–31 At present, many classic strategies to decrease lipophilicity
have been extensively adopted by introducing hydrophilic group
and scaffold hopping with polar skeleton.28,32–34 With these
perspectives in mind, our previous study identified a series of polar
skeletons such as thiazole,35 pyrrole,36 oxime ether37 and phenoxy-
acetamide linker38 to improve the lipophilicity of TAK-875. The
applications of click chemistry are increasingly found in every
aspects of drug discovery, ranging from hit-to-lead finding by using
combinatorial chemistry and target labeling, to proteomics or DNA
research.39–41 In this study, the hydrophilic triazole ring was intro-
duced via click chemistry as a bioisostere of benzene ring to
decrease the lipophilicity (Fig. 2). After systematic exploration of
SAR, the compound 26 was discovered as an orally bioavailable
lead compound with a considerable 21.4% and 14.2% decrease of
blood glucose levels in normal ICR mice and type 2 diabetic
C57BL/6 mice, respectively.
Recently, a variety of FFA1 agonists have been reported in the
literature (Fig. 1),13–21 and the TAK-875 and AMG-837 were both
in clinical trials. Unfortunately, Takeda decided to terminate the
development of TAK-875 due to concerns about liver toxicity.22,23
⇑
Corresponding authors. Tel./fax: +86 25 83271302.
E-mail addresses: ydhuangwenlong@126.com (W. Huang), qianhai24@163.com
(H. Qian).
http://dx.doi.org/10.1016/j.bmc.2016.08.068
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
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Z. Li et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Figure 1. Selected examples of FFA1 agonists.
Figure 2. Our strategy to decrease the molecular weight and lipophilicity of TAK-875.
methanol, followed by basic hydrolysis, afforded the target car-
boxylic acids 2–26.
2. Results and discussion
2.1. Chemistry
2.2. FFA1 agonistic activity and SAR study
The synthetic routes of target compounds 2–26 are summarized
in Scheme 1. The intermediates 1a, 5a and 5b were synthesized via
published procedures.13,15 The alkynyl compounds 2a, 6a and 6b
were derived from Williamson ether synthesis of starting material
(1a, 5a or 5b) and propargyl bromide in the presence of K2CO3. Azi-
dobenzenes 4a or 8a were obtained by treating commercially
available anilines 3a or 7a with NaNO2 followed by NaN3 in 50%
acetic acid. The intermediates 2a, 6a or 6b reacted smoothly with
azidobenzenes 4a or 8a at room temperature in the presence of
catalytic amount of sodium ascorbate and copper sulfate in 80%
The synthetic compounds were screened on human FFA1 by the
fluorometric imaging plate reader (FLIPR) assay. Besides overall
activity, the lipophilicity (clogP and LogD7.4) and molecular weight
were applied to evaluate the analogues. As shown in Table 1, the
compound 2, initially designed to decrease the lipophilicity
(clogP = 2.70), leads to a significant decrease in potency compared
with TAK-875 (clogP = 4.69). We speculated that the diminished
affinity was associated with the need of enough lipophilicity for
ligands to enter the binding pocket via the lipid bilayer.42 A slight
Scheme 1. Synthesis of target compounds 2–26. Reagents and conditions: (a) Propargyl bromide, K2CO3, DMF, rt, 12 h; (b) NaNO2, 50% acetic acid, NaN3, 0 °C, 2 h;
(c) CuSO4Á5H2O, sodium ascorbate, CH3OH, rt, 24 h; (d) LiOHÁH2O, THF/MeOH/H2O, rt, 4 h.
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Z. Li et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
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Table 1
In vitro agonistic activities and selected parameters of target compounds
N
N
6
5
N
O
O
2
R1
3
COOH
Mw
c
Compd.
R1
Act% (100 nM)a
clogPb
LogD7.4
TAK-875
2
3
4
5
6
7
8
65.32
0.76
6.69
524.63
351.36
369.35
385.80
385.80
385.80
381.39
457.49
420.25
399.83
4.697
2.702
3.017
3.587
3.587
3.587
3.182
4.698
4.356
4.337
2.43
ND
ND
ND
1.09
ND
ND
ND
1.96
2.08
H
2-F
2-Cl
3-Cl
9.34
22.84
24.69
19.36
3.67
21.69
33.82
4-Cl
3-OMe
4-OBn
2, 6-diCl
2-Me-5-Cl
9
10
a
b
c
Agonist activities mean values at a screening concentration of 100 nM were obtained from three independent experiments.
clogP values were estimated with ChemDraw Ultra, version 12.0.
LogD7.4 values were determined by shake-flask procedure. ND = Not determined.
improvement in potency was obtained by adding an additional
halogen in compound 2 to achieve compound 3 (2-F) and com-
pound 4 (2-Cl). From the results of chlorine scan in the terminal
benzene ring (compounds 4–6), the meta-position and para-posi-
tion seemed to be better than the ortho-position. Besides elec-
tron-withdrawing group, the electron-donating methoxy group
(compound 7) was also tolerated in meta-position. The bulkier
benzyloxy group on the para-position (8), however, showed a
markedly reduced potency, indicating that the introduction of
steric group at the para-position of terminal phenyl is unfavorable.
Borrowing elements of ortho-disubstituted FFA1 agonists, the
(Table 2).13,14 The compound 11, a hybrid structure containing
both phenoxyacetic acid scaffold and the triazole moiety of com-
pound 2, turned out a drastic loss of activity as compound 2. With
the beneficial experience to increased potency using meta-sub-
stituent and para-substituent, the compounds 12–15 were synthe-
sized and evaluated. The results indicated that the phenoxyacetic
acid series (compound 14) are slightly inferior to the dihydroben-
zofuran series (compound 6).
We have previously observed a robust improvement of potency
in the phenoxyacetic acid series after introduction of 2-fluoro just
as compound 1.13 The same effect was observed on the introduc-
tion of 2-fluoro at compounds 16, 19 and 20, which exhibited a
marked improvement on potency in comparison with the corre-
sponding compounds 11, 14 and 15. As the dihydrobenzofuran ser-
ies, chlorine scan in the terminal benzene ring (compounds 17–19)
revealed that the agonistic activity of para-position > meta-
position > ortho-position. The same effect was also observed upon
methoxy substituted analogues (compounds 23 and 24). Moreover,
obtained compound
9 exhibited a significantly enhanced
affinity in comparison with the parent compound 4. Interestingly,
re-position the chlorine from 6-position to 5-position obtained
the compound 10, which revealed a marked improvement on
potency compared to the compounds 5 and 9, respectively.
These promising results prompted a comprehensive evaluation
on our previously reported phenoxyacetic acid scaffold
Table 2
Agonistic activities and selected index of designed compounds
6
N
N
N
5
R1
O
2
R2
3
COOH
O
Act% (100 nM)a
Mw
clogPb
LogD7.4
c
Compd.
R1
R2
TAK-875
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
65.32
0.64
524.63
325.32
339.35
339.35
359.77
343.31
343.31
377.76
377.76
377.76
361.30
399.42
387.37
373.34
373.34
412.20
391.78
4.697
2.753
3.253
3.253
3.638
3.068
2.768
3.653
3.653
3.653
3.083
4.594
3.525
2.996
2.996
4.422
4.152
2.43
ND
ND
0.89
ND
ND
-0.23
ND
ND
1.37
0.92
ND
ND
ND
0.52
2.16
1.95
H
H
H
H
H
F
F
F
F
F
F
F
F
F
F
F
H
3-Me
4-Me
4-Cl
4-F
H
2-Cl
3-Cl
4-Cl
4-F
4-tBu
4-OEt
2-OMe
3-OMe
2,6-diCl
2-Me-5-Cl
11.85
16.37
19.42
13.79
5.83
11.35
22.71
26.15
20.39
4.53
7.62
14.61
24.38
29.67
36.15
a
b
c
Agonist activities mean values at a screening concentration of 100 nM were obtained from three independent experiments.
clogP values were estimated with ChemDraw Ultra, version 12.0.
LogD7.4 values were determined by shake-flask procedure. ND = Not determined.
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Z. Li et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
the bulkier groups on the para-position (21 and 22) may introduce
unfavorable steric effect in the binding pocket. Unlike the
phenoxyacetic acid series, the 2-fluoro analogues (compounds
16–19 and 24–26) revealed a better activity than the correspond-
ing dihydrobenzofuran series (compounds 2, 4–7 and 9–10).
Among them, the optimal lead compound 26 showed a moderate
lipophicity (LogD7.4 = 1.95) and molecular weight (Mw = 391.78),
which is still enough space for avoiding undue increase on
lipophilicity and molecular size in the further optimization.
2.5. Hypoglycemic effects of 26 explored in type 2 diabetic mice
To assess glucose lowering effects of compound 26 in the dia-
betic state, an OGTT in STZ-induced type 2 diabetic C57BL/6 mice
was performed.43,44 As shown in Figure 5, the hyperglycemia was
markedly controlled with a 14.2% decrease of glucose AUC0–2h in
compound 26 (50 mg/kg) treated mice despite the reduction was
less than that of TAK-875 (20 mg/kg, À32.5% glucose AUC0–2h).
The promising results indicated the compound 26 holds potential
for further optimization as an orally bioavailable lead compound.
2.3. Molecular modeling study
3. Conclusion
To further understand the interaction mode of the triazole
series, a docking study of compound 26 based on the X-ray struc-
ture of FFA1 (PDB accession code: 4PHU) was performed.42 As
shown in Figure 3, the compound 26 docked well to the same
binding pocket for TAK-875. Just as TAK-875, an edge-on interac-
tion was also formed between the residue Trp174 and the
fluorobenzene ring of compound 26. The head acid moiety, how-
ever, just form two hydrogen bonds with Tyr91 and Arg2258
(Fig. 3B), which explained the reason why the agonistic activity
of compound 26 is inferior to TAK-875 (three hydrogen bonds).
Moreover, the chlorine of terminal phenyl in compound 26 has
a hydrophobic interaction with the residues Leu158 and Pro80.
It was likely suggested that the hydrophobic interaction was cru-
cial for the superior agonistic activity of compound 26 rather
than other un-substituted compounds (such as 2, 11 and 16).
With the purpose of hunting potent lead compound with
reduced lipophilicity and molecular weight, we have identified a
new series of triazole-based FFA1 agonists via click chemistry. Sys-
tematic exploration of SAR in the triazole scaffold leads to the iden-
tification of lead compound 26, a potent FFA1 agonist with
relatively low lipophicity (LogD7.4 = 1.95) and molecular weight
(Mw = 391.78) suitable for further optimization. Moreover, com-
pound 26 showed a great potential for decreasing the plasma glu-
cose levels in normal ICR mice (À21.4% of glucose AUC0–2h) and
type
2
diabetic C57BL/6 mice (À14.2% of glucose AUC0–2h).
Although the potency of lead compound 26 was inferior to TAK-
875, the information obtained from our SAR and molecular model-
ing studies might help to design more competitive FFA1 agonists
with distinct advantage in physicochemical property. Based on
the promising lead compound 26, a more targeted combinatorial
chemical library using click chemistry will also provide more
active candidates suitable for clinical development.
2.4. Effect of optimized compounds on OGTT
In order to select an orally bioavailable lead compound, the
optimized compounds 6, 10, 19, 24, 25 and 26 (50 mg/kg) were
selected for pharmacological evaluation in normal ICR mice by oral
glucose tolerance test (OGTT). The time-dependent changes of
plasma glucose levels and the area under the curve (AUC0–2h) are
shown in Figure 4. The hypoglycemic activities of the selected
compounds were in accordance with the FFA1 agonistic activities.
Among them, compounds 10, 25 and 26 revealed a significant
improvement in the glucose tolerance with a 16.8%, 17.7% and
21.4% reduction in glucose AUC0–2h, respectively. The compound
26, an orally bioavailable lead compound with the strongest ago-
nistic activity and minimal lipophicity as well as molecular weight
in the above three compounds, was selected for further pharmaco-
logical evaluation.
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). Melt-
ing 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 (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
Figure 3. The interaction mode of compound 26 bound to FFA1. Key residues are labeled in red, and hydrogen bonding interactions are represented by purple dashed lines.
(A) Overlay of TAK-875 and 26 (blue) bound to FFA1. (B) Compound 26 bound to FFA1.
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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 6 0.05, ⁄⁄p 6 0.01 compared to vehicle mice by Student’s t-test.
code: 4PHU) was retrieved from Protein Data Bank. Prior to dock-
ing, 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 Pharma-
cophore Query Editor. The structure of compound 26 was docked
into the binding pocket with Pharmacophore method and then
ranked with the London dG scoring function. For the energy mini-
mization of ligand in the active site, MOE Forcefield Refinement
was used and ranked with London dG scoring function.
Figure 5. Effects of 26 on blood glucose levels during an OGTT in fasting type 2
diabetic C57BL/6 mice. Values are mean SEM (n = 6). ⁄p 6 0.05 and ⁄⁄p 6 0.01
4.4. Biological methods
compared to vehicle diabetic mice by Student’s t test.
4.4.1. Ca2+ influx activity of CHO cells stably expressing human
FFA1 (FLIPR assay)
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.15
CHO cells stably expressing human FFA1 (accession no.
NM_005303) were plated at a density of 15 K cells/well and incu-
bated 12 h in 5% CO2 at 37 °C. Subsequently, culture medium was
removed and washed with Hank’s Balanced Salt Solution
(100
medium containing 2.5
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 -linole-
lL). Then, cells were incubated in loading buffer (recording
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.
l
g/mL fluorescent calcium indicator Fluo
c
nic acid (Sigma) were added into the cells, and the intracellular
Ca2+ flux signals after addition were monitored by FLIPR Tetra sys-
tem (Molecular Devices) for 90 s. The agonistic activities of test
compounds on human FFA1 were expressed as [(AÀB)/(CÀB)] Â
100 (increase of the intracellular calcium concentration (A) in the
test compounds-treated cells and (B) in vehicle-treated cells, and
4.2. Determination of LogD7.4
In 10 mL glass vial, 40
added 1980 L phosphate buffer solution (0.01 M, pH = 7.4) and
1980 L 1-octanol (Sigma), obtaining 100 M final concentration
lL of 10 mM stock solution in DMSO was
l
l
l
(C) in 10 lM c-linolenic acid-treated cells).
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
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 pel-
lets 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 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 com-
mittee of China Pharmaceutical University.
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.
4.3. Molecular modeling
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.
The molecular docking study of compound 26 was performed
by using MOE (version 2008.10, The Chemical Computing Group,
Montreal, Canada). The crystal structure of FFA1 (PDB accession
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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, TAK-875 (20 mgÁkgÀ1) or selected
compounds (50 mgÁkgÀ1) were orally administered 30 min before
oral glucose load (3 gÁkgÀ1). Blood samples were collected from
the tail vein immediately before drug administration (À30 min),
0 min (just before glucose load), and at 15, 30, 45, 60 and
120 min after glucose load. The blood glucose levels were mea-
sured by using blood glucose test strips (SanNuo ChangSha, China).
4.4.2.2. Hypoglycemic effects of compound 26 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 Medi-
science 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 mLÁkgÀ1; 80 mgÁkgÀ1). The C57BL/6 mice
were fed with high-fat diet for another 4 weeks, and mice with
fasting blood glucose level P11.1 mmol/L were used as type 2 dia-
betic mice model.43,44
Type 2 diabetic C57BL/6 mice were fasted 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 as negative con-
trol. A single doses of TAK-875 (20 mgÁkgÀ1), vehicle or compound
26 (50 mgÁkgÀ1) were orally administered 30 min before oral glu-
cose load (2 gÁkgÀ1). Blood samples were collected from the tail
vein immediately before drug administration (À30 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).
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Conflict of interest
The authors have no conflicts of interest to declare.
Acknowledgements
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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), innovation project of Jiangsu Province (Grant
KYLX16_1175) and the Fundamental Research Funds for the Cen-
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Please cite this article in press as: Li, Z.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.068