Design, synthesis, and biological evaluation of novel pan agonists of FFA1, PPARγ and PPARδ

Article abstract of DOI:10.1016/j.ejmech.2018.09.071

The free fatty acid receptor 1 (FFA1) and peroxisome proliferator-activated receptors (PPARs) have attracted interest as potent targets for the treatment of metabolic syndrome such as type 2 diabetes. Based on the hypothesis that the dual agonists of PPARs and FFA1 would act as insulin sensitizers and secretagogues by simultaneous activation of PPARs and FFA1, we developed the design strategy to obtain dual PPARs/FFA1 agonist by hybrid FFA1 agonist 1 with PPARδ agonist 2 in consideration of their structural similarity. As expected, systematic exploration of structure-activity relationship and molecular modeling, results in the discovery of lead compound 15, a pan agonist with relative balanced activities between FFA1, PPARγ and PPARδ. The dose-response relationship studies suggested that the pan agonist 15 suppressed the excursion of blood glucose levels in a dose-dependent manner. During a 5-days treatment in ob/ob mice, the pan agonist 15 (100 mg/kg) revealed sustained hypoglycemic effect, even proximity to the most advanced FFA1 agonist (TAK-875, 40 mg/kg), which might be attributed to its pan PPARs/FFA1 activities to simultaneous regulate the mechanism of insulin secretion and resistance. These positive results suggest that the dual PPARs/FFA1 agonists such as lead compound 15 might be novel therapeutic strategy to modulate the complex pathological mechanisms of type 2 diabetes.

Full text of DOI:10.1016/j.ejmech.2018.09.071

Accepted Manuscript
Design, synthesis, and biological evaluation of novel pan agonists of FFA1, PPARγ
and PPARδ
Zheng Li, Zongtao Zhou, Fengjian Deng, Yuyi Li, Danjun Zhang, Luyong Zhang
PII:
S0223-5234(18)30860-2
10.1016/j.ejmech.2018.09.071
EJMECH 10782
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 July 2018
Revised Date: 27 September 2018
Accepted Date: 29 September 2018
Please cite this article as: Z. Li, Z. Zhou, F. Deng, Y. Li, D. Zhang, L. Zhang, Design, synthesis, and
biological evaluation of novel pan agonists of FFA1, PPARγ and PPARδ, European Journal of Medicinal
Chemistry (2018), doi: https://doi.org/10.1016/j.ejmech.2018.09.071.
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ACCEPTED MANUSCRIPT
Design, synthesis, and biological evaluation of novel pan agonists of FFA1, PPARγ and
PPARδ
Zheng Li1, 2*, Zongtao Zhou1, Fengjian Deng1, Yuyi Li1, Danjun Zhang1, Luyong Zhang1, 2, 3, 4*
1 School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China.
2 Key Laboratory of New Drug Discovery and Evaluation, Guangdong Pharmaceutical University,
Guangzhou 510006, PR China.
3
Guangzhou Key Laboratory of Construction and Application of New Drug Screening Model
Systems, Guangdong Pharmaceutical University, Guangzhou 510006, PR China.
4 Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009,
PR China.
Graphical Abstract
Aiming to develop potent dual PPARs/FFA1 agonists, we have hybrid the Sanofi’s FFA1 agonist 1
with PPARδ agonist 2 based on their structural similarity, exemplified by the orally bioavailable
FFA1/PPARγ/PPARδ pan agonist 15.
*
Corresponding author.
E-mail addresses: li.zheng.sky@163.com (Z. Li), lyzhang@cpu.edu.cn (L. Zhang).

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Design, synthesis, and biological evaluation of novel pan agonists of FFA1, PPARγ and
PPARδ
Zheng Li1, 2*, Zongtao Zhou1, Fengjian Deng1, Yuyi Li1, Danjun Zhang1, Luyong Zhang1, 2, 3, 4*
1 School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China.
2 Key Laboratory of New Drug Discovery and Evaluation, Guangdong Pharmaceutical University,
Guangzhou 510006, PR China.
3
Guangzhou Key Laboratory of Construction and Application of New Drug Screening Model
Systems, Guangdong Pharmaceutical University, Guangzhou 510006, PR China.
4 Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009,
PR China.
Abstract
The free fatty acid receptor 1 (FFA1) and peroxisome proliferator-activated receptors (PPARs)
have attracted interest as potent targets for the treatment of metabolic syndrome such as type 2
diabetes. Based on the hypothesis that the dual agonists of PPARs and FFA1 would act as insulin
sensitizers and secretagogues by simultaneous activation of PPARs and FFA1, we developed the
design strategy to obtain dual PPARs/FFA1 agonist by hybrid FFA1 agonist 1 with PPARδ agonist
2 in consideration of their structural similarity. As expected, systematic exploration of
structure-activity relationship and molecular modeling, results in the discovery of lead compound
15, a pan agonist with relative balanced activities between FFA1, PPARγ and PPARδ. The
dose-response relationship studies suggested that the pan agonist 15 suppressed the excursion of
blood glucose levels in a dose-dependent manner. During a 5-days treatment in ob/ob mice, the
pan agonist 15 (100 mg/kg) revealed sustained hypoglycemic effect, even proximity to the most
advanced FFA1 agonist (TAK-875, 40 mg/kg), which might be attributed to its pan PPARs/FFA1
activities to simultaneous regulate the mechanism of insulin secretion and resistance. These
positive results suggest that the dual PPARs/FFA1 agonists such as lead compound 15 might be
*
Corresponding author.
E-mail addresses: li.zheng.sky@163.com (Z. Li), lyzhang@cpu.edu.cn (L. Zhang).
1

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novel therapeutic strategy to modulate the complex pathological mechanisms of type 2 diabetes.
Keywords: FFA1; PPAR; Type 2 diabetes; Hybrid; Pan agonist.
1. Introduction
Type 2 diabetes mellitus (T2DM) is a metabolic syndrome characterized by chronic
hyperglycemia attribute to insufficient insulin secretion and the peripheral insulin resistance.[1, 2]
Agents that improve insulin secretion and resistance such as sulfonylureas and thiazolidinediones
are commonly used for the administration of T2DM. However, most of current anti-diabetic drugs
are associated with the high risk of hypoglycemia, weight gain, and gastrointestinal side
effects.[3-6] Hence, a novel drug that has excellent safety and efficacy in controlling
hyperglycemic state would be advantageous.[7-9]
The long-chain free fatty acid receptor 1 (FFA1/GPR40) is mainly expressed in pancreatic
β-cells, which promotes the glucose-stimulated insulin secretion without the risk of
hypoglycemia.[10-13] Recently, many FFA1 agonists have been reported in literature (Figure 1),
and several candidates such as TAK-875, AMG-837 and LY2881835 have reached to clinical trials.
Readers interested in more structure-activity relationship of FFA1 agonists may refer to our recent
reviews.[14, 15] To explore structurally diverse FFA1 agonists, various structure moieties were
also introduced in our previous researches.[16-24]
The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that play a
critical role in regulating energy metabolism by the modulation of related gene expression.[25]
PPARs have three subtypes: PPARα, PPARγ, and PPARδ. The agonists of PPARα or PPARγ
revealed potent therapeutic benefit for the T2DM and dyslipidemia.[26, 27] Particularly, PPARγ
has been identified as a key regulator for the improvement of insulin resistance, and its agonist
rosiglitazone has demonstrated clinical success for the treatment of T2DM.[28] The role of PPARδ
is involved in lipid metabolism and inflammation, and thereby plays an important role in obesity,
insulin resistance and atherosclerosis.[29-31] Therefore, the dual agonists of PPARs and FFA1
would act as insulin sensitizers and secretagogues by simultaneous activation of PPARs and FFA1.
It is worth mentioning that their pharmacophoric features are very similar and their endogenous
ligands are free fatty acids.[32] Based on these features, several groups have reported some lead
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compound with dual agonistic activities for FFA1 and PPARγ, though their activities are limited to
one digit µM.[33-35]
After systematic investigation, we found several interesting compounds in the field of PPARs
and FFA1. As shown in Figure 1, the Sanofi’s FFA1 agonist 1 [14, 15] shared the same scaffold
(red lable in Figure 1) with the PPARδ agonist 2.[36] Hence, we speculate that the hybrid of FFA1
agonist 1 and PPARδ agonist 2 would be a rational strategy to obtain the dual PPARs/FFA1
agonist. In consideration of the similarity of acid head between compound 2 and TAK-875, the
acid head of TAK-875 was also introduced to the hybrid compounds to maintain the PPARδ
activity of compound 2 as much as possible (Figure 2). After systematic exploration of
structure-activity relationship (SAR) in this hybrid series, the compound 15 was identified as a
FFA1/PPARγ/PPARδ pan agonist with potent glucose-lowering effect in ob/ob mice.
2. Results and Discussion
2.1. Chemistry
The synthetic routes of target compounds 3-15 are shown in Scheme 1. The commercially starting
material substituted phenols were treated with 1,2-dibromoethane or 1,3-Dibromopropane to
afford the intermediates 2a-L, which were further converted to target compounds 3-14 by
Williamson ether synthesis with 3a [37] and basic hydrolysis. The racemate 4a was synthesized
via reported procedures,[37] which was further converted to optically pure 6a by a novel and
simple chiral resolution with R-phenethylamine. Condensation of intermediate 6a with 2g by
Williamson ether synthesis, followed by basic hydrolysis, furnished the target compound 15. The
compounds 16-18 were synthesized starting from intermediates 2g and 2L as summarized in
Scheme 2. The intermediates 7a-b were synthesized via our previous published procedures.[38]
The intermediate 2g or 2L was treated with 7a-b in acetonitrile at the presence of potassium
carbonate to give ester in high yield, which was further converted to the desired carboxylic acids
16-18 under hydrolysis conditions of lithium hydrate.
The target compounds 19 and 20 were depicted in Scheme 3. The intermediate 9a was
prepared by Knoevenagel condensation of p-hydroxy benzaldehyde and Meldrum’s acid, followed
by treating with sodium borohydride at room temperature. The intermediate 9a were further
derived into deuterated compound 10a by decarboxylation and esterification. The commercially
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starting material 11a was treated with methyl chloroacetate to obtain 12a, followed by oxidation
with Oxone to provide intermediate 13a, which was treated with boron tribromide to give 14a.
Subsequently, condensing phenols 10a or 14a with 2g in the presence of potassium carbonate,
followed by basic hydrolysis, furnished the target compounds 19 and 20.
2.2. PPARs/FFA1 agonistic activity and SAR study
In order to explore the SAR of hybrid compounds, the FFA1 agonistic activities were evaluated by
a fluorometric imaging plate reader (FLIPR) assay, and the agonistic activities of PPARs were
investigated in Gal4 receptor cell-based assay with positive controls GW7647, Rosiglitazone, and
GW0742, respectively (Table 1). The hybrid compound 4, a direct analog of selective PPARδ
agonist 2, revealed an approximately 20-fold decrease in PPARδ agonistic activity while the
potency on PPARγ has improved at certain degree. Shortening the linker of compound 4 by a
methylene provided compound 3, which led to a significant drop of potency both in PPARγ and
PPARδ, suggesting that the length of linker is crucial to the agonistic activity of PPARs.
Interestingly, the compound 3 and 4 were almost inactive for FFA1 though their analog Sanofi’s
FFA1 agonist 1 has highly agonistic activity at nanomolar level (EC50 = 10 nM). Methyl scanning
in the terminal benzene ring (compounds 5, 6, and 7) indicated that substitution is preferred in the
3-position and tolerated in 4-position for FFA1 agonistic activity, while have little effect on the
PPARs. The 2-position substituted derivatives (5 and 8) revealed better agonistic activities than
parent compound 4 for PPARγ and PPARδ, but there was no significant improvement on the
potency of FFA1.
Based on the obtained SAR above, we therefore selected the 3-position as structural
modification site in further SAR to maintain a better balance between FFA1, PPARγ and PPARδ
(Table 1). For the FFA1 and PPARγ, various substituents (compounds 9-13) were well tolerated in
the 3-position. For the PPARδ, the agonistic activity of 9 (3-Cl, 1.75 Å) ＞ 6 (3-Me, 1.80 Å) ＞
11 (3-tBu) ＞ 13 (3-Ph) suggested that the steric effect in the 3-position might affect the potency
of PPARδ, which could be attributed to the limited ligand-binding pocket in this position. Notably,
4-position phenyl substituted derivative 14 revealed improved agonistic activity on PPARα
compared to other analogs.
In consideration of the better balance between FFA1, PPARγ and PPARδ, the compound 9
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was selected to provide the optimal enantiomer by chiral separation (Table 2). As expected, the
optically pure compound 15 (FFA1: 2.38 µM; PPARγ: 1.43 µM; PPARδ: 1.75 µM) revealed a
significant improvement on potency compared with the racemate compound 9 (FFA1: 3.07 µM;
PPARγ: 1.69 µM; PPARδ: 2.16 µM). With compound 15 as a pan agonist, we decided to focus on
the exploration of acid moiety to further extend the SAR (compounds 16-20). The 2-methyl
phenoxyacetic acid analog 16 turned out to be more potent than the dihydrobenzofuran series in
terms of PPARγ and PPARδ, but the potency on FFA1 was found to be rather low (> 10 µM).
Similar to compound 14, the incorporation of 4-position phenyl (17) gave a further increase in the
activity of PPARα but unfortunately also significantly decreased potency on PPARγ. The 2-fluoro
analog 18 resulted in an approximately 2-fold decrease in PPAR agonistic activity compared to the
analog 16. Consistent with our previously reported FFA1 agonists with 2-fluoro phenoxyacetic
acid moiety,[38] compound 18 revealed a significant improvement on the agonistic activity of
FFA1. The deuterated phenylpropionic acid (19) and phenylsulfonyl acetic acid (20), two
previleged scaffolds in our reported FFA1 agonists,[22, 23] were also introduced to explore their
potential on PPARs and FFA1 by hybrid with compound 15. Compound 19 indicated an increased
FFA1 activity back to the level of compound 9 but, nevertheless, led to 2-fold reduced potency on
PPARδ. Interestingly, phenylsulfonyl acetic acid analog 20 appeared to diminish the potency both
in PPARs and FFA1, implying that our previously explored SAR could not transfer to this series.
After systematic structural optimization, a clear SAR picture of this series was developed and
summarized in Figure 3.
2.3. Docking study
Based on the crystal complexes of FFA1 (PDB code: 4PHU), PPARγ (2Q8S) and PPARδ (1GWX),
we performed an induced-fit docking study of compound 15 to better clarify the SAR of this series.
As shown in Figure 4, compound 15 fitted very well into the binding pocket of FFA1, PPARδ and
PPARγ. In the binding pocket of PPARδ (Figure 4A and 4B), the acidic moiety of compound 15
formed hydrogen-bonding interaction with residues Tyr473 and His449. Moreover, there is an
open cavity around the terminal benzene ring, which reasonably explained that the methyl was
tolerated in different position (compounds 5-7). Notably, the chlorine is accommodated well into
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the small hydrophobic pocket of PPARδ, which could form steric hindrance with bulkier
substituents (compounds 11-13). In the binding pocket of FFA1 (Figure 4C and 4D), the
carboxylic acid of compound 15 formed strong hydrogen-bonding network with Arg2258 and
Arg183, while the α-methylene of acid generate an σ-π interaction with Phe87. Because the steric
effect around the 2-position of terminal benzene ring, the substituent was not tolerated in the
2-position (compound 5). In contrast, the 3-position substituent was preferred due to the
hydrophobic interaction with the hydrophobic surface of binding pocket. In the binding site of
PPARγ (Figure 4E and 4F), the acidic moiety of compound 15 formed three hydrogen-bonding
interactions with Ser289, Tyr473 and Tyr327. Similar to the binding pocket of PPARδ, there is a
large hydrophobic cavity around the terminal benzene ring, rationally explained that this area
could be occupied by different hydrophobic substituents.
2.4. Dose-response relationship of 15 in normal mice
Based on the better balance between FFA1, PPARγ and PPARδ, compound 15 (10, 30 and 100
mg/kg) was selected to evaluate its hypoglycemic effect and dose-response relationship. As shown
in Figure 5, compound 15 (10, 30 and 100 mg/kg) suppressed the excursion of plasma glucose
levels in a dose-dependent manner in normal mice, with a decrease in the AUC0−120min values of
1.1%, 8.1% and 18.6%, respectively. Moreover, compound 15 revealed significantly
hypoglycemic effect at the dose of 100 mg/kg though it is still inferior to TAK-875 (20 mg/kg,
-26.9% AUC0−120min).
2.5. Hypoglycemic effects of 15 in ob/ob mice
To further investigate hypoglycemic effect in diabetic state, compound 15 was evaluated for its
therapeutic potential to improve glucose tolerance in ob/ob mice, a type 2 diabetes model that
develops obese and insulin resistance.[39, 40] In this research, TAK-875 (40 mg/kg), compound
15 (100 mg/kg), or vehicle was orally administered to ob/ob mice twice daily for five days, and
the oral glucose tolerance tests (OGTTs) were determined on day 0 and 5. As shown in Figure 6,
the acute administration of TAK-875 and compound 15 resulted in a 13.1% and 7.6% reduction on
AUC0−120min of glucose at the day 0 of treatment. On day 5, TAK-875 or compound 15 provided
robust glucose-lowering effects, with a reduction in the AUC0−120min values of 20.2% and 17.3%,
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respectively. These results indicated that the glucose-lowering effect of compound 15 could be
maintained and even improved throughout the treatment without the indication of desensitization
or tachyphylaxis. Notably, at the 5th days of treatment, the hypoglycemic effect of compound 15
(100 mg/kg, -17.3% AUC0−120min) was similar to that of TAK-875 (40 mg/kg, -20.2% AUC0−120min).
This positive result might be attributed to the dual effects of compound 15 to improve the insulin
secretion and sensitivity by simultaneous activation of FFA1, PPARγ and PPARδ.
3. Conclusion
Aiming to develop potent dual PPARs/FFA1 agonists, we have hybrid the Sanofi’s FFA1 agonist 1
with the PPARδ agonist 2 based on their structural similarity, exemplified by the orally
bioavailable FFA1/PPARγ/PPARδ pan agonist 15. The important aspects that led to the
identification of lead compound 15 were as follows: (1) the hybrid strategy not only maintained
the potency on FFA1 and PPARδ to some extent, but also unexpectedly increased the activity of
PPARγ; (2) systematic SAR and molecular modeling studies allowed us to explore a better
balance of potency between FFA1, PPARγ and PPARδ. The dose-response relationship studies
indicated that compound 15 reduced plasma glucose levels in a dose-dependent manner. During a
5-days treatment in ob/ob mice, compound 15 (100 mg/kg) revealed sustained glucose-lowering
effects, even proximity to that of TAK-875 (40 mg/kg), which might be attributed to its pan
PPARs/FFA1 agonistic activities to simultaneous regulate the mechanism of insulin secretion and
resistance. All of these results suggested that pan agonist 15 was meaningful for further evaluation
as a tool compound, and the information acquired from the SAR and molecular modeling studies
might help to explore more competitive FFA1/PPARγ/PPARδ pan agonist.
4. Experimental section
4.1. General chemistry
All starting materials, reagents and solvents were obtained from commercial sources and used
without further purification unless otherwise indicated. Purifications by column chromatography
were carried out over silica gel (200-300 mesh) and monitored by thin layer chromatography
performed on GF/UV 254 plates and were visualized using UV light at 254 and 365 nm. NMR
spectra were recorded on a Bruker ACF-300Q instrument (300 MHz for 1H NMR and 75 MHz for
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13C NMR spectra), chemical shifts are expressed as values relative to tetramethylsilane as internal
standard, and coupling constants (J values) were given in hertz (Hz). LC/MS spectra were
recorded on a Waters liquid chromatography-mass spectrometer system (ESI). Optical rotation
was determined on a JASCO P-1030 polarimeter. Elemental analyses were performed by the
Heraeus CHN-O-Rapid analyzer. Compound 2[36] and TAK-875[37] were synthesized as
previously reported.
1
The physical characteristics, H NMR, 13C NMR, MS and elemental analysis data for all
intermediates and target molecules, were reported in the supporting information.
4.2. Ca2+ influx activity of CHO cells expressing human FFA1 (FLIPR Assay)
CHO cells stably expressing human FFA1 were seeded into 96-well plates at a density of 15K
cells/well and incubated 16 h in 5% CO2 at 37 °C. After, the culture medium in the wells was
removed and washed with 100 µL of Hank’s Balanced Salt Solution. Then, cells were incubated in
loading buffer (containing 2.5 µg/mL fluorescent calcium indicator Fluo 4-AM, 2.5 mmol/L
probenecid and 0.1% fatty acid-free BSA) for 1 h at 37 °C. Various concentrations of test
compounds or γ-linolenic acid (Sigma) were added into the cells and the intracellular calcium flux
signals were monitored by FLIPR Tetra system (Molecular Devices). The agonistic activities of
test compounds on human FFA1 were expressed as [(A−B)/(C−B)]×100% (increase of the
intracellular Ca2+ 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 value of tested compound was
obtained with Prism 5 software (GraphPad).
4.3. Cell-based GAL4 transactivation assay for PPARγ and PPARδ
For transfection, HEK293 cells were plated onto 96-well plates at 5 × 104 cells/well in DMEM
(Gibco) without antibiotics 1 day prior to transfection. Transfections were performed using
FuGENE HD Transfection Reagent (Roche) according to the manufacturer’s protocol. The cells
were transiently transfected with 25 ng/well pBIND-PPARδ or PPARγ, 25 ng/well pG5Luc, and
0.15 µl/well FuGENE HD. After transfection for 24 hours, various concentrations of test
compounds or positive controls were added into the cells and incubated for 18 hours. Cells from
each well were lysed with 20 µl lysis buffer, and each well was added 30 µl Luciferase Assay
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Reagent II. The firefly and renilla luciferase signal is assayed by Promega’s Dual Luciferase
Reporter Assay System. Envision is used as the luminometer. The data values are normalize by
dividing the firefly signal with renilla signal. “F/R” means “Firefly/Renilla”. This normalization
eliminates the discrepancy of different cell number and transfection efficiency in each well.
The %Activation value is calculated by the following equation: %Activation
=
[(X-Min)/( Max-Min)]×100% (X is the “F/R” value in each concentration point. Min is the mean
“F/R” value from no compound control. Max is the mean “F/R” value from reference compound
control). EC50 value of tested compound was obtained with Prism 5 software (GraphPad).
4.4. Cell-based GAL4 transactivation assay for PPARα
For transfection, HepG2 cells were plated onto 96-well plates at 6 × 104 cells/well in MEM (Gibco)
without antibiotics 1 day prior to transfection. The similar protocol was used as described for the
cell-based GAL4 transactivation assay for PPARγ and PPARδ.
4.5. Molecular modeling studies
MOE (version 2014.0901, The Chemical Computing Group, Montreal, Canada) was used to
perform the docking modeling based on the reported crystal structure of FFA1 (PDB ID: 4PHU),
PPARγ (2Q8S) and PPARδ (1GWX). Prior to molecular docking, other crystallized ligands and
water were removed, and the obtained protein was performed by Protonate 3D prior to the
Gaussian Contact surface was draw around the binding pocket of ligand. Subsequently, the
binding pocket with Gaussian Contact surface was isolated. Compound 15 was induced-fit docked
into the binding pocket and ranked with London dG scoring function. For energy minimization in
binding pocket, MOE Forcefield Refinement was performed and ranked with London dG scoring
function. The binding free energy calculations in the docking studies of positive controls and
compound 15 were represented as ∆G value.
4.6. Animals and statistical analysis of the data
Male ICR mice (18-22 g) were purchased from Guangdong Medical Laboratory Animal Center
(Guangdong, China), and six weeks old male ob/ob mice were purchased from Model Animal
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Research Center of nanjing university (Jiangsu, China). All animals were acclimatized for 1 week
before the experiments. The animal room was maintained under a constant 12 h light/black cycle
with temperature at 23 ± 2 °C and relative humidity 50 ± 10% throughout the experimental period.
Mice were allowed ad libitum access to standard pellets and water unless otherwise stated, and the
vehicle used for drug administration was 0.5% sodium salt of Carboxy Methyl Cellulose aqueous
solution for all animal studies. All animal experimental protocols were approved by the ethical
committee at Guangdong 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 performed using GraphPad InStat version 5.00 (San Diego, CA,
USA). General effects were analyzed by Student’s t test or using a one-way ANOVA with Tukey’s
multiple-comparison post hoc test.
4.7. Oral Glucose Tolerance Test in male ICR mice
Normal male ICR mice after adaptation one week were fasted for 12 h, weighted, bled via the tail
tip, and randomized into 5 groups (n = 6 per group). Compound 15 (10, 30, and 100 mg/kg),
TAK-875 (20 mg/kg), or vehicle (0.5% methylcellulose aqueous solution) was orally administered
30 min before oral glucose load (3 g/kg). Blood samples were collected immediately at -30 min
(just before drug administration), 0 min (just before glucose challenge), and 15, 30, 60 and 120
min after glucose load. The blood glucose was measured by plasma glucose test strips (SanNuo
ChangSha, China).
4.8. Oral Glucose Tolerance Test in male ob/ob mice
The male ob/ob mice were dosed twice daily with the vehicle (0.5% methylcellulose aqueous
solution), TAK-875 (40 mg/kg) or compound 15 (100 mg/kg) by gavage administration for 5 days.
Mice were dosed at fixed time daily, and the OGTTs were performed on days 0 and 5 of treatment.
Mice were fasted overnight prior to treatment with a single doses of vehicle, TAK-875 (40 mg/kg)
or compound 15 (100 mg/kg) and subsequently dosed orally with glucose aqueous solution (3 g/kg)
after 30 min. Mice were bled via tail tip immediately before drug administration (-30 min), before
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glucose challenge (0 min), and at 15, 30, 60 and 120 min post-dose and the blood glucose was
measured by blood glucose test strips (SanNuo ChangSha, China).
Acknowledgements
This study was supported by the Natural Science Foundation of Guangdong Province (Grant
2018A030313445), the Starting Foundation for Ph.D., Guangdong Pharmaceutical University
(Grant 43540073) and Guangdong Province Medical Science and Technology Research Fund
(Grant A2018273).
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Table 1: In vitro activities of the human PPARs and FFA1 a
Compd.
R1
n
hFFA1
hPPARα
hPPARγ
hPPARδ
(LBD)-GAL4
EC50 (µM)
EC50 (µM)
(LBD)-GAL4 (LBD)-GAL4
EC50 (µM)
EC50 (µM)
0.045
ND b
ND
ND
ND
ND
ND
TAK-875
0.007
ND
ND
0.12
ND
GW7647
ND
Rosiglitazone
ND
ND
0.013
0.17
7.93
3.26
1.93
2.42
1.95
2.09
2.16
2.37
4.84
9.57
5.49
1.98
GW0742
> 10
> 10
> 10
> 10
3.59
7.62
> 10
3.07
3.45
3.02
3.85
2.76
4.26
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
7.31
> 10
7.75
5.26
2.02
1.75
1.81
2.67
1.69
1.67
1.75
2.03
1.85
2.97
2
3
H
0
1
1
1
1
1
1
1
1
0
1
1
H
4
2-CH3
3-CH3
4-CH3
2-F
5
6
7
8
3-Cl
3-Br
3-tBu
3-tBu
3-Ph
4-Ph
9
10
11
12
13
14
a EC50 value represent the mean of three determinations, which is the concentration giving 50% of the maximal
activity determined for the tested compound.
b ND: not determined.
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Table 2: In vitro activities of designed compounds with various acid moieties a
Compd.
R1
R
hFFA1
EC50
hPPARα
hPPARγ
hPPARδ
(LBD)-GAL4 (LBD)-GAL4 (LBD)-GAL4
(µM)
EC50 (µM)
EC50 (µM)
EC50 (µM)
0.045
ND b
ND
ND
ND
ND
TAK-875
GW7647
Rosiglitazone
GW0742
2
0.007
ND
ND
0.12
ND
ND
ND
ND
ND
0.013
0.17
2.16
> 10
3.07
> 10
> 10
> 10
1.69
3-Cl
3-Cl
3-Cl
4-Ph
3-Cl
3-Cl
3-Cl
9
O
2.38
> 10
> 10
8.45
3.28
> 10
> 10
> 10
5.26
> 10
> 10
> 10
1.43
1.54
4.56
3.17
1.92
> 10
1.75
1.73
1.52
3.35
4.56
> 10
15
16
17
18
19
20
COOH
a EC50 value represent the mean of three determinations, which is the concentration giving 50% of the maximal
activity determined for the tested compound.
b ND: not determined.
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Figure 1: Structure of FFA1 or PPARs agonists, the common scaffold was labeled in red.
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Figure 2: Our strategy to obtain dual PPARs/FFA1 agonists by hybrid the common scaffold of PPARs and FFA1
agonists.
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Figure 3: Structure-activity relationship summary.
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Figure 4: The frontal view and side-view in docking model of compound 15 (cyan carbon) in crystal structure of
PPARδ (1GWX, A and B), FFA1 (4PHU, C and D) and PPARγ (2Q8S, E and F). Key residues are labeled in white,
and interactions are represented by dashed lines. G represents the binding free energy calculations in the docking
studies of positive controls and compound 15, respectively.
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Figure 5: Dose–response relationship of compound 15 explored in normal mice. (A) represented time-dependent
changes of blood glucose levels after oral administration of compound 15, followed by oral glucose load (3 g/kg),
respectively. (B) represented AUC0−120min of plasma glucose levels. Results are mean ± SD (n = 6 per group). *p≤
0.05 and **p≤0.01 was analyzed using a one-way ANOVA with Tukey’s multiple-comparison post hoc test.
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Figure 6: Effects of compound 15 on plasma glucose levels and corresponding AUC0−120min of glucose during an
OGTT in fasting ob/ob mice after five days treatment. TAK-875, compound 15, or vehicle was orally administered
to ob/ob mice twice daily for 5 days, and the OGTTs were determined on treatment day 0 and 5. Values are mean ±
SD (n = 6 per group). *P≤0.05 and **p≤0.01 compared to vehicle mice by Student’s t test.
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Scheme 1. Synthesis of compounds 3-15. Reagents and conditions: (a) 1,2-dibromoethane or 1,3-Dibromopropane,
K2CO3, acetone, 45 °C, 12 h; (b) K2CO3, acetonitrile, KI, 45 °C, 12 h; (c) LiOH·H2O, THF/MeOH/H2O, r.t., 4 h;
(d) R-Phenethylamine, acetone, reflux; (e) 1 M HCl; (f) H2SO4, MeOH, reflux; (g) 2g, K2CO3, acetonitrile, KI,
45 °C, 12 h.
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Scheme 2. Synthesis of compounds 16-18. Reagents and conditions: (a) K2CO3, acetonitrile, KI, 45 °C, 12 h; (b)
LiOH·H2O, THF/MeOH/H2O, r.t., 4 h.
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Scheme 3. Synthesis of target compounds 19-20. Reagents and conditions: (a) Meldrum’s acid, water, 75 °C, 2 h;
(b) NaBH4, r.t.; (c) D2O, DMF, 100 °C, 12 h, then MeOH, H2SO4, reflux, 3 h; (d) Methyl chloroacetate, K2CO3,
acetone, reflux, 2 h; (e) Oxone, MeOH, H2O, 0 °C to r.t.; (f) BBr3, CH2Cl2; (g) 2g, K2CO3, acetonitrile, KI, 45 °C,
12 h; (h) LiOH·H2O, THF/MeOH/H2O, r.t., 4 h.
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Highlights
‹
‹
‹
‹
‹
A therapeutic strategy that simultaneous targets insulin secretion and resistance.
FFA1/PPARγ/PPARδ pan agonist was identified by the hybrid of FFA1 and PPARδ agonists.
Compound 15 revealed relative balanced activities between FFA1, PPARγ and PPARδ.
Compound 15 suppressed the excursion of glucose levels in a dose-dependent manner.
Compound 15 exhibited similar glucose-lowering effect as TAK-875 after 5-days treatment.