Design, synthesis, and biological evaluation of novel dual PPARα/δ agonists for the treatment of T2DM

Article abstract of DOI:10.1016/j.bioorg.2020.103963

Dual PPARα/δ agonists have been considered as potential therapeutics for the treatment of type 2 diabetes mellitus. After comprehensive structure–activity relationship study based on GFT505, a novel dual PPARα/δ agonist compound 6 was identified with highly activities on PPARα/δ and higher selectivity against PPARγ than that of GFT505. The modeling study revealed that compound 6 binds well to the binding pockets of PPARα and PPARδ, which formed multiple hydrogen bonds with key residues related to the activation of PPARα and PPARδ. Moreover, oral glucose tolerance test exhibited that compound 6 exerts dose-dependent anti-diabetic effects in ob/ob mice and reveals similar potency to that of GFT505, the most advanced candidate in this field. These findings suggested that compound 6 is a promising candidate for further researches, and the extended chemical space might help us to explore better PPARα/δ agonist.

Full text of DOI:10.1016/j.bioorg.2020.103963

Journal Pre-proofs
Design, synthesis, and biological evaluation of novel dual PPARα/δ agonists
for the treatment of T2DM
Qiang Ren, Liming Deng, Zongtao Zhou, Xuekun Wang, Lijun Hu, Rongrong
Xie, Zheng Li
PII:
S0045-2068(20)30455-7
DOI:
https://doi.org/10.1016/j.bioorg.2020.103963
YBIOO 103963
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To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
26 February 2020
2 May 2020
20 May 2020
Please cite this article as: Q. Ren, L. Deng, Z. Zhou, X. Wang, L. Hu, R. Xie, Z. Li, Design, synthesis, and
biological evaluation of novel dual PPARα/δ agonists for the treatment of T2DM, Bioorganic Chemistry (2020),
doi: https://doi.org/10.1016/j.bioorg.2020.103963
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Design, synthesis, and biological evaluation of novel dual PPARα/δ agonists for the treatment
of T2DM
Qiang Ren1, 2, Liming Deng1, Zongtao Zhou1, Xuekun Wang3, Lijun Hu1, Rongrong Xie1*, Zheng
Li1, 2*
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 College of pharmacy, Liaocheng University, Liaocheng 252059, China.
Abstract
Dual PPARα/δ agonists have been considered as potential therapeutics for the treatment of type 2
diabetes mellitus. After comprehensive structure-activity relationship study based on GFT505, a
novel dual PPARα/δ agonist compound 6 was identified with highly activities on PPARα/δ and
higher selectivity against PPARγ than that of GFT505. The modeling study revealed that compound
6 binds well to the binding pockets of PPARα and PPARδ, which formed multiple hydrogen bonds
with key residues related to the activation of PPARα and PPARδ. Moreover, oral glucose tolerance
test exhibited that compound 6 exerts dose-dependent anti-diabetic effects in ob/ob mice and reveals
similar potency to that of GFT505, the most advanced candidate in this field. These findings
suggested that compound 6 is a promising candidate for further researches, and the extended
chemical space might help us to explore better PPARα/δ agonist.
Keywords: Dual agonist; PPAR; T2DM; GFT505; Selectivity.
1. Introduction
Type 2 diabetes mellitus (T2DM), known as a progressive metabolic disorder, is characterized by
elevated levels of blood glucose due to relatively insufficient insulin secretion and resistance [1, 2].
If blood glucose of type 2 diabetic patients cannot be effectively controlled, it may cause a
* Corresponding author.
E-mail addresses: li.zheng.sky@163.com (Z. Li), happyyunyun_1985@126.com (R. Xie).

consequence of chronic complications, such as retinopathy, renal disease, heart disease,
hypertension and peripheral vascular disease [3]. Currently, the continuing emergence of the
insufficient potency, hypoglycemia, and gastrointestinal side effects, motivates the search for novel
anti-diabetic drugs, especially for the multi-target drugs [4-7].
The peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor
superfamily as a class of ligand-inducible transcription factors. The crucial roles in glucose and lipid
metabolism make it possible for PPARs to become drug targets for T2MD and a series of metabolic
syndrome [8-10]. PPARs can be subdivided into three subtypes including PPARα, PPARδ and
PPARγ. Each subtype is pharmacologically distinct in that they are encoded separately, and the
residues sequence is various at the ligand-binding site [11, 12]. The activation of PPARα not only
decrease triglyceride and increase HDL cholesterol but also stabilizes glucose homeostasis and
ameliorates insulin resistance [13-15]. Therefore, PPARα agonists have been widely used for
hyperlipidemia in clinical trials, such as fenofibrate (Figure 1) [16]. In addition to improving
dyslipidemia, the activation of PPARδ can increase insulin sensitivity and thereby synergized with
PPARα for the treatment of T2DM [17-21]. In previous researches, we have identified several
PPARδ agonists with great potential on glucolipid metabolism [22-26]. The pharmacological role
of PPARγ is associated with adipogenesis, lipid storage, and glucose homeostasis [27]. Over the
years, PPARγ (rosiglitazone) and dual PPARα/γ (ragaglitazar) agonists (Figure 1) have been
developed to treat T2DM [28-30]. However, further research on this series of agonists is prevented
by multiple adverse effects related to PPARγ, including weight gain, fluid reaction, hemodilution,
and edema effect [31, 32]. Therefore, the development of dual PPARα/δ agonists with minimum
activity on PPARγ might be provided a better benefit/risk ratio for the treatment of T2DM.
GFT505 (Figure 1) is a dual PPARα/δ agonist, and the selectivity against PPARγ is still to be
improved. Furthermore, no literature has been reported to disclose the structure-activity relationship
(SAR) of GFT505. In this study, we carried out SAR study on the three parts of GFT505 (Figure
2) and finally got compound 6 with higher selectivity to PPARα/δ and low agonistic activity on
PPARγ. Moreover, compound 6 revealed dose-dependent hypoglycemic effects in the oral glucose
tolerance test in vivo.
2. Results and discussion

2.1 Chemistry
The synthetic route for compounds 1-10 is depicted in Scheme 1. Initial, compounds 1a-1c are
etherified with iodides under basic conditions by classical Williamson ether synthesis. These ethers
and thioethers (2a-2d and 4a) are subsequently aldol-condensed with a series of p-
hydroxybenzaldehyde derivatives under the catalysis of sulfuric acid to obtain intermediates 3a-3f
and 5a. Finally, these phenolic hydroxyl intermediates and bromo alkyl methyl ester combined
under heating and basic by Williamson ether synthesis, and then hydrolysis at room temperature
using lithium hydroxide monohydrate as a catalyst to provide target compounds 1-10.
2.2 SAR study
GFT505, a typical PPARs agonist, consists of three parts: a carboxylic acid head, a lipophilic tail,
and a linker. In this study, we tried to modify the three parts of GFT505 to clarify the SAR, and to
obtain more promising dual PPARα/δ agonist (Figure 2). In vitro activities data for all compounds
are listed in Table 1. First, GFT505 was replaced methylthio with methoxy and introducing fluoro
at R6-position led to compound 1, whose potency is 1.5 fold higher to PPARγ and approximately
half lower to PPARα/δ than GFT505. Modifying the lipophilic tail to 2,3-dihydrobenzofuran
(compound 4) gave the same trend as compound 1 on potency to PPARs. The potency of compound
4 is further increased on PPARγ and reduced on PPARα/δ. Substituting the methoxy of compound
1 by deuteration and deuterated methylthio provided compounds 2 and 3, are reduced potency on
PPARγ compared to compound 1. Among compounds 1-4, compound 3 has the best agonistic effect
on PPARα/δ, while the agonistic activity on PPARα/δ is still lower than GFT505. These
modifications at the lipophilic tail are not conducive to enhancing the agonistic activity of PPARα/δ
and have little effect on the agonistic activity of PPARγ.
Next, we try to modify the benzene ring at the right hand side. Interestingly, mono-substitution
of the right hand benzene ring with halogen provided compound 5 (fluorine) and 6 (chlorine), which
almost completely loss the agonistic effect on PPARγ while maintaining great potency on PPARα/δ.
Among them, the potency of chlorine substitution (compound 6) is better than that of fluorine
substitution (compound 5). Based on compound 6, we continue to modify the substituents at the
alpha position of the carboxylic acid. The results show that the more methyl substituents at the alpha
position of the carboxylic acid of these compounds (compound 6 vs 8 and 9), the better potency on

PPARα/δ. Similar to the above findings, it is also shown that two methyl groups at the alpha position
of the carboxylic acid are essential for the potency of PPARs (compound 5 vs 7, GFT505 vs
compound 10). Therefore, we conducted further research on the optimal compound 6 based on its
considerable potencies on PPARα/δ and better selectivity against PPARγ (PPARα EC50 = 0.468
μM, PPARδ EC50 = 0.362 μM and PPARγ > 10 μM).
2.3 Molecular docking study
As shown in Figure 3, the binding pocket of PPARα and PPARδ are a Y-shaped cavity with three
arms, and compound 6 can bind well to these two binding pockets with a similar binding pattern.
Compound 6 occupies arm-I and arm-II regions in these both of Y-shaped cavities. In the arm-I
region, carboxylic acid head of compound 6 forms hydrogen bonds with TYR-314, HIS-440 and
TYR-464 at PPARα, and HIS-323, HIS-449 and TYR-473 at PPARδ. Two methyl substituents at
the alpha position of the carboxylic acid are located on the hydrophobic region and form
indispensable hydrophobic interaction with hydrophobic residue. The hydrophobic interaction may
be crucial for the agonistic potency of PPARα and PPARδ [33-35]. Because, in the SAR study,
when there are no two methyl groups, the potency on PPARα/δ is reduced by 6-fold compared to
the presence of two methyl groups. Moreover, the chlorine substituent points to arm-III region but
is not inserted, and can form a hydrophobic interaction with adjacent hydrophobic residues in the
binding pocket of PPARα and PPARδ. Under the same conditions, the hydrophobicity of the
chlorine substituent is slightly stronger than fluorine substituent, which explains that the potency of
compound 6 on PPARα/δ is slightly better than compound 5 in vitro activities data. However, there
is a difference. The conformation of compound 6 in the binding pocket of PPARδ is U-shaped, but
in the binding pocket of PPARα, compound 6 is twisted into S-shaped due to hydrogen bonding
with THR-283.
2.4 Oral glucose tolerance test
The hypoglycemic effect of compound 6 was evaluated by oral glucose tolerance test in vivo in
ob/ob mice. During the oral glucose tolerance test, compound 6 revealed dose-dependent
hypoglycemic effects after five days treatment (Figure 4). Moreover, the hypoglycemic effect of
compound 6 is equivalently to that of GFT505 (10mg/kg), and is slightly better in compound 6

group at the dose of 20mg/kg. These results indicated that compound 6 might be a potential
candidate as effective as GFT505 in vivo, despite lower potencies of compound 6 on PPARs
compared to GFT505 in vitro.
3. Conclusion
A novel dual PPARα/δ agonist was discovered through the SAR study of GFT505. Compound 6 is
a highly potent PPARα/δ agonist (PPARα EC50: 0.468 μM, PPARδ EC50: 0.362 μM), and its
selectivity against PPARγ (PPARγ EC50 > 10 μM) is better than that of GFT505. Moreover,
molecular docking study shows that compound 6 binds well to the binding pockets of PPARα and
PPARδ and can form multiple hydrogen bonds with key residues related to the activation of PPARα
and PPARδ. During oral glucose tolerance test, compound 6 exerts dose-dependent anti-diabetic
effects in ob/ob mice and reveals similar potency to that of GFT505. In summary, we described the
SAR study based on the modification of GFT505, and compound 6 was identified as a promising
candidate for further researches.
4. Experimental section
4.1 General Chemistry
All reagents solvents were purchased from commercial sources and used as received without further
purification. Normal phase column chromatography was carried out on silica gel (200-300 mesh),
and TLC analysis was carried out on GF254 plates. NMR spectra were recorded in DMSO-d6 on a
Bruker ACF-300Q, and chemical shifts are calibrated internal tetramethylsilane standard. Mass
spectrometry was performed on liquid chromatography-tandem mass spectrometry (Waters)
equipped with electrospray ionization (ESI) probe. Melting points were measured by RY-1 melting-
point apparatus.
General synthetic procedure for intermediates 2a-d
To a solution of 1a-c (1 equiv) in acetonitrile was added K2CO3 (3 equiv), and
trideuterio(iodo)methane (3 equiv). The mixture was stirred at room temperature for 12h. The
mixture was filtered and the filtrates were concentrated to 2a-d.

General synthetic procedure for intermediates 3a-c and 5a
2a-c (or 4a) (1 equiv) and 4-hydroxy-3,5-dimethylbenzaldehyde (or 4-hydroxybenzaldehyde) (1
equiv) were dissolved in MeOH, and H2SO4 (0.05 equiv) was added dropwise to the mixture. The
mixture was stirred at room temperature for 16h. The solution was concentrated to give the crude
product. Water (20 mL) was added to the crude product and extracted with ethyl acetate (3×10mL).
After the solvent was concentrated under reduced pressure, it was purified by column
chromatography to afford to 3a-c (or 5a).
General synthetic procedure for intermediates 3d-f
2d (1 equiv) and 3-Fluoro-4-hydroxybenzaldehyde (3-Chloro-4-hydroxybenzaldehyde or 4-
Hydroxy-3-methylbenzaldehyde) (1 equiv) were dissolved in MeOH, and H2SO4 （0.05 equiv）
was added dropwise to the mixture. The mixture was stirred at room temperature for 16h. The
solution was concentrated to give the crude product. Water (20 mL) was added to the crude product
and extracted with ethyl acetate (3×10 mL). After the solvent was concentrated under reduced
pressure, it was purified by column chromatography to afford to 3d (3e or 3f).
(E)-3-(3-chloro-4-hydroxyphenyl)-1-(4-(methylthio)phenyl)prop-2-en-1-one (3e)
1H NMR (300 MHz, DMSO-d6) δ: 10.84 (s, 1H), 8.11 (d, J = 8.3 Hz, 2H), 8.04 (s, 1H), 7.95 – 7.75
(m, 1H), 7.72 – 7.55 (m, 2H), 7.39 (d, J = 8.3 Hz, 2H), 7.02 (d, J = 8.3 Hz, 1H), 2.56 (s, 3H).
General synthetic procedure for targets 1-6
To a solution of 3a-e (or 5a) in acetonitrile was added K2CO3 (3 equiv), and methyl 2-bromo-2-
methylpropanoate (1.1 equiv). The mixture was stirred at room temperature for 12h. The mixture
was filtered and the filtrate was concentrated to afford solid. The residue was purified by silica gel
column chromatography to afford a white solid, which was dissolved in the mixed solvent of
THF/MeOH/H2O (18 mL, 2:3:1), and added lithium hydroxide (1.5 equiv). After stirring at room
temperature for 4 h, the volatiles were removed under reduced pressure. The residue was acidified
with 1N hydrochloric acid solution and then filtered and the filter cake was washed with cold water
(5 mL), dried in vacuum to afford a white powder. Recrystallization from 75% ethanol provides
pure compound 1-6.

(E)-2-(4-(3-(3-fluoro-4-methoxyphenyl)-3-oxoprop-1-en-1-yl)-2,6-dimethylphenoxy)-2-
methylpropanoic acid (1)
Yield 45%; m.p. 123-125 °C; 1H NMR (300 MHz, DMSO-d6) δ: 13.00 (s, 1H), 8.23 – 8.00 (m, 2H),
7.83 (d, J = 15.5 Hz, 1H), 7.69 – 7.53 (m, 3H), 7.31 (t, J = 8.4 Hz, 1H), 3.95 (s, 3H), 2.22 (s, 6H),
1.38 (s, 6H). 13C NMR (75 MHz, DMSO-d6) δ: 188.12 (C15), 176.35 (C1), 155.25 (C5), 153.09
(C19), 152.63 (C18), 144.68 (C13), 131.85 (C16), 130.58 (C8), 130.43 (C21), 129.38 (C20), 128.65
(C7, C9), 126.34 (C6, C10), 121.49 (C14), 115.73 (C17), 81.53 (C2), 55.87 (C22), 25.68 (C3, C4),
18.12 (C11, C12). (The carbon numbers of the compound use the default atom numbers in
Chemdraw.) ESI-MS m/z: 385.1 [M-H]-. Anal. calcd. For C22H23FO5: C, 68.38; H, 6.00; Found: C,
68.23; H, 6.15.
(E)-2-(4-(3-(3-fluoro-4-(methoxy-d3)phenyl)-3-oxoprop-1-en-1-yl)-2,6-dimethylphenoxy)-2-
methylpropanoic acid (2)
Yield 38%; m.p. 125-127 °C; 1H NMR (300 MHz, DMSO-d6) δ: 12.93 (s, 1H), 8.16 – 7.94 (m, 2H),
7.84 (d, J = 15.0 Hz, 1H), 7.68 – 7.51 (m, 3H), 7.41 – 7.21 (m, 1H), 2.23 (s, 6H), 1.39 (s, 6H). 13C
NMR (75 MHz, DMSO-d6) δ: 187.79 (C15), 176.21 (C1), 155.43 (C5), 153.16 (C19), 152.67 (C18),
144.65 (C13), 131.86 (C16), 130.76 (C8), 130.52 (C21), 129.43 (C20), 128.76 (C7, C9), 126.38
(C6, C10), 121.46 (C14), 114.86 (C17), 81.53 (C2), 25.65 (C3, C4), 18.07 (C11, C12). ESI-MS m/z:
388.1 [M-H]-. Anal. calcd. For C22H20D3FO5: C, 67.85; H, 6.73; Found: C, 67.68; H, 6.79.
(E)-2-(4-(3-(3-fluoro-4-((methyl-d3)thio)phenyl)-3-oxoprop-1-en-1-yl)-2,6-dimethylphenoxy)-
2-methylpropanoic acid (3)
Yield 42%; m.p. 128-130 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.03 (dd, J = 8.2, 1.8 Hz, 1H), 7.97
(dd, J = 11.3, 1.8 Hz, 1H), 7.82 (d, J = 15.5 Hz, 1H), 7.66 (d, J = 15.5 Hz, 1H), 7.55 (s, 2H), 7.53 –
7.45 (m, 1H), 2.27 (s, 6H), 1.34 (s, 6H). 13C NMR (75 MHz, DMSO-d6) δ: 188.13 (C15), 179.25
(C1), 160.58 (C18), 153.25 (C5), 145.18 (C13), 135.29 (C16), 132.65 (C19), 128.19 (C20), 126.33
(C8), 124.58 (C7, C9, C21), 124.43 (C6, C10), 121.37 (C14), 114.92 (C17), 81.55 (C2), 25.63 (C3,
C4), 18.05 (C11, C12). ESI-MS m/z: 404.1 [M-H]-. Anal. calcd. For C22H20D3FO4S: C, 65.16; H,
6.46; Found: C, 65.03; H, 6.32.

(E)-2-(4-(3-(2,3-dihydrobenzofuran-5-yl)-3-oxoprop-1-en-1-yl)-2,6-dimethylphenoxy)-2-
methylpropanoic acid (4)
Yield 49%; m.p. 148-150 °C; 1H NMR (300 MHz, DMSO-d6) δ: 8.14 – 7.94 (m, 2H), 7.81 (d, J =
15.5 Hz, 1H), 7.63 – 7.43 (m, 3H), 6.90 (d, J = 8.4 Hz, 1H), 4.66 (t, J = 8.8 Hz, 2H), 3.27 (t, J = 8.8
Hz, 2H), 2.23 (s, 6H), 1.38 (s, 6H). 13C NMR (75 MHz, DMSO-d6) δ: 187.51 (C15), 175.80 (C1),
164.42 (C19), 155.51 (C5), 143.12 (C13), 133.62 (C21), 131.36 (C20), 130.80 (C18), 130.61 (C22),
129.60 (C8), 128.76 (C7, C9), 126.45 (C6, C10), 121.42 (C14), 109.32 (C23), 81.51 (C2), 72.62
(C16), 28.89 (C17), 25.67 (C3, C4), 18.05 (C11, C12). ESI-MS m/z: 379.1 [M-H]-. Anal. calcd. For
C23H24O5: C, 72.61; H, 6.36; Found: C, 72.52; H, 6.28.
(E)-2-(2-fluoro-4-(3-(4-(methylthio)phenyl)-3-oxoprop-1-en-1-yl)phenoxy)-2-
methylpropanoic acid (5)
Yield 44%; m.p. 118-120 °C; 1H NMR (300 MHz, DMSO-d6) δ: 8.11 (d, J = 8.5 Hz, 2H), 7.99 –
7.77 (m, 3H), 7.71 – 7.65 (m, 1H), 7.41 – 7.33 (m, 2H), 6.99 (t, J = 8.6 Hz, 1H), 2.56 (s, 3H), 1.55
(s, 6H). 13C NMR (75 MHz, DMSO-d6) δ: 197.37 (C13), 188.16 (C1), 174.79 (C6), 146.01 (C5),
145.87 (C11, C17), 134.22 (C14), 133.51 (C8), 129.52 (C15, C19), 129.14 (C16, C18), 125.38
(C10), 125.28 (C9, C12), 121.62 (C7), 81.07 (C2), 25.55 (C3, C4), 14.38 (C20). ESI-MS m/z: 373.1
[M-H]-. Anal. calcd. For C20H19FO4S: C, 64.16; H, 5.12; Found: C, 64.35; H, 5.26.
(E)-2-(2-chloro-4-(3-(4-(methylthio)phenyl)-3-oxoprop-1-en-1-yl)phenoxy)-2-
methylpropanoic acid (6)
Yield 47%; m.p. 132-134 °C; 1H NMR (300 MHz, DMSO-d6) δ: 8.18 – 8.09 (m, 3H), 7.96 – 7.85
(m, 1H), 7.78 – 7.61 (m, 2H), 7.39 (d, J = 8.6 Hz, 2H), 6.93 (d, J = 8.6 Hz, 1H), 2.56 (s, 3H), 1.58
(s, 6H). 13C NMR (75 MHz, DMSO-d6) δ: 188.15 (C13), 174.79 (C1), 153.35 (C5), 146.02 (C11,
C17), 142.48 (C14), 134.21 (C15, C19), 130.40 (C8), 129.55 (C9), 129.49 (C16, C18), 125.36 (C7),
124.69 (C6), 121.66 (C12), 118.01 (C10), 80.83 (C2), 25.55 (C3, C4), 14.39 (C20). ESI-MS m/z:
389.1 [M-H]-. Anal. calcd. For C20H19ClO4S: C, 61.46; H, 4.90; Found: C, 61.58; H, 4.76.
General synthetic procedure for targets 7 and 8

To a solution of 3d (or 3e) in acetonitrile was added K2CO3 (3 equiv), and methyl bromoacetate (1.1
equiv). The mixture was stirred at room temperature for 12h. The mixture was filtered and the filtrate
was concentrated to afford solid. The residue was purified by silica gel column chromatography to
afford a white solid, which was dissolved in the mixed solvent of THF/MeOH/H2O (18 mL, 2:3:1),
and added lithium hydroxide (1.5 equiv). After stirring at room temperature for 4 h, the volatiles
were removed under reduced pressure. The residue was acidified with 1N hydrochloric acid solution
and then filtered and the filter cake was washed with cold water (5 mL), dried in vacuum to afford
a white powder. Recrystallization from 75% ethanol provides pure compound 7 (or 8).
(E)-2-(2-fluoro-4-(3-(4-(methylthio)phenyl)-3-oxoprop-1-en-1-yl)phenoxy)acetic acid (7)
Yield 46%; m.p. 206-208 °C; 1H NMR (300 MHz, DMSO-d6) δ: 13.23 (s, 1H), 8.11 (d, J = 8.5 Hz,
2H), 8.00 – 7.82 (m, 2H), 7.72 – 7.54 (m, 2H), 7.40 (d, J = 8.5 Hz, 2H), 7.15 (t, J = 8.7 Hz, 1H),
4.88 (s, 2H), 2.56 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ: 188.16 (C11), 170.03 (C1), 151.67
(C4), 146.00 (C9, C15), 142.93 (C3), 134.24 (C12), 129.53 (C6), 129.13 (C13, C17), 127.20 (C14,
C16), 125.38 (C7, C8), 121.51 (C10), 115.13 (C5), 65.45 (C2), 14.39 (C18). ESI-MS m/z: 345.1
[M-H]-. Anal. calcd. For C18H15FO4S: C, 62.42; H, 4.37; Found: C, 62.25; H, 4.23.
(E)-2-(2-chloro-4-(3-(4-(methylthio)phenyl)-3-oxoprop-1-en-1-yl)phenoxy)acetic acid (8)
Yield 41%; m.p. 203-205 °C; 1H NMR (300 MHz, DMSO-d6) δ: 8.15 (d, J = 8.6 Hz, 2H), 7.99 –
7.84 (m, 2H), 7.76 – 7.58 (m, 1H), 7.48 – 7.33 (m, 3H), 7.11 (d, J = 8.7 Hz, 1H), 4.91 (s, 2H), 2.57
(s, 3H). 13C NMR (75 MHz, DMSO-d6) δ: 189.07 (C11), 170.06 (C1), 153.58 (C3), 145.35 (C15),
142.85 (C9), 134.36 (C12), 128.95 (C6, C13, C17), 128.33 (C7), 127.45 (C14, C16), 127.34 (C5),
122.51 (C4), 121.32 (C10), 112.15 (C8), 65.47 (C2), 14.52 (C18). ESI-MS m/z: 361.1 [M-H]-. Anal.
calcd. For C18H15ClO4S: C, 59.59; H, 4.17; Found: C, 59.43; H, 4.35.
General synthetic procedure for targets 9 and 10
To a solution of 3e (or 3f) in acetonitrile was added K2CO3 (3 equiv), and methyl methyl 2-
bromopropionate (1.1 equiv). The mixture was stirred at room temperature for 12h. The mixture
was filtered and the filtrate was concentrated to afford solid. The residue was purified by silica gel
column chromatography to afford a white solid, which was dissolved in the mixed solvent of

THF/MeOH/H2O (18 mL, 2:3:1), and added lithium hydroxide (1.5 equiv). After stirring at room
temperature for 4 h, the volatiles were removed under reduced pressure. The residue was acidified
with 1N hydrochloric acid solution, and then filtered and the filter cake was washed with cold water
(5 mL), dried in vacuum to afford a white powder. Recrystallization from 75% ethanol provides
pure compound 9 (or 10).
(E)-2-(2-chloro-4-(3-(4-(methylthio)phenyl)-3-oxoprop-1-en-1-yl)phenoxy)propanoic acid (9)
Yield 35%; m.p. 150-152 °C; 1H NMR (300 MHz, DMSO-d6) δ: 8.11 (d, J = 8.5 Hz, 2H), 7.96 –
7.80 (m, 2H), 7.76 – 7.57 (m, 2H), 7.39 (d, J = 8.5 Hz, 2H), 7.00 (d, J = 8.8 Hz, 1H), 4.99 (q, J =
6.7 Hz, 1H), 2.56 (s, 3H), 1.55 (d, J = 6.7 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ: 188.14 (C12),
173.00 (C1), 155.30 (C4), 145.95 (C16), 142.63 (C10), 134.25 (C13), 130.29 (C14, C18), 130.10
(C7), 129.54 (C8), 128.99 (C15, C17), 125.35 (C6), 122.54 (C5), 121.28 (C11), 114.47 (C9), 73.52
(C2), 18.72 (C3), 14.40 (C19). ESI-MS m/z: 375.2 [M-H]-. Anal. calcd. For C19H17ClO4S: C, 60.56;
H, 4.55; Found: C, 60.34; H, 4.41.
(E)-2-(2,6-dimethyl-4-(3-(4-(methylthio)phenyl)-3-oxoprop-1-en-1-yl)phenoxy)propanoic
acid (10)
Yield 40%; m.p. 132-134 °C; 1H NMR (300 MHz, DMSO-d6) δ: 8.09 (d, J = 8.6 Hz, 2H), 7.87 –
7.73 (m, 1H), 7.66 – 7.50 (m, 3H), 7.39 (d, J = 8.6 Hz, 2H), 4.49 (q, J = 6.7 Hz, 1H), 2.55 (s, 3H),
2.28 (s, 6H), 1.42 (d, J = 6.7 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ: 188.24 (C14), 173.63 (C1),
157.39 (C4), 145.85 (C18), 143.97 (C12), 134.35 (C15), 131.50 (C16, C20), 130.30 (C17, C19),
130.16 (C7), 129.46 (C6, C8), 125.37 (C5, C9), 120.96 (C13), 77.45 (C2), 18.99 (C3), 17.25 (C10,
C11), 14.40 (C21). ESI-MS m/z: 369.1 [M-H]-. Anal. calcd. For C21H22O4S: C, 68.09; H, 5.99;
Found: C, 68.23; H, 5.85.
4.2 Molecular docking
Molecular docking simulations of compounds 6 were performed using AutoDock vina1.1.2[36].
The crystal structures of PPARα (PDB code: 3VI8) and PPARδ (PDB code: 1GWX) were obtained
from Protein Data Bank. Before the docking study, we conducted a re-dock study to verify the
accuracy of the docking method. In the re-dock study of PPARα and PPARδ, the RMSDs (root-

mean-square-deviations) calculated by VMD 1.9.3 is 0.6718 and 1.601, respectively. These results
show that our docking method and parameter settings are reliable in molecular docking simulations.
After that, we have docked compound 6 with PPARα and PPARδ, and the docking pictures were
drawn by Pymol 2.3.1.
4.3 Evaluation for PPARα, PPARγ and PPARδ
Detailed descriptions on transfection and cell-based evaluation for PPARα, PPARγ and PPARδ
were given in our previously reported literature [22]. Briefly, HepG2 or HEK293 cells were
transfected with pBIND-PPARα, PPARδ or PPARγ according to the manufacturer’s protocol. After
transfection, tested compounds were added and incubated for 18 h, then lysed with lysis buffer, and
added Luciferase Assay Reagent II. The luciferase signals of firefly and renilla were measured using
Dual Luciferase Reporter Assay System (Promega). EC50 values were obtained from GraphPad 5.00
(San Diego, USA).
4.4 Animals and statistical analysis of the data
Six weeks old male ob/ob mice were purchased from Model Animal 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 using a one-way ANOVA with Tukey’s multiple-comparison post
hoc test.
4.5 Oral Glucose Tolerance Test in male ob/ob mice

The male ob/ob mice were dosed once daily with the vehicle, GFT505 (10 mg/kg) or compound 6
(3, 10, and 20 mg/kg) by gavage administration for 5 days. Mice were dosed at fixed time daily, and
OGTT was performed on day 6. Mice were fasted overnight prior to treatment with a single doses
of vehicle, GFT505 or compound 6 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 glucose challenge (0 min), and at 30, 60 and 120 min post-dose and the blood glucose was
measured by blood glucose test strips (SanNuo ChangSha, China).
Conflicts of interest
The authors declare no conflict of interest. The authors are solely responsible for the content and
results presented in this paper.
Acknowledgments
This study was supported by the Guangdong Basic and Applied Basic Research Foundation (Grant
2019A1515011036), the National Natural Science Foundation of China (Grant 81803341), the
Natural Science Foundation of Guangdong Province, China (Grant 2018A030313445), the
Innovative strong school project of Guangdong Pharmaceutical University (Grant 2018KTSCX111),
the projects of Guangzhou key laboratory of construction and application of new drug screening
model systems (No. 201805010006) and Key Laboratory of New Drug Discovery and Evaluation
of ordinary universities of Guangdong province (No. 2017KSYS002), the Innovation Team Projects
in Universities of Guangdong Province (No. 2018KCXTD016).

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Table 1: In vitro activities of the human PPARs a
O
R3
O
R5
R6
O
OH
R
R4
1 R2
O
O
COOH
4
O
Compd.
R1
R2 R3 R4
R5
R6
hPPARα
EC50
hPPARγ
EC50
(μM)
ND
hPPARδ
EC50 (μM)
(μM)
0.013
ND
GW7647
ND
Rosiglitazone
0.115
ND
ND
GW0742
ND
0.021
0.253
0.557
0.528
0.396
0.739
0.516
0.362
3.195
2.509
0.381
0.437
GFT505
Me Me Me Me MeS
Me Me Me Me MeO
Me Me Me Me D3CO
Me Me Me Me D3CS
H
F
F
F
0.355
0.627
0.735
0.503
1.257
0.871
0.468
6.285
3.274
0.583
0.516
3.622
1.374
2.183
4.715
0.926
> 10
1
2
3
4
5
Me Me
F
H
H
H
H
H
MeS
MeS
MeS
MeS
MeS
H
H
H
H
H
H
6
Me Me Cl
> 10
7
H
H
H
H
H
H
F
> 10
8
Cl
Cl
> 10
9
Me
Me
> 10
10
Me Me MeS
5.625
a EC50 value represent the mean of three determinations, which is the concentration giving 50% of
the maximal activity determined for the tested compound.

Figure 1：Structure of PPARs agonists.

Figure 2: The design strategy for the modification of GFT505.

Figure 3: Predicted binding modes of compound 6 in the PPARα/δ. Compound 6 (yellow) in the
PPARα (A, C) and PPARδ (B, D). The protein backbone is shown in cartoon, residues and
compound 6 are in stick. Hydrogen bonds are shown as green dashes.

Figure 4: Effects of compound 6 on plasma glucose levels and corresponding AUC0−120min of
glucose during an OGTT in fasting ob/ob mice after five days treatment. GFT505, compound 6, or
vehicle was orally administered to ob/ob mice once daily for 5 days, and the OGTT was determined
on treatment day 6. Values 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.

Scheme 1: Synthesis of target compounds 1-10. Reagent and condition: (a) K2CO3, KI, acetonitrile,
r.t., 12h; (b) H2SO4, MeOH, r.t., 16h; (c) Methyl 2-bromo-2-methylpropanoate (methyl 2-
bromopropionate, or methyl bromoacetate), K2CO3, KI, acetonitrile, 50 ℃, 16h; (d) LiOH·H2O,
THF/MeOH/H2O, r.t., 4h.

Highlights
1. Compound 6 shows higher selectivity to PPARα/δ and lower agonistic activity on PPARγ.
2. Compound 6 exerts dose-dependent anti-diabetic effects in ob/ob mice.
3. Compound 6 reveals similar potency to GFT505, the most advanced candidate in this field.
4. The obtained SAR in this study might help us to explore better PPARα/δ agonist.

Graphical Abstract
Carboxylic acid head
R3
Lipophilic tail
O
R5
R6
O
COOH
S
O
OH
SAR study
R1 R2
R4
O
O
6
Examplified by compound
Right hand benzene
GFT505
PPAR EC50 = 0.468M
PPAR EC50 = 0.362M
PPAR EC50 > 10M
PPAR EC50 = 0.355M
PPAR EC50 = 0.253M
PPAR EC50 = 3.622M
Excellent hypoglycemic effect in ob/ob mice
From the SAR study based on GFT505, a dual PPARα/δ agonist compound 6 was identified, which
has lower agonistic activity on PPARγ and exerted excellent hypoglycemic effect in ob/ob mice.

Conflict of interest
The authors declare no competing financial interest.