Discovery of novel dual PPARα/δ agonists based on benzimidazole scaffold for the treatment of non-alcoholic fatty liver disease

Article abstract of DOI:10.1016/j.bioorg.2020.103803

Many peroxisome proliferator-activated receptors (PPARs) agonists have been developed for the treatment of metabolic disorders, while several PPARs agonists were discontinued in clinical trials because of PPARγ related side effects. In order to increase the selectivity against PPARγ, we performed a structure-activity relationship study based on PPARα/γ/δ agonist MHY2013. These efforts eventually led to the identification of compound 4, a dual PPARα/δ agonist with considerable potencies on PPARα/δ and high selectivity against PPARγ. In the Western Diet and CCl₄-induced non-alcoholic steatohepatitis model, compound 4 alleviates the hepatic steatosis, inflammation, and fibrosis. These results indicated that dual PPARα/δ agonist 4 might be a promising lead compound for further investigations.

Full text of DOI:10.1016/j.bioorg.2020.103803

BioorganicChemistry99(2020)103803
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Discovery of novel dual PPARα/δ agonists based on benzimidazole scaffold
for the treatment of non-alcoholic fatty liver disease
⁎
Zheng Lia,b, , Yawen Xua, Zongyu Caia, Xuekun Wangc, Qiang Rena, Zongtao Zhoua,
⁎
Rongrong Xiea,
a School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China
b Key Laboratory of New Drug Discovery and Evaluation of Ordinary Universities of Guangdong Province, Guangdong Pharmaceutical University, Guangzhou 510006, PR
China
c College of Pharmacy, Liaocheng University, Liaocheng 252059, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Dual agonist
Fatty liver
Fibrosis
Many peroxisome proliferator-activated receptors (PPARs) agonists have been developed for the treatment of
metabolic disorders, while several PPARs agonists were discontinued in clinical trials because of PPARγ related
side effects. In order to increase the selectivity against PPARγ, we performed a structure-activity relationship
study based on PPARα/γ/δ agonist MHY2013. These efforts eventually led to the identification of compound 4, a
dual PPARα/δ agonist with considerable potencies on PPARα/δ and high selectivity against PPARγ. In the
Western Diet and CCl4-induced non-alcoholic steatohepatitis model, compound 4 alleviates the hepatic steatosis,
inflammation, and fibrosis. These results indicated that dual PPARα/δ agonist 4 might be a promising lead
compound for further investigations.
NASH
PPAR
1. Introduction
clinical trials because of PPARγ related side effects such as weight gain,
fluid retention, and cardiovascular risk [25,26]. As a potent PPARα/γ/δ
Non-alcoholic fatty liver disease (NAFLD) is one of the most pre-
valent liver diseases, which includes different course of the disease,
from hepatic steatosis to non-alcoholic steatohepatitis (NASH) followed
by fibrosis [1,2]. Although various factors including obesity, insulin
resistance and inflammation have been attributed to the development
of NAFLD, the exact pathogenesis is still unclear [3,4]. To date, there
are no approved drugs for NAFLD [5,6].
agonist, MHY2013 reduces insulin resistance, plasma lipid level and
hepatic steatosis [27]. In order to decrease the risk of PPARγ related
side effects, a lot of research has been done to obtain dual PPARα/δ
agonist. Herein, we describe the structure-activity relationship study
based on MHY2013 to further increase selectivity against PPARγ
(Fig. 2).
The peroxisome proliferator-activated receptors (PPARs), including
PPARα/δ/γ, have an important role in energy metabolism [7–9].
PPARα is primarily expressed in the liver and modulates plasma lipid
levels, fatty acid β-oxidation and transport [10–12]. PPARδ is primarily
expressed in muscle tissue, which modulates fatty acid transport and β-
oxidation [13,14]. Moreover, the activation of PPARδ improves plasma
lipid levels and insulin sensitivity, and provides anti-inflammatory ef-
fects [15–17]. PPARγ is primarily expressed in adipocytes, which in-
creases the storage of fatty acids and improves insulin resistance [18].
At present, many PPARs agonists (Fig. 1) have been developed for the
treatment of dyslipidemia, NAFLD and diabetes [19–21]. In our pre-
vious study, we have also discovered several PPARδ agonists with a
therapeutic effect on glucose and lipid metabolism [22–24]. Although
the field is promising, several PPARs agonists were discontinued in
2. Results and discussion
2.1. Chemistry
The designed compounds 1–12 were synthesized as shown in
Scheme 1. Treatment of commercially available B with phenol A using
Williamson ether synthesis provided intermediate C, which was con-
verted into benzimidazole with good yields [28], followed by hydro-
lysis provided the target compounds 1–12.
2.2. Structure-activity relationship study
In vitro activities of target compounds on PPARα/γ/δ were eval-
uated by using cell-based assays. As shown in Table 1, replacing
⁎ Corresponding authors at: School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China (Z. Li).
E-mail addresses: li.zheng.sky@163.com (Z. Li), happyyunyun_1985@126.com (R. Xie).
https://doi.org/10.1016/j.bioorg.2020.103803
Received 4 February 2020; Received in revised form 16 March 2020; Accepted 26 March 2020
Availableonline31March2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

Z. Li, et al.
BioorganicChemistry99(2020)103803
Fig. 1. Structure of representative PPARα, PPARγ and PPARδ agonists.
Fig. 2. Our design strategy and structure-activity relationship study to obtain dual PPARα/δ agonist from PPARα/γ/δ agonist MHY2013.
Scheme 1. Synthesis of target compounds 1–11. Reagents and conditions: (a) K2CO3, acetonitrile, 45 °C, 12 h; (b) H2O (10 vol% in DMF), 80 °C, open flask, and then
LiOH·H2O, THF/MeOH/H2O, r.t., 4 h.
reported in PPAR modulators [29–31]. Exploration of other hydro-
phobic substituents (CF3 and Cl) at the benzimidazole scaffold provided
compounds 2 and 3, which have less effect on the selectivity against
PPARγ. Notably, replacing methyl at right benzene with chlorine af-
forded compound 4, which revealed better activities on PPARα/δ and
higher selectivity against PPARγ compared to parent compound 1.
Further replacement with fluorine (compound 5) significantly de-
creased potencies on PPARα/γ/δ, indicating that hydrophobic inter-
action in this site might be crucial to agonistic activity. Next, we ex-
plored the importance of carboxylic acid moiety. Removing a methyl
group in acid moiety reduced activity on PPARα/γ/δ (compounds 6 and
7), but enhanced selectivity against PPARγ (compound 7 vs 2). The
structure-activity relationship of phenoxy acetic acid series (compounds
8–12) was also explored, while both of them revealed lower potencies
on PPARα/γ/δ compared to their parent compounds (compound 8 vs 1,
9 vs 3, 10 vs 2, 11 vs 5, and 12 vs 4). Among all of these compounds, the
optimal compound 4 revealed the best agonistic activity on PPARα/δ
and high selectivity against PPARγ.
Table 1
In
vitro
activities
R1
of
target
compounds
on
PPARα/δ/
γ.
Compd.
R2
R3
R4
EC50 (nM)
PPARαa
PPARγ
PPARδ
MHY2013
217
185
163
1
Me
CF3
Cl
Me
Me
Me
Cl
Me
Me
Me
Me
Me
Me
Me
H
Me
Me
Me
Me
Me
H
349
736
278
2
385
684
296
3
481
1075
2052
2839
2571
2137
3124
4016
3363
5932
3769
451
4
Me
Me
H
307
214
5
F
891
1035
717
6
Me
Me
Me
Me
Me
F
953
7
CF3
Me
Cl
H
579
341
8
H
1035
1479
1437
3725
859
848
9
H
H
1327
1206
1507
684
10
11
12
CF3
Me
Me
H
H
H
H
Cl
H
H
a
2.3. Docking study
EC50 value represents the mean of three determinations.
benzothiazole of MHY2013 with benzimidazole provided compound 1
with decreased activities on PPARα/γ/δ, while increasing selectivity
against PPARγ. Indeed, the benzimidazole scaffold has been widely
To better understand the binding model of compound 4, we per-
formed the modeling studies based on the complex structures of PPARα
(PDB code: 3VI8) and PPARδ (PDB code: 1GWX). As shown in Fig. 3,
2

Z. Li, et al.
BioorganicChemistry99(2020)103803
Fig. 3. The 3-D binding diagram (A) and 2-D interaction map (B) of compound 4 (green carbon) in the crystal structure of PPARα (3VI8), and 3-D binding diagram
(C) and 2-D interaction map (D) of compound 4 (green carbon) in the crystal structure of PPARδ (1GWX). Key residues are labeled, and interactions are represented
by yellow dashed lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
compound 4 fitted very well with the binding pocket of PPARα and
PPARδ. The carboxylic acid of compound 4 generated hydrogen-
bonding interactions with His440, Tyr314, and Ser280 in the binding
pocket of PPARα (Fig. 3A and B). Moreover, the benzimidazole scaffold
of compound 4 is inserted into the hydrophobic pocket of PPARα. The
binding mode of compound 4 in PPARδ is slightly different from
PPARα, which exhibited a rotation angle between benzimidazole and
the right hand benzene of compound 4 (Fig. 3C). The carboxylic acid of
compound 4 formed three hydrogen-bonding with His449, Tyr473 and
His323. Moreover, hydrogen-bonding was also generated between the
oxygen atom of compound 4 and His449.
3. Conclusion
In order to decrease the risk of PPARγ related side effects, we per-
formed a structure-activity relationship study based on PPARα/γ/δ
agonist MHY2013. All of these efforts resulted in the discovery of dual
PPARα/δ agonist 4, which revealed the best agonistic activity on
PPARα/δ and high selectivity against PPARγ in our researches. Further
modeling studies illuminated that compound 4 fitted very well with the
binding pocket of PPARα and PPARδ, and formed multiple interactions
with key residues related to the agonistic activity. Moreover, the he-
patic steatosis, inflammation, and fibrosis were significantly alleviated
in the NASH model after treated with compound 4. In summary, we
described the discovery process of novel dual PPARα/δ agonists, and
compound 4 was identified as a promising lead compound for further
researches.
2.4. Effects on fatty liver
The effects of compound 4 on the fatty liver were evaluated in WD/
CCl4 mice, a Western Diet and CCl4-induced NASH model [32]. As
shown in Fig. 4, the hepatic steatosis and inflammation were sig-
nificantly increased WD/CCl4 mice, which were alleviated in MHY2013
and compound 4 groups. Plasma levels of AST and ALT, the markers of
hepatic injury, were raised in WD/CCl4 mice compared to normal mice.
Similar to the improvement of fatty liver, AST and ALT levels were
notably diminished by the treatment of compound 4 (Fig. 4). Moreover,
liver fibrosis induced by the Western Diet and CCl4 was also amelio-
rated in compound 4-treated group (Fig. 5). These results indicated that
compound 4 might be a promising dual PPARα/δ agonist for the
treatment of fatty liver.
4. Experimental section
4.1. General chemistry
All starting materials, reagents, and solvents were obtained from
commercial sources. Purifications of chromatography were performed
by silica gel and detected by thin layer chromatography using UV light
at 254 and 365 nm. Melting points were measured on RY-1 melting-
point apparatus. NMR spectra were recorded on a Bruker ACF-300Q
instrument (300 MHz for 1H NMR and 75 MHz for 13C NMR spectra),
chemical shifts are expressed as values (ppm) relative to
3

Z. Li, et al.
BioorganicChemistry99(2020)103803
Fig. 4. Effect of compound 4 on the fatty liver after 30 days of treatment in WD/CCl4 mice. Representative photomicrographs of histological alterations in liver
stained with Hematoxylin-Eosin at 400× magnification. Empty/hollow spaces show fat globules or accumulation (macro-vesicular steatosis). Steatosis: blue arrow,
inflammation: red arrow. Histograms show the plasma levels of Aspartate transaminase (AST) and Alanine transaminase (ALT). All the values are expressed as
mean
SD (n = 6). *p ≤ 0.05 compared to model control were analyzed using a one-way ANOVA with Tukey’s multiple-comparison post hoc test. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
tetramethylsilane as internal standard, and coupling constants (J va-
lues) were given in hertz (Hz). LC/MS spectra were recorded on a
Waters LC-MS system (ESI). Elemental analyses were performed by the
Heraeus CHN-O-Rapid analyzer and were within 0.4% of the theoretical
values. MHY2013 was synthesized by published procedures [27].
open flask, and the reaction progress was monitored by TLC. Then the
reaction mixture was poured into water (60 mL) and extracted with
ethyl acetate (3 × 25 mL). The combined organic phases were washed
with water (25 mL), dried over anhydrous sodium sulfate and filtered.
The filtrate was evaporated and the residue was purified by silica gel
column chromatography (petroleum ether/ethyl acetate, 10:1, v/v). To
a solution of the obtained solid (1 equiv) in 2:3:1 THF/MeOH/H2O
(18 mL) was added LiOH·H2O (3 equiv). After stirring at room tem-
perature for 4 h, the volatiles were removed under reduced pressure.
The residue was acidified with 1 N hydrochloric acid solution and then
filtered and the filter cake was washed with 5 mL of water, dried in
vacuum to afford a white powder. The white powder was purified by
column chromatography using a mixture of petroleum ether/ethyl
acetate (2:1–1:2, v/v) as eluent to afford the target compounds as solid.
4.1.1. General synthetic procedure for target compounds 1–12
To a stirred solution of A (1 equiv) in acetonitrile was added po-
tassium carbonate (3 equiv) and B (2 equiv). The mixture was stirred at
45 °C for 12 h, and filtered. The filtrate was concentrated and the re-
sidue was dissolved in ethyl acetate. The organic layers were washed
with brine (2 × 20 mL), dried over anhydrous sodium sulfate and fil-
tered. The filtrate was concentrated to give intermediate C, which was
used for the next reaction without further purification. The inter-
mediate C (1.0 equiv) and D (1.0 equiv) were dissolved in DMF (90 vol
% in H2O). The resulting reaction mixture was stirred at 80 °C in an
Fig. 5. Effect of compound 4 on liver fibrosis (Masson’s trichrome staining) of WD/CCl4 mice.
4

Z. Li, et al.
BioorganicChemistry99(2020)103803
4.1.2. 2-Methyl-2-(2-methyl-4-(6-methyl-1H-benzo[d]imidazol-2-yl)
phenoxy)propanoic acid (1)
4.1.8. 2-(2-Methyl-4-(6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)
phenoxy)propanoic acid (7)
Yield 32%; melting point, 247–249 °C; 1H NMR (300 MHz, D2O) δ:
10.53–10.29 (m, 2H), 9.87 (d, J = 8.4 Hz, 1H), 9.77 (s, 1H), 9.56–9.47
(m, 1H), 9.10 (d, J = 8.7 Hz, 1H), 5.38 (s, 1H), 4.70 (s, 3H), 4.49 (s,
3H), 3.85 (s, 6H). 13C NMR (75 MHz, DMSO‑d6) δ: 174.89, 158.54,
151.73, 135.12, 132.69, 131.45, 129.69, 128.05, 126.43, 125.65,
122.25, 115.73, 115.38, 114.69, 79.65, 25.63, 21.35, 16.76. ESI-MS m/
z: 323.1 [M−H]−. Anal. calcd. For C19H20N2O3: C, 70.35; H, 6.21; N,
8.64; Found: C, 70.46; H, 6.33; N, 8.72.
Yield 31%; melting point, 235 °C (carbonize); 1H NMR (300 MHz,
DMSO‑d6) δ 13.38 (s, 1H), 8.25–8.02 (m, 2H), 8.04 (d, J = 6.0 Hz, 1H),
7.98–7.93 (m, 1H), 7.77–7.72 (m, 1H), 7.09 (d, J = 8.5 Hz, 1H), 5.09
(q, J = 6.8 Hz, 1H), 2.31 (s, 3H), 1.59 (d, J = 6.8 Hz, 3H). 13C NMR
(75 MHz, DMSO‑d6) δ: 174.78, 158.57, 151.68, 135.76, 132.59, 131.62,
129.37, 128.12, 126.35, 126.18, 125.27, 122.29, 115.53, 115.47,
111.58, 65.69, 18.74, 16.65. ESI-MS m/z: 363.1 [M−H]−. Anal. calcd.
For C18H15F3N2O3: C, 59.34; H, 4.15; N, 7.69; Found: C, 59.15; H, 4.07;
N, 7.56.
4.1.3. 2-Methyl-2-(2-methyl-4-(6-(trifluoromethyl)-1H-benzo[d]imidazol-
2-yl)phenoxy)propanoic acid (2)
4.1.9. 2-(2-Methyl-4-(6-methyl-1H-benzo[d]imidazol-2-yl)phenoxy)acetic
acid (8)
Yield 37%; melting point, 230 °C (carbonize); 1H NMR (300 MHz,
D2O) δ: 10.67–10.44 (m, 2H), 10.26 (s, 1H), 10.17 (d, J = 8.5 Hz, 1H),
10.00 (d, J = 8.5 Hz, 1H), 9.09 (d, J = 8.7 Hz, 1H), 5.37 (s, 1H), 4.47
(s, 3H), 3.84 (s, 6H). 13C NMR (75 MHz, DMSO‑d6) δ: 174.87, 158.57,
151.79, 135.46, 132.90, 131.31, 129.53, 128.01, 126.47, 126.05,
125.62, 122.27, 115.79, 115.35, 111.67, 79.69, 25.60, 16.82. ESI-MS
m/z: 377.1 [M−H]−. Anal. calcd. For C19H17F3N2O3: C, 60.32; H, 4.53;
N, 7.40; Found: C, 60.15; H, 4.38; N, 7.52.
Yield 45%; melting point, 254–256 °C; 1H NMR (300 MHz,
DMSO‑d6) δ 8.28–8.16 (m, 2H), 8.05 (s, 1H), 7.95 (d, J = 8.5 Hz, 1H),
7.78 (dd, J = 8.7, 1.8 Hz, 1H), 7.19 (d, J = 8.5 Hz, 1H), 4.91 (s, 2H),
2.32 (s, 3H), 2.26 (s, 3H). 13C NMR (75 MHz, DMSO‑d6) δ: 174.62,
158.38, 151.51, 135.30, 132.21, 131.35, 129.43, 128.01, 126.55,
125.46, 122.12, 115.65, 115.34, 114.58, 65.75, 21.65, 16.76. ESI-MS
m/z: 295.1 [M−H]−. Anal. calcd. For C17H16N2O3: C, 68.91; H, 5.44;
N, 9.45; Found: C, 68.78; H, 5.35; N, 9.38.
4.1.4. 2-(4-(6-Chloro-1H-benzo[d]imidazol-2-yl)-2-methylphenoxy)-2-
methylpropanoic acid (3)
4.1.10. 2-(4-(6-Chloro-1H-benzo[d]imidazol-2-yl)-2-methylphenoxy)
acetic acid (9)
Yield 34%; melting point, 248 °C (carbonize); 1H NMR (300 MHz,
DMSO‑d6) δ: 8.26 (d, J = 1.6 Hz, 1H), 8.19 (dd, J = 8.7, 2.1 Hz, 1H),
7.80–7.70 (m, 2H), 7.46, 7.43 (dd, J = 8.7, 1.9 Hz, 1H), 6.86 (d,
J = 8.7 Hz, 1H), 2.26 (s, 3H), 1.62 (s, 6H). 13C NMR (75 MHz,
DMSO‑d6) δ: 174.98, 157.94, 150.87, 134.98, 132.87, 130.89, 129.43,
129.33, 127.39, 125.38, 117.00, 115.76, 115.60, 114.05, 79.57, 25.62,
16.87. ESI-MS m/z: 343.1 [M−H]−. Anal. calcd. For C18H17ClN2O3: C,
62.70; H, 4.97; N, 8.12; Found: C, 62.79; H, 4.89; N, 8.07.
Yield 42%; melting point, 276–278 °C; 1H NMR (300 MHz,
DMSO‑d6) δ: 8.27 (d, J = 1.8 Hz, 1H), 8.21 (dd, J = 8.6, 2.2 Hz, 1H),
7.82–7.74 (m, 2H), 7.45, 7.42 (dd, J = 8.8, 1.8 Hz, 1H), 6.88 (d,
J = 8.8 Hz, 1H), 4.60 (s, 2H), 2.25 (s, 3H). 13C NMR (75 MHz,
DMSO‑d6) δ: 174.87, 157.96, 150.85, 134.96, 132.85, 130.87, 129.46,
129.37, 127.35, 125.39, 117.03, 115.78, 115.62, 114.03, 65.77, 16.81.
ESI-MS m/z: 315.1 [M−H]−. Anal. calcd. For C16H13ClN2O3: C, 60.67;
H, 4.14; N, 8.84; Found: C, 60.48; H, 4.07; N, 8.68.
4.1.5. 2-(2-Chloro-4-(6-methyl-1H-benzo[d]imidazol-2-yl)phenoxy)-2-
methylpropanoic acid (4)
4.1.11. 2-(2-Methyl-4-(6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)
phenoxy)acetic acid (10)
Yield 27%; melting point, 250 °C (carbonize); 1H NMR (300 MHz,
DMSO‑d6) δ: 8.59 (d, J = 2.1 Hz, 1H), 8.35 (dd, J = 8.8, 2.1 Hz, 1H),
7.64 (d, J = 8.3 Hz, 1H), 7.54 (s, 1H), 7.28 (d, J = 8.4 Hz, 1H), 7.08 (d,
J = 8.8 Hz, 1H), 2.45 (s, 3H), 1.65 (s, 6H). 13C NMR (75 MHz,
DMSO‑d6) δ: 174.32, 154.83, 147.57, 135.63, 133.42, 131.43, 130.15,
128.18, 127.22, 124.78, 118.42, 117.95, 114.07, 113.80, 81.08, 25.50,
21.65. ESI-MS m/z: 343.1 [M−H]−. Anal. calcd. For C18H17ClN2O3: C,
62.70; H, 4.97; N, 8.12; Found: C, 62.56; H, 4.85; N, 8.23.
Yield 47%; melting point, 221–223 °C; 1H NMR (300 MHz,
DMSO‑d6) δ 12.57 (s, 1H), 7.99 (d, J = 2.3 Hz, 1H), 7.91 (dd, J = 8.5,
2.3 Hz, 1H), 7.39–7.33 (m, 2H), 7.05–7.00 (m, 2H), 4.91 (s, 2H), 2.30
(s, 3H). 13C NMR (75 MHz, DMSO‑d6) δ: 174.73, 158.55, 151.76,
135.43, 132.82, 131.35, 129.34, 128.07, 126.23, 126.12, 125.56,
122.38, 115.68, 115.32, 111.65, 65.64, 16.82. ESI-MS m/z: 349.1
[M−H]−. Anal. calcd. For C17H13F3N2O3: C, 58.29; H, 3.74; N, 8.00;
Found: C, 58.12; H, 3.63; N, 8.09.
4.1.6. 2-(2-Fluoro-4-(6-methyl-1H-benzo[d]imidazol-2-yl)phenoxy)-2-
methylpropanoic acid (5)
4.1.12. 2-(2-Fluoro-4-(6-methyl-1H-benzo[d]imidazol-2-yl)phenoxy)
acetic acid (11)
Yield 29%; melting point, 217–218 °C; 1H NMR (300 MHz, D2O) δ:
10.74–10.57 (m, 1H), 10.44 (d, J = 8.7 Hz, 1H), 9.89 (d, J = 8.4 Hz,
1H), 9.80 (s, 1H), 9.56 (d, J = 8.4 Hz, 1H), 9.38 (t, J = 8.6 Hz, 1H),
5.37 (s, 1H), 4.69 (s, 3H), 3.85 (s, 6H). 13C NMR (75 MHz, DMSO‑d6) δ:
174.23, 147.62, 147.38, 136.27, 132.54, 130.41, 127.75, 125.41,
119.85, 116.52, 113.90, 113.69, 81.16, 25.43, 21.64. ESI-MS m/z:
327.1 [M−H]−. Anal. calcd. For C18H17FN2O3: C, 65.85; H, 5.22; N,
8.53; Found: C, 65.67; H, 5.35; N, 8.58.
Yield 43%; melting point, 196–198 °C; 1H NMR (300 MHz,
DMSO‑d6) δ 12.39 (s, 1H), 8.22 (d, J = 2.2 Hz, 1H), 8.06 (dd, J = 8.7,
2.2 Hz, 1H), 7.46 (d, J = 8.2 Hz, 1H), 7.36 (s, 1H), 7.22 (d, J = 8.7 Hz,
1H), 7.02 (dd, J = 8.2, 1.6 Hz, 1H), 4.92 (s, 2H), 2.42 (s, 3H). 13C NMR
(75 MHz, DMSO‑d6) δ: 174.63, 157.45, 149.93, 135.15, 132.73, 131.15,
125.95, 125.71, 124.65, 117.01, 115.35, 115.12, 114.32, 65.73, 21.65.
ESI-MS m/z: 299.1 [M−H]−. Anal. calcd. For C16H13FN2O3: C, 64.00;
H, 4.36; N, 9.33; Found: C, 64.21; H, 4.23; N, 9.18.
4.1.7. 2-(4-(1H-Benzo[d]imidazol-2-yl)-2-methylphenoxy)propanoic acid
(6)
4.1.13. 2-(2-Chloro-4-(6-methyl-1H-benzo[d]imidazol-2-yl)phenoxy)
acetic acid (12)
Yield 25%; melting point, 241 °C (carbonize); 1H NMR (300 MHz,
DMSO‑d6) δ 13.24 (s, 1H), 8.23–8.00 (m, 2H), 7.77–7.72 (m, 2H),
7.46–7.41 (m, 2H), 7.09 (d, J = 8.7 Hz, 1H), 5.08 (q, J = 6.8 Hz, 1H),
2.31 (s, 3H), 1.60 (d, J = 6.8 Hz, 3H). 13C NMR (75 MHz, DMSO‑d6) δ:
173.07, 159.42, 150.05, 135.35, 131.35, 130.37, 127.81, 127.56,
125.00, 114.46, 112.87, 72.34, 18.72, 16.64. ESI-MS m/z: 295.1
[M−H]−. Anal. calcd. For C17H16N2O3: C, 68.91; H, 5.44; N, 9.45;
Found: C, 68.73; H, 5.58; N, 9.33.
Yield 38%; melting point, 243–245 °C; 1H NMR (300 MHz,
DMSO‑d6) δ 13.44 (s, 1H), 8.47 (d, J = 2.3 Hz, 1H), 8.28 (dd, J = 8.8,
2.3 Hz, 1H), 7.65 (d, J = 8.3 Hz, 1H), 7.55 (s, 1H), 7.39 (d, J = 8.8 Hz,
1H), 7.32–7.28 (m, 1H), 5.01 (s, 2H), 2.48 (s, 3H). 13C NMR (75 MHz,
DMSO‑d6) δ: 174.42, 157.45, 149.93, 135.13, 132.55, 131.95, 128.45,
125.83, 123.42, 116.25, 115.32, 115.16, 65.75, 21.63. ESI-MS m/z:
315.1 [M−H]−. Anal. calcd. For C16H13ClN2O3: C, 60.67; H, 4.14; N,
8.84; Found: C, 60.52; H, 4.05; N, 8.71.
5

Z. Li, et al.
BioorganicChemistry99(2020)103803
4.2. Evaluation for PPARα, PPARγ and PPARδ
exsanguination after ketamine and xylazine anesthesia. Tissue and
serum samples were collected and processed for histological and ser-
ological analysis. Alanine aminotransferase (ALT) and aspartate trans-
aminase (AST) levels were determined by the automatic biochemical
analyzer (Beckman Coulter, AU5811, Tokyo, Japan). The liver of each
experimental group was isolated immediately after sacrifice and wa-
shed with ice-cold saline before fixed in 10% (v/v) formalin. The sec-
tions were embedded in paraffin after dehydrate. Four-micron sections
were cut and stained with Hematoxylin-Eosin and Masson’s trichrome
staining for histopathological assessment at 400× magnification.
Detailed descriptions on transfection and cell-based evaluation for
PPARα, PPARγ, and PPARδ were given in our previously reported lit-
erature [22]. Briefly, HepG2 or HEK293 cells were transfected with
pBIND-PPARα, PPARδ or PPARγ according to the manufacturer’s pro-
tocol. After transfection, positive controls or compounds with different
concentrations 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).
Declaration of Competing Interest
4.3. Molecular modeling
The authors declare no competing financial interest.
MOE (version 2014.0901, The Chemical Computing Group,
Montreal, Canada) was used to perform the docking modeling based on
the reported crystal structure of PPARα (3VI8) 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. Subsequently, the binding pocket with the Gaussian Contact
surface was isolated and the backbone was deleted. The ligand poses in
binding site was filtered by using Pharmacophore Query Editor.
Acknowledgments
This study was supported by the Natural Science Foundation of
Guangdong Province, China (Grant 2018A030313445), the Guangdong
Basic and Applied Basic Research Foundation, China (Grant
2019A1515011036), the Innovative strong school project of
Guangdong Pharmaceutical University, China (Grant 2018KTSCX111),
Guangdong Province Medical Science and Technology Research Fund,
China (Grant B2018053), and Research Projects of the Chinese
Medicine Council of Guangdong Province, China (Grant 20191199 and
20192043).
Compound
4 was docked into the binding pocket with the
Pharmacophore method and ranked with London dG scoring function.
For energy minimization in the binding pocket, MOE Forcefield
Refinement was performed and ranked with London dG scoring func-
tion. All protocols were validated by re-docking of the crystal ligands
into the binding pocket of PPARα and PPARδ. The RMSD between the
co-crystal ligand and the performed experiment must be less than 2.0 Å
in all cases (0.85 Å and 1.56 Å, respectively). These values indicate that
the parameters used for calculations agree by replicating the con-
formation and orientation in the X-ray coordinates of the receptors.
MOE 2014.0901 and Pymol 2.3 were employed for adequate visuali-
zation.
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