Anti-inflammatory effects of an optimized PPAR-γ agonist via NF-κB pathway inhibition

Article abstract of DOI:10.1016/j.bioorg.2020.103611

In our previous study, a PPAR-γ agonist (+)-(R,E)-6a1 was elaborated as an anti-inflammatory lead. However, in silico analysis showed that (+)-(R,E)-6a1 lacks key hydrogen bonding with Tyr⁴⁷³ of PPAR-γ LBD (ligand binding domain). To facilitate

Full text of DOI:10.1016/j.bioorg.2020.103611

Journal Pre-proofs
Anti-inflammatory effects of an optimized PPAR-γ agonist via NF-κB path-
way inhibition
Zhiran Ju, Mingzhi Su, Jongki Hong, Eun La Kim, Jee H. Jung
PII:
S0045-2068(19)31531-7
DOI:
https://doi.org/10.1016/j.bioorg.2020.103611
YBIOO 103611
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
15 September 2019
14 January 2020
20 January 2020
Please cite this article as: Z. Ju, M. Su, J. Hong, E. La Kim, J.H. Jung, Anti-inflammatory effects of an optimized
PPAR-γ agonist via NF-κB pathway inhibition, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/
j.bioorg.2020.103611
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Anti-inflammatory effects of an optimized PPAR-γ agonist
via NF-κB pathway inhibition
Zhiran Ju a,†, Mingzhi Su a,†, Jongki Hong b, Eun La Kim a, Jee H. Jung a,*
a College of Pharmacy, Pusan National University, Busan 46241, Republic of Korea
b College of Pharmacy, Kyunghee University, Seoul 02447, Republic of Korea
*Correspondence: jhjung@pusan.ac.kr (J.H.J.); +82-51-510-2803 (J.H.J.).
†These authors equally contributed to this work
Abstract: In our previous study, a PPAR-γ agonist (+)-(R,E)-6a1 was elaborated as an anti-
inflammatory lead. However, in silico analysis showed that (+)-(R,E)-6a1 lacks key hydrogen
bonding with Tyr473 of PPAR-γ LBD (ligand binding domain). To facilitate additional
hydrogen bonding with Tyr473, a more polar head group was introduced to the structure of (+)-
(R,E)-6a1, and we also attempted to synthesize enzymatically stable derivatives. Of the
synthetic derivatives, compound (+)-(R,E)-5f showed highest PPAR-γ transcriptional activity
and reasonable metabolic stability. Compound (+)-(R,E)-5f suppressed the expression of pro-
inflammatory mediators such as inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2),
interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α). Reduction of nitric oxide (NO),
and ROS was also observed. Compound (+)-(R,E)-5f was found to suppress the NF-κB
pathway by inhibiting phosphorylation of IKK (IκB kinase), and this may lead to subsequent
inhibition of IκBα (inhibitor of NF-κBα) phosphorylation and inhibition of NF-κB activation.
These results indicate that (+)-(R,E)-5f exerts anti-inflammatory activity via NF-κB pathway
inhibition, and may serve as a potential anti-inflammatory lead.
Keywords: PPAR-γ agonist, anti-inflammatory, NF-κB pathway, cyclooxygenase-2
1. Introduction
Inflammation, the fundamental defense mechanism of the immune system, can protect
against injuries caused by harmful stimuli such as pathogens and poisons. However, excessive
1

inflammation, especially chronic inflammation, can cause immune system abnormalities and
ultimately, pathogenesis [1]. The nuclear factor-κB (NF-κB) pathway has long been
considered a prototypical pro-inflammatory signaling pathway, largely based on NF-κB
activation in the expression of pro-inflammatory cytokines such as interleukin 6 (IL-6) and
tumor necrosis factor α (TNFα), and its role in the expression of other pro-inflammatory
genes including chemokines and adhesion molecules [2-4]. The NF-κB pathway has long
been considered a preferred target for the development of new anti-inflammatory drugs.
Peroxisome proliferator-activated receptor γ (PPAR-γ), which can be modulated by
direct binding of endogenous 15-deoxy-∆12,14-prostaglandin J2 (15d-PGJ2), is a member of the
nuclear receptor superfamily. 15d-PGJ2 and its derivatives, which possess an α,β-unsaturated
ketone moiety can covalently bind to the thiol group of a cysteine residue (Cys285) in the
PPAR-γ LBD through Michael addition reaction, and this additional covalent bonding
contribute to the activation of PPAR-γ [5,9]. Binding of synthetic or natural ligands to PPAR-
γ sequentially leads to activation, nuclear translocation, and transcriptional activation to
modulate glucose and lipid metabolism [6]. Further research revealed that PPAR-γ also works
as a negative regulator of inflammation, especially in macrophages [7,8]. In our previous
study, we designed an exocyclic eneone jasmonate derivative (+)-(R,E)-6a1, which displayed
significant PPAR-γ transcriptional activity and subsequent anti-inflammatory activity via
inhibition of the NF-κB pathway in macrophages [9,10]. Typical PPAR-γ ligands such as
rosiglitazone and 15d-PGJ2 comprise three distinct partial structures including a polar head,
linker, and hydrophobic tail (Figure 1) [10]. The polar head and hydrophobic tail play
important roles in H-bonding and hydrophobic interaction, respectively, with the PPAR-γ
LBD (ligand binding domain). Compound (+)-(R,E)-6a1 also contained these partial
structures and showed remarkable PPAR-γ transactivation; in silico analysis showed that (+)-
(R,E)-6a1 binds to PPAR-γ in a manner similar to rosiglitazone and 15d-PGJ2 (Figure 5).
However, the binding pose of (+)-(R,E)-6a1 lacks the key hydrogen bonding with tyrosine-
473 (Tyr473). Hydrogen bonding with Tyr473 is important for the stabilization and activation of
PPAR-γ [9-12]. Therefore, it would be beneficial if a more polar head, as a hydrogen bond
acceptor, is introduced to the structure of (+)-(R,E)-6a1. Moreover, the head group of (+)-
2

(R,E)-6a1 may be labile to enzymatic hydrolysis in the biological system. An obvious
decrease in PPAR-γ activity with the free acid form of a similar compound has been observed
previously [9].
A
B
polar
OCH2CH3
head
C
polar head
O
O
polar
head
COOH
CH3
N
HN
tail
O
S
N
linker
linker
linker
O
O
O
tail
tail
Rosiglitazone
15d-PGJ2
6a1
(+)-(R,E)-
Figure 1. Structures and pharmacophoric regions of (A) rosiglitazone, (B) 15-deoxy-∆12,
14 prostaglandin J2 (15d-PGJ2), and (C) (+)-(R,E)-6a1 [diagram from ref. 10]. Typical
PPAR-γ agonists comprise a polar head and hydrophobic tail connected by a linker
group.
In this study, to facilitate hydrogen bonding with Tyr473 and to improve metabolic
stability, the head group of (+)-(R,E)-6a1 was modified to various substituents (Figure 2),
and evaluated for biological activity.
polar head
ester head
OR
amide head
NR
O
OCH2CH3
O
O
1) to facilitate H-bonding with Tyr473
2) to improve enzymatic stability
O
O
O
B
A
C
R = linear alkyl, branched alkyl,
hydroxyalkyl, methoxyalkyl
Figure 2. Design of (+)-(R,E)-6a1 derivatives as PPAR-γ agonists with enhanced hydrogen
bonding with Tyr473 and improved enzymatic stability. (A) (+)-(R,E)-6a1. (B) Designed
analogs with an ester head. (C) Designed analogs with an amide head.
2. The strategy to design PPAR-γ activators based on (+)-(R,E)- 6a1
Based on our original design, the polar head ethyl ester moiety was replaced with
appropriately designed substituents that would facilitate additional hydrogen bonding with
Tyr473 and/or resist enzymatic degradation in the biological system, retaining the desired
potency of the parent molecule. To facilitate more hydrogen bonding with Tyr473, an
3

additional hydrogen bond acceptor such as hydroxyl, ether, or methoxyl group may be
introduced. To improve metabolic stability, steric hindrance may be induced by introducing a
long alkyl group, branched alkyl group, or other bulky groups that may suppress binding to
hydrolytic enzymes. Based on this hypothesis, the metabolically labile ethyl ester moiety of
(+)-(R,E)-6a1 was replaced with linear alkyl, branched alkyl, hydroxyalkyl, or methoxyalkyl
esters and amide derivatives (Schemes 1 and 2).
3. Results and discussion
3.1. Chemistry
The synthetic routes of exocyclic enone derivatives are outlined in Schemes 1-3. The
endocyclic ketones 2 were prepared from cyclopent-2-en-1-one 1 via a Baylis-Hillman
reaction. Subsequent orthoester Claisen rearrangement [with MeC(OMe)3 and propionic acid
at 110°C] gave the exocyclic ketone 3 as a mixture of (E)/(Z) geometric isomers and
inseparable R/S racemates of each geometric isomer with a yield of 80% (Scheme 1)
[9,13,14]. Hydrolysis of 3 by LiOH in aqueous THF yielded the corresponding acid 4
(Scheme 1), which was further coupled with various alcohols in EtOAc using the coupling
agent 2-(1H-benzotriazole-1-yl)-1,2,3,3-tetramethyluronium tetrafluoroborate (TBTU) to
generate esters 6a, 5b-5l as mixtures of geometric and enantiomeric isomers. Compounds 7a-
7b were synthesized from 3 via three steps. The reactive acyl chloride 6 was prepared by
treatment of acid 4 with oxalyl chloride in dichloromethane. The final amides 7a-7b were
consequently obtained as mixtures of geometric and enantiomeric isomers by coupling the
acyl chloride 6 with isopropylamine or N-methylethanolamine, respectively, in the presence
of N,N’-Dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBT), and
triethylamine (TEA) in dichloromethane (Scheme 2) [15,16]. The (E) and (Z) isomers were
interconvertible by a deconjugation/conjugation process under acidic reaction conditions.
However, the thermodynamically more stable (E) isomer was the predominant product in
most reactions (Table 1) [9]. The esters 6a, 5b-5l, and amides 7a-7b were initially obtained as
mixtures with various (E)/(Z) ratios, and the (E) or (Z) isomers can be separated by HPLC in a
reasonably stable state in most cases, with the exception of other unstable isomers [9,17-19].
4

The geometric configurations of (E) and (Z) were determined by comparing the 1H and
13C-NMR data with the reported data of methyl (2E)-3-oxo-2-pentylidenecyclopentaneacetate
[13]. In order to investigate the relative biological activity of each enantiomer of the most
potent derivative of (E)-5f (Figure 3), the racemate of (E)-5f was further resolved into each
enantiomer by chiral separation using a chiral cellulose column (Scheme 3). Each enantiomer
20
20
was defined as (+)-(R,E)-5f ([α]D = +5.5, c = 0.02, CHCl3) and (-)-(S,E)-5f ([α]D = -6.0, c =
0.02, CHCl3) by comparing their specific rotations with those reported for similar compounds:
20
methyl (+)-(R,E)-3-oxo-2-pentylidenecyclopentaneacetate ([α]D = +21.3, c = 1.0, CHCl3) and
20
methyl (-)-(S,Z)-3-oxo-2-pentylidenecyclopentan acetate ([α]D = -1.4, c = 1.0, CHCl3).
O
*
OCH3
O
*
OH
O
*
i
ii
iii
O
O
HO
O
O
1
2
3
4
iv
(E)-6a
(E)-5b
(E)-5c
(E/Z)-5d R
(E)-5f
R
(Z)-6a
(Z)-5b
(Z)-5c
R
R
R
= CH2CH3
= CH2CH3
= (CH2)3CH3
= (CH2)4CH3
R
R
= (CH2)3CH3
= (CH2)4CH3
O
OR
(E/Z)-5e R
= (CH2)5CH3
= (CH2)2OH
= (CH2)2OCH3
= (CH2)2OCH2CH2OH
= CH2CH2OCH2CH2OCH3
= (CH2)2(OCH2CH2)2OH
= CH(CH3)2
= (CH2)2OH
R
(Z)-5f
(Z)-5h
(Z)-5j
R
R
*
(E/Z)-5g R
(E)-5h
(E/Z)-5i R
(E)-5j
(E/Z)-5k R
R
= (CH2)2OCH2CH2OH
O
5
R
R
= (CH2)2(OCH2CH2)2OH
(E/Z)-5l R
= (CH2)2(OCH2CH2)2OCH3
= CH2CH(OH)CH3
Scheme 1: Reagents and conditions: i) Aldehyde, Bu3P, BINOL, THF, N2, rt, 15 h; ii)
MeC(OMe)3, propionic acid, 110°C, 15 h; iii) LiOH, THF-Water (3:2), rt, 12 h; iv) ROH,
TBTU, TEA, EtOAc, rt, 12 h;
O
*
OCH3
O
*
OH
O
*
Cl
O
*
R
iii
v
vi
O
O
O
O
3
4
6
7
(E/Z)-7a R
(E/Z)-7b R
= NHCH(CH3)2
= NCH3(CH2)2OH
Scheme 2: Reagents and conditions: iii) LiOH, THF-Water (3:2), rt, 12 h; v) oxalyl chloride,
CH2Cl2, 40°C, 1 h; vi) RNH2, DDC, HOBT, TEA, CH2Cl2, 40°C, 24 h.
5

O
*
OCH2CH2OH
O
OCH2CH2OH
O
OCH2CH2OH
R
S
E
E
E
O
O
O
5f
5f
(+)-(R,E)-
(E)-
5f
(-)-(S,E)-
Scheme 3: Conditions: Shimadzu LC-20A HPLC with a YMC chiral cellulose-C column,
22% Acetonitrile, flow rate: 1 ml/min, UV: 210, 254 nm. Retention time: (+)-(R,E)-5f at 180
min; (-)-(S,E)-5f at 190 min (R:S = 1:1).
3.2. PPAR-γ transactivation assay
The synthetic derivatives 6a, 5b-5l, and 7a-7b were first screened for their in vitro
PPAR-γ transactivation potency using Ac2F rat liver cells. Prior to the PPAR-γ
transactivation assay, these derivatives were determined for their cytotoxicity to Ac2F cells to
gauge the suitable concentration for performing the PPAR-γ transactivation assay. At
concentrations of 5 and 10 μM, these derivatives were mostly non-toxic to Ac2F cells, but 5l,
7a, and 7b showed mild cytotoxicity in the tested cell lines (See SI). Therefore, the
concentrations of 5 and 10 μM were employed for the PPAR-γ transactivation assay. PPAR-γ
agonistic activity was measured using Ac2F cells that had been transiently transfected with
pcDNA3/pFlag-PPAR-γ1 and PPRE. As shown in Figure 3, the differential activities of
derivatives indicated that their PPAR-γ agonistic activity could be modulated by differential
substitution at the carboxyl polar head group. Of these derivatives, the (E)-isomers frequently
showed higher PPAR-γ transcriptional activity than (Z)-isomers as in the cases of (E)-6a, (E)-
5b, (E)-5c, (E)-5f, and (E)-5h. This result was identical to that of our previous study [9], i.e.,
the trans-isomer was more active than the cis-isomer. The collected data also indicated that it
is beneficial to introduce a hydroxyl group rather than a methoxyl group as shown by the
significant activities of (E)-5f, (E)-5h, and (E)/(Z)-5l. Compound (E)-5f showed stronger
PPAR-γ activation than rosiglitazone at the concentrations of 10 μM, and its potency was
enhanced compared to the original compound 6a1. Despite improved metabolic stability
(Table 1), the amide derivatives (E)/(Z)-7a and -7b showed decreased PPAR-γ agonistic
6

activity compared to 6a1, possibly due to higher cytotoxicity than other compounds (see SI).
3
5 μM
10 μM
**
***
**
**
***
***
**
2
1
0
i
i
j
j
l
f
f
5
-
3
a
a
c
c
e
k
a
7
n
b
b
d
g
h
h
b
7
C
C
s
5
5
5
5
-
-
5
6
6
5
5
5
5
5
5
5
5
5
5
-
-
-
o
)
-
N
o
-
-
-
-
-
-
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)
-
-
-
-
-
-
)
)
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)
-
)
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)
)
)
)
)
Z
)
)
)
)
)
)
/
Z
Z
Z
/
R
Z
E
(
E
Z
Z
Z
/
Z
Z
/
E
Z
E
Z
Z
Z
(
(
Z
/
E
(
E
(
(
/
/
(
(
/
/
E
(
(
(
(
E
E
(
E
E
E
(
E
E
(
(
E
(
(
(
(
(
Figure 3. In vitro assay of PPAR-γ activation by compounds 3, 6a, 5b-5l, 7a, 7b, and by
rosiglitazone at 5 μM or 10 μM in rat liver Ac2F cells. NC: negative control, transfected with
a plasmid containing PPRE and pcDNA3. Con: control, transfected with a plasmid containing
PPRE and pFlag-PPAR-γ1. Rosi: rosiglitazone. Rosiglitazone was used as the positive
reference control to monitor activation of the luciferase reporter. Compound-treated cells
were transiently transfected with PPRE plus pFlag-PPAR-γ1. Luciferase expression (folds of
the control) is presented as the mean ± SD (n = 3). ** p < 0.01, *** p < 0.001.
The most potent derivative, (E)-5f, was further resolved into each enantiomer, (+)-(R,E)-
5f and (-)-(S,E)-5f. The isomer (+)-(R,E)-5f showed almost equal activity to (-)-(S,E)-5f
(Figure 4). Docking simulation suggested a higher binding affinity of (+)-(R,E)-5f (-8.2
Kcal/mol) than (-)-(S,E)-5f (-7.4 Kcal/mol) (Figure 6).
3
5 μM
10 μM
**
***
***
*
***
2
1
0
***
i
s
f
f
n
C
5
-
5
-
o
N
o
)
)
E
C
R
E
,
,
S
R
(
(
Figure 4. In vitro assay of PPAR-γ activation by (+)-(R,E)-5f, (-)-(S,E)-5f and by
rosiglitazone at 5 μM or 10 μM in rat liver Ac2F cells. NC: negative control, transfected with
a plasmid containing PPRE and pcDNA3. Con: control, transfected with a plasmid containing
PPRE and pFlag-PPAR-γ1. Rosi: rosiglitazone. Rosiglitazone was used as the positive
7

reference control to monitor activation of the luciferase reporter. Compound-treated cells
were transiently transfected with PPRE plus pFlag-PPAR-γ1. Luciferase expression (folds of
the control) is presented as the mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.
The PPAR-γ agonistic potency of (+)-(R,E)-5f was further compared with rosiglitazone
and 15dPGJ2 at low concentrations (10 – 100 nM) (Figure 5). However, compound (+)-(R,E)-
5f did not show higher potency than rosiglitazone and 15dPGJ2 at lower concentrations.
2.0
10 nM
100 nM
1000 nM
1.5
1.0
0.5
0.0
i
f
2
J
n
C
s
o
5
o
-
)
N
G
P
C
R
E
,
d
R
(
5
1
Figure 5. In vitro assay of PPAR-γ activation by (+)-(R,E)-5f, rosiglitazone, and 15dPGJ2 at
10, 100, and 1000 nM in rat liver Ac2F cells. NC: negative control, transfected with a plasmid
containing PPRE and pcDNA3. Con: control, transfected with a plasmid containing PPRE and
pFlag-PPAR-γ1. Compound-treated cells were transiently transfected with PPRE plus pFlag-
PPAR-γ1. Luciferase expression (folds of the control) is presented as the mean ± SD (n = 3).
* p < 0.05, ** p < 0.01, *** p < 0.001.
3.3. In vitro metabolic stability assay
The synthesized esters 3, 6a, 5b-5l, and amides 7a-7b were evaluated for in vitro
metabolic stability using porcine liver esterase (PLE). The compounds were subjected to
hydrolysis by incubating with PLE under special physiological conditions (phosphate buffer,
pH 7.4, and 37°C) [15]. After 30 min and 60 min incubation, the reaction product was
collected and analyzed by RP-HPLC to quantify the amount of the intact ester/amide and its
corresponding metabolite (acid). The in vitro metabolic stability (ratio of esters/acids,
amides/acids), and calculated log P value were employed as criteria for further lead selection
(Table 1).
8

As shown in Table 1, the in vitro metabolic stability seems to be substantially modulated
by polarity and steric hindrance of the ester/amide moiety. The linear alkyl esters 3, 6a, and
5b-5d with a rather lipophilic property were highly susceptible to enzymatic hydrolysis, and
the original peaks of esters were mostly replaced by peaks of their corresponding acids at 30
min and 60 min incubation with PLE. These results also suggested that extension of the linear
alkyl chain is detrimental to their enzymatic stability. In contrast, branched ester 5e was much
more stable than linear alkyl esters 3, 6a, and 5b-5d, even though it has similar lipophilicity.
Further, the metabolic stability of the hydroxyl alkyl esters (5f, 5h, 5j, and 5l) and the
methoxyl alkyl esters (5g, 5i, and 5k) was significantly improved. In particular, significant
improvement of metabolic stability was observed in hydroxyl alkyl esters 5f, 5h, 5j, and 5l,
suggesting that resistance to enzymatic hydrolysis increases with increasing hydrophilicity. In
addition, amides 7a-7b showed stronger enzymatic stability compared to linear alkyl esters,
and 7b displayed the highest metabolic stability indicating that the amide bond is more
resistant to enzymatic degradation. All these results suggest that branched alkyl esters or
amides are more resistant to enzymatic hydrolysis than linear alkyl ones, and that
hydrophilicity is an important factor for metabolic stability.
According to the collected results, (+)-(R,E)-5f was the most potent for PPAR-γ
transactivation without notable cytotoxicity in normal cell lines and showed reasonably high
enzymatic stability. Therefore, compound 5f was selected for further biological evaluation.
Table 1. In vitro metabolic stability and (E:Z) ratio of synthetic derivatives
Compound.
Ratio
(E:Z)
15:1
NT
Log P
Stability
30 min
0
60 min
(E)-6a/(Z)-6a
(E/Z)-3
3.20
2.83
4.26
4.77
5.27
3.56
2.10
2.62
1.90
2.42
1.69
0
0
0
(E)-5b/(Z)-5b
(E)-5c/(Z)-5c
(E/Z)-5d
33:1
5:1
0
0
0
0
8:1
0
0
(E/Z)-5e
6:1
0.51±0.05
2.21±0.31
0.27±0.14
1.71±0.25
0.04±0.01
1.02±0.16
0
(E)-5f/(Z)-5f
(E/Z)-5g
4:1
1.40±0.25
4:1
0
(E)-5h/(Z)-5h
(E/Z)-5i
8:1
0.43±0.10
0
9:1
(E)-5j/(Z)-5j
7:1
0.33±0.07
9

(E/Z)-5k
(E/Z)-5l
(E/Z)-7a
(E/Z)-7b
5:1
7:1
10:1
6:1
2.21
2.56
3.05
1.99
3.19±0.31
4.28±0.42
1.40±0.39
17.50±0.34
1.41±0.07
1.77±0.20
0.43±0.15
2.88±0.40
Note: In vitro metabolic stability was determined by the esterase-catalyzed hydrolysis assay
and was analyzed by HPLC. Data are presented as the ratio of intact esters (or amides) /
hydrolyzed acids at 30 and 60 min, and are expressed as the mean ± SD (n=3) of three
independent experiments; log P (octanol-water partition coefficient) values were calculated
using Molinspiration software. For assessment of druglikeness of drug candidates, log P < 5
was considered a prerequisite for good bioavailability. NT: not tested.
3.4. Binding mode of (+)-(R,E)-5f to PPAR-γ
To gain insight into the interaction between compound (+)-(R,E)-5f and PPAR-γ,
molecular docking simulation was performed using human PPAR-γ (PDB code: 2PRG) [20 -
22].
The best docking poses of compound (+)-(R,E)-6a1, (+)-(R,E)-5f, (+)-(R,Z)-5f, and (-)-
(S,E)-5f are illustrated in Figure 6. Compound (+)-(R,E)-6a1 formed hydrogen bonds with
key amino acid residues, Ser289, His323, and Tyr327 on helices H3 and H4 in the PPAR-γ LBD
(Figure 6A), but did not show direct hydrogen bonding with Tyr473 on the H12 helix. In
contrast, compound (+)-(R,E)-5f showed hydrogen bonding with Tyr473 in addition to Tyr327,
Ser289, Gln286, His323, and His449 on helices H3, H4, H11, and H12, in a similar pattern as
rosiglitazone, but the affinity value was lower than that of rosiglitazone (-9.4 Kcal/mol)
(Figure 6B). Additional interaction between (+)-(R,E)-5f and Tyr473 in the PPAR-γ LBD may
lead to induction of H12 into the active conformation and thereby improve PPAR-γ
transcriptional activity compared to (+)-(R,E)-6a1. It is reported that the Y473F mutant still
retains the ability to bind covalently to 15d-PGJ2, but loses the ligand-induced transcriptional
activity [9,11,12]. Therefore, it is essential for a PPAR-γ agonist to form a hydrogen bond
with Tyr473 for effective PPAR-γ activation. The geometric isomer (+)-(R,Z)-5f showed a
similar binding pose as (+)-(R,E)-5f, but its affinity value (-7.4 Kcal/mol) was lower than that
of (+)-(R,E)-5f (-8.2 Kcal/mol) (Figure 6C). Meanwhile, the enantiomer (-)-(S,E)-5f lost the
10

key interactions with amino acids Tyr327, Ser289, Gln286, His323, and Tyr473 (Figure 6D).
The docking calculation also showed that (+)-(R,E)-5f locates in the active cavity of
PPAR-γ LBD in a similar manner as 15d-PGJ2. Moreover, the thiol group of Cys285 is
substantially close to the α,β-unsaturated enone group of (+)-(R,E)-5f, to facilitate a possible
covalent agonism [11,12] (Figure 6B).
Figure 6. Docking structures of ligand/PPAR-γ binding. (A) Zoomed view of hydrogen
bonding interactions (dotted lines) between the (+)-(R,E)-6a1 and surrounding amino acids,
Ser289, His323, and Tyr327. The key helices are labeled as H3, 4, 10, 11, and 12. (B) Zoomed
view of hydrogen bonding interactions between (+)-(R,E)-5f and surrounding amino acids
Tyr327, Ser289, Gln286, His323, His449, and Tyr473. (C) Zoomed view of hydrogen bonding
interactions between (+)-(R,Z)-5f and surrounding amino acids Tyr327, Ser289, Gln286, His323,
His449, and Tyr473. (D) Zoomed view of hydrogen bonding interactions between (-)-(S,E)-5f
11

and surrounding amino acid, His449.
3.5. (+)-(R,E)-5f inhibited LPS-induced expression of pro-inflammatory factors in RAW264.7
cells
Prior to the in vitro anti-inflammatory assay, the cytotoxicity of (+)-(R,E)-5f to murine
macrophages (RAW 264.7) was first determined to gauge the suitable concentration for cell-
based anti-inflammatory assays. The result indicated that (+)-(R,E)-5f showed no significant
toxicity to RAW 264.7 cells at concentrations of 10 and 50 μΜ for 24 h (See SI). Therefore, a
concentration lower than 30 μΜ for (+)-(R,E)-5f was applied to RAW 264.7 cells for the anti-
inflammatory assay.
Some studies have reported that PPAR-γ protein can be expressed in macrophages, and
that activated PPAR-γ translocates to the nucleus and binds NF-κB. Binding with PPAR-γ
leads to NF-κB inhibition, thereby inhibiting the transcription of pro-inflammatory mediators
such as iNOS, COX, TNF-, and IL-6 [23]. Here, we used western blot analysis to assess the
protein level of PPAR-γ translocated to the nucleus through activation by (+)-(R,E)-5f. As
expected, the nuclear PPAR-γ protein level was significantly and concentration-dependently
increased by (+)-(R,E)-5f treatment, and its potency was comparable to that of rosiglitazone at
a concentration of 30 μM (Figure 7).
Figure 7. Endonuclear and cytoplasmic PPAR-γ protein levels in RAW264.7 macrophages
treated with (+)-(R,E)-5f for 24 h as determined by western blotting. Nuclear levels of
transcription Factor II B (TF II B) were used for reference purposes. The ratio of
Nuclear/cytoplasmic PPAR-γ protein levels were shown as bar graph. Rosi: rosiglitazone. The
results shown are representative of three independent experiments. # p < 0.05 compared with
the control group.
12

At a very early stage of inflammation, macrophages are recruited to the inflammatory
site and produce a large amount of pro-inflammatory molecules. For instance, nitric oxide
(NO) and prostaglandin E2 (PGE2) are among these molecules, with a role in promoting the
inflammation process [24]. Therefore, in order to investigate the anti-inflammatory activity of
(+)-(R,E)-5f, the protein levels of pro-inflammatory factors iNOS and COX-2 were examined
by western blot, employing RAW 264.7 cells. As shown in Figures 8A and 8B, LPS
stimulation markedly increased the iNOS and COX-2 protein levels, but this increase was
diminished in a dose-dependent manner after treatment with (+)-(R,E)-5f. The potency of (+)-
(R,E)-5f at 10 μΜ was comparable to that of dexamethasone. We used dexamethasone as a
positive control in evaluation of anti-inflammatory activity because it is clinically used anti-
inflammatory drug. Meanwhile, suppression of iNOS by (+)-(R,E)-5f is considered to cause
decreased NO production in macrophages. As shown in Figure 9A, compound (+)-(R,E)-5f
significantly and concentration-dependently decreased NO production.
Figure 8. Effects of (+)-(R,E)-5f on LPS-induced iNOS (A) and COX-2 (B) protein
expression in RAW264.7 cells. The cells were treated with different concentrations of (+)-
(R,E)-5f, and then cultured in the presence or absence of LPS (1 μg/ml) for 24 h.
Dexamethasone (DEX) was employed as a positive control (10 μM). The results are shown as
the mean ± SD (n = 3) of three independent experiments. ## p < 0.01 compared with the
control group; * p < 0.05, ** p < 0.01 compared with the LPS-stimulated group.
IL-6 and TNF-α are two cytokines that contribute to the progression of inflammation
[25]. In acute and chronic inflammation, TNF-α is produced mainly by activated macrophages
and plays a critical role in both tissue destruction and damage recovery, maintaining the
13

reversibility of inflammation. TNF-α may initiate an inflammatory cascade consisting of other
inflammatory cytokines. Other studies have also indicated that TNF-α also acts as an inducer
for nuclear factor-κB (NF-κB), which shows pro-inflammatory activity. So, inhibition of IL-6
and TNF-α production is effective strategy to suppress the progress of inflammation. The
produced amounts of IL-6 and TNF-α were detected by enzyme-linked immunosorbent assay
(ELISA). The results show that the amounts of IL-6 and TNF-α were markedly increased
when murine macrophages RAW264.7 were exposed to LPS. However, these increases were
inhibited by (+)-(R,E)-5f in a dose-dependent manner (Figures 9B and 9C), suggesting that
(+)-(R,E)-5f as a PPAR-γ agonist could participate in a signaling pathway activated by LPS in
macrophages [10,26,27].
Figure 9. Inhibitory activity of (+)-(R,E)-5f on LPS-induced NO and cytokine production.
(A) The production of nitric oxide (NO) in the medium of RAW264.7 cells after treatment
with (+)-(R,E)-5f for 1 h, and then with LPS (25 ng/mL, 24 h). The concentration of NO in
the medium was determined using the Griess method. (B) Production of tumor necrosis factor
α (TNF-α) in RAW264.7 cells after treatment with (+)-(R,E)-5f for 1 h, and then with LPS
(25 ng/mL, 3 h). (C) Production of interleukin-6 (IL-6) in RAW264.7 cells after treatment
with (+)-(R,E)-5f for 1 h, and then with LPS (25 ng/mL, 24 h). Dexamethasone was employed
as a positive control (10 μM). The results are shown as the mean ± SD (n=3) of three
independent experiments. ## p < 0.01, ### p < 0.001 compared with the control group; * p <
0.05, ** p < 0.01 compared with the LPS-stimulated group.
3.6. (+)-(R,E)-5f reduced ROS levels in RAW264.7 cells
In a biological system, reactive oxygen species (ROS), which include hydroxyl radical,
superoxide anion, hydrogen peroxide, and singlet oxygen, play an important role in
14

inflammatory responses. COX-2 has also been implicated in ROS generation [28].
Overabundant ROS can oxidize and therefore damage endogenous molecules, which can lead
to inflammation. ROS can activate redox-sensitive transcription factors such as NF-κB and
then influence pro-inflammatory responses. NF-κB is involved in the regulation of pro-
inflammatory genes, which represents a key step in the production of pro-inflammatory
mediators such as TNF-α, IL-6, iNOS, and COX-2. Herein, we detected and quantified
cellular oxidative stress. As shown in Figures 10A and 10B, compound (+)-(R,E)-5f dose-
dependently reduced ROS production compared to the LPS-treatment group. Notably, at a
concentration of 30 μΜ, (+)-(R,E)-5f significantly decreased ROS production with a potency
comparable to that of 10 μΜ dexamethasone.
Figure 10. Reduction of ROS in RAW264.7 cells by treatment with (+)-(R,E)-5f. (A) LPS (1
g/mL) induced intracellular ROS, which is shown as green fluorescence using the
fluorescent probe DCFH-DA. By treatment with (+)-(R,E)-5f or dexamethasone (DEX) (10
M), ROS expression was significantly decreased. (B) Fluorescence intensity was quantified
using a fluorescence microplate reader. ##p < 0.01 vs untreated controls. *p < 0.05, ***p < 0.001
vs. LPS-treated cells.
3.7. (+)-(R,E)-5f inhibited LPS-induced NF-κB signaling pathway in RAW264.7 cells
NF-κB is a critical promoter involved in inflammation and in the induction of pro-
inflammatory cytokines such as iNOS, COX-2, IL-6, and TNF-α. These cytokines play
important roles in inflammation [29]. It has been recognized that conventional PPAR-γ
ligands exert anti-inflammatory activity via binding to NF-κB. Ligand-activated PPAR-γ may
15

be translocated into the nucleus to bind with NF-κB. The NF-κB/PPAR-γ complex cannot
bind to the promotor region of DNA, leading to suppressed gene expression of pro-
inflammatory mediators [10]. In an alternative pathway, PPAR-γ agonists such as 15d-PGJ2
inhibit the NF-κB activation pathway in the cytoplasm. In the cytoplasm, NF-κB generally
exists in an inactive NF-κB-IκB complex. Extracellular stimuli such as cytokines (i.e., TNF-
α), pathogens, and endotoxin (i.e., lipopolysaccharides [LPS]) trigger the activation of IKK by
phosphorylation. Activated IKK then phosphorylates IκB with its subsequent ubiquitination
and proteolytic degradation. After IκB degradation, NF-κB is released and translocates to the
nucleus where it binds to the promoter regions of its target inflammatory genes. We intended
to examine the effect of (+)-(R,E)-5f on the alternative pathway. As shown in Figure 11, the
phosphorylation levels of NF-κB increased significantly after LPS treatment, but the pre-
treatment of (+)-(R,E)-5f obviously decreased the NF-κB p65 phosphorylation in a dose-
dependent manner (Figures 11A and 11B). As a result, the phosphorylated protein level of
NF-κB in the nucleus was also significantly decreased by (+)-(R,E)-5f treatment (Figure 11F).
Meanwhile, the phosphorylation of IKK and IκBα was decreased (Figures 11C and 11D) and
IκBα degradation was prevented in a dose-dependent manner (Figure 11E) by (+)-(R,E)-5f
treatment. These results indicated that (+)-(R,E)-5f can inhibit the phosphorylation of IKK,
and this may lead to subsequent inhibition of phosphorylation of IκBα and NF-κB.
To further investigate the downstream events of NF-κB inhibition by (+)-(R,E)-5f, we
examined the effect on the nuclear level of NF-κB. In immunofluorescence assay, (+)-(R,E)-
5f significantly reduced the nuclear level of phosphorylated NF-κB (Figure 11G). Our
findings suggest that (+)-(R,E)-5f may exert anti-inflammatory activity by inhibition of NF-
κB activation like PGJ2 [10].
16

Figure 11. Inhibitory effect of (+)-(R,E)-5f on NF-κB p65 activation in RAW 264.7
macrophages. (A) Phosphorylated NF-κB p65 (total), (B) total NF-κB p65, (C)
phosphorylation of IKK, (D) phosphorylation of IκBα, (E) intact IκBα protein, and (F)
phosphorylated nuclear NF-κB p65 was determined by western blot. Cells were pretreated
with (+)-(R,E)-5f for 1 h, and then stimulated with lipopolysaccharide (LPS, 25 ng/mL) for 30
min. β-actin was used as an internal control. (G) Immunofluorescence assay, NF-κB p65
shows green fluorescence, and cell nuclei show red fluorescence by PI staining in confocal
microscopy. The results shown are representative of three independent experiments. # p <
0.05, ## p < 0.01 compared with the control group; * p < 0.05, ** p < 0.01, ***p < 0.001
compared with the LPS-stimulated group.
4. Conclusion
17

In this study, we designed and synthesized a series of potential PPAR-γ activators by
further modifying the polar head of the previous lead (+)-(R,E)-6a1. The synthesized analogs
were tested for their PPAR-γ transcriptional activity and in vitro metabolic stability. The
differential PPAR-γ transcriptional activities of the geometric and enantiomeric isomers of
selected compounds were also compared. The results indicated that (+)-(R,E)-5f might
function as a potential PPAR-γ activator at the cellular level, and showed more potent PPAR-
γ transcriptional activity and higher metabolic stability than (+)-(R,E)-6a1. Docking of (+)-
(R,E)-5f to PPAR-γ showed that it locates in the active cavity of PPAR-γ LBD in a similar
manner as rosiglitazone and 15d-PGJ2. Moreover, the thiol group of Cys285 is substantially
close to the α,β-unsaturated enone group of (+)-(R,E)-5f, to facilitate a possible covalent
agonism like 15d-PGJ2 [11,12]. In vitro anti-inflammatory assay indicated that (+)-(R,E)-5f
exerted a significant anti-inflammatory activity through suppressed expression of pro-
inflammatory factors including iNOS, COX-2, TNF-α, and IL-6 in LPS-stimulated murine
RAW264.7 macrophages. Reduction of NO and ROS was also observed. The phosphorylation
of IKK and IκBα was decreased and IκBα degradation was prevented in a dose-dependent
manner by (+)-(R,E)-5f treatment. Treatment by (+)-(R,E)-5f obviously decreased the NF-κB
p65 phosphorylation and nuclear level of NF-κB. Therefore, it is speculated that (+)-(R,E)-5f
suppressed LPS-induced phosphorylation of IKK, leading to inhibition of IκBα
phosphorylation, and finally inhibition of NF-κB activation. The upgraded PPAR-γ agonist
(+)-(R,E)-5f is expected to serve as a lead for the study of potential anti-inflammatory agents.
5. Experimental section
5.1. Chemistry
All reagents used were commercially available; some organic solvents were redistilled
under positive pressure if necessary. Reactions were monitored by thin-layer chromatography
(TLC) on glass plates coated with silica gel using a fluorescent indicator (GF254). The 1H-
and 13C-NMR spectral data were recorded on Varian Unity 500 MHz and 400 MHz NMR
spectrometer, respectively. High-resolution fast-atom bombardment mass spectrometry
(HRFABMS) data were obtained using an Agilent 1200 UHPLC accurate-mass Q-TOF MS
18

spectrometer. The optical rotation data were obtained using a Jasco P-1020 Polarimeter.
HPLC analysis was performed on a YMC ODS-Triart C18 column (250 × 10 mm, 5 µm, 120
Å) and a YMC chiral cellulose-C column (250 × 10 mm, 5 µm, 100 Å) using a Shodex RI-71
detector and Shimadzu LC-20A HPLC. All chemical reagents were purchased from Sigma-
Aldrich and Alfa Aesar.
5.1.1. Synthesis of compounds 6a, 5b-5l
General Procedure A: Baylis-Hillman reaction. A solution of cyclopent-2-en-1-one (1.0 mol-
equiv.), the appropriate aldehyde (1.5 mol-equiv.), [1,1 -binaphthalene]-2,2-diol (0.1 mol-
equiv.), and Bu3P (0.2 mol-equiv.) in THF (25 ml) was stirred at room temperature in the
presence of N2 for 15 h. General Procedure B: Orthoester Claisen rearrangement reaction. A
reaction product from Baylis-Hillman reaction, trimethyl orthoacetate (6 ml), was heated with
propionic acid (a few drops) at 110°C for 18 h. The crude mixture was evaporated, and the
residue was subjected to HPLC (Shodex RI-71 Detector, 65% Methanol, flow rate: 2 ml/min)
to separate the desired product 3, from the apolar aldehyde, Bu3P, and polar compound [1,1 -
binaphthalene]-2,2-diol. General Procedure C: Hydrolysis reaction. Lithium hydroxide
(LiOH) (1N, 2 equiv) was added to a solution of compound 3 (1 equiv) in aqueous THF
(THF: water = 3:2), and the reaction mixture was stirred at 40°C for 12 h. The THF was
evaporated under vacuum and the water layer was neutralized with (1N, 2 equiv) HCl. The
resulting suspension was extracted with EtOAc (three times). The combined organic layer
was washed with brine, dried over MgSO4, concentrated to yield the acid 4, which was used
without purification for the next step. 2-(1H-Benzotriazole-1-yl)-1,2,3,3-tetramethyluronium
tetrafluoroborate (TBTU, 1 equiv) was added to a solution of compound 4 and triethylamine
(TEA, 2 equiv) in EtOAc. After 10 min of stirring at room temperature, alcohols (2 equiv)
were added separately and the reaction mixture was stirred for 12 h. The mixture was washed
with water, dried, and concentrated to give a residue that was purified by reversed-phase
HPLC (YMC ODS-Triart C18 column) to yield the target esters 6a–5l.
5.1.2. Ethyl-2-(2-(cyclohexylmethylene)-3-oxocyclopentyl)acetate (E)-6a
Yellow oil; 45% yield; 1H-NMR (500 MHz, CD3OD) δ 6.40 (dd, J = 10.7, 1.9 Hz, 1H), 4.22
19

(q, J = 7.1 Hz, 2H), 3.50 (m, 1H), 2.57 (m, 2H), 2.50 (m, 1H), 2.34 (m, 2H), 2.11 (m, 1H),
1.95 (m, 1H), 1.83 (m, 2H), 1.70 (m, 2H), 1.44 (m, 2H), 1.20-1.36 (m, 4H), 1.33 (m, 3H), 13C-
NMR (125 MHz, CD3OD) δ 209.0, 173.4, 143.6, 139.4, 61.6, 40.3, 39.5, 36.6, 36.2, 33.1,
33.0, 26.8, 26.4 (×2), 26.3, 14.5. HRFABMS m/z 265.1798 [M+H] + (calcd for C16H24O3,
265.1798).
5.1.3. Ethyl-2-(2-(cyclohexylmethylene)-3-oxocyclopentyl)acetate (Z)-6a
Yellow oil; 3% yield; 1H-NMR (500 MHz, CD3OD) δ 5.82 (dd, J = 9.6, 1.0 Hz, 1H), 4.17 (q,
J = 7.1 Hz, 2H), 3.45 (m, 1H), 2.63 (m, 1H), 2.43 (m, 1H), 2.35 (m, 1H), 2.28 (m, 1H), 2.19
(m, 1H), 2.06 (d, J = 1.2 Hz, 2H), 1.73 (m, 2H), 1.67 (m, 2H), 1.62 (m, 2H), 1.36 (m, 2H),
1.28 (m, 3H), 1.10 (m, 2H). 13C-NMR (125 MHz, CD3OD) δ 209.6, 173.8, 147.7, 137.8, 61.5,
40.4, 39.9, 39.0, 36.9, 33.7, 33.4, 27.5, 26.9, 26.6 (×2), 14.5. HRFABMS m/z 265.1798
[M+H] + (calcd for C16H24O3, 265.1798).
5.1.4. Butyl-2-(2-(cyclohexylmethylene)-3-oxocyclopentyl)acetate (E)-5b
Yellow oil; 66% yield; 1H-NMR (500 MHz, CD3OD) δ 6.39 (dd, J = 10.7, 1.6 Hz, 1H), 4.14
(t, J = 6.6 Hz, 2H), 3.49 (m, 1H), 2.54 (m, 2H), 2.47 (m, 1H), 2.32 (m, 2H), 2.08 (m, 1H),
1.92 (m, 1H), 1.80 (m, 2H), 1.66 (m, 4H), 1.49-1.20 (m, 8H), 0.99 (t, J = 7.4 Hz, 3H). 13C-
NMR (125 MHz, CD3OD) δ 209.1, 173.5, 143.6, 139.5, 65.5, 40.4, 39.6, 36.6, 36.3, 33.1,
33.0, 31.8, 26.8, 26.4, 26.4, 26.3, 20.2, 14.0. HRFABMS m/z 293.2112 [M+H] + (calcd for
C18H28O3, 293.2111).
5.1.5. Butyl-2-(2-(cyclohexylmethylene)-3-oxocyclopentyl)acetate (Z)-5b
Yellow oil; 2% yield; 1H-NMR (500 MHz, CD3OD) δ 5.80 (dd, J = 9.6, 2.1 Hz, 1H), 4.11 (t, J
= 6.6 Hz, 2H), 3.45 (m, 1H), 2.61 (m, 2H), 2.43 (m, 1H), 2.31 (m, 1H), 2.19 (m, 1H), 1.63-
1.51 (m, 6H), 1.39 (m, 6H), 1.22 (m, 2H), 1.08 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H). 13C-NMR
(125 MHz, CD3OD) δ 209.6, 173.8, 147.7, 137.9, 65.4, 40.4, 39.9, 39.0, 36.9, 33.7, 33.4,
31.8, 27.5, 27.0, 26.6 (×2), 20.2, 14.0. HRFABMS m/z 293.2112 [M+H] + (calcd for C18H28O3,
293.2111).
5.1.6. Pentyl-2-(2-(cyclohexylmethylene)-3-oxocyclopentyl)acetate (E)-5c
20

Yellow oil; 50% yield; 1H-NMR (500 MHz, CD3OD) δ 6.38 (dd, J = 10.4, 1.5 Hz, 1H), 4.12
(t, J = 6.7 Hz, 2H), 3.49 (m, 1H), 2.56 (m, 2H), 2.46 (m, 1H) 2.31 (m, 2H), 2.07 (m, 1H), 1.92
(m, 1H), 1.76 (m, 2H), 1.67 (m, 4H), 1.39 (m, 6H), 1.26 (m, 4H), 0.95 (t, J = 7.6 Hz, 3H).
13C-NMR (125 MHz, CD3OD) δ 209.3, 173.6, 143.7, 139.5, 65.8, 40.5, 39.6, 36.6, 36.3, 33.1,
33.0, 29.4, 29.3, 26.8, 26.4 (×2), 26.3, 23.3, 14.3. HRFABMS m/z 307.2268 [M+H] + (calcd
for C19H30O3, 307.2268).
5.1.7. Pentyl-2-(2-(cyclohexylmethylene)-3-oxocyclopentyl)acetate (Z)-5c
Yellow oil; 10% yield; 1H-NMR (500 MHz, CD3OD) δ 5.81 (dd, J = 9.6, 2.2 Hz, 1H), 4.12 (t,
J = 6.7 Hz, 2H), 3.46 (m, 1H), 2.60 (m, 2H), 2.50 (m, 1H) 2.31 (m, 2H), 2.18 (m, 1H), 1.92
(m, 1H), 1.77 (m, 2H), 1.66 (m, 4H), 1.35 (m, 6H), 1.25 (m, 2H), 1.09 (m, 2H), 0.95 (t, J =
7.8 Hz, 3H). 13C-NMR (125 MHz, CD3OD) δ 208.0, 172.6, 146.5, 136.7, 64.5, 39.1, 38.7,
37.8, 35.7, 32.5, 32.1, 28.2, 28.0, 26.3, 25.7, 25.4 (×2), 22.1, 13.0. HRFABMS m/z 307.2268
[M+H] + (calcd for C19H30O3, 307.2268).
5.1.8. Hexyl-2-(2-(cyclohexylmethylene)-3-oxocyclopentyl)acetate (E/Z)-5d
Yellow oil; 60% yield; 1H-NMR (500 MHz, CD3OD) δ 6.38 (dd, J = 10.6, 1.4 Hz, 1H), 4.12
(t, J = 6.4 Hz, 2H), 3.49 (m, 1H), 2.54 (m, 2H), 2.45 (m, 1H) 2.31 (m, 2H), 2.06 (m, 1H), 1.90
(m, 1H), 1.79 (m, 2H), 1.65 (m, 4H), 1.39 (m, 8H), 1.26 (m, 4H), 0.94 (t, J = 7.6 Hz, 3H).
13C-NMR (125 MHz, CD3OD) δ 209.2, 173.6, 143.7, 139.5, 65.8, 40.5, 39.6, 36.6, 36.3, 33.1,
33.0, 32.5, 29.7, 26.8, 26.7, 26.4 (×2), 26.3, 23.6, 14.3. HRFABMS m/z 321.2424 [M+H] +
(calcd for C20H32O3, 321.242).
5.1.9. Isopropyl-2-(2-(cyclohexylmethylene)-3-oxocyclopentyl)acetate (E/Z)-5e
Yellow oil; 45% yield. 1H-NMR (500 MHz, CD3OD) δ 6.34 (dd, J = 10.6, 1.9 Hz, 1H), 4.98
(qd, J = 12.6, 6.2 , Hz, 1H), 3.43 (m, 1H), 2.47 (m, 2H), 2.41 (m, 1H) 2.27 (m, 2H), 2.03 (m,
1H), 1.88 (m, 1H), 1.76 (m, 2H), 1.65 (m, 4H), 1.36 (m, 2H), 1.25 (d, J = 3.1 Hz, 3H), 1.23
(d, J = 3.1 Hz, 3H), 1.18 (m, 2H). 13C-NMR (125 MHz, CD3OD) δ 209.3, 173.1, 143.7, 139.5,
69.4, 40.7, 39.6, 36.7, 36.2, 33.1, 33.0, 26.8, 26.4, 26.3, 26.3, 22.1, 22.0. HRFABMS m/z
279.1955 [M+H] + (calcd for C17H26O3, 279.1955).
21

5.1.10. 2-Hydroxyethyl-2-(2-(cyclohexylmethylene)-3-oxocyclopentyl)acetate (E)-5f
Yellow oil; 40% yield; 1H-NMR (500 MHz, CD3OD) δ 6.38 (dd, J = 10.7, 1.9 Hz, 1H), 4.20
(t, J = 6.7 Hz, 2H), 3.77 (t, J = 6.7 Hz, 2H), 3.47 (m, 1H), 2.67 (m, 1H), 2.46 (m, 1H), 2.33
(m, 2H), 2.21 (m, 1H), 1.66 (m, 8H), 1.73 (m, 6H), 1.25 (m, 5H). 13C-NMR (125 MHz,
CD3OD) δ 209.5, 173.6, 143.8, 139.5, 67.1, 60.9, 40.2, 39.6, 36.7, 36.2, 33.1, 33.0, 26.8, 26.4
(×2), 26.3. HRFABMS m/z 281.1746 [M+H] + (calcd for C16H24O4, 281.1747).
5.1.11. 2-Hydroxyethyl-2-(2-(cyclohexylmethylene)-3-oxocyclopentyl)acetate (Z)-5f
Yellow oil; 10% yield; 1H-NMR (500 MHz, CD3OD) δ 5.84 (dd, J = 9.6, 2.1 Hz, 1H), 4.18 (t,
J = 6.8 Hz, 2H), 3.75 (t, J = 6.8 Hz, 2H), 3.45 (m, 1H), 2.67 (m, 1H), 2.46 (m, 2H) 2.33 (m,
2H), 2.21 (m, 1H), 1.66 (m, 8H), 1.35 (m, 1H), 1.25 (m, 2H), 1.11 (m, 1H). 13C-NMR (125
MHz, CD3OD) δ 209.6, 173.8, 147.8, 137.8, 66.9, 60.9, 40.2, 39.9, 39.0, 36.9, 33.6, 33.3,
27.5, 27.0, 26.6 (×2). HRFABMS m/z 281.1746 [M+H] + (calcd for C16H24O4, 281.1747).
5.1.12. 2-Methoxyethyl -2-(2-(cyclohexylmethylene)-3-oxocyclopentyl)acetate (E/Z)-5g
Yellow oil; 50% yield; 1H-NMR (500 MHz, CD3OD) δ 6.39 (dd, J = 10.7, 1.9 Hz, 1H), 4.27
(t, J = 6.8 Hz, 2H), 3.64 (t, J = 6.6 Hz, 2H), 3.49 (m, 1H), 3.40 (s, 3H), 2.56 (m, 2H), 2.49 (m,
1H) 2.32 (m, 2H), 2.08 (m, 1H), 1.94 (m, 1H), 1.80 (m, 2H), 1.67 (m, 2H), 1.21-1.48 (m, 6H).
13C-NMR (125 MHz, CD3OD) δ 209.3, 173.4, 143.8, 139.4, 71.4, 64.6, 59.0, 40.2, 39.6, 36.6,
36.2, 33.1, 33.0, 26.8, 26.4, 26.3, 26.3. HRFABMS m/z 295.1903 [M+H] + (calcd for
C17H26O4, 295.1904).
5.1.13. 2-(2-Hydroxyethoxy)ethyl-2-(2-(cyclohexylmethylene)-3-oxocyclopentyl)acetate (E)-
5h
Yellow oil; 48% yield;1H-NMR (500 MHz, CD3OD) δ 6.35 (dd, J = 10.7, 1.8 Hz, 1H), 4.24
(t, J = 6.8 Hz, 2H), 3.70 (t, J = 6.6 Hz, 2H), 3.66 (t, J = 6.6 Hz, 2H), 3.56 (t, J = 6.6 Hz, 2H),
3.45 (m, 1H), 2.53 (m, 2H), 2.45 (m, 1H) 2.30 (m, 2H), 2.04 (m, 1H), 1.90 (m, 1H), 1.75 (m,
2H), 1.63 (m, 2H), 1.19-1.48 (m, 6H). 13C-NMR (125 MHz, CD3OD) δ 209.3, 173.4, 143.7,
139.5, 73.6, 70.0, 64.8, 62.1, 40.2, 39.6, 36.6, 36.2, 33.1, 33.0, 26.8, 26.4, 26.3 (×2).
HRFABMS m/z 325.2011 [M+H] + (calcd for C18H28O5, 325.2010).
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5.1.14. 2-(2-Hydroxyethoxy)ethyl-2-(2-(cyclohexylmethylene)-3-oxocyclopentyl)acetate (Z)-
5h
Yellow oil; 6% yield; 1H-NMR (500 MHz, CD3OD) δ 5.81 (dd, J = 9.6, 2.0 Hz, 1H), 4.24 (t, J
= 6.8 Hz, 2H), 3.70 (t, J = 6.6 Hz, 2H), 3.65 (t, J = 6.6 Hz, 2H), 3.56 (t, J = 6.4 Hz, 2H), 3.43
(m, 1H), 2.64 (m, 1H), 2.44 (m, 1H), 2.29 (m, 2H) 2.17 (m, 1H), 1.78-1.56 (m, 6H), 1.32 (m,
2H), 1.21 (m, 2H), 1.07 (m, 2H). 13C-NMR (125 MHz, CD3OD) δ 209.7, 173.7, 147.8, 137.8,
73.6, 70.0, 64.8, 62.2, 40.3, 39.9, 39.0, 36.9, 33.7, 33.3, 27.5, 27.01, 26.6 (×2). HRFABMS
m/z 325.2011 [M+H] + (calcd for C18H28O5, 325.2010).
5.1.15. 2-(2-Methoxyethoxy)ethyl-2-(2-(cyclohexylmethylene)-3-oxocyclopentyl)acetate
(E/Z)-5i
Yellow oil; 54% yield; 1H-NMR (500 MHz, CD3OD) δ 6.40 (dd, J = 10.7, 1.7 Hz, 1H), 4.29
(m, 2H), 3.73 (m, 2H), 3.68 (m, 2H), 3.59 (m, 2H), 3.51 (m, 1H), 3.40 (s, 3H), 2.60 (m, 2H),
2.49 (m, 1H) 2.34 (m, 2H), 2.09 (m, 1H), 1.96 (m, 1H), 1.80 (m, 2H), 1.69 (m, 2H), 1.40 (m,
2H), 1.27 (m, 4H). 13C-NMR (125 MHz, CD3OD) δ 209.2, 173.4, 143.7, 139.5, 72.9, 71.3,
70.0, 64.7, 59.1, 40.2, 39.6, 36.6, 36.2, 33.1, 33.0, 26.8, 26.4, 26.3 (×2). HRFABMS m/z
339.2166 [M+H] + (calcd for C19H30O5, 339.2166).
5.1.16. 2-(2-(2-Hydroxyethoxy)ethoxy)ethyl-2-(2-(cyclohexylmethylene)-3-
oxocyclopentyl)acetate (E)-5j
Yellow oil; 56% yield; 1H-NMR (500 MHz, CD3OD) δ 6.39 (dd, J = 10.7, 1.7 Hz, 1H), 4.29
(t, J = 6.8 Hz, 2H), 3.75 (t, J = 6.6 Hz, 2H), 3.69 (m, 6H), 3.58 (t, J = 6.4 Hz, 2H), 3.50 (m,
1H), 2.57 (m, 2H), 2.50 (m, 1H) 2.34 (m, 2H), 2.09 (m, 1H), 1.95 (m, 1H), 1.79 (m, 2H), 1.69
(m, 2H), 1.42 (m, 2H), 1.27 (m, 4H). 13C-NMR (125 MHz, CD3OD) δ 209.2, 173.4, 143.7,
139.4, 73.6, 71.5, 71.4, 70.0, 64.7, 62.2, 40.2, 39.6, 36.6, 36.2, 33.1, 33.0, 26.8, 26.4, 26.3
(×2). HRFABMS m/z 369.2273 [M+H] + (calcd for C20H32O6, 369.2272).
5.1.17. 2-(2-(2-Hydroxyethoxy)ethoxy)ethyl-2-(2-(cyclohexylmethylene)-3-
oxocyclopentyl)acetate (Z)-5j
Yellow oil; 8% yield; 1H-NMR (500 MHz, CD3OD) δ 5.83 (dd, J = 9.6, 2.0 Hz, 1H), 4.29 (t, J
23

= 6.8 Hz, 2H), 3.73 (t, J = 6.6 Hz, 2H), 3.67 (m, 6H), 3.58 (t, J = 6.4 Hz, 2H), 3.45 (m, 1H),
2.67 (m, 1H), 2.45 (m, 1H) 2.33 (m, 2H), 2.20 (m, 1H), 1.74 (m, 2H), 1.64 (m, 2H), 1.34 (m,
4H), 1.24 (m, 2H), 1.10 (m, 2H). 13C-NMR (125 MHz, CD3OD) δ 209.4, 173.7, 147.7, 137.7,
73.7, 71.5, 71.4, 70.1, 64.6, 62.2, 40.2, 39.9, 39.0, 36.9, 33.7, 33.3, 27.5, 27.0, 26.6 (×2).
HRFABMS m/z 369.2273 [M+H] + (calcd for C20H32O6, 369.2272).
5.1.18. 2-(2-(2-Methoxyethoxy)ethoxy)ethyl-2-(2-(cyclohexylmethylene)-3-
oxocyclopentyl)acetate (E/Z)-5k
Yellow oil; 30% yield; 1H-NMR (500 MHz, CD3OD) δ 6.37 (dd, J = 10.7, 1.8 Hz, 1H), 4.25
(m, 2H), 3.71 (m, 2H), 3.65 (m, 6H), 3.56 (m, 2H), 3.45 (m, 1H), 3.37 (s, 3H), 2.54 (m, 2H),
2.47 (m, 1H) 2.31 (m, 2H), 2.06 (m, 1H), 1.92 (m, 1H), 1.78 (m, 2H), 1.66 (m, 2H), 1.39 (m,
2H), 1.25 (m, 4H). 13C-NMR (125 MHz, CD3OD) δ 209.2, 173.4, 143.7, 139.5, 72.9, 71.5,
71.5, 71.3, 70.0, 64.8, 59.1, 40.2, 39.6, 36.6, 36.2, 33.1, 33.0, 26.8, 26.4, 26.3, 26.3 (×2).
HRFABMS m/z 405.2247 [M+H] + (calcd for C21H34O6, 405.2248).
5.1.19. 2-Hydroxypropyl-2-(2-(cyclohexylmethylene)-3-oxocyclopentyl)acetate (E/Z)-5l
Yellow oil; 64% yield; 1H-NMR (500 MHz, CD3OD) δ 6.38 (dd, J = 10.7, 1.7 Hz, 1H), 4.05
(m, 1H), 3.99 (m, 2H), 3.50 (m, 1H), 2.57 (m, 2H), 2.49 (m, 1H) 2.33 (m, 2H), 2.07 (m, 1H),
1.93 (m, 1H), 1.79 (m, 2H), 1.66 (m, 2H), 1.38 (m, 2H), 1.30 (m, 4H), 1.21 (d, J = 1.9 Hz,
3H). 13C-NMR (125 MHz, CD3OD) δ 209.3, 173.4, 143.7, 139.5, 70.4, 66.3, 40.2, 39.6, 36.6,
36.2, 33.1, 33.0, 26.8, 26.4 (×2), 26.3, 19.6. HRFABMS m/z 295.1904 [M+H] + (calcd for
C17H26O4, 295.1904).
5.1.20. Synthesis of compounds 7a-7b
Acyl chloride 6, which was prepared by reaction of acid 4 with an excess amount of oxalyl
chloride in dichloromethane at 45°C for 1 h, was treated with isopropylamine and N-
methylethanolamine in the presence of TEA (1 equiv), N,N-dicyclohexylcarbodiimide (DCC)
(0.1 equiv), 1-hydroxybenzotriazole (HOBT) (0.1 equiv) in dichloromethane at room
temperature for 12 h to yield the intermediate esters 7a–7b. The mixture was washed with
water, dried, concentrated to give a residue and was then purified by reversed-phase HPLC
(YMC ODS-Triart C18 column) to yield the target amides 7a–7b.
24

5.1.21. 2-(2-(Cyclohexylmethylene)-3-oxocyclopentyl)-N-isopropylacetamide (E/Z)-7a
Yellow oil; 55% yield; 1H-NMR (500 MHz, CD3OD) δ 6.44 (dd, J = 10.7, 1.8 Hz, 1H), 3.99
(m, 1H), 3.52 (m, 1H), 2.67 (m, 1H), 2.44 (m, 1H) 2.26 (m, 1H), 2.07 (m, 1H), 1.77 (m, 3H),
1.70 (m, 4H), 1.38 (m, 3H), 1.25 (m, 4H), 1.16 (d, J = 6.6 Hz, 3H), 1.12 (d, J = 6.6 Hz, 3H).
13C-NMR (125 MHz, CD3OD) δ 197.1, 172.5, 144.7, 141.9, 43.3, 39.9, 34.0, 33.2 (×2), 32.5,
26.8, 26.5, 26.4, 25.1 (×2), 22.8 (×2). HRFABMS m/z 278.2115 [M+H] + (calcd for
C17H27NO2, 278.2115).
5.1.22. 2-(2-(Cyclohexylmethylene)-3-oxocyclopentyl)-N-(2-hydroxyethyl)-N-methylacetami-
de (E/Z)-7b
Yellow oil; 56% yield; 1H-NMR (500 MHz, CD3OD) δ 6.36 (dd, J = 10.9, 1.8 Hz, 1H), 3.71
(m, 2H), 3.56 (m, 2H), 3.50 (m, 1H), 3.00 (s, 3H), 2.75 (m, 1H), 2.54 (m, 1H), 2.48 (m, 1H)
2.29 (m, 2H), 2.02 (m, 1H), 1.89 (m, 1H), 1.79 (m, 2H), 1.67 (m, 2H),1.39 (m, 2H), 1.25 (m,
4H). 13C-NMR (125 MHz, CD3OD) δ 209.9, 174.3, 143.3, 140.5, 60.1, 53.1, 39.6, 39.0, 37.6,
36.7, 36.5, 34.0, 33.1 (×2), 26.8, 26.4 (×2). HRFABMS m/z 294.2061 [M+H] + (calcd for
C17H27NO3, 294.2064).
5.2. Biological methods
5.2.1. Cell culture and cell viability
RAW264.7 murine macrophages were purchased from the Korean Cell Line Bank
(KCLB®, Seoul, Korea). Rat liver Ac2F cells were obtained from the American Type Culture
Collection (ATCC, Rockville, MD, USA). Cells were cultured at 37°C in a 5% CO2
humidified incubator and maintained in high glucose Dulbecco’s Modified Eagle Medium
(DMEM, Nissui, Tokyo, Japan) containing 100 mg/mL streptomycin, 2.5 mg/L amphotericin
B, and 10% heat-inactivated fetal bovine serum (FBS). Suspensions of tested cell lines (cal.
1.0 × 104 cells/well) were seeded in a 96-well culture plates and cultured for 12 h followed by
treatment with various diluted concentrations of test compounds for 24 h. Control cultures
were treated with culture medium alone. The tested compounds were evaluated at two
dilutions and the highest concentration was 50 μM. Cell viabilities were evaluated using a
25

water soluble tetrazolium (WST) reagent (EZ-CyTox, Daeil Lab Service Co., Ltd., Seoul,
Korea), which was added to each well (10 μL) and incubated at 37oC for 1 h. Absorbances
were read using an iMark Microplate Absorbance Reader (Bio-Rad Laboratories, Hercules,
CA, USA) at a wavelength of 450 nm. Cells in the exponential phase were used for all
experiments.
5.2.2. Luciferase assay
Rat liver Ac2F cells were obtained from the American Type Culture Collection (ATCC,
Rockville, MD, USA). Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM,
Nissui, Tokyo) containing 2 mM L-glutamine, 100 mg/mL streptomycin, 2.5 mg/L
amphotericin B and 10% fetal bovine serum (FBS) and maintained in a humidified
atmosphere containing 5% CO2 at 37°C. The TK-PPRE × 3-luciferase reporter plasmid,
containing 3 copies of the PPRE in the acyl CoA oxidase promoter was generously donated
by Dr. Chistopher K. Glass (University of California at San Diego, La Jolla, CA, USA). The
pcDNA3 expression vector and full-length human PPARγ1 expression vector (pFlag-
PPARγ1) were generously donated by Dr. Chatterjee (the University of Cambridge,
Addenbrooke’s Hospital at Cambridge, UK). The Lipofectamine™ 2000 transfection reagent
was generously donated by Invitrogen Co. (Carlsbad, CA, USA). For the luciferase assays,
proper plasmids were transfected into Ac2F cells in a 48-well plate (5 × 104 cells/well) with
effector plasmids, and TK-PPRE × 3-luciferase reporter plasmid, pcDNA3, and pFlag-
PPARγ1 using Lipofectamine™ 2000 according to the manufacturer’s instructions. After
transfection for 4 h, the conditioned media was removed and replaced with complete media,
and the cells were incubated for an additional 20 h. The medium was then removed, and cells
were exposed in serum-free media to rosiglitazone or test compounds for 6 h. Washed with
PBS and assayed using Steady-Glo Luciferase Assay System (Promega, Madison, WI, USA).
Luciferase activity was measured using a GloMax-Multi Microplate Multimode Reader
(Promega Corporation, CA, USA).
5.2.3. In vitro stability assessment assay
26

The mixture of the synthesized compounds (0.5 mg) and 50 μL of porcine liver esterase
(Sigma Chemical; Lot no. E2884-1KU: 1000U/0.34Ml; 1 unit (1U) of the enzyme hydrolyzes
1 μmol of an ester) in 0.01 M phosphate buffer solution (pH 7.4, 1 mL) and acetone (12 μL)
was incubated at 37ºC. At regular intervals (30 and 60 min), the reacted products (500 μL)
were withdraw and directly injected into Shimadzu LC-20A HPLC with a YMC ODS-Triart
C18 column (250 × 10 mm, 5 µm, 120 Å) for analysis using 5-95% gradient MeOH as the
mobile phase, with a flow rate of 1 mL/min, and the UV detection at 210 and 254 nm. The
ratio of intact esters/amides and their corresponding acids were analyzed by calculating the
areas size.
5.2.4. Production levels of NO and cytokines released into the medium
RAW264.7 macrophages (cal. 1 × 104 cells/well) were seeded in a 96-well culture plate
and cultured for 12 h. Cells were pre-treated with various concentrations of the drugs for 1 h
and then co-incubated with 25 ng/mL of LPS for 24 h. NO concentrations in the medium were
determined using Griess assay. Griess reagent (80 μL) was added to the media supernatants
(80 μL) and then incubated at 37°C for 15 min in the dark. Absorbance was measured at 520
nm using an iMark Microplate Absorbance Reader (Bio-Rad Laboratories, Hercules, CA,
USA). NO concentrations were calculated using 0-100 μM sodium nitrite standards. TNF-α
and IL-6 expression levels in culture medium were quantified using a sandwich-type ELISA
kit (Biolegend, San Diego, CA, USA). Absorbance was measured at 450 nm.
5.2.5. Immunofluorescence staining of NF-κB p65 in RAW264.7 cells
Cells were grown on a confocal dish and treated with the compound for 24 h and then
co-incubated with 1 g/mL of LPS for 30 min. Cells were then fixed in 10% formalin solution
for 15 min, washed thrice with PBS, treated with 0.5% (v/v) Triton X-100/PBS for 15 min,
washed thrice with PBS, and then blocked at room temperature for 30 min in 10% FBS/PBS.
Cells were then incubated with rabbit anti-NFκB-p65 antibody (Cell signaling technology,
USA) at 4ºC overnight, washed thrice with PBS, incubated for 30 min at room temperature
with secondary antibody anti-rabbit Alexa 488 (Cell signaling technology, USA) as the
27

molecular probe, washed thrice with PBS, and then incubated with PI/Rnase (10 µg/mL) at
room temperature for 20 min. The location of NFκB-p65 was determined by confocal
microscopy FluoView FV10i (Olympus, Australia) using an excitation wavelength of 488 nm
and an emission wavelength of 537 nm.
5.2.6. Western blot assay
RAW264.7 cells were harvested and suspended in lysis buffer containing protease and
phosphatase inhibitor cocktails. Protein concentrations were determined using a BCA protein
assay (Thermo Scientific, Rockford, IL, USA). Equal amounts of protein were resolved by
10% SDS-polyacrylamide gel electrophoresis and were electrophoretically transferred to
polyvinylidene difluoride (PVDF) membranes; these were then blocked in Tris-buffered
saline containing 0.1% Tween 20 (TBS-T) and 5% skimmed milk for 1 h at room
temperature, and then incubated with specific primary antibodies (Cell Signaling Technology,
Danvers, MA, USA) overnight at 4°C. Anti-rabbit IgG-HRP was used as the secondary
antibody. Signals were developed using the ChemiDoc™Touch Imaging System (Bio-Rad
Laboratories, Hercules, CA, USA).
5.2.7. Reactive oxygen species (ROS) measurement
RAW264.7 macrophages were grown on a confocal dish and treated with the test
compound for 1 h. LPS solution was added into the dish at a final concentration of 1 μg/mL
and continually cultured for 24 h. The medium was removed and washed with PBS; DCFH-
DA diluted in FBS-free medium to a final concentration of 100 μM was added and cultured at
37°C for 30 min. The medium was removed and the cells were washed once with PBS. The
fluorescence was observed using a confocal microscope (FluoView FV10i; Olympus,
Australia) with an excitation wavelength of 485 nm and an emission wavelength of 520 nm.
Quantification of cellular oxidative stress was performed by using a previously reported
method [30,31]. RAW264.7 macrophages (cal. 1 × 104 cells/well) were seeded into black 96-
well cell culture plates and cultured for 12 h. The cells were pre-treated with the test
compound for 1 h and then co-incubated with 1 μg/mL LPS for 24 h. The medium was
removed and washed with PBS; 100 μL DCFH-DA diluted in FBS-free medium to a final
28

concentration of 100 μM was added and cultured at 37°C for 30 min. The medium was then
removed, 100 μL PBS was added to each well, and fluorescence was detected at an excitation
wavelength of 485 nm and an emission wavelength of 520 nm using the TriStar LB 941
Multimode Microplate Reader (Bad Wildbad, Germany).
5.2.8. Statistical analysis
The significance of intergroup differences was determined by one-way analysis of
variance (ANOVA) using SPPS 13.0. Results are expressed as the mean ± SD of the indicated
numbers of independent experiments. Values of p < 0.05 were considered statistically
significant.
5.2.9. Molecular Modeling
Docking calculations were performed using Auto Dock Vina 1.1.2 software. The default
settings and scoring function of Vina were applied. The AMBER force field was applied for
energy minimization. For ligand preparation, Chem3D Ultra 8.0 software was used to convert
the 2D structures of the candidates into 3D structural data and to prepare the chemical
structures with minimized energy. Protein coordinates were downloaded from the Protein
Data Bank (accession code 2PRG). Chain A was prepared for docking within the molecular
modeling software package, Chimera 1.5.3, by removing chain B and all ligands and water
molecules (except water molecules 308, 399, 444, and 467), and by calculating the
protonation state of the protein. Addition of polar hydrogen and setting of grid box parameters
was performed using MGLTools 1.5.4. PyMol v1.5 was used to analyze and visually
investigate the ligand-protein interactions of the docking poses.
Disclosure statement
No potential conflict of interest was reported by the authors.
29