Synthesis of phthalimide derivatives as potential PPAR-γ ligands

Article abstract of DOI:10.3390/md14060112

Paecilocin A, a phthalide derivative isolated from the jellyfish-derived fungus Paecilomyces variotii, activates PPAR-γ (Peroxisome proliferator-activated receptor gamma) in rat liver Ac2F cells. Based on a SAR (Structure-activity relationships) study and in silico analysis of paecilocin A-mimetic derivatives, additional N-substituted phthalimide derivatives were synthesized and evaluated for PPAR-γagonistic activity in both murine liver Ac2F cells and in human liver HepG2 cells by luciferase assay, and for adipogenic activity in 3T3-L1 cells. Docking simulation indicated PD6 was likely to bind most strongly to the ligand binding domain of PPAR-γ by establishing crucial H-bonds with key amino acid residues. However, in in vitro assays, PD1 and PD2 consistently displayed significant PPAR-activation in Ac2F and HepG2 cells, and adipogenic activity in 3T3-L1 preadipocytes.

Full text of DOI:10.3390/md14060112

marine drugs
Communication
Synthesis of Phthalimide Derivatives as Potential
PPAR-γ Ligands
So Hyeon Eom 1,†, Sen Liu , Mingzhi Su , Tae Hwan Noh , Jongki Hong , Nam Deuk Kim ,
1,†
1
1
2
1
1
1,
1,
Hae Young Chung , Min Hye Yang * and Jee H. Jung *
1
College of Pharmacy, Pusan National University, Busan 46241, Korea; god5425um@naver.com (S.H.E.);
ls691392008@sina.com (S.L.); sumingzhi0310@gmail.com (M.S.); notea151@naver.com (T.H.N.);
nadkim@pusan.ac.kr (N.D.K.); hyjung@pusan.ac.kr (H.Y.C.)
College of Pharmacy, Kyung Hee University, Seoul 02447, Korea; jhong@khu.ac.kr
2
*
Correspondence: jhjung@pusan.ac.kr (J.H.J.); mhyang@pusan.ac.kr (M.H.Y.); Tel.: +82-51-510-2803 (J.H.J.);
82-51-510-2811 (M.H.Y.); Fax: +82-51-513-6754 (J.H.J. & M.H.Y.)
These authors equally contributed to this work.
+
†
Academic Editor: Orazio Taglialatela-Scafati
Received: 1 April 2016; Accepted: 30 May 2016; Published: 8 June 2016
Abstract: Paecilocin A, a phthalide derivative isolated from the jellyfish-derived fungus
Paecilomyces variotii, activates PPAR-γ (Peroxisome proliferator-activated receptor gamma) in rat
liver Ac2F cells. Based on a SAR (Structure-activity relationships) study and in silico analysis of
paecilocin A-mimetic derivatives, additional N-substituted phthalimide derivatives were synthesized
and evaluated for PPAR-
cells by luciferase assay, and for adipogenic activity in 3T3-L1 cells. Docking simulation indicated
PD6 was likely to bind most strongly to the ligand binding domain of PPAR- by establishing crucial
H-bonds with key amino acid residues. However, in in vitro assays, PD1 and PD2 consistently
displayed significant PPAR- activation in Ac2F and HepG2 cells, and adipogenic activity in
T3-L1 preadipocytes.
γ agonistic activity in both murine liver Ac2F cells and in human liver HepG2
γ
γ
3
Keywords: PPAR-γ agonist; paecilocin A; type 2 diabetes; phthalimide; adipogenesis; 3T3-L1
1
. Introduction
Type 2 diabetes mellitus (T2DM) is one of the most common serious metabolic diseases.
T2DM results in insulin resistance and damages
impaired insulin secretion, increased hepatic glucose production, and decreased insulin-stimulated
glucose uptake by peripheral tissues [ ]. Peroxisome proliferator-activated receptors (PPARs) are
ligand-activated nuclear receptors that regulate transcription factors. The three known PPAR isoforms
are PPAR- , - , and - , and PPAR- is known to increase insulin sensitivity in adipose, muscle, and
]. Thiazolidinediones (TZDs) (e.g., troglitazone, pioglitazone, and rosiglitazone) are
agonists that are used as anti-diabetic drugs [ ] and improve insulin resistance in liver
-cell function [ ]. However, TZDs have been associated
with undesirable side effects, such as hepatotoxicity and edema [ ]. Therefore, research interest in
β-cell function [1]. Consequently, T2DM exhibits
2
α
β/
δ
γ
γ
hepatic tissues [
3–5
a class of PPAR-
γ
6
and skeletal muscle and preserve pancreatic
β
7
8,9
this area is focused on the discovery of novel PPAR-γ agonists with fewer adverse effects [10].
In our previous study, paecilocin A (Figure 1), a new phthalide derivative isolated from the
jellyfish-derived fungus Paecilomyces variotii, showed PPAR-γ agonistic activity [11]. Notably, the
plant-derived phthalides 3-butylphthalide, 3-butylidenephthalide, and ligustilide have been patented
as effective agents for the treatment of diabetes mellitus type 2 [12]. The mechanism responsible for
their activities was surmised to be PPAR-γ activation. The findings of our previous pharmacophore
study suggested the 3-hydroxy phthalide moiety of paecilocin A serves as a hydrophilic head that binds
Mar. Drugs 2016, 14, 112; doi:10.3390/md14060112
www.mdpi.com/journal/marinedrugs

Mar. Drugs 2016, 14, 112
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to the ligand binding domain (LBD) of PPAR-
γ
and forms H-bonds with key amino acid residues [13].
Based on analyses of the structures of paecilocin A and TZDs, N-substituted phthalimide derivatives
(PD1–PD4) were synthesized and evaluated [14].
In the present study, additional N-substituted phthalimide derivatives PD5 and PD6 were
synthesized based on in silico analysis. In a docking simulation, PD5 and PD6, which are hydroxylated
at C-4 rather than at C-3, showed high affinity and formed H-bonds with the key amino acid residues
of the LBD of PPAR-
and compared with PD1
of adipogenic marker genes [15], these analogues were also evaluated for adipogenic activity on
γ
. Therefore, PD5 and PD6 were synthesized as potential PPAR-
γ agonists
–
PD4. Since PPAR- is a key transcriptional factor for the inductions
γ
3
T3-L1 preadipocytes.
O
paecilocin A
O
OH
Figure 1. Chemical structure of paecilocin A.
2
. Results and Discussion
2
.1. Docking Simulation
In our previous study, a series of N-substituted phthalimide derivatives, including PD1–PD4,
were synthesized based on the potential pharmacophore of paecilocin A, and PD1 was proposed
as an interesting lead [14]. Further docking analysis of derivatives (PD5 and PD6) hydroxylated at
C-4 showed that they both bind to LBD of PPAR-
γ
and form H-bonds with key amino acid residues
(
Figure 2). In particular, docking analysis showed PD6 forms H-bonds with Tyr473, Ser289, and Gln286
in the PPAR-γ binding pocket with higher binding affinity than PD1. Therefore, PD5 and PD6 were
synthesized as potential PPAR-γ agonists and compared with PD1–PD4.
Figure 2. Docking simulations of rosiglitazone, PD1, PD5, and PD6 with the LBD of PPAR-γ.
A) Rosiglitazone interacts with key amino acid residues (Tyr473, His , His , Ser , and Gln286) in
449
323
289
(
the PPAR-
γ
binding pocket (
´
8.6 kcal/moL). The docking pose of rosiglitazone was simulated using
PD1 8.3 kcal/moL), interacts with key amino acid
binding pocket; ( PD5 8.1 kcal/moL) interacts
binding pocket; ( PD6
the reported co-crystal structure (PDB: 2PRG); (
B
)
γ
(´
residues (Tyr4 , His , and Ser ) in the PPAR-
73
323
289
C
)
(´
with key amino acid residues (Tyr , Gln286, and Ser289) in the PPAR-
473
γ
D)
´10.0 kcal/moL) interacts with key amino acid residues (Tyr , Ser289, Gln286 and Arg288) in the
473
(
PPAR-γ binding pocket.

Mar. Drugs 2016, 14, 112
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2
.2. Synthesis of Analogues
N-substituted phthalimide can be prepared by heating anhydride with various N-containing
reagents [16]. According to the previous study, the introduction of hydroxyl groups would allow the
phthalimide moiety to be more effective as a polar head group and this modification could be further
extended using linker and tail groups to generate potential PPAR-γ ligands. Compounds PD1–PD4
were prepared according to the protocol in our previous report [14]. Then 3-Hydroxyphthalic
anhydride was treated with tyramine in aqueous glacial acetic acid to afford phthalimide
derivative PD1. Compound PD1 was further derivatized by treating with iodopropane, iodobutane, or
benzyl chloride to afford PD2, PD3, and PD4, respectively. Commercially available 4-hydroxyphthalic
acid was added to acetic anhydride and then treated with pyridine to afford 4-hydroxyphthalic
anhydride. Then 4-Hydroxyphthalic anhydride was subsequently treated with tyramine in aqueous
glacial acetic acid to afford PD5 (Schemes 1 and 2).
Scheme 1. Synthesis of phthalimide derivatives. Reagents and conditions: (a) Ac O, pyridine, rt, 12 h;
2
˝
b) tyramine, aqueous CH COOH, reflux 85 C, overnight.
3
(
Compound PD6 was synthesized by treating PD5 with benzyl chloride at room temperature for
4
h (Scheme 2).
Scheme 2. Synthesis of phthalimide derivatives. Reagents and conditions: (a) RI (n-PrI, n-BuI), Ag O,
2
CH CN, 12h; (b) benzyl chloride, K CO , NaI, DMF, rt, 4 h.
3
2
3
2
.3. Biological Evaluation
2
.3.1. PPAR-γ Activation
PPARs are ligand-activated transcription factors. PPAR-γ binding of ligands results in up-regulation
of the expression levels of mRNAs encoded by PPAR target genes [17]. In the luciferase assay,
activation of a receptor by ligands leads to the induction of reporter enzyme expression, which can be

Mar. Drugs 2016, 14, 112
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assayed using a photometric plate reader. Synthesized PD5 and PD6 were subsequently evaluated
for PPAR- activation using a luciferase assay in rat liver Ac2F cells. Initially, they were compared
with rosiglitazone and PD1 PD4 with respect to PPAR- activation at concentrations of 10 and 25
Figure 3). Compound PD5 induced significantly more PPAR- activation than untreated controls,
γ
–
γ
µM
(
γ
but it was not as potent as PD1. Furthermore, PD6 was not as potent as was indicated by docking
simulation. It would appear that the hydroxyl at C-4 of the phthalimide moiety is not as useful for
binding to the LBD of PPAR-γ in Ac2F cells as hydroxyl at C-3.
Figure 3. PPAR-
phthalimides PD1
Cells were transiently transfected by PPRE together with pFlag-PPAR-
γ
activation by PD1–PD6 in Ac2F cells. In vitro assay of PPAR-
PD6 and rosiglitazone at concentrations of 10 and 25 M in rat liver Ac2F cells.
1. Rosiglitazone was used as
γ activation by
~
µ
γ
positive control to monitor the activation of luciferase reporter. Control cells were transfected with
plasmid containing PPRE and pcDNA3. Luciferase expressions (folds of control) are presented as
means ˘ SDs (n = 3). * p < 0.05 versus control. *** p < 0.001 versus control.
Analogues PD1
at concentrations of 10 and 25 µM (Figure 4). PD1 and PD2 induced significant PPAR-
M. Although PD1 PD6 were not as potent as rosiglitazone in HepG2 or Ac2F cells, PD1 consistently
significantly activated PPAR-γ in both cell lines.
–PD6 were further evaluated for PPAR-
γ
activation using human liver HepG2 cells
γ
activation at
25
µ
~
Figure 4. PPAR-
PD1 PD6 and rosiglitazone were investigated at 10
transiently transfected with PPRE plus pFlag-PPAR- 1. Rosiglitazone was used as positive control to
γ
activation by PD1–PD6 in HepG2 cells. In vitro PPAR-
γ activations by phthalimides
–
µ
M and 25 M in HepG2 cells. Cells were
µ
γ
monitor the activation of luciferase reporter. Luciferase expressions (folds of untreated controls) are
presented as means ˘ SDs (n = 3). * p < 0.05 versus untreated controls.

Mar. Drugs 2016, 14, 112
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2
.3.2. Effect on Adipocyte Differentiation in 3T3-L1 Cells
PPAR- is a key transcription factor for the induction of adipogenic marker genes, and PPAR-
agonists induce adipogenesis of preadipocytes into mature adipocytes. To examine whether PD1
γ
γ
–
PD6
induce adipocyte differentiation, 3T3-L1 preadipocytes were treated with insulin and dexamethasone
in the presence of various concentrations of PD1 PD6 for eight days. Rosiglitazone was used as a
positive control, as it is known to have a marked adipogenic effect on preadipocytes in vitro. Significant
induction of adipocyte differentiation by rosiglitazone was observed at a concentration of 1 M by
Oil red O staining (Figure 5A), and this correlated well with observed increases in lipid accumulation
–
µ
(
(
Figure 5B). PD1 and PD2 significantly stimulated adipogenesis in a concentration-dependent manner
1~20 M), whereas PD3 PD6 did not significantly increase adipogenesis. Furthermore, the adipogenic
µ
–
profiles of PD1–PD6 correlated fairly well with PPAR-γ activations in Ac2F and HepG2 cells.
Figure 5. Effects of PD1–PD6 on adipocyte differentiation of 3T3-L1 cells. The 3T3-L1 cells were
exposed to 10% FBS/DMEM containing 10 g/mL insulin and 1 M dexamethasone in the presence
of various concentrations (1, 10, or 20 M) of PD1 PD6 or rosiglitazone (1 M). After eight days,
treated cells were stained with Oil red O for morphological assessment by microscopy ( ) and lipid
contents was determined using an iMark Microplate Absorbance Reader ( ). The results shown are
SDs (n = 3). * p < 0.05
µ
µ
µ
–
µ
A
B
representative of three independent experiments, and are presented as means
versus untreated controls.
˘

Mar. Drugs 2016, 14, 112
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3
. Experimental Section
3
.1. Chemistry
The H and 13C NMR spectra were recorded on a Varian Unity 400 MHz NMR spectrometer,
1
and chemical shifts are reported with respect to residual solvent or deuterated solvent peaks (
δ 3.30
H
and
δ 49.0 for CD OD, δ 7.24 and δ 76.8 for CDCl ). ESI MS data were obtained using a Agilent
C 3 H C 3
6
530 accurate-mass Q-TOF MS spectrometer .HPLC was performed using a YMC ODS-H80 column
(
250 10 mm, 4 m, 80 Å) or a C18-5E Shodex packed column (250 10 mm, 5 m, 100 Å) and
ˆ
µ
ˆ
µ
a Shodex RI-71 detector. All reagents were purchased from Sigma-Aldrich and used as received.
A mixture of tyramine (1.2 equivalents) and 3-hydroxypthalic anhydride in aqueous glacial acetic
acid (1 M) was stirred under reflux overnight. Products were precipitated by adding water, filtering,
and washing thoroughly with water. Residues were diluted with MeOH, dried using MgSO , and
4
evaporated to provide the crude products [12].
1
3
δ
7
-Hydroxy-N-(p-hydroxy-phenethyl)phthalimide (PD1). White powder; H NMR (400 MHz, CD OD):
3
2.82 (t, J = 7.2 Hz, 2H), 3.75 (t, J = 7.6 Hz, 2H), 6.64 (d, J = 8.4 Hz, 2H), 6.98 (d, J = 8.4 Hz, 2H),
+
.08 (d, J = 8.0 Hz, 1H), 7.24 (d, J = 7.2 Hz, 1H),
δ
7.53 (dd, J = 7.2, 7.6 Hz, 1H). ESI MS m/z 284.0912 [M + H] .
To a solution of 3-hydroxy-N-(p-hydroxy-phenethyl)phthalimide PD1 (13 mg, 0.046 mM) in
CH CN (1.5 mL), RI (CH (CH ) I: 9 L, ca. 0.09 mM; CH (CH ) I: 10 L, ca. 0.09 mM) and Ag O
µ
µ
3
3
2 2
3
2 3
2
(10 mg, 0.04 mM) were added. The mixture was then heated under reflux with stirring for 12 h. Solid
material was removed by filtration and solvent by evaporation. The solid material so obtained was
purified by RP-HPLC using 90% aqueous MeOH as eluent to give PD2 and PD3.
1
3
δ
δ
δ
-Hydroxy-N-(p-propoxy-phenethyl)phthalimide (PD2). White powder; H NMR (400 MHz, CDCl ):
3
1.09 (t, J = 7.2 Hz, 3H),
6.74 (d, J = 6.4 Hz, 2H),
7.61 (t, J = 7.8 Hz, 1H); 13C NMR (100 MHz, CDCl3):
δ
1.92 (m, 2H),
δ
2.89 (m, 2H),
7.16 (d, J = 8.0 Hz, 1H),
168.2, 167.1, 156.4, 154.4, 136.0, 134.4, 130.4,
δ
3.82 (t, J = 6.0 Hz, 2H),
δ
4.13 (t, J = 6.0 Hz, 2H),
δ
7.11 (d, J = 6.8 Hz, 2H),
δ
δ 7.39 (d, J = 7.2 Hz, 1H),
δ
1
30.2, (2C overlapped), 118.7, 117.5, 115.5, (2C overlapped), 115.4, 70.9, 39.5, 33.9, 22.5, 10.5. ESI MS
+
m/z 326.1381 [M + H] .
3
δ
2
-Hydroxy-N-(p-butoxy-phenethyl)phthalimide (PD3). White powder; 1H NMR (400 MHz, CDCl3):
0.99 (t, J = 7.4 Hz, 3H), 1.52 (m, 2H), 1.87 (m, 2H), 2.89 (m, 2H), 3.82 (m, 2H), 4.17 (t, J = 6.8 Hz
6.75 (d, J = 8.0 Hz, 2H), 7.12 (d, J = 8.2 Hz, 2H), 7.17 (d, J = 8.6 Hz, 1H), 7.39 (d, J = 7.6 Hz,
7.61 (m, 1H); 13C NMR (100 MHz, CDCl3):
168.2, 167.1, 156.4, 154.4, 136.0, 134.4, 130.4, 130.2,
δ
δ
δ
δ
δ
,
H),
H),
δ
δ
δ
δ
δ
1
δ
(
3
2C overlapped), 118.6, 117.5, 115.5, (2C overlapped), 115.4, 69.2, 39.5, 33.9, 31.1, 19.3, 14.0. ESI MS m/z
40.1543 [M + H] .
+
To a suspension of 3-hydroxy-N-(p-hydroxy-phenethyl) phthalimide PD1 (13.4 mg, 0.047 mM),
K CO (6.5 mg, 0.047 mM), and NaI (2.6 mg, 0.02 mM) in DMF (2 mL) was added benzyl chloride
2
3
˝
(
6.0 µL, ca. 0.06 mM). The mixture was then stirred for 30 min at 0 C and for 4 h at room temperature,
acidified with aqueous 6 M HCl, and extracted with EtOAc. The organic layer was sequentially washed
with H O and brine, dried with MgSO , and evaporated to give the crude product, which was purified
2
4
by RP-HPLC using 85% aqueous MeOH as eluent to give PD4.
1
3
-Hydroxy-N-(p-benzyl-phenethyl)phthalimide (PD4). White powder; H NMR (400 MHz, CDCl ):
3
δ
2.91 (t, J = 8.0 Hz, 2H),
δ
3.85 (t, J = 7.2 Hz, 2H),
δ
5.33 (s, 2H),
δ
6.75 (d, J = 8.0 Hz, 2H),
δ
7.12 (d,
J = 8.4 Hz, 2H),
δ
7.19 (d. J = 8.4 Hz, 1H),
δ
7.33 (m, 1H),
δ
7.40 (t, J = 7.2 Hz, 3H), δ 7.49 (d, J = 7.6 Hz,
2H), δ δ 168.0, 166.9, 155.7, 154.4, 136.0, 136.0,
7.58 (t, J = 8.4 Hz, 1H); 13C NMR (100 MHz, CDCl3):
1
1
34.4, 130.4, 130.2, (2C overlapped), 128.9, (2C overlapped), 128.3, 127.0, (2C overlapped), 119.5, 118.1,
15.9, 115.5, (2C overlapped), 71.0, 39.5, 33.9 . ESI MS m/z 374.1387 [M + H] .
+
Next 4-Hydroxy phthalic acid (182 mg, 1.0 mM) was added to acetic anhydride (5 mL) in a flask,
and then 1 mL of pyridine was added with stirring in an iced-water bath. After 10 min, the reaction
mixture was stirred for 12 h at room temperature, and the resultant solution was slowly acidified with

Mar. Drugs 2016, 14, 112
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0
.01 M HCl with stirring in an iced-water bath. The large amount of precipitate produced was collected
by filtration [18]. A mixture of tyramine (164 mg, 1.2 mM) and this precipitate in aqueous glacial acetic
acid (1 M) was then stirred under reflux overnight. Solvent was removed by evaporation, and the solid
material so obtained was purified by RP-HPLC using 65% aqueous MeOH as eluent to give PD5.
1
4
δ
2
-Hydroxy-N-(p-hydroxy-phenethyl)phthalimide (PD5). White powder; H NMR (400 MHz, CD OD):
3
2.83 (m, 2H),
δ
3.78 (m, 2H),
δ
6.66 (d, J = 8.8 Hz, 2H),
δ
7.00 (d, J = 8.4 Hz, 2H),
7.64 (d, J = 8.0 Hz, 1H); 13C NMR (100 MHz, CDCl3):
δ
7.07 (dd, J = 8.2,
169.7,
.2 Hz, 1H), 7.14 (d, J = 2.0 Hz, 1H),
δ
δ
δ
1
4
69.6, 164.9, 157.1, 136.0, 130.8, (2C overlapped), 130.3, 126.1, 123.6, 121.3, 116.2, (2C overlapped), 110.9,
0.5, 34.6. ESI MS m/z 284.0913 [M + H] .
+
To a suspension of 4-hydroxy-N-(p-hydroxy-phenethyl) phthalimide PD5 (30.2 mg, 0.106 mM),
K CO (14.7 mg, 0.106 mM), and NaI (6.89 mg, 0.053 mM) in DMF (5 mL) was added benzyl chloride
2
3
˝
(
13.5
µL, ca. 0.13 mM). The mixture was then stirred for 30 min at 0 C and for 4 h at room temperature,
acidified with aqueous 6 M HCl, and extracted with EtOAc. The organic layer was sequentially washed
with H O and brine, dried with MgSO and evaporated to give the crude product, which was purified
2
4
by RP-HPLC using 85% aqueous MeOH as eluent to give PD6.
1
4
δ
-Hydroxy-N-(p-benzyl-phenethyl)phthalimide (PD6). White powder; H NMR (400 MHz, DMSO-d6):
2.77 (t, J = 7.2 Hz, 2H), 3.70 (t, J = 7.2 Hz, 2H), 5.29 (s, 2H), 6.62 (d, J = 8.4 Hz, 2H), 6.95 (d,
7.35 (m, 2H), 7.41 (m, 3H), 7.48 (m, 2H), 7.76 (d, J = 8.4 Hz, 1H), 9.21 (s, 1H);
C NMR (100 MHz, DMSO-d6): 167.4, 167.3, 163.4, 155.8, 136.1, 134.1, 129.5, (2C overlapped), 128.6,
δ
δ
δ
δ
J = 8.4 Hz, 2H),
δ
δ
δ
δ
δ
13
δ
(
7
2C overlapped), 128.3, 128.2, 127.9, (2C overlapped), 124.9, 123.5, 120.5, 115.2, (2C overlapped), 108.9,
0.2, 39.8, 32.9. ESI MS m/z 384.1385 [M + H] .
+
3
.2. Cell Culture
Rat liver (Ac2F) cells were obtained from the American Type Culture Collection (ATCC, Rockville,
MD, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Nissui, Tokyo, Japan)
containing 2 mM l-glutamine, 100 g/mL streptomycin (HyClone, Logan, UT, USA), 2.5 mg/L
amphotericin B (HyClone, Logan, UT, USA), and 10% heat-inactivated fetal bovine serum (FBS, Gibco,
µ
˝
Grand island, NE, USA) at 37 C in a humidified 5% CO . Human hepatoma (HepG2) cells were
2
from the ATCC and maintained in DMEM supplemented with 10% FBS, 100 units/mL penicillin, and
˝
µg/mL streptomycin at 37 C with 5% CO . Preadipocytes (3T3-L1) was purchased from ATCC
2
1
00
CL-173 and was grown and maintained in DMEM containing 10% Bovine calf serum (BCS, Gibco,),
˝
00 units/mL penicillin, and 100 µg/mL streptomycin at 37 C in a humidified 5% CO .
2
1
3
.3. Luciferase Assay
TK-PPRE 3-luciferase reporter plasmid containing three copies of PPAR response element
ˆ
(
(
PPRE) in acyl CoA oxidase promoter was generously donated by Dr. Christopher K. Glass
University of California, San Diego, CA, USA). pcDNA3 expression vector and full-length
human PPAR-
γ
1 expression vector (pFlag-PPAR-γ1) were generously donated by Dr. Chatterjee
(
7
University of Cambridge, Addenbrooke’s Hospital, Cambridge, UK). For luciferase assays, cells at
4
0%–80% confluence were transfected in a 48-well plate (5
3-luciferase reporter plasmid (1 g/well) plus pcDNA3 (0.1
g/well) using Lipofectamine 2000 (Invitrogen Co., Carlsbad, CA, USA), according to the
ˆ
10 cells/well) with effector plasmids
and TK-PPRE
0.1
ˆ
µ
µg/well) or pFlag- PPAR-γ1
(
µ
™
manufacturer’s instructions. After transfection for 4 h, conditioned media were replaced with complete
medium, and cells were incubated for an additional 20 h. Media were then removed, and cells
were treated with rosiglitazone or test compounds in serum-free media for 6 h. Cells were then
lysed and assayed using the ONE-Glo
Luciferase activities were measured using a GloMax -Multi Microplate Multimode Reader (Promega
Co., Sunny Vale, CA, USA).
™
Luciferase Assay System (Promega, Madison, WI, USA).
®

Mar. Drugs 2016, 14, 112
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3
.4. Molecular Docking Study
Docking calculations were performed using AutoDock Vina 1.1.2 software (The Scripps Research
Institute, La Jolla, CA, USA). Default settings and the Vina scoring function were applied. For PPAR
γ
ligand preparation, Chem3D Ultra 8.0 software (Cambridge Soft Corporation, Cambridge, MA, USA)
was used to convert the 2D structures of candidates into 3D structural data. Protein coordinates were
downloaded from the Protein Data Bank (accession code: 2PRG). Chain A was prepared for docking
using the Chimera 1.5.3 molecular modeling software package (National Institute of Health, Bethesda,
MD, USA) by removing chain B, all ligands and water molecules (except water molecules 308, 399, 444,
and 467), and calculating protein protonation states. Polar hydrogen and setting grid box parameters
were added using MGLTools 1.5.4 (The Scripps Research Institute, La Jolla, CA, USA). The analysis
and visual investigation of ligand-protein interactions of docking poses were performed using PyMol
v1.5 (Schrodinger LLC, New York, NY, USA).
3
.5. Adipocyte Differentiation Assay
4
Preadipocytes (3T3-L1) were seeded in 48 well plates at a density of 5
ˆ
10 cells/well and
maintained until two days post-confluence. After two days of confluency, medium was replaced with
0% FBS/DMEM containing 10 µg/mL insulin, 1 M dexamethasone, and test samples at various
concentrations (day 2). After two days, the medium was replaced with 10% FBS/DMEM containing
1
µ
1
1
0 µg/mL insulin and test samples (1, 10, and 20 µM) (day 4). The culture was then maintained in
0% FBS/DMEM for an additional four days.
3
.6. Quantification of Lipid Contents in 3T3-L1 Cells
After eight days of differentiation in the presence of samples, medium was removed, cells were
carefully rinsed with PBS, fixed with 70% ethanol for 30 min, stained with Oil red O solution for
, and washed with dH O. Pictures were taken using a Optinity (Gyeonggi-Do, Korea) microscope.
1
h
2
Oil red O was eluted by adding 200
transferred to a 96-well plate and ODs were measured using a iMark Microplate Absorbance Reader
Bio-Rad Laboratories, Hercules, CA, USA) at 544 nm.
µL of isopropanol on a rotary shaker for 5 min. Eluents were
(
4
. Conclusions
Several PPAR-
γ
agonists, such as troglitazone, pioglitazone, and rosiglitazone, have been
developed to improve insulin sensitivity and stimulate glucose metabolization in muscle and adipose
tissues [10]. Paecilocin A, isolated from jellyfish-derived fungus Paecilomyces variotii, previously
showed good activity in a PPAR-
were designed as pharmacophores for PPAR-
γ
binding assay [11]. Subsequently, new phthalimide derivatives
activation based on core structures of paecilocin A
γ
derivatives and TZDs, including rosiglitazone [14].
In the present study, enhancement of the PPAR-
γ
agonistic activity of PD1 was pursued by in silico
analysis of PD1-derived structures, and PD5 and PD6 were selected for synthesis and biological
evaluation. In contrast to in silico predictions, PD1 consistently exhibited higher PPAR- agonistic
activity than PD5 or PD6 in Ac2F and HepG2 cells. PD1 PD6 were also evaluated with respect to the
γ
–
induction of adipocyte differentiation using murine 3T3-L1 cells, and PD1 and PD2 were found to
stimulate adipogenesis in a concentration-dependent manner. Furthermore, the adipogenic activity
profiles of PD1
In summary, we designed new phthalimide PPAR-
experimental data. The design was aided by SAR analysis and computer simulations of ligand-binding
to the LBD of PPAR- protein. Synthesized analogues were evaluated for in vitro PPAR- activation
using both murine liver cells (Ac2F) and human liver cells (HepG2). PPAR- activation by the
compounds was further gauged by induced adipogenesis in 3T3-L1 cells. The presented information
~PD6 correlated fairly well with their activations of PPAR-γ in Ac2F and HepG2 cells.
γ
ligands based on our previous experience and
γ
γ
γ

Mar. Drugs 2016, 14, 112
9 of 10
on SAR of these phthalimide derivatives would serve as a basis for future study and development of
PPAR-γ ligands.
Acknowledgments: This study was supported by the Korean National Research Foundation (Grant No.
2
0090083538). This research was a part of the project entitled ‘Omics based fishery disease control technology
development and industrialization’ funded by the Ministry of Oceans and Fisheries, Korea.
Author Contributions: Conceived and designed the experiments: So Hyeon Eom, Sen Liu and Jee H. Jung.
Performed the experiments: So Hyeon Eom, Sen Liu, Mingzhi Su, Jongki Hong, Tae Hwan Noh and Min Hye Yang
.
Analyzed the data: Sen Liu, Nam Deuk Kim, Hae Young Chung, Min Hye Yang and Jee H. Jung. Wrote the paper:
Sen Liu and So Hyeon Eom.
Conflicts of Interest: The authors declare no conflict of interest.
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