Design and synthesis of marine fungal phthalide derivatives as PPAR-γ agonists

Article abstract of DOI:10.1016/j.bmc.2012.06.039

On the basis of a marine fungal phthalide (paecilocin A) skeleton, we synthesized 20 analogs and evaluated them for peroxisome proliferator-activated receptor gamma (PPAR-γ) binding and activation. Among these analogs, 6 and 7 had significant PPAR-γ binding activity, and 7 showed further PPAR-γ activation in rat liver Ac2F cells. In docking simulation, 7 formed H bonds with key amino acid residues of the PPAR-γ binding domain, and the overall positioning was similar to rosiglitazone. This new phthalide derivative is considered an interesting new molecular class of PPAR-γ ligands.

Full text of DOI:10.1016/j.bmc.2012.06.039

Bioorganic & Medicinal Chemistry 20 (2012) 4954–4961
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of marine fungal phthalide derivatives as PPAR-c agonists
Bin Xiao a, Jun Yin b, Minhi Park a, Juan Liu c, Jian Lin Li a, Eun La Kim a, Jongki Hong d, Hae Young Chung a,
Jee H. Jung a,
⇑
a College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea
b College of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China
c South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China
d College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
On the basis of a marine fungal phthalide (paecilocin A) skeleton, we synthesized 20 analogs and evalu-
Received 31 May 2012
Revised 21 June 2012
Accepted 21 June 2012
Available online 29 June 2012
ated them for peroxisome proliferator-activated receptor gamma (PPAR-
Among these analogs, 6 and 7 had significant PPAR- binding activity, and 7 showed further PPAR-
vation in rat liver Ac2F cells. In docking simulation, 7 formed H bonds with key amino acid residues of the
PPAR- binding domain, and the overall positioning was similar to rosiglitazone. This new phthalide
derivative is considered an interesting new molecular class of PPAR- ligands.
c
) binding and activation.
c
c
acti-
c
c
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
PPAR-
c
Phthalide
Marine fungus
Binding assay
Luciferase assay
1. Introduction
L-tyrosine analogs, natural fatty acids such as docosahexaenoic
acid (DHA), eicosapentaenoic acid (EPA), and cyclopentenone pros-
taglandin (15d-PGJ2), are also reported to be PPAR- agonists with
relatively low affinities.11 PPAR-
agonists consist of a hydrophilic
head group tethered to an aromatic center linked to a hydrophobic
tail. The hydrophilic head group typically has hydroxyl, carbonyl,
or carboxyl oxygen atoms (e.g., carboxylic acid and 2, 4-thiazolid-
inedione), which form H bonds with the key amino acid residues
Peroxisome proliferator-activated receptors (PPARs) are mem-
bers of the nuclear receptor superfamily of ligand-activated tran-
c
c
scription factors and include three isoforms:
a
, b/d, and c 1–4
.
PPAR- is the most abundant isoform in adipose tissue, macro-
c
phages, monocytes, intestinal cells, skeletal muscle, and endothe-
lium, and plays an important role in the regulation of insulin
sensitivity, lipid metabolism, adipogenesis, and glucose homeosta-
sis.5 Thiazolidinediones (TZDs) such as rosiglitazone, pioglitazone,
(Tyr473, His449, His323, and Ser289) of the PPAR-c LBD. These H
bonds stabilize PPAR- in the proper conformation, which is cru-
c
and ciglitazone are high-affinity ligands and full agonists of PPAR-
that are used in the treatment of type 2 diabetes mellitus.6–8 Be-
cause if the adverse effects asociated with PPAR- ligands, such
as weight gain, edema, and fluid retention, discovery of new
PPAR- ligands that do not have adverse effects is essential in
c
cial for successful co-activator recruitment.12,13 The aromatic cen-
ter forms a variety of Van der Waals interactions with various
hydrophobic residues in the LBD, and the hydrophobic tail toler-
ates a more diverse set of substituents that are free to interact with
the fairly large hydrophobic pocket as well as water molecules in
it.14 These are graphically illustrated in Figure 1 as a key pharma-
cophore concept in terms of rosiglitazone, a potent and selective
c
c
the development of new therapeutics for insulin resistance and
type 2 diabetes mellitus.9
A ternary complex structure (2PRG) composed of the PPAR-
c
PPAR-c agonist.
ligand-binding domain (LBD), rosiglitazone, and human steroid
In the course of our search for bioactive marine natural prod-
ucts, new phthalide derivatives paecilocins A–C (Fig. 2A) were iso-
lated from the jellyfish-derived fungus Paecilomyces variotii.15 In a
receptor co-activating factor-1 (SRC-1) was described in 1998.10
The PPAR-c LBD contains a large binding pocket that allows diverse
types of ligands to enter and search for their proper laying confor-
subsequent screening for PPAR-c competitive binding activity,
mations for ligand–receptor complexes. In addition to TZDs and
paecilocin A showed activity comparable to that shown by rosiglit-
azone (Fig. 2B). Other similar structures 3-butyl-1(3H)-isobenzof-
uranone, 3-butylidenephthalide, and senkyunolide B are covered
in a patent as effective agents for the prevention or treatment of
⇑
Corresponding author. Tel.: +82 51 5102803; fax: +82 51 5136754.
E-mail address: jhjung@pusan.ac.kr (J.H. Jung).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.06.039

B. Xiao et al. / Bioorg. Med. Chem. 20 (2012) 4954–4961
4955
bromide to generate a mixture of differently alkylated products
2–7 (Scheme 1). Monoalkyl derivatives (2 and 3) were obtained
as the major products. Simultaneous alkylation did not occur on
both carbonyls. However, 2 alkyl chains easily attached to a single
carbonyl carbon (4 and 5). Continuous treatment with HCl pro-
duced dehydroxy derivatives 6 and 7 (paecilocin A racemate).
Compound 3 was treated with benzyl chloride to yield hydroxy-
substituted analogs. In addition, a mixture of benzyl derivatives
(9 and 10) was obtained. Further treatment of 10 with MeI gener-
ated derivative 11, with the free hydroxyl group blocked as a meth-
oxyl group. Compound 8 was produced from 7 by using the same
procedure. Compounds 13 and 14, without hydroxyls in the ben-
zene ring, were obtained in a mixture by the reaction of commer-
cially available phthalic anhydride (12) with octylmagnesium
bromide. In this case, the dialkyl derivative (14) was obtained as
a major product and the monoalkyl derivative (13) was obtained
as a minor component (Scheme 2).
Figure 1. Graphical illustration of the key pharmacophore concept of rosiglitazone.
The rosiglitazone skeleton is divided into the head, linker, and tail by dashed line
boxes. Key helices and sheets (light blue), amino acid residues (purple), hydrogen
bonds (gray dashed line), and water molecule (in green) of PPAR-c LBD are shown.
In the presence of H2O and heat, commercially available phtha-
lic anhydrides (1 and 16) were hydrolyzed to ortho-phthalic acids
(Scheme 2).19 To retain the free carboxyl on the benzene ring as
a ligand head, the ortho-phthalic acids were treated with 1-bromo-
octane, and the octyl phthalates 15 and 17 were produced
(Scheme 2). The etherification selectively proceeded on the meta-
carboxyl group, possibly due to the intra-molecular H bond
formation between the phenolic hydroxyl or nitro group with the
ortho-carboxyl group. Treatment of 1 with 1-bromooctane yielded
phthalic anhydride 3-octyl ether. Subsequent treatment with
MeOH and heat yielded a mixture of methyl esters (18 and 19)
in similar yields. Compound 20 was produced by treating 1 with
decanoyl chloride to yield a TZD-mimic pharmacophore with dione
as a head and a long acyl chain as the hydrophobic tail (Scheme 2).
A
H3CO
OH
H
O
O
OCH3
O
OH
paecilocin A (3S)
paecilocin B(1S*)
paecilocin C(1R*)
B
2.2. PPAR-c binding activity
All synthesized compounds were compared with rosiglitazone
for their binding affinity to the PPAR- LBD (Fig. 3). Compounds
c
6 and 7 showed significant binding, comparable to that shown by
rosiglitazone. Of the three different phthalic anhydride heads (1,
12, and 16), only 1 showed activity while 12 and 16 showed no
notable activity. This suggested the importance of the hydroxyl
group on the benzene ring. When the hydroxyl group was blocked
by benzyl or methyl groups (8, 9, 10, and 11), the binding ability
diminished. Additionally, the substitution of a bulky group at the
stereo-center (2–5 and 9, 10, and 11) was detrimental to the activ-
ity, indicating that introduction of a third arm on the benzene ring
did not enhance activity.6–8
Figure 2. Chemical structures (A) and PPAR-
phthalides. Activity was measured using LanthaScreen TM TR-FRET PPAR-
competitive binding assay. BL (blank control, without PPAR- LBD), Ros (rosiglit-
azone). Rosiglitazone and phthalide analogs a–c (paecilocins A–C, respectively)
were assayed at 100 M. The fluorescence ratio at 520/495 nm is shown.
c binding activity (B) of fungal
c
c
l
Among the open phthalate analogs (15, 17, 18, and 19), com-
pounds 15 and 17 exhibited better activity, indicating that the free
carboxyl moiety and additional H bond donor or acceptor (such as
the hydroxyl or nitro group on the head) were valuable for binding
diabetes mellitus and the mechanism of action was speculated to
be PPAR-
activation.16 Motivated by these preliminary findings,
we synthesized paecilocin A racemate and its analogs to character-
ize a new PPAR- agonistic skeleton.
c
the PPAR-c LBD. The imitation structure (20) of classic pharmaco-
c
phore of TZDs did not show activity enhancement compared to the
simple phthalic anhydride (1). The preliminary SAR of each phthal-
ide derivative is summarized in Figure 4.
Interestingly, compared to rosiglitazone, the synthetic racemate
(7) displayed better binding activity than natural paecilocin A
(Figs. 2B and 3). This suggests that the enantiomer (R-isomer, 7-
R) of paecilocin A (S-isomer, 7-S) might be a better ligand for
PPAR-c. Therefore, enantiomeric structures of paecilocin A (7-R
and 7-S) as well as 6 and rosiglitazone were evaluated for receptor
binding by docking simulation to collect supplementary data.
2. Results and discussion
2.1. Chemistry
Paecilocin A can be synthesized in at least 5 steps by applying a
reported scheme.17 For a preliminary study, a racemic mixture of
paecilocin A as well as a series of derivatives were synthesized
according to a reported single-step scheme.18 Considering the
pharmacophore analyses of rosiglitazone, the carbonyls or hydrox-
yls and the hydrophobic alkyl chain were retained on the head
group and as a tail group, respectively.
2.3. Molecular docking study
Commercially available 3-hydroxy phthalic anhydride (1) was
treated with the nucleophilic Grignard reagent octylmagnesium
PPAR-
c has been a challenging target for molecular modeling
for several reasons. First, the binding site is large, and the ligand

4956
B. Xiao et al. / Bioorg. Med. Chem. 20 (2012) 4954–4961
O
O
O
OH
1
a
O
O
O
HO
O
O
O
+
O
+
O
+
+
+
O
HO
O
O
OH
O
OH
OH
OH
6 (10%)
OH
OH
5 (12%)
2 (23%)
4 (11%)
7 (12%)
3 (24%)
c
b
O
O
HO
O
O
O
OCH3
+
O
O
8 (98%)
O
O
9 (20%)
10 (75%)
d
H3CO
O
O
O
11 (98%)
Scheme 1. Reagents and conditions: (a) Me(CH2)7MgBr, THF, À20 °C, stir, 2 h; 1 M HCl, stir, 0.5 h; (b) benzyl chloride, K2CO3, NaI, DMF, stir, rt, 4 h; (c) MeI, Ag2O, stir, 12 h; (d)
MeI, Ag2O, stir, 12 h.
O
O
O
O
OH
O
O
+
R
O
13 (25%)
14 (73%)
15 R=OH (80%)
17 R=NO2 (80%)
a
c
b
d
O
O
O
O
O
R1
R2
O
R
O
1
R=OH
O
O
O
12 R=H
16 R=NO2
O
18 R1=OCH3, R2=OH (45%)
19 R1=OH, R2=OCH3 (40%)
20 (70%)
Scheme 2. Reagents and conditions: (a) Me(CH2)7MgBr, THF, À20 °C, stir, 2 h; 1 M HCl, stir, 0.5 h; (b) aq. CH3CN Me(CH2)7Br, Ag2O, aq. CH3CN 80 °C, stir, 12 h; (c) Me(CH2)7Br,
Ag2O, 80 °C, stir, 12 h; Methanol, 35 °C 0.3 h; (d) decanoyl chloride, stir, 0–15 °C, 4 h.
tail is always located in a big hydrophobic pocket that does not
interact with amino acid residues to contribute to its biological
activity. Next, the binding is considered flexible and in particular,
the side chains move significantly upon ligand binding. Finally,
the binding site is highly lipophilic, which presents a challenge
for structure-based approaches.10,14,20,21
we did not rely only on the RMSD value for validating the docking
workflow. Instead, we used PyMol v1.5 for analysis and investiga-
tion of the ligand–protein interactions of the docking poses.22
The crystal form of 2PRG contains 2 molecules in the asymmet-
ric unit, denoted A and B. Only chain A was proposed to represent a
realistic model for the activated PPAR-
c
protein.20 For chain B,
On the basis of these characteristics and the large amount of
available experimental data, we were able to apply more sophisti-
cated docking workflow. The crystal structure of the 2PRG complex
was re-docked for validation. Because a low root mean square
deviation (RMSD) value does not necessarily correspond to a dock-
ing pose that renders the most crucial ligand–protein interactions,
most re-docking modes lacked the key interactions with His449
and Glu286. However, re-docking with chain A allowed most cru-
cial ligand–protein interactions to occur. Therefore, chain A was se-
lected for the PPAR-
c agonist docking workflow. Re-docking of
rosiglitazone was performed with different water molecules in
the hydrophobic binding pocket, and H2O308, H2O339, H2O444,

B. Xiao et al. / Bioorg. Med. Chem. 20 (2012) 4954–4961
4957
Figure 3. Measurement of the in vitro PPAR-
receptor control, without PPAR-
fluorescence ratio at 520/495 nm is shown as the mean + s.d. (n = 3).
c
binding activity of phthalide analogs by using the LanthaScreenTM TR-FRET PPAR-
c
competitive binding assay. NRC (no
c
LBD), NC (negative control, with solvent DMSO), Ros (rosiglitazone). Rosiglitazone and phthalide analogs were assayed at 50 lM. The
-OH:
3rd arm:
small subst. >large subst.
6 and 7 were subsequently evaluated for PPAR-
Ac2F cells.
c activation in
presence >absence
7-OH ~ 4-OH
Free >blocked
Side chain:
2.4. PPAR-c activation
single chain >double chain
HO
O
Compounds 6 and 7 were evaluated using luciferase transacti-
vation assays in rat liver Ac2F cells transiently transfected with
pcDNA3/pFlag-PPAR-c1 and PPRE. In contrast to the binding assay
results (Fig. 3), 6 and 7 were less active than rosiglitazone. How-
ever, compound 7 had activity that increased in a concentration-
dependent manner (Fig. 6).
Bioisostere:
phthalide >phthalic anhydride
O
Open-ring phthalate:
-COOH with ortho -OH or -NO2
are valuable
Figure 4. Structure–activity relationship of phthalide derivatives.
The intra-molecular H bond of compound 7 might modulate
lipophilicity and lead to cell permeability differences between 6
and 7 in Ac2F cells. Moreover, competitive binding and activation
of PPAR-
binding conditions, 6 and 7 could similarly displace the fluorescent
pan PPAR- agonist from the PPAR- LBD. In the transactivation as-
say, however, the changed conformation of the PPAR- protein by
different ligands (6 and 7) should be different, and this could lead
to different degrees of co-activator recruitment. Other factors
might be also involved in bioavailability modulation of these mol-
ecules in vivo. Therefore, further optimization and in vivo evalua-
tion of these molecules would provide valuable data.
c might involve different mechanisms. Under competitive
and H2O467 were finally selected. This generated top-ranked and
reproducible binding modes that are close to those in the 2PRG
crystal.
The enantiomer 7-R ranked over 7-S (paecilocin A) and was
likely to form H bonds with Tyr473, His449, His343, Ser289, and
c
c
c
Glu286 in the PPAR-c LBD. This is highly similar to the H bonding
network of rosiglitazone in 2PRG. The enantiomer 7-S lacked inter-
action with His449. However, their affinities were not significantly
different (-7.0 kcal/mol for 7-S and -6.9 kcal/mol for 7-R), but were
higher than 6 (-6.4 kcal/mol) (Fig. 5). Not only binding affinity but
also other factors such as H bonding with key amino acid residues
3. Conclusion
In this study, a series of fungal phthalide-based analogs were
would be important for activation of PPAR-c. More H bonds would
prepared and evaluated for PPAR-
pound 7 showed significant PPAR-
c
c
binding and activation. Com-
binding and moderate PPAR-
be helpful to ‘lock’ PPAR- protein in an active conformation which
c
is preferable for the co-activator recruitment and further activa-
tion. Compound 7-S lack two H bonds compared to 7-R, especially
the H bond between hydroxyl oxygen and His449 on the helix 10,
which is crucial for the lock of this helix as well as the whole pro-
tein. The enantiomer 6-R ranked over 6-S and formed H bonds with
c
activation in rat liver Ac2F cells. Compound 7 formed H bonds
with key amino acid residues of the PPAR- LBD, and its overall
c
positioning in the binding pocket was similar to that of rosiglitaz-
one. The slightly higher binding activity of the synthetic racemate
(7) over the natural molecule (paecilocin A, 7-S) was verified by
molecular docking experiments and the 7-R isomer was considered
a more favorable ligand. These new phthalide derivatives are con-
sidered an interesting new molecular class of PPAR-c ligands, and
the asymmetric synthesis of pure enantiomers and further biolog-
ical evaluation would provide valuable information.
Tyr473, His343, and Ser289 of the PPAR-
c LBD. Simulation data re-
vealed that 7-R and 7-S bind PPAR- more preferentially than 6,
c
and 7–R was a better ligand than 7-S. The enantiomer 7-R is con-
sidered to be anchored by the H bonding network in the Y-shaped
binding pocket of PPAR-c and stabilizes the protein like a mouse
trap, which is the preferable conformation for co-activator binding.
The asymmetric carbon atom acts like a switch, and the R configu-
ration could force the lipophilic alkyl chain to fill the hydrophobic
portions of the pocket in a preferable pose as well as stabilize the
head group in a direction to furnish the bonding network.23 Be-
cause the correlation of experimental affinity data and scoring
4. Experimental section
4.1. Chemistry
functions is controversial, the best docking modes for the PPAR-
c
The 1H and 13C NMR spectra were recorded on Varian Unity
400 MHz NMR spectrometer. The chemical shifts are reported with
reference to the respective residual solvent or deuterated solvent
ligands were selected by taking the docking scores as well as the
results of the visual investigation into account.24–26 Compounds

4958
B. Xiao et al. / Bioorg. Med. Chem. 20 (2012) 4954–4961
Figure 5. The 3D putative binding modes of rosiglitazone, 6-R, and 7-R/S with PPAR-c LBD. (A) Rosiglitazone interacts with key amino acid residues (Tyr473, His449, His343,
Ser289, and Glu286) in the PPAR-
S (À7.0 kcal/mol).
c
binding pocket (À8.2 kcal/mol); (B) binding mode of 6-R (À6.4 kcal/mol); (C) binding mode of 7-R (À6.9 kcal/mol); (D) binding mode of 7-
4.2. General procedure for the synthesis of phthalide derivatives
4.2.1. Preparation of 2–7
To a solution of 3-hydroxyphthalic anhydride (1) in THF at
À20 °C, octylmagnesium bromide (2.5 equiv) in THF was added,
and the mixture was stirred for 15 min and warmed to room tem-
perature for another 2 h. The reaction was quenched with 1 M HCl
and concentrated in vacuo. The residue was diluted with EtOAc and
successively washed with H2O and brine. The organic layer was
dried with MgSO4 and evaporated to obtain a crude product, which
was purified by RP HPLC eluting with 80% aqueous MeOH to give
2–7. Compound
2
(23%): White powder; 1H NMR (CD3OD,
400 MHz): d 0.83 (t, J = 7.2 Hz, 3H), 1.18–1.26 (m, 12H), 2.32 (brs,
2H), 7.05 (d, J = 8.4 Hz, 1H), 7.27 (d, J = 7.2 Hz, 1H), 7.36 (t,
J = 7.6 Hz, 1H); 13C NMR (CD3OD, 100 MHz): d 169.8, 153.4,
148.1, 133.8, 131.5, 129.4, 120.6, 36.9, 31.8, 29.2, 29.1, 29.0, 23.4,
22.5, 13.2; FABMS m/z 279.3 [M+H]+. Compound 3 (24%): White
powder; 1H NMR (CD3OD, 400 MHz): d 0.86 (t, J = 7.2 Hz, 3H),
1.18–1.26 (m, 12H), 2.10 (brs, 2H), 6.98 (d, J = 8.0 Hz, 2H), 7.5
(brs, 1H); 13C NMR (CD3OD, 100 MHz): d 169.8, 158.3, 150.8,
136.1, 131.5, 118.4, 113.6, 36.9, 31.8, 29.2, 29.1, 29.0, 23.4, 22.5,
13.2; FABMS m/z 279.3 [M+H]+. Compound 4 (11%): White powder;
1H NMR (CD3OD, 400 MHz): d 0.82 (t, J = 7.2 Hz, 6H), 1.19–1.24 (m,
24H), 2.01–2.03 (m, 2H), 2.14–2.16 (m, 2H), 7.03 (d, J = 7.6 Hz, 1H),
7.25 (d, J = 7.6 Hz, 1H), 7.34 (t, J = 7.6 Hz, 1H); 13C NMR (CDCl3,
100 MHz): d 170.7, 150.1, 138.2, 130.6, 129.7, 120.1, 118.0, 90.8,
37.1, 32.0, 29.9, 29.7, 29.5, 29.4, 23.5, 22.8, 14.3; FABMS m/z
Figure 6. In vitro PPAR-
transiently transfected with pcDNA or PPRE with pFlag-PPAR-
control, without transfection of plasmid), NC (negative control, transfected with
plasmid with PPRE and pcDNA), Ros (rosiglitazone). Rosiglitazone was used as
positive control to monitor the activation of the luciferase reporter. Luciferase
expression (RLU/well) is shown as the mean + s.d. (n = 5). RLU; relative light unit.
c
activation by 6 and 7 in rat liver Ac2F cells. Cells were
1. NRC (no receptor
c
peaks (dH 3.30 and dC 49.0 for CD3OD, dH 7.24 and dC 76.8 for
CDCl3). The FAB MS data was obtained on a JEOL JMS SX-102A
spectrometer. HPLC was performed on a YMC ODS-H80 column
(250 Â 10 mm, 4
l
l
m, 80 Å) and a C18-5E Shodex packed column
m, 100 Å) by using a Shodex RI-71 detector.
(250 Â 10 mm, 5
All chemical reagents were purchased from Sigma-Aldrich and
375.5 [M+H]+. Compound
5
(12%): White powder; 1H NMR
used as received.

B. Xiao et al. / Bioorg. Med. Chem. 20 (2012) 4954–4961
4959
(CD3OD, 400 MHz): d 0.83 (t, J = 7.2 Hz, 6H), 1.19–1.26 (m, 24H),
1.87 (m, 2H), 1.98 (m, 2H), 6.85 (m, 2H), 7.25 (t, J = 8.0 Hz, 1H);
13C NMR (CDCl3, 100 MHz): d 156.6, 153.3, 137.0, 136.9, 115.2,
112.7, 112.0, 92.8, 37.1, 32.0, 29.9, 29.7, 29.5, 29.4, 23.5, 22.8,
14.3; FABMS m/z 375.5 [M+H]+. Compound 6 (10%): White powder;
1H NMR (CD3OD, 400 MHz): d 0.83 (t, J = 7.2 Hz, 3H), 1.25 (m, 12H),
1.67 (m, 1H), 2.0 (m, 1H), 5.46 (dd, J = 4.4, 8.0 Hz, 1H), 6.83 (d,
J = 8.0 Hz, 1H), 6.94 (d, J = 7.2 Hz, 1H), 7.52 (t, J = 8.0 Hz, 1H); 13C
NMR (CDCl3, 100 MHz): d 170.8, 157.0, 152.5, 136.4, 115.4, 112.6,
81.5, 34.5, 31.8, 29.3, 29.2, 29.1, 24.5, 22.5, 13.2; FABMS m/z
263.3 [M+H]+. Compound 7 (12%): White powder; 1H NMR (CDCl3,
400 MHz): d 0.84 (t, J = 7.2 Hz, 3H), 1.25 (m, 12H), 1.68 (m, 1H),
2.01 (m, 1H), 5.49 (dd, J = 3.6, 8.0 Hz, 1H), 6.91 (m, 2H), 7.54 (dd,
J = 6.0, 6.4 Hz, 1H), 7.80 (s, 1H); 13C NMR (CD3OD, 100 MHz): d
172.4, 156.8, 150.7, 137.0, 115.5, 113.2, 111.4, 83.1, 34.8, 32.0,
29.9, 29.5, 29.5, 29.4, 25.0, 22.8, 14.3; FABMS m/z 263.3 [M+H]+.
129.7, 128.43, 128.38, 128.25, 120.9, 120.0, 67.2, 56.3, 43.6, 31.8,
29.3, 29.1, 28,9, 23.1, 22.5, 13.2; FABMS m/z 263.3 [M+H]+.
4.2.5. Preparation of 13 and 14
To a solution of phthalic anhydride (12) in THF at À20 °C, octyl-
magnesium bromide (2.5 equiv) in THF was added, and the reac-
tion was stirred for 15 min and then warmed to room
temperature for another 2 h. The reaction was quenched with
1 M HCl and concentrated in vacuo. The residue was diluted with
EtOAc and successively washed with H2O and brine. The organic
layer was dried with MgSO4 and evaporated to give a crude prod-
uct, which was chomatographed on silica gel eluting with CHCl3–
MeOH to give 13 and 14. Compound 13 (25%): Yellow oil; 1H
NMR (CDCl3, 400 MHz): d 0.84 (t, J = 7.2 Hz, 3H), 1.23 (m, 12H),
1.72 (m, 1H), 2.01 (m, 1H), 5.45 (dd, J = 4.1, 7.6 Hz, 1H), 7.41 (d,
J = 7.6 Hz, 1H), 7.5 (t, J = 7.2 Hz, 1H), 7.65 (t, J = 7.6 Hz, 1H), 7.88
(d, J = 7.2 Hz, 1H); 13C NMR (CDCl3, 100 MHz): d 170.7, 152.9,
134.1, 129.0, 125.8, 121.3, 90.6, 39.0, 32.0, 30.0, 29.8, 29.5, 29.3,
23.3, 22.8, 14.3; HRFABMS m/z 247.2738 [M+H]+. Compound 14
(73%): Yellow oil; 1H NMR (CDCl3, 400 MHz): d 0.82 (t, J = 7.2 Hz,
6H), 1.15 (m, 24H), 1.81 (m, 2H), 2.01 (m, 2H), 7.29 (d, J = 8.0 Hz,
1H), 7.47 (t, J = 8.0 Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H), 7.83 (d,
J = 8.0 Hz, 1H); 13C NMR (CDCl3, 100 MHz): d 170.7, 152.9, 134.1,
129.0, 125.8, 121.3, 90.6, 39.0, 32.0, 30.0, 29.8, 29.5, 29.3, 23.3,
22.8, 14.3. FABMS m/z 359.3 [M+H]+.
4.2.2. Preparation of 8
To
a
solution of 3-octyl-7-hydroxyphthalide (7; 5 mg,
l, ca. 0.04 mmol) and
0.018 mmol) in CH3CN (1.5 ml), CH3I (2.5
l
Ag2O (8.5 mg, 0.04 mmol) were added, and the mixture was heated
under reflux with stirring for 12 h. The solid material was removed
by filtration, and the filtrate was evaporated to obtain a solid,
which was purified by RP HPLC and eluted with 90% aqueous
MeOH to give 8 (98%): white powder; 1H NMR (CDCl3, 400 MHz):
d 0.85 (t, J = 7.2 Hz, 3H), 1.23 (m, 12H), 1.68 (m, 1H), 1.97 (m,
1H), 3.97 (s, 3H), 5.35 (dd, J = 4.4, 8.0 Hz, 1H), 6.89 (d, J = 8.4 Hz,
2H), 6.93 (d, J = 7.2 Hz, 2H), 7.57 (t, J = 7.2, 8.4 Hz, 1H); 13C NMR
(CD3OD, 100 MHz): d 172.4, 156.8, 150.7, 137.0, 115.5, 113.2,
111.4, 83.1, 57.0, 34.8, 32.0, 29.9, 29.5, 29.5, 29.4, 25.0, 22.8,
14.3; FABMS m/z 277.3 [M+H]+.
4.2.6. Preparation of 15 and 17
To a solution of 1 (16.4 mg, 0.1 mmol) or 16 (21 mg, 0.1 mmol)
in aqueous CH3CN (2 ml), 1-bromooctane (20 ll, ca. 0.12 mmol)
and Ag2O (46 mg, 0.2 mmol) were added, and the mixture was
heated under reflux with stirring for 12 h. The solid material was
removed by filtration, and the filtrate was evaporated to give a so-
lid, which was purified by RP HPLC eluting with 90% aqueous
MeOH to give 15 or 17. Compound 15 (80%): white powder; 1H
NMR (CD3OD, 400 MHz): d 0.87 (t, J = 7.2 Hz, 3H), 1.28 (m, 10H),
1.70 (m, 2H), 4.23 (t, J = 6.8 Hz, 2H), 7.02 (d, J = 8.4 Hz, 1H), 7.06
(d, J = 7.2 Hz, 1H), 7.38 (dd, J = 7.6, 8.0 Hz, 1H); 13C NMR (CD3OD,
100 MHz): d 170.9, 169.0, 158.9, 134.1, 132.4, 119.1, 118.8, 116.2,
65.7, 31.8, 29.1, 29.0, 28.3, 25.9, 22.5, 13.2; FABMS m/z 295.3
[M+H]+. Compound 17 (80%): white powder; 1H NMR (CD3OD,
400 MHz): d 0.87 (t, J = 6.8, 7.2 Hz, 3H), 1.29 (m, 10H), 1.75 (m,
2H), 4.29 (t, J = 6.8 Hz, 2H), 7.58 (t, J = 7.2, 8.4 Hz, 1H), 8.13 (d,
J = 7.6 Hz, 1H), 8.16 (dd, J = 8.4 Hz, 1H); 13C NMR (CD3OD,
100 MHz): d 171.1, 165.7, 146.8, 134.5, 130.1, 130.0, 128.2, 127.4,
66.0, 31.8, 29.2, 29.1, 28.4, 25.9, 22.5, 13.2; HRFABMS m/z
324.1314 [M+H]+.
4.2.3. Preparation of 9 and 10
To a suspension of 3 (26 mg, 0.088 mmol), K2CO3 (12.3 mg,
0.088 mmol) and NaI (4.4 mg, 0.03 mmol) in DMF (3 ml) was
added to benzyl chloride (20 ll, ca. 0.09 mmol), and the mixture
was stirred for 30 min at 0 °C and for 4 h at room temperature.
The mixture was acidified with aqueous 6 M HCl and extracted
with EtOAc. The organic layer was successively washed with H2O
and brine, dried with MgSO4, and evaporated to give a crude prod-
uct, which was purified by RP HPLC eluting with 85% aqueous
MeOH to give 9 and 10. Compound 9 (20%): White powder; 1H
NMR (CD3OD, 400 MHz): d 0.87 (t, J = 7.2 Hz, 3H), 1.26 (m, 10H),
1.45 (m, 2H), 2.68 (dd, J = 7.2, 7.6 Hz, 2H), 5.12 (s, 2H), 5.24 (s,
2H), 7.35 (m, 12H), 7.58 (d, J = 8.0 Hz, 1H); 13C NMR (CD3OD,
100 MHz): d 167.0, 154.1, 135.8, 129.7, 128.4, 128.4, 128.3, 120.9,
120.0, 67.2, 43.6, 31.8, 29.3, 29.1, 28,9, 23.1, 22.5, 13.2; FABMS
m/z 459.4 [M+H]+. Compound 10 (75%): White powder; 1H NMR
(CD3OD, 400 MHz): d 0.88 (t, J = 6.8 Hz, 3H), 1.25 (m, 10H), 1.52
(m, 2H), 2.72 (dd, J = 7.2, 7.6 Hz, 2H), 5.23 (s, 2H), 7.03 (d,
J = 8.0 Hz, 1H), 7.35 (m, 7H); 13C NMR (CD3OD, 100 MHz): d
166.3, 154.1, 135.8, 129.7, 128.4, 128.4, 128.3, 120.9, 120.0, 67.2,
43.6, 31.8, 29.3, 29.1, 28,9, 23.1, 22.5, 13.2; FABMS m/z 369.3
[M+H]+.
4.2.7. Preparation of 18 and 19
To a solution of 1 (16.4 mg, 0.1 mmol) in aqueous CH3CN (2 ml),
1-bromooctane (20 ll, ca. 0.12 mmol) and Ag2O (46 mg, 0.2 mmol)
were added, and the mixture was heated under reflux with stirring
for 12 h. After this time, the solid material was removed by filtra-
tion, and the filtrate was evaporated to give a solid. After methanol
was added, the mixture was heated again at 35 °C for 30 min, evap-
orated to get the crude products, and purified by RP HPLC eluting
with 90% aqueous MeOH to give 18 and 19. Compound 18 (45%):
white powder; 1H NMR (CDCl3, 400 MHz): d 0.86 (t, J = 7.2 Hz,
3H), 1.26 (m, 8H), 1.40 (m, 2H), 1.75 (m, 2H), 3.89 (s, 3H), 3.99
(t, J = 6.4 Hz, 2H), 7.12 (d, J = 8.4 Hz, 1H), 7.39 (t, J = 8.0 Hz, 1H),
7.63 (d, J = 8.4 Hz, 1H); 13C NMR (CD3OD, 100 MHz): d 170.9,
166.4, 158.9, 128.2, 126.8, 121.3, 119.1, 116.8, 69.1, 51.5, 31.8,
29.1, 29.0, 28.3, 25.9, 22.5, 13.2; FABMS m/z 309.5 [M+H]+. Com-
pound 19 (40%): white powder; 1H NMR (CD3OD, 400 MHz): d
0.85 (t, J = 7.2 Hz, 3H), 1.23 (m, 8H), 1.42 (m, 2H), 1.80 (m, 2H),
3.89 (s, 3H), 4.06 (t, J = 6.4, 6.8 Hz, 2H), 7.11 (d, J = 8.0 Hz, 1H),
7.42 (t, J = 8.0 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H); 13C NMR (CD3OD,
4.2.4. Preparation of 11
To a solution of 10 (10 mg, 0.027 mmol) in CH3CN (2 ml), CH3I
(3.4 ll, ca. 0.054 mmol) and Ag2O (12.6 mg, 0.054 mmol) were
added, and the mixture was heated under reflux with stirring for
12 h. After this time, the solid material was removed by filtration,
and the filtrate was evaporated to obtain a solid, which was puri-
fied by RP HPLC eluting with 90% aqueous MeOH to give 11
(98%): white powder; 1H NMR (CD3OD, 400 MHz): d 0.88 (t,
J = 6.8 Hz, 3H), 1.25 (m, 10H), 1.52 (m, 2H), 2.72 (dd, J = 7.2,
7.6 Hz, 2H), 3.81 (s, 3H), 5.23 (s, 2H), 7.03 (d, J = 8.0 Hz, 1H), 7.35
(m, 7H); 13C NMR (CD3OD, 100 MHz): d 166.3, 154.1, 135.8,

4960
B. Xiao et al. / Bioorg. Med. Chem. 20 (2012) 4954–4961
100 MHz): d 169.2, 167.2, 156.1, 130.3, 129.7, 125.6, 121.7, 116.5,
69.0, 51.7, 31.8, 29.5, 29.2, 29.1, 29.0, 25.8, 22.5, 13.2; FABMS m/z
309.5 [M+H]+.
activity was measured using a GloMaxÒ-Multi Microplate Multi-
mode Reader (Promega Corporation, CA, USA).
ANOVA was used to determine significant differences between
groups. Differences among the mean of individual groups were as-
sessed by the Fisher’s protected LSD post hoc test. Values of p <0.05
were considered statistically significant.
4.2.8. Preparation of 20
Decanoyl chloride (20 ll, ca. 0.1 mmol) in THF (2 ml) was
cooled to 0 °C, and a solution of 1 (16.4 mg, 0.1 mmol) in THF
(1.5 ml) was added dropwise; the mixture was stirred under
15 °C for 4 h. After this time, water was added and extracted with
dichloromethane, then successively washed with H2O and brine.
The organic layer was dried with MgSO4, and evaporated to give
a crude product, which was chomatographed on silica gel eluting
with CHCl3-MeOH to give 20 (70%): white oil; 1H NMR (CDCl3,
400 MHz): d 0.84 (t, J = 7.2 Hz, 3H), 1.23 (m, 12H), 1.72 (m, 2H),
2.24 (t, J = 6.4 Hz, 2H), 6.62 (d, J = 7.6 Hz, 1H), 6.64 (d, J = 8.0 Hz,
1H), 7.14 (t, J = 8.0 Hz, 1H); 13C NMR (CDCl3, 100 MHz): d 172.3,
163.7, 163.5, 154, 143.1, 134.3, 130.1, 127.0, 126.7, 33.5, 31.9,
29.7, 29.4, 29.1, 25.1, 22.8, 14.1; HRFABMS m/z 319.1381 [M+H]+.
4.4. Molecular modeling
Docking calculations were performed using AutoDock Vina
1.1.2 software.27 The default settings and scoring function of Vina
were applied. For ligand preparation, Chem3D Ultra 8.0 software
was used to convert the 2D structures of the candidates into 3D
structural data. Protein coordinates were downloaded from the
Protein Data Bank, accession code 2PRG. Chain A was prepared
for docking within the molecular modeling software package Chi-
mera 1.5.3 by removing chain B, as well as all ligands and water
molecules (except water molecules 308, 399, 444, and 467) and
by calculating the protonation state of the protein. Addition of po-
lar hydrogen and setting grid box parameters was performed by
MGLTools 1.5.4. PyMol v1.5 was used for analyzing and visually
investigating the ligand–protein interactions of the docking poses.
4.3. Biological evaluations
4.3.1. Competitive binding assays
PPAR-
instructions (LanthaScreen, Invitrogen). In brief, 40
reaction kit contained 0.5 nM PPAR- LBD (GST), 5 nM terbium-
tagged anti-GST antibody, 5 nM Fluormone™ Pan-PPAR Green,
and varying concentrations of rosiglitazone (10 nM–1000 M).
The negative control did not include the agonist. After 4-h incuba-
tion in the dark, TR-FRET measurements were performed using a
TriStar LB941 spectrofluorometer (Berthold Technologies, Calmb-
acher, Germany) with the following settings: optical module, Lan-
c
binding was assessed according to the manufacturer’s
ll of the total
Acknowledgements
c
This study was supported by a Grant from the Marine Biotech-
nology Program funded by the Ministry of Land, Transport, and
Maritime Affairs, and by a Grant from the National Research Foun-
dation (No. 20090083538), Korea.
l
Supplementary data
thaScreen; counting time, 0.1 s; and integration time, 20 ls. The
ratiometric emission at 520/495 nm was plotted against various
agonist concentrations. The data were analyzed using Prism soft-
ware (GraphPad Software, Inc., San Diego, CA, USA).
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.06.039.
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