Design, synthesis, biological evaluation and docking studies of pterostilbene analogs inside PPARα

Article abstract of DOI:10.1016/j.bmc.2008.01.051

Pterostilbene, a naturally occurring analog of resveratrol, has previously shown PPARα activation in H4IIEC3 cells and was found to decrease cholesterol levels in animals. In this study, analogs of pterostilbene were synthesized and their ability to activ

Full text of DOI:10.1016/j.bmc.2008.01.051

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3800–3808
Design, synthesis, biological evaluation and docking studies
of pterostilbene analogs inside PPARa
Cassia S. Mizuno,a Guoyi Ma,b Shabana Khan,b Akshay Patny,c
Mitchell A. Averyc and Agnes M. Rimandoa,*
aUSDA-ARS, Natural Products Utilization Research Unit, PO Box 8048, University, MS 38677, United States
bNCNPR, School of Pharmacy, University of Mississippi, University, MS 38677, United States
cDepartment of Medicinal Chemistry and Laboratory for Applied Drug Design and Synthesis, School of Pharmacy,
University of Mississippi, University, MS 38677, United States
Received 3 December 2007; revised 25 January 2008; accepted 25 January 2008
Available online 31 January 2008
Abstract—Pterostilbene, a naturally occurring analog of resveratrol, has previously shown PPARa activation in H4IIEC3 cells and
was found to decrease cholesterol levels in animals. In this study, analogs of pterostilbene were synthesized and their ability to acti-
vate PPARa was investigated. Among analogs that was synthesized (E)-4-(3,5-dimethoxystyryl)phenyl dihydrogen phosphate
showed activity higher than pterostilbene and control drug ciprofibrate. Docking of the stilbenes inside PPARa showed the presence
of important hydrogen bond interactions for PPARa activation.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Resveratrol has been extensively investigated because its
occurrence in wine has been linked to lower risk of car-
diovascular diseases among wine drinking popula-
tion.13,14 The antioxidant activity of resveratrol has
been proven in a number of studies.1,15–17 In addition
to its antioxidant activity, resveratrol has also been dem-
onstrated to reduce blood lipid levels in animals.18
Pterostilbene 2, a naturally occurring dimethylether ana-
log of 1, has shown in previous studies to lower plasma
lipid levels when fed to hamsters.19 Significant activation
of PPARa was also observed with 2 in the same study,
whereas none of the other naturally occurring stilbenes
tested, including 1, showed activation of PPARa.19
The stilbene scaffold is known to hold several biological
activities considered beneficial for human health and for
use in agriculture. The potential health benefits of this
class of compounds have been extensively investigated
in the past several years. Pharmacological properties
of hydroxylated stilbenes include antioxidative,1 anti-
inflammatory,2 antileukemic,3 antibacterial,4 anti-
fungal,5 antiplatelet aggregation,6 vasodilator,7 and
antitumor8 activities. Due to the variety of their benefi-
cial effects, including chemopreventive9 and chemopro-
tective10 activities, this relatively structurally simple
group of compounds has attracted attention as a novel
potential therapeutic class. Resveratrol 1 [(E)-1-(40-
hydroxyphenyl)-2-(3,5-dihydroxyphenyl)ethene, Fig. 1],
the most widely studied stilbene, is a naturally occurring
polyphenol stilbenoid found in grapes,11 peanuts, and
berries.12 This molecule is synthesized by plants in re-
sponse to environmental stress and fungal infections
and is known to be involved in defense mechanisms of
plants.
The peroxisome proliferator activated receptors
(PPARs) are members of the nuclear receptor superfam-
ily of ligand-activated transcription factors that play
important role in carbohydrate and lipid metabolism.
PPARa is expressed in the liver, heart, and muscles
and is involved in the metabolism of fatty acids and lip-
ids. PPARa is a known target for the treatment of dysl-
ipidemia. Among the commonly used drugs for
hypercholesterolemia (or hyperlipidemia), fibrates are
known PPARa agonists. The triglyceride lowering and
HDL-cholesterol increasing effects of fibrates are attrib-
uted to the activation of PPARa, which plays a role in
lipoprotein catabolism.20
Keywords: Pterostilbene; Resveratrol; Peroxisome proliferator acti-
vated receptor; Dyslipidemia.
*
Corresponding author. Tel.: +1 662 915 1037; fax: +1 662 915
1035; e-mail: arimando@olemiss.edu
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.01.051

C. S. Mizuno et al. / Bioorg. Med. Chem. 16 (2008) 3800–3808
3801
This study was undertaken to investigate the effect of
OMe
11a
chemical modification of pterostilbene on PPARa acti-
vation. We kept the methoxy groups at the 3 and 5 posi-
tions and introduced different groups at 40 position. The
docking of some of the natural and synthetic stilbenes
into PPARa is also reported.
MeO
NH2
a
8a, 8b
NH2
2. Chemistry
OMe
11b
Pterostilbene derivatives 8a,b–10a,b were synthesized
through Wittig reaction according to Pettit et al.21
(Scheme 1). Coupling of phosphonium salt 421 and aro-
matic aldehydes 5, 6, and 7 gave a mixture of cis and
trans isomers, which were isolated by column chroma-
tography. The configurations of the molecules were
determined by comparing their spectral data with those
reported in the literature.22 The nitro derivatives 8a and
8b were treated with sodium dithionite22 to give com-
pounds 11a and 11b (Scheme 2). Hydrolysis of esters
9a and 9b afforded acids 12a and 12b (Scheme 3). To
study the role of the vinyl double bond of 2 in the acti-
vation of PPARa, reduction was accomplished with Pd/
C in MeOH generating the saturated compound 13.
Phosphate derivative 15 was synthesized through reac-
tion of 2 and dibenzylphosphite using DMAP, DIEA,
followed by deprotection of the benzyl groups21
(Scheme 4). Wittig reaction between aldehyde 17 and
phosphonium salt 1921 afforded cis and trans isomers
21 and 22 (Scheme 5).
MeO
Scheme 2. Synthesis of 11a and 11b. Reagents and condition: (a)
Na2S2O4, acetone/H2O, 50 ꢁC.
3. Results and discussion
Based on previous observations of its significant PPARa
activation, 2 was used as a lead compound for designing
analogs with the aim of identifying molecules more ac-
tive toward PPARa activation than the parent natural
compound 2.
The crystal structure of PPARa ligand-binding domain
(LBD) (PDB code 1K7L) revealed that the ligand
OMe
OH
MeO
HO
2
1
OH
OH
Figure 1. Chemical structure of resveratrol 1 and pterostilbene 2.
OMe
MeO
R
8a R= NO2
9a R= COOMe
10a R= OMe
P(Ph3)Br
OMe
Br
O
a
b
+
R
MeO
MeO
OMe
OMe
3
4
R
5 R= NO2
6 R= COOMe
7 R= OMe
MeO
8b R= NO2
9b R= COOMe
10b R= OMe
Scheme 1. Synthesis of 8a,b–10a,b. Reagents and conditions: (a) PPh3, toluene, reflux, overnight; (b) n-BuLi, THF, ꢀ78 ꢁC ! rt, overnight.

3802
C. S. Mizuno et al. / Bioorg. Med. Chem. 16 (2008) 3800–3808
scriptional activation of the receptor via recruitment of
co-activator proteins.
OMe
In addition, the tyrosine moiety of GW409544 is ori-
ented toward the hydrophobic pocket formed by helices
3, 6, and 10, adjacent to the C-terminal AF-2 helix.23
Based on these observations we decided to introduce
acidic and polar groups at the 40 position of 2. These po-
lar groups are anticipated to form hydrogen bond and
interact with the critical polar amino acid residues on
the AF-2 helix such as Tyr464 considered important
for PPARa activation.
MeO
12a
COOH
a
9a, 9b
COOH
OMe
12b
Analogs (8–22) were evaluated for their activity as
PPARa agonist in H4IIEC3 cells at concentrations
ranging from 0.1 lM to 100 lM. Their activities were
compared with ciprofibrate (Fig. 3).
MeO
Scheme 3. Synthesis of 12a and 12b. Reagents and condition: (a)
NaOH, MeOH, reflux, 4 days.
The nitro compounds (8a and 8b) and the amine com-
pounds (11a and 11b) did not exhibit a significant acti-
vation of PPARa. Among the cis isomers, compound
10b had significant activity as PPARa agonists (>2-fold
activation at 1–100 lM). Introduction of an ester group
(9a) also resulted in increase in activity (>2-fold activa-
tion at 10–100 lM). Interestingly, reduction of the vinyl
double bond in 2 resulted in loss of its activity as repre-
sented by 13, indicating that the double bond is essential
for PPARa activation for stilbenes. Saturation of the
double bond causes conformation changes of the mole-
cule resulting in loss of its activity. The most active com-
pound was the phosphate derivative 15, which exhibited
a sixfold induction of PPARa at the highest concentra-
tion tested (100 lM) with twofold or more induction at
GW409544 (Fig. 2) makes several hydrogen bond inter-
actions with Ser280, Tyr314, Tyr464, and His440 that
are known to be the key interactions for PPARa ago-
nists.23 GW409544 is a L-tyrosine analog of PPARc ago-
nist farglitazar (Fig. 2) and its synthesis was first
described in 2000 by Collins et al.24 for treatment of dia-
betes and cardiovascular diseases. This molecule is a full
PPARa (EC50 = 2.3 nM) and PPARc (EC50 = 0.28 nM)
agonist.23 GW409544 adopts a conformation where the
acidic head group of the ligand functions as hydrogen
bond donor and interacts with Tyr314 on helix 5 and
Tyr464 on the AF-2 helix. These interactions stabilize
the receptor in a conformation that leads to the tran-
OMe
MeO
2
OH
b
a
OMe
OMe
MeO
MeO
O
P
13
14
OH
O
OBz
OBz
c
OMe
MeO
O
P
15
O
OH
OH
Scheme 4. Synthesis of 13 and 15. Reagents and conditions: (a) Pd/C MeOH, overnight; (b) DMAP, DIEA, CH3CN, dibenzyl phosphite, CCl4,
ꢀ10 ꢁC ! rt, 12 h; (c) BrSi(CH)3, DCM 0 ꢁC ! rt, 2 h.

C. S. Mizuno et al. / Bioorg. Med. Chem. 16 (2008) 3800–3808
3803
OMe
OH
21
OH
16
OTBS
a
HO
O
O
HO
TBSO
OTBS
17
c
d
P(Ph)3Br
Br
TBSO
OH
20
OMe
b
HO
OMe
19
OMe
18
OMe
22
Scheme 5. Synthesis of 21 and 22. Reagents and conditions: (a) TBDMSCl, DIEA, DMF, 18 h, rt, 60%; (b) PPh3, toluene, reflux, overnight; (c) n-
BuLi, THF, 15 ꢁC ! rt, 12 h; (d) TBAF, THF, 15 min.
CO2H
H
HN
CO2H
H
HN
O
N
O
N
O
O
O
O
Farglitazar
GW409544
Figure 2. Chemical structure of Farglitazar and GW409544.
7
6
5
4
3
2
1
0
0.1uM
1uM
10uM
100uM
DMSO
2
8a
8b
9a
9b 10a 10b 11a 11b 12a 12b
13
15
21
22 ciprofibrate
Figure 3. Effects of stilbenes on PPARa activation in a cell-based assay at different concentrations.
the lower concentrations. This observation shows that
introduction of an acidic group leads to a compound
more active than 2. It is possible that phosphate 15
may serve as a prodrug with improved cell permeability
that leads to the observed increase in its activity. In gen-
eral, compounds 9a, 10a, and 10b were also more effec-
tive than ciprofibrate in their ability to activate PPARa.
Considering that compounds 8–12 and 15 all have meth-
oxy groups at 3 and 5 positions, the different groups at
40 position determine the activity. Thus methoxy, ester,
and phosphate are preferred. In the case of compounds
with hydroxyl groups at 3 and 5 positions compound 21
did not show significant activation of PPARa at any
concentration tested. Compound 22 showed activation

3804
C. S. Mizuno et al. / Bioorg. Med. Chem. 16 (2008) 3800–3808
Figure 4. Proposed binding pose of cis isomer 21 (colored by atom type) inside PPARa ligand-binding domain. The protein backbone is shown as
green ribbon. Important residues are shown in yellow.
of PPARa (>2-fold) only at the highest concentration
indicating that methoxy groups at 3 and 5 positions
are important for activity. Among the active compounds
9a, 10a, 10b, and 15, compound 10b was the only cis iso-
mer suggesting that the configuration trans is more
favorable for activating PPARa.
gen bond interaction with this amino acid residue
(Fig. 5).
The phosphate derivative 15 was the most active com-
pound of this series. Docking of this molecule showed
that the phosphate group forms hydrogen bonds and
interacts with Ser280 and His440, with the two phenyl
rings docked into the hydrophobic pocket formed by
helices 20, 3, and b sheet (Fig. 6). As the compounds pre-
sented similar interactions with amino acids in the
PPARa active site and the activities among the com-
pounds were close, docking in this particular case could
not explain the difference in activity between analogs but
shall shed light on the structural basis for structure–
activity relationship of stilbenes in general.
To investigate in detail these molecules with different
scaffolds from the known PPARa agonists, the stilbenes
were docked into the PPARa ligand-binding domain.23
Docking studies revealed that the inactive cis isomers
were mostly docked in the hydrophobic pocket formed
by helices 20, 3, and b sheet and did not interact with po-
lar amino acids located on the left most part of the T-
shaped cavity known to be important for activation of
the receptor (Fig. 4). This lack of polar interactions
can possibly explain the lack of activity of the cis iso-
mers. Compound 10b was the only cis isomer that
showed activity but the docking results are not consis-
tent with the observed activity of this molecule in partic-
ular, as it was docked in very similar manner as the
other inactive cis isomers. However, although active,
10b showed only moderate activity and it may bind to
the receptor in different mode.
4. Conclusion
The stilbene comprises a unique template as PPARa
agonist. Here we have described the synthesis of 14 stilb-
enes and their activities as PPARa agonists. Phosphate
15 was discovered as PPARa agonist with potency high-
er than ciprofibrate. In general, the trans analogs were
more active than the cis isomers. Docking studies re-
vealed that the trans isomers interact with amino acids
residues essential for activation of the receptor while
the cis isomers do not. Based on previous results with
2, we succeeded to design a stilbene (15) which is more
active as PPARa agonist.
In general, docking of the trans isomers revealed very
similar interactions to 2 as shown in Figure 5. The two
methoxy groups at 3 and 5 positions of 2 make hydro-
gen bond interactions with Ser280 and Tyr464 which
were considered as key interactions for activation of
the receptor. The remaining part of the molecule fitted
very well into the hydrophobic pocket formed by helices
3, 6, and 10 adjacent to the AF-2 helix, also known as
‘benzophenone’ pocket.25 Unlike GW409544 that forms
favorable hydrophobic interactions with Phe351, the hy-
droxy group on the 40 position of 2 displayed a hydro-
5. Experimental
All solvents were redistilled prior to use. All reactions
occurred under inert atmosphere. All round bottom

C. S. Mizuno et al. / Bioorg. Med. Chem. 16 (2008) 3800–3808
3805
Figure 5. Proposed binding pose of 2 (colored by atom type) superimposed on the cocrystal structure of GW409544 (magenta) inside PPARa ligand-
binding domain. The protein backbone is shown as green ribbon. Important residues are shown in yellow and hydrogen bond interactions are shown
as yellow dotted line.
Figure 6. Proposed binding pose of 15 (colored by atom type) inside PPARa ligand-binding domain. The protein backbone is shown as green ribbon.
Important residues are shown in yellow and hydrogen bond interactions are shown as yellow dotted line.
flasks were kept inside the oven or dried under vacuum
and heated prior to use. Reactions were monitored by
thin layer chromatography (TLC) using silica gel (Kie-
selgel 60 F254, Merck). NMR data were recorded on a
Bruker 400 MHz 50 MM, Bruker 400 MHz Ultra Shield
or Varian AS400 and the analyses conducted in CDCl3,

3806
C. S. Mizuno et al. / Bioorg. Med. Chem. 16 (2008) 3800–3808
DMSO-d6, or MeOD. Purification was performed using
Automated Flash Purification (Biotage Horizon) and
flash chromatography using silica gel (Sorbent Technol-
ogies 40–60 lM). Mass spectra were collected using a
JEOL AccuTOF JMS-T100LC.
5.1.3. 1,3-Dimethoxy-5-(4-methoxystyryl)benzene 10a
and 10b. Reaction of (3,5-dimethoxybenzyl)triphenyl-
phosphonium 4 (300 mg, 0.608 mmol) and 4-methoxy-
benzaldehyde 7 (75 lL, 0.608 mmol) 10b and 10a.
Compound 10a as a white solid: 15 mg (9%). 1H
NMR (CDCl3, 400 MHz): d 3.83 (s, 9H); 6.39 (s, 1H);
6.67 (s, 2H); 6.89 (s, 2H); 6.92 (d, 1H, J = 8 Hz); 7.02
(d, 1H, J = 8 Hz); 7.45 (d, 2H, J = 8 Hz). 13C NMR
(CDCl3, 400 MHz): d 55.3 (3C), 99.6, 104.3 (2C), 114.1
(2C), 126.5, 127.8 (2C), 128.7, 129.9, 139.7, 159.4,
161.0 (2C). HRMS: Calcd for [2M+Na] C34H36NaO6
563.24096, found 563.24419. Compound 10b as a
viscous liquid: 58 mg (35%). 1H NMR (CDCl3,
400 MHz): d 3.69 (s, 6H); 3.78 (s, 3H); 6.37 (s, 1H);
6.46–6.49 (m, 3H); 6.55 (d, 1H, J = 12 Hz); 6.80 (d,
2H, J = 8 Hz); 7.26 (d, 2H, J = 8 Hz). 13C NMR
(CDCl3, 400 MHz): d 55.1 (2C), 99.6, 106.6 (2C), 113.5
(2C), 128.7, 129.5, 130.2 (2C), 130.3 (2C), 139.5, 158.8,
160.6 (2C). HRMS: Calcd for [2M+Na] C34H36NaO6
563.24096, found 563.24396.
5.1. General procedure for stilbenes synthesis
To a cold solution (ꢀ78 ꢁC) of phosphonium salt (1.0
equiv) in THF was added n-butyllithium (1.6 mol in hex-
anes, 1.0 equiv) and the resulting solution was stirred
under inert atmosphere for 2 h. A solution of aldehyde
(1.0 equiv) in THF was added dropwise, and the mixture
was stirred for 12 h at room temperature. The resulting
suspension was poured into water and extracted with
dichloromethane. The organic phase was combined
and dried over MgSO4 and concentrated under reduced
pressure. The crude product was purified through auto-
mated flash purification eluting with hexanes/ethyl ace-
tate (97:3). The cis isomer was eluted first followed by
the trans isomer.
5.1.4. 5-(4-Methoxystyryl)benzene-1,3-diol 21 and 22.
Reaction of 3,5-bis(tert-butyldimethylsilyloxy)benzalde-
hyde 17 (408 mg, 1.11 mmol) and (4-methoxybenzyl)tri-
phenylphosphonium bromide 19 (500 mg, 1.11 mmol)
afforded 452 mg (86% yield) of mixture of cis and trans
stilbenes 20. Due to difficulties found in separating the
two isomers, deprotection of TBS group was followed
without isolation of the isomers. Tetrabutylammonium
fluoride (2.5 mL, 2.5 mmol) was added to a mixture of
(E) and (Z)-(5-(4-methoxystyryl)-1,3-phenylene)bis(oxy)
bis(tert-butyldimethylsilane) 20 (452 mg, 0.96 mmol) in
anhydrous tetrahydrofuran (10 mL). The solution was
stirred for 45 min, poured into water, and extracted with
ether. After removal of the solid, the resulting crude
mixture was purified using flash chromatography (7:3
hexanes/ethyl acetate) and afforded cis (21) and trans
(22). Compound 22 23 mg (8%). 1H NMR (MeOD,
400 MHz): d 3.73 (s, 3 H); 6.19 (s, 1H); 6.47 (s, 2H);
6.79–6.85 (m, 3H); 6.96 (d, 1H, J = 16 Hz); 7.38 (d,
2H, J = 12 Hz). 13C NMR (DMSO, 400 MHz): d 58.5,
105.6, 108.7 (2C), 117.9 (2C), 130.5, 131.5 (2C), 131.9,
134.2, 144.0, 162.4 (2C), 163.5. HRMS: Calcd for
[MꢀH] C15H13O3 241.08647, found 241.08863. Com-
5.1.1. 1,3-Dimethoxy-5-(4-nitrostyryl)benzene (8a and
8b). Reaction of (3,5-dimethoxybenzyl)triphenylphos-
phonium 4 (300 mg, 0.608 mmol) and 4-nitrobenzalde-
hyde (92 mg, 0.608 mmol) 3 afforded 8b and 8a.
Compound 8a as a yellow solid: 71 mg (41%). 1H
NMR (CDCl3, 400 MHz): d 3.83 (s, 6H); 6.45 (s, 1H);
6.68 (s, 2H); 7.07–7.16 (m, 1H); 7.23 (d, 1H,
J = 36 Hz); 7.61 (d, 2H, J = 8 Hz); 8.20 (d, 2H,
J = 8 Hz). 13C NMR (CDCl3, 400 MHz): d 55.4 (2C),
100.9, 105.0 (2C), 124.1 (2C), 126.7, 126.9 (2C), 133.2,
138.1, 143.6, 146.7, 161.0 (2C). HRMS: Calcd for
[2M+Na] C32H30N2O8Na 593.18998, found 593.18332.
Compound 8b 56 mg (32% yield) 1H NMR (CDCl3,
400 MHz): d 3.66 (s, 6H); 6.34–6.35 (m, 3H); 6.58 (d,
1H, J = 12 Hz); 6.74 (d, 1H, J = 16 Hz); 7.39 (d, 2H,
J = 8 Hz); 8.07 (d, 2H, J = 8 Hz). 13C NMR (CDCl3,
400 MHz): d 55.4 (2C), 100.3, 106.9 (2C), 123.6 (2C),
128.5, 129.9 (2C), 134.1, 138.2, 144.2, 146.7, 161.0
(2C). HRMS: Calcd for [2M+Na] C32H30N2O8 Na
593.18998, found 593.18987.
5.1.2. Methyl 4-(3,5-dimethoxystyryl)benzoate 9a and 9b.
Reaction of (3,5-dimethoxybenzyl)triphenylphospho-
nium 4 (300 mg, 0.608 mmol) and methyl 4-form-
ylbenzoate 6 (99 mg, 0.608 mmol) afforded 9b and 9a.
Compound 9a as a white solid: 50.2 mg (27%). 1H
NMR (CDCl3, 400 MHz): d 3.83 (s, 6H); 3.92 (s, 3H);
6.42 (s, 1H); 6.68 (s, 2H); 7.07–7.16 (m, 2H); 7.55 (d,
2H, J = 8 Hz); 8.02 (d, 2H, J = 8 Hz). 13C NMR
(CDCl3, 400 MHz): d 52.3, 55.5 (2C), 100.7, 105.0
(2C), 126.6 (2C), 128.2, 129.1, 130.2 (2C), 131.4, 138.9,
141.8, 161.2 (2C), 167. HRMS: Calcd for [2M+Na]
C36H36NaO8 619.23079, found 619.22594. Compound
1
pound 21 57 mg (22%). H NMR (MeOD, 400 MHz):
d 3.70 (s, 3H); 6.18 (s, 1H); 6.32 (s, 2H); 6.33 (d, 1H,
J = 12); 6.47 (d, 1H, J = 12); 6.73 (d, 2H, J = 8 Hz);
7.18 (d, 2H, J = 8 Hz). 13C NMR (DMSO, 400 MHz):
d 58.4, 105.2, 111.1, 117.3 (2C), 132.5, 133.5, 133.7
(2C), 134.1 (2C), 143.9, 162 (2C), 162.9. HRMS: Calcd
for [MꢀH] C15H13O3 241.08647, found 241.08735.
5.1.5. General procedure for the reduction of nitro
derivatives 8a and 8b. A solution of trans nitro derivative
8a (45 mg, 0.15 mmol) in acetone/water (10:5 mL) was
heated to 50 ꢁC for 30 min. Sodium dithionite (686 mg,
3.75 mmol) was slowly added and the mixture was
heated to reflux for 1 h. After cooled to room tempera-
ture the mixture was poured into water and extracted
with ethyl acetate. The organic phase was combined
and dried over MgSO4 and solvent was removed under
reduced pressure. The crude mixture was purified using
automated flash chromatography eluting with hexanes/
1
9b as a viscous liquid: 95 mg (52%). H NMR (CDCl3,
400 MHz): d 3.63 (s, 6H); 3.88 (s, 3H); 6.33 (9s, 1H);
6.36 (s, 2H); 6.57 (dd, 2H, J1,2 = 12 Hz, J1,3 = 8 Hz);
7.32 (d, 2H, J = 8 Hz); 7.90 (d, 2H, J = 8 Hz). 13C
NMR (CDCl3, 400 MHz): d 52.0, 55.1 (2C), 100.0,
106.7 (2C), 128.6, 128.9 (2C), 129.4 (2C), 129.5, 132.1,
138.4, 142.0, 160.6 (2C), 166.8. HRMS: Calcd for
[2M+Na] C36H36NaO8 619.23079, found 619.22729.

C. S. Mizuno et al. / Bioorg. Med. Chem. 16 (2008) 3800–3808
3807
ethyl acetate (75:25). Compound 11a as a yellow solid:
14.7 mg (36%). H NMR (CDCl3, 400 MHz): d 3.82 (s,
DIEA (245 lL, 1.4 mmol). The mixture was left stirring
at ꢀ10 ꢁC for 30 min and dibenzyl phosphate (224 lL,
1 mmol) was added. The solution was stirred for 12 h
at room temperature and poured into 0.5 M monobasic
potassium phosphate solution. The mixture was ex-
tracted with ethyl acetate and the organic phase was
combined and dried over MgSO4. The solvent was evap-
orated, the crude mixture was purified by column chro-
matography eluting with hexanes/ethyl acetate (7:3) and
gave 360 mg (80%) of (E)-dibenzyl 4-(3,5-dimethoxysty-
1
6H); 6.37 (s, 1 H); 6.65–6.68 (m, 4H); 6.86 (d, 1H,
J = 16 Hz); 7.01 (d, 1H, J = 16 Hz); 7.33 (d, 2H,
J = 8 Hz). 13C NMR (CDCl3, 400 MHz): d 55.5 (2C),
99.6, 104.4 (2C), 115.5 (2C), 125.2, 128, 128.1 (2C),
129.5, 140.2, 146.4, 161.2 (2C). HRMS: Calcd for
[M+H] C16H18NO2 256.13375, found 256.14321. Com-
1
pound 11b as a yellow solid (47%). H NMR (CDCl3,
400 MHz): d 3.68 (s, 6H); 6.32 (s, 1H); 6.39 (s, 1H);
6.47 (m, 3H); 6.54 (d, 2H, J = 8 Hz); 7.11 (d, 2H,
J = 8 Hz). 13C NMR (CDCl3, 400 MHz): d 55.4 (2C),
99.7, 106.8 (2C), 114.8 (2C), 127.6, 127.7, 130.4 (2C),
130.8, 140.1, 145.7, 160.7 (2C). HRMS: Calcd for
[M+H] C16H18NO2 256.13375, found 256.10757.
1
ryl)phenyl phosphate 14. H NMR (CDCl3, 400 MHz):
d 3.83 (s, 6 H); 5.13 (d, 4H, J = 8 Hz); 6.41 (s, 1H);
6.67 (s, 2H); (dd, 2H, J1,2 = 16 Hz, J1,3 = 33 Hz); 7.15
(d, 2H, J = 8 Hz); 7.34 (s, 10H); 7.43 (d, 2H,
J = 8 Hz). 13C NMR (CDCl3, 400 MHz): d 55.6 (2C),
70.2 (2C), 100.3, 104.8 (2C), 120.5 (2C), 128 (2C),
128.2 (4C), 128.8 (4C), 128.9 (2C), 129, 134.5, 135.6,
135.7, 139.4, 150.2, 150.3, 161.2 (2C). HRMS: Calcd
for [M+H] C30H30O6P 517.17800, found 517.17444.
5.1.6. General procedure for hydrolysis of esters 9a and
9b. To a solution of 9a (50 mg, 0.16 mmol) in THF was
added NaOH solution (1 N, 335 lL). The reaction was
left stirring at reflux for 3 days and NaOH was added
from time to time until starting material disappeared.
The mixture was poured into water, the pH was brought
to neutral and the mixture was extracted with ethyl ace-
tate. The organic phase was combined and dried over
MgSO4 and concentrated. The crude material was puri-
fied using automated flash chromatography eluting with
chloroform/methanol (9:1). Compound 12a as a white
solid: 19 mg (44%). 1H NMR (DMSO, 400 MHz): d
3.74 (s, 6 H); 6.41 (s, 1H); 6.78 (s, 2H); 7.29 (d, 2H,
J = 4 Hz); 7.67 (d, 2H, J = 8 Hz); 7.91 (d, 2H,
J = 8 Hz). 13C NMR (DMSO, 400 MHz): d 55.9 (2C),
101, 105.4 (2C), 127.8 (2C), 128.6, 130.2, 130.4 (2C),
131.6, 139.3, 142, 161.3 (2C), 167.8 HRMS: Calcd for
[MꢀH]. C17H15O4283.09703, found 283.09726. Com-
5.1.9. (E)-4-(3,5-dimethoxystyryl)phenyl dihydrogen
phosphate 15. Bromotrimethylsilane (80 lL, 0.61 mmol)
was added to a solution of dibenzyl phosphate 14
(150 mg, 0.29 mmol) in anhydrous dichloromethane
(10 mL) at 0 ꢁC. After 2 h stirring at room temperature,
water was added and the solution was stirred for 1 h.
The mixture was extracted with ethyl acetate and the or-
ganic phase was combined and dried over MgSO4. The
1
solvent was removed to afford 15, 38.6 mg (39%). H
NMR (CDCl3, 400 MHz): d 3.63 (s, 6H); 6.23 (s, 1H);
6.40 (s, 2H); 6.60 (d, 1H, J = 16 Hz); 6.71 (d, 1H,
J = 16 Hz); 7.02–7.14 (m, 4H); 9.11 (br s, 2H). 13C
NMR (CDCl3, 400 MHz): d 58.6 (2C), 103.5, 108.2
(2C), 124.4, 131.4 (3C), 131.8, 132.1, 137.2, 143.6, 156,
165.1 (2C). HRMS: Calcd for [MꢀH] C16H17O6P
335.06845, found 335.07300.
1
pound 12b as a white solid (24%). H NMR (DMSO,
400 MHz): d 3.56 (s, 6H); 6.31–6.34 (m, 3H); 6.64 (s,
2H); 7.30 (d, 2H, J = 8 Hz); 7.80 (d, 2H, J = 8 Hz).
13C NMR (DMSO, 400 MHz): d 55.4 (2C), 100, 107
(2C), 129.1 (2C), 129.7 (3C), 130.3, 132, 138.5, 141.7,
160.7 (2C), 167.8. HRMS: Calcd for [MꢀH] C17H15O4
283.09703, found 283.09681.
5.1.10. 3,5-Bis(tert-butyldimethylsilyloxy)benzaldehyde
17. To a solution of 3,5 dihydroxybenzaldehyde 16
(500 mg, 3.62 mmol) in DMF (10 mL) was added DIEA
(126 lL, 7.24 mmol). The solution was stirred for 15 min
when TBDMSCl was added dissolved in DMF. After
stirring for 18 h, the mixture was poured into water
and extracted with ethyl acetate. The organic phase
was combined and dried over MgSO4 and the solvent re-
moved under reduced pressure. The crude mixture was
purified using column chromatography eluting with hex-
anes/ethyl acetate (95:5) and afforded 800 mg, (60%) of
3,5-bis(tert-butyldimethylsilyloxy)benzaldehyde 17. 1H
NMR (CDCl3, 400 MHz): d 0.21 (s, 12H); 0.98 (s,
18H); 6.59 (s, 1H); 6.94 (d, 2H, J = 4 Hz); 9.85 (s, 1H).
13C NMR (CDCl3, 400 MHz): d ꢀ4.20 (4C), 18.4 (2C),
25.8 (6C), 114.6 (2C), 118.6, 138.5, 157.4, 192 (2C). IR
5.1.7. 4-(3,5-Dimethoxyphenethyl)phenol 13. Pd/C (cata-
lytic) was added to a solution of pterostilbene 2
(50 mg, 0.195 mmol) in methanol. The mixture was left
stirring overnight under H2 at room temperature. The
mixture was filtered and the solvent was evaporated un-
der reduced pressure. The crude mixture was purified
using flash chromatography eluting with hexanes/ethyl
acetate (7:3) to afford 49 mg (98%) of 4-(3,5-dimethoxy-
1
phenethyl)phenol 13. H NMR (CDCl3, 400 MHz): d
2.84 (s, 4H); 3.78 (s, 6H); 6.34–6.35 (m, 4H); 6.76 (d,
2H, J = 8 Hz); 7.05 (d, 2H, J = 8 Hz). 13C NMR
(CDCl3, 400 MHz): d 36.7, 38.4, 55.2 (2C), 97.9, 106.6
(2C), 115.1 (2C), 129.5 (2C), 133.8, 144.2, 153.7, 160.6
(2C). HRMS: Calcd for [MꢀH] C16H17O3 257.11777,
found 257.11889.
(neat) 2956, 2931, 2887, 2859, 1704 cmꢀ1
.
5.2. Molecular docking
The compounds were sketched and converted into three-
dimensional structures using the program Sybyl. Mini-
mization was carried out for these compounds, which
employed 1000 steps each of steepest descents followed
by conjugate gradients and finally BFGS method up
5.1.8. (E)-Dibenzyl 4-(3,5-dimethoxystyryl)phenyl phos-
phate 14. To a cold mixture (ꢀ10 ꢁC) of 2 (200 mg,
0.67 mmol) and N,N-(dimethylamino)pyridine (10.2 mg,
0.083 mmol) in anhydrous acetonitrile (10 mL) was
added carbon tetrachloride (323 lL, 3.35 mmol) and
˚
to a gradient of 0.01 kcal/mol/A. Docking of pterostil-

3808
C. S. Mizuno et al. / Bioorg. Med. Chem. 16 (2008) 3800–3808
bene analogs was performed in the PPARa ligand-bind-
ing domain (PDB code: 1K7L). Protein preparation was
done using the program Maestro, which involved addi-
tion of hydrogen atoms, minimization of the protein
and assigning of formal charges to the amino acid resi-
dues. The X-ray crystal bound pose of GW409544 was
used to define binding site during protein preparation.
Docking of the compounds was performed using the
program GOLD. The binding site for docking was de-
fined using the sulfur atom of Cys276 and all amino acid
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were plated in 96-well plate at
a
density of
5 · 104 cells/well and grown for 24 h. The cells were trea-
ted with different concentrations of test compounds for
24 h followed by addition of 40 lL 1:1 mixture of Luc-
Lite reagent and PBS containing 1 mM calcium and
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of Light output measured on a TopCount microplate
reader (Packard Instrument Co. Meriden, CT) in a sin-
gle photon counting mode.
Acknowledgments
USDA Agricultural Research Service Specific Coopera-
tive Agreement No. 58-6408-2-0009 is acknowledged for
partial support of this work. We thank Guilherme Fave-
rao for technical support and his dedication to this
project.
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U.S. Patent 6,498,174 BI, 2000.
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