Design, synthesis and in vitro evaluation of a series of α-substituted phenylpropanoic acid PPARγ agonists to further investigate the stereochemistry-activity relationship

Article abstract of DOI:10.1016/j.bmc.2012.08.061

We previously demonstrated that the α-benzylphenylpropanoic acid-type PPARγ-selective agonist 6 exhibited a reversed stereochemistry-activity relationship, that is, the (R)-enantiomer is a more potent PPARγ agonist than the (S)-enantiomer, compared with s

Full text of DOI:10.1016/j.bmc.2012.08.061

Bioorganic & Medicinal Chemistry 20 (2012) 6375–6383
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and in vitro evaluation of a series of
phenylpropanoic acid PPAR agonists to further investigate
the stereochemistry–activity relationship
a-substituted
c
Masao Ohashi a, Izumi Nakagome b, Jun-ichi Kasuga c, Hiromi Nobusada a, Kenji Matsuno a,
Makoto Makishima d, Shuichi Hirono b, Yuichi Hashimoto c, Hiroyuki Miyachi a,
⇑
a Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan
b School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
c Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
d School of Medicine, Nihon University, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We previously demonstrated that the
a-benzylphenylpropanoic acid-type PPARc-selective agonist 6
Received 3 August 2012
Revised 28 August 2012
Accepted 29 August 2012
Available online 11 September 2012
exhibited a reversed stereochemistry–activity relationship, that is, the (R)-enantiomer is a more potent
PPAR agonist than the (S)-enantiomer, compared with structurally similar -ethylphenylpropanoic
acid-type PPAR agonists. Here, we designed, synthesized and evaluated the optically active -cyclo-
hexylmethylphenylpropanoic acid derivatives 7 and -phenethylphenylpropanoic acid derivatives 8,
respectively. Interestingly, -cyclohexylmethyl derivatives showed reversal of the stereochemistry–
activity relationship [i.e., (R) more potent than (S)], like -benzyl derivatives, whereas -phenethyl
c
a
a
a
a
Keywords:
a
a
Peroxisome proliferator-activated receptor
PPAR
derivatives showed the ‘normal’ relationship [(S) more potent than (R)]. These results suggested that
the presence of a branched carbon atom at the b-position with respect to the carboxyl group is a critical
determinant of the reversed stereochemistry–activity relationship.
PPAR
c
Phenylpropanoic acid
Ó 2012 Elsevier Ltd. All rights reserved.
SAR
1. Introduction
hypothesis concerning the NR ligand superfamily,13 and we have
already successfully designed many optically active
phenylpropanoic acids, including a human PPAR -selective agonist
(1),14,15 PPAR /d dual agonist (2, 3),16,17 PPARd-selective agonist
(4),18,19 and PPAR pan agonist (5) (Fig. 1).20
/d/
Previous structure–activity relationship (SAR) data on our com-
pounds indicated that the (S)-enantiomers are more potent human
PPAR transactivators than the antipodal (R)-enantiomers, in good
a-substituted
The peroxisome proliferator-activated receptors (PPARs) are li-
gand-dependent transcription factors belonging to the nuclear
receptor (NR) superfamily; they are activated by endogenous fatty
acids and their metabolites, and by synthetic ligands.1–3 In the
presence of a ligand, PPARs heterodimerize with another nuclear
receptor, retinoid X receptor (RXR), and the heterodimers modu-
late transcription of target genes by binding to PPAR response ele-
ments in the promoter region of the target genes.4 The three
a
a
a
c
agreement with data reported for other series of PPAR
nists.21,22 However, we recently found a notable exception. The
-benzylphenylpropanoic acid-type PPAR -selective agonist, 6
exhibited a reversed stereochemistry–activity relationship, that
c ago-
subtypes (PPAR
a
, PPARd, and PPAR
c
) identified to date5 are differ-
a
c
entially expressed in a tissue-specific manner,6 and play pivotal
roles in not only metabolic homeostasis,7 but also various kinds
of biological responses.8–12 Therefore, PPARs have been recognized
as molecular targets for drug development since the 1990s.
We have been interested in structural development of NR ligands
(agonists and antagonists) for over 10 years, based on our working
is, the (R)-enantiomer is a more potent hPPARc agonist than the
(S)-enantiomer (Table 1).23 This interesting discrepancy was inves-
tigated by means of X-ray crystallographic analysis and computa-
tional chemistry based on the obtained X-ray crystallographic
data.23 It was concluded that, although the stereochemistry at
the
and (S)-6, the benzyl side chains of both enantiomers are hosted
in the same hydrophobic pocket of hPPAR LBD, formed by the
H3, H5, H7, H11, and H12 helixes (referred to as the benzyl pocket),
and in the case of (S)-6, this involves a short contact between the
2-position hydrogen atom of the benzyl group and the oxygen
a-position of the carboxyl group is opposite between (R)-6
Abbreviations: NR, nuclear receptor; hPPAR, human peroxisome proliferator-
activated receptor; RXR, retinoid X receptor; LBD, ligand-binding domain; PDB,
protein data bank.
c
⇑
Corresponding author. Tel.: +81 086 251 7930.
E-mail address: miyachi@pharm.okayama-u.ac.jp (H. Miyachi).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.08.061

6376
M. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 6375–6383
O
O
F
O
O
CO2H
CO2H
CO2H
F
CO2H
N
H
N
H
N
H
F
F
O
O
O
O
F
1
3
5
F
O
O
CO2H
CO2H
N
H
N
H
N
H
F
F
O
O
O
2
F
4
6
Figure 1. Representatives of our previously developed PPAR agonists. (1) hPPAR
a-selective agonist. (2,3) hPPARad dual agonists. (4) hPPARd-selective agonist. (5) hPPAR pan
agonist. (6) hPPARc-selective agonist.
Table 1
carboxyl group afforded the ethyl derivative 5, which exhibited
(S)-enantiomer preference. Conversely, introduction of a phenyl
group afforded the benzyl derivative 6, which exhibited the oppo-
site preference. On the basis of this result, we hypothesized that
the introduction of ring-structure at the same position might result
in reversal of the ‘normal’ stereochemistry–activity relationship.
hPPARs transactivation activities of the present series of
anoic acid derivatives
a
-substituted phenylprop-
O
N
CO2H
Accordingly, we designed and synthesized the optically active
a-
H
R
O
cyclohexylmethylphenypropanoic acids 7 and -phenethylpheny-
a
propanoic acids 8. The cyclohexylmethyl group was selected to
examine the influence of the aromaticity of the benzyl ring of 5,
while the phenethyl group was selected to examine the influence
of a branched carbon atom at the b-position (Fig. 3).
a
Compd.
R
EC50 (nm)
2.2. Chemistry
hPPAR
a
hPPARd
hPPARc
The synthetic route to the desired compounds is shown in
Scheme 1. Benzyl 3-bromomethyl benzoate derivative 12, which
was prepared by our previous method,23 was used as the starting
material. It was treated with (S)-N-3-acyl-4-benzyloxazolidin-2-
one according to Evan’s asymmetric alkylation protocol,24 followed
by hydrogenolysis to afford benzoic acid derivative 14. This was re-
duced with BH3–THF, then oxidation with PDC afforded the benz-
aldehyde derivative 12. This was amide-alkylated with
adamantan-1-yl benzamide in the presence of trifluoroacetic acid
and triethylsilane as the reducing agent, followed by removal of
the chiral auxiliary with LiOOH to afford the desired (R)-configura-
tion products (R)-7 and (R)-8 with 98% ee.25 The antipodal (S)
enantiomers were similarly prepared from (R)-N-3-acyl-4-benzyl-
oxazolidin-2-one and showed equivalent optical purity.
(R)-6
(S)-6
(R)-7
(S)-7
(R)-5
(S)-5
(R)-8
(S)-8
Benzyl
Benzyl
Cyclohexylmethyl
Cyclohexylmethyl
Ethyl
Ethyl
Phenethyl
Phenethyl
ia
ia
ia
ia
ia
ia
ia
ia
3.60 1.1
22.0 4.0
13.0 1.6
22.0 3.0
83.0 10
41.0 9.0
34.0 2.2
9.2 0.9
500 50
54
1760 83
770 18
870 70
130 40
ia
ia
8
a
Compounds were screened for agonist activity towards PPAR-GAL4 chimeric
receptors in transiently transfected HEK-293 cells. The EC50 value is the molar
concentration of the test compound that affords 50% of the maximal reporter
activity.
atom of the side chain of Ser289. Consequently (S)-6 is energeti-
cally less stable and somewhat distorted in the complex as com-
pared to (R)-6 (Fig. 2),23 and this may be the main reason why
the (R)-enantiomer of 6 exhibits more potent hPPAR
the (S)-enantiomer.
2.3. hPPARs transactivation activities of the present series of
compounds
c activity than
Nevertheless, it remained unclear whether a similar situation
might hold for other -substituted phenylpropanoic acids. There-
fore, we designed and synthesized a series of novel optically active
-substituted phenylpropanoic acid hPPAR -selective agonists to
further examine the stereochemistry–activity relationship of these
compounds.
To investigate the cell-level hPPARs transactivation activities,
we adopted a previously reported hPPARs-responsive reporter
gene assay with CMX-GAL4N-hPPARs LBD as the recombinant
receptor gene, TK-MH100Â4-LUC as the reporter gene and the
CMX b-galactosidase gene for normalization.14 The hPPARs trans-
activation activities of the new compounds, together with the re-
sults for (R)-5, (S)-5, (R)-6 and (S)-6, are summarized in Figures 4
and 5 and Table 1.
a
a
c
2. Results and discussion
2.1. Molecular design
As expected, all the enantiomers of
propanoic acids 7 and -phenethylphenylpropanoic acids 8 exhib-
ited potent and selective hPPAR transactivation activity. These
compounds showed about 240-fold and 50-fold selectivity for
hPPAR over the other two hPPAR subtypes, respectively. In addi-
tion, as shown in Figure 4, the enantiomers showed the maximum
transactivation activities towards hPPAR at 1 M concentration,
a-cyclohexylmethylphenyl-
a
c
First of all, we focused on the structural difference at the b-posi-
tion with respect to the carboxyl group of 5 and 6, which exhibited
opposite stereochemistry-activity relationships. The introduction
of a methyl group at the b-position methylene carbon atom of the
c
c
l

M. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 6375–6383
6377
Figure 2. (A) Superposition of the crystal structures of hPPAR
and (R)-6 are depicted as cylinder models in orange ((S)-6 complex) and green ((R)-6 complex). (B) Superposition of the bound structures of (S)-6 and (R)-6. (C) Zoomed views
of the benzyl pocket of hPPAR LBD (S)-6 complex. (D) Zoomed views of the benzyl pocket of hPPAR LBD (R)-6 complex. The four interacting amino acids are depicted as
c LBD (S)-6 complex and hPPARc LBD (R)-6 complex. Proteins are represented as wireframe models, and (S)-6
c
c
white ((S)-6 complex) and yellow cylinders ((R)-6 complex), and the benzyl side chains are depicted in orange ((S)-6) and green ((R)-6). (E) Superposition of the bound
structure of (S)-6 and the energy-minimized structure of (S)-6. (F) Superposition of the bound structure of (R)-6 and the energy-minimized structure of (R)-6. The calculated
ligand energies of the bound ligands are shown below the structures. The yellow arrows indicate the directions of distortion of the benzyl groups.
O
H
H
O
O
O
CO2H
CO2H
H
H
H
N
H
CO2H
CO2H
CO2H
N
H
N
H
α
O
O
O
β
(S)-8
(S)-5
(S)-7
O
O
H
CO2H
N
H
O
N
α
N
H
H
O
β
O
(R)-8
(R)-6
(R)-7
Figure 3. Molecular design of our compounds.
whereas much higher concentrations were needed for apparent
activation of the hPPAR and hPPARd subtypes. These results
clearly indicate that the change of the -position substituent from
benzyl to cyclohexylmethyl or phenethyl does not alter the
selectivity for hPPAR , and show that the -cyclohexylmethylphe-
nylpropanoic acid derivatives 7 and -phenethylphenylpropanoic
acid derivatives 8 are both potent hPPAR -selective full agonists.
The EC50 values of these enantiomers for hPPAR transactivation
toward PPARc than did the antipodal (S)-7 (EC50 values for hPPARc
a
were 12.0 and 22.0 nM, respectively). As regards the dose-response
relationship, (R)-7 exhibited the maximum transactivation activity
at about 50 nM, whereas a much higher concentration of (S)-7 was
a
c
a
needed for full activity (Fig. 5). On the other hand,
nylpropanoic acid 8 showed similar SAR to -ethylphenylpropa-
noic acid 5. The EC50 values of (R)-8 and (S)-8 for hPPAR were
a-phenethylphe-
a
a
c
c
c
34.0 and 9.20 nM, respectively; also, (S)-8 exhibited the maximum
transactivation activity at about 50 nM, whereas a much higher
concentration of (R)-8 was needed for full activity (Fig. 5).
The observed SAR indicated that the presence of a branched car-
bon atom at the b-position with respect to the carboxyl group is
important for reversal of the stereochemistry–transactivation
were somewhat larger to that of (R)-6, suggesting that the (R)-ben-
zyl group has the most favorable hydrophobic interaction with the
benzyl pocket among these substituents.
It is of particular interest that (R)-7, like
a-benzylphenylpropa-
noic acid (R)-6, exhibited more potent transactivation activity

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M. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 6375–6383
BnO2C
BnO2C
O
BnO2C
CHO
c
HO2C
HO
CHO
b
a
OH
Br
O
O
9
10
11
12
O
O
O
O
O
Bn
Bn
N
N
O
Bn
d
N
e
f
H
R
H
H
HO2C
O
OHC
BnO2C
O
O
O
O
R
R
O
13
14
15
O
O
H
O
Bn
CO2H
N
O
N
H
H
g
h
R
O
N
H
O
R
O
(R)-7 : R = cyclohexylmethyl
(R)-8 : R = phenetyl
16
Scheme 1. Synthetic route to the present series of compounds. Reagents and conditions: (a) (1) BnBr, KHCO3, DMF, rt, overnight, 80%, (2) n-PrI, K2CO3, DMF, 60 °C, 16 h, 91%;
(b) NaBH4, EtOH, rt, 2 h, 92%; (c) PBr3, diethylether, 0 °C, 2 h, 95%; (d) (S)-4-benzyl-3-(3-cyclohexylpropanoyl)oxazolidin-2-one or (S)-4-benzyl-3-(4- phenylbutanoyl)oxaz-
olidin-2-one, LiHMDS, À50 °C to 0 °C, 2 h, 53–58%; (e) H2, 10% Pd/C, AcOEt, rt, 2 h, 61–69%; (f) (1) BH3-THF, THF, 0 °C to rt, overnight, 88–98%, (2) PDC, CH2Cl2, rt, overnight,
61–73%; (g) 4-adamantylbenzamide, (Et)3SiH, TFA, toluene, reflux, 48 h, 42–67%; (h) LiOH-H2O, 30%H2O2, THF:H2O = 4:1 (V/V), 0 °C to rt, 83–86%; (i) (S)-4-benzyl-3-(3-
cyclohexyl- propanoyl)oxazolidin-2-one or (S)-4-benzyl-3-(4-phenylbutanoyl)oxazolidin-2-one, LiHMDS, À50 °C to 0 °C, 2 h, 50–62%.
activity relationship, rather than the presence of an aromatic ring-
containing moiety. For example, in the cases of 6 and 7, which pos-
sess a branched b-carbon, the (R)-enantiomers were more potent
the
in the binding region of the hPPAR
group of the ligand. In the case of (R)-7, the carbon atom at the
a
-position of (S)-7 is located very close to Ser289 and Arg288
c
LBD that hosts the carboxyl
hPPAR
the case of 5 and 8, which possess an unbranched b-carbon, the
(S)-enantiomers were more potent hPPAR transactivators than
c
transactivators than the (S)-enantiomers. Conversely, in
a
-position of (R)-7 is located in the center of the binding pocket.
On the other hand, none of the energy parameters appears to
c
contribute to destabilization of the (R)-8-hPPAR
c LBD complex as
the (R)-enantiomers. However, the reason for this behavior re-
mained unclear. Therefore, we conducted further studies of the
reversal of the stereochemistry–activity relationship in this series
by means of computational analysis.
compared to the enantiomeric (S)-8-hPPAR LBD complex. How-
c
ever, as mentioned above, we have already reported that distortion
of the ligand structure in the form bound to the receptor is an
important factor in the reversal of the stereochemistry–activity
relationship. For example, the benzyl group of (S)-6 is substantially
2.4. Construction of the binding model and calculation of the
interaction energies
distorted when (S)-6 is bound to the hPPAR
dral angle of the bound benzyl group of (S)-6 in the hPPAR
c
LBD, that is, the dihe-
LBD
c
differs by 131° from that of the corresponding energy-minimized
In order to understand the structure-dependent reversal of the
stereochemistry–activity relationship, a computational study was
performed. First, we constructed binding models of each enantio-
structure of (S)-6 itself.23 Therefore, we examined the distortion
of each enantiomer of 8 in the bound form to hPPARc LBD. These
results are summarized in Figure 8. The root-mean-square diame-
ter (rmsd) of (S)-8 is only 1.04 Å, which indicates that the bound
structure of (S)-8 closely resembles the energy-minimized struc-
ture. However, in the case of (R)-8, the rmsd differs by 2.46 Å from
that of the energy-minimized structure of (R)-8.
In conclusion, our results show that the structure of the substi-
tuent at the b-position with respect to the carboxyl group in the
present series of a-substituted phenylpropanoic acid hPPARc-
mer of 7 and 8 with our hPPARc LBD structure (PDB: 3AN3,
3AN4), using the computational ligand docking program Glide
5.5 in the Schrödinger Suite. Then, we calculated the interaction
energies of each enantiomer of 7 and 8 with the hPPAR
c LBD.
The results are summarized in Figure 626 and Table 2. The calcu-
lated GlideScores27,28 were À12.39 and À11.85 for the (R)-7-
hPPAR
c
LBD complex and (S)-7-hPPAR
c
LBD complex, respectively.
In the case of 8, the calculated GlideScores were À12.48 and
selective agonists is critically important in determining the stereo-
chemistry–activity relationship. However, the reasons for the
reversal of the stereochemistry–activity relationship might not
À12.60 for the (R)-8- hPPAR
c
LBD complex and (S)-8- hPPAR
c
LBD complex, respectively. Therefore, the (R)-7 complex and the
(S)-8 complex were calculated to be more stable than the corre-
sponding enantiomer complexes, in accordance with the finding
be precisely the same for the
a-cyclohexylmethyl derivative 7
and the phenethyl derivative 8. An X-ray crystallographic study
that (R)-7 and (S)-8 exhibited more potent hPPAR
c
transactivation
is in progress to resolve this question.
activity than their enantiomers. This indicates that glide score is a
good predictor of which enantiomer is more stable (potent).
In the case of 7, the greatest contributor to destabilization of
3. Experimental
(S)-7-hPPAR
c
LBD complex appeared to be Van der Waals energy
3.1. General methods
(evdw). The difference in this hydrophobic parameter indicates
that the contribution of the side chains of the amino acids of
Melting points were determined with a Yanagimoto hot-stage
melting point apparatus and are uncorrected. NMR spectra were
recorded on a Varian VNMRS-400 (1H 400 MHz) spectrometer.
hPPARc LBD to the binding is critically different between (R)-7
and (S)-7. Based on Figure 7, we found that the carbon atom at

M. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 6375–6383
6379
(S)-7
PPARδ
12000
9000
6000
3000
PPARα
PPARγ
0
cont. feno 2.5 10
50 200 1000
cont. GW 2.5 10 50 200 1000
cont. pio 2.5 10 50 200 1000 (nM)
)
)
0)
0
1
M
0
(
u
1
(2
)
5
)
)
0)
1
e
.
(2
50)
(
00)
cl
i
(
2
(
uM
000
1
(
S
S
6S
6S
6S
MO
10
(
6S
veh
o (
pi
6S
MO
O
M
O6
M
o
(MRO)-7
MO
O6
M
en
f
12000
9000
6000
3000
0
cont. feno 2.5
1
)
0
50 200 1000
cont. GW 2.5 10
5)
2.
5
)
0
200 1000
000)
cont. pio 2.5 10 50 200 1000 (nM)
)
0)
0
1
M)
0
(
2
u
1
(
7R
R
)
)
0
0
0
e
1
l
c
i
0
2
(
(5
(
7R
uM
(
R
1
(
0
7R
7
1
7R
veh
o (
i
7
MO
(
7R
MO
MO
p
MO
MO
no
e
MO
f
MO
(S)-8
12500
10000
7500
PPARα
PPARδ
PPARγ
5000
2500
0
cont. feno 2.5 10
50 200 1000
cont. GW 2.5 10 50 200 1000
cont. pio 2.5 10 50 200 1000 (nM)
12000
(R)-8
8000
4000
0
cont. feno 2.5 10
)
50 200 1000
cont. GW 2.5 10 50 200 1000
cont. pio 2.5 10 50 200 1000 (nM)
)
)
)
5
.
2
)
0
(5
)
0
)
M
0
)
M
u
0
1
(
0
0
e
l
c
hi
ve
00)
2
(
(1
0
0
(1
R
O5
(2
R
R
R
5
0 u
R (
O5
5
(1
io
p
1
5R
O5
5R
O
M
(
o
MO
M
MO
M
MO
en
f
M
Figure 4. Dose-response relationship of the hPPARs transactivation activities of (R)-7, (S)-7, (R)-8 and (S)-8. Abbreviations used are as follows: feno, 10À5 M fenofibrate
(PPAR
-selective agonist); GW, 10À7 M 2-(2-methyl-4-((4-methyl-2-(4-(trifluoromethyl)phenyl)thiazol-5-yl)methylthio)phenyl)acetic acid (GW-501516) (PPARd-selective
agonist); pio, 10À6 M pioglitazone (PPAR
-selective agonist).
a
c

6380
M. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 6375–6383
cont.
pio
(S)-5
(R)-5
cont.
pio
(S)-6
(R)-6
330000000
30000
16000
1166000000
12000
1122000000
220000000
20000
8000
88000000
110000000
10000
4000
44000000
0
0
0
00
0
vehicle
pio
1
(S)-6 (S)-6 (S)-6 (S)-6 (S)-6 (S)-6
(R)-6 (R)-6 (R)-6 (R)-6 (R)-6 (R)-6
1
10 30 100 300 1000 3000
10
30 100 300 1000 3000 (nM)
1
3
10 100 1000 10000
1
3
10 100 1000 10000 (nM)
cont. pio
(S)-8
(R)-8
cont.
pio
(S)-7
(R)-7
112200000
12000
12000
1122000000
8
8
0
0
0
0
0
0
8000
8800000
8000
4000
4
4
0
0
0
0
0
0
4000
4400000
0
0
0
0
0
0
vehicle
pio
1
(S)-8
0.5
(S)-8
10
(S)-8
50
(S)-8
(S)-8
(R)-8
0.5
(R)-8
10
(R)-8
50
(R)-8
(R)-8
200 1000
200 1000 (nM)
vehicle
pio (1uM)
1
(S)-7 (0.5)
(S)-7 (10)
(S)-7 (200)
(R)-7 (2.5)
(R)-7 (50) (R)-7 (1000)
0.5 2.5 10 50 200 1000
0.5 2.5 10 50 200 1000 (nM)
Figure 5. Dose-response relationship of the hPPAR
(PPAR -selective agonist).
c
transactivation activities of both enantiomers of 5, 6, 7 and 8. Abbreviations used are as follows: pio, 10À6 M pioglitazone
c
Figure 6. Docking structures of each enantiomer of 7 and 8, complexed with our hPPARc LBD structure obtained from the 6-hPPARc complex (PDB: 3AN3A).
Proton chemical shifts were referenced to TMS internal standard.
Elemental analysis was carried out with a Yanagimoto MT-5 CHN
recorder elemental analyzer and results were within 0.4% of the
theoretical values. FAB-MS was carried out with a VG70-SE.
(1.98 g, 19.8 mmol), and 30 mL of N,N-dimethylformamide was
stirred at room temperature for 7 h. The reaction mixture was
poured into ice-water and extracted with ethyl acetate. The organic
layer was washed with water and brine, dried over anhydrous
magnesium sulfate and concentrated. The residue was purified by
silica gel column chromatography (eluant; n-hexane/ethyl ace-
tate = 7:2 v/v) to afford 3.71 g (80%) as a pale yellow solid. A mix-
ture of this product (3.71 g, 14.5 mmol), iodopropane (2.14 ml,
18.0 mmol), potassium carbonate (2.60 g, 18.9 mmol), and 30 mL
of N,N-dimethylformamide was stirred at 60 °C for 16 h, then
3.2. Chemistry
3.2.1. Benzyl 5-formyl-2-propoxybenzoate (10)
A mixture of 5-formylsalicylic acid (8) (3.00 g, 18.0 mmol), ben-
zyl bromide (2.14 ml, 18.0 mmol), potassium hydrogencarbonate

M. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 6375–6383
6381
Table 2
Binding energy (GlideScore) calculations for each enantiomer of the phenylpropanoic acid hPPAR
c ligands, 7 and 8. Gscore: GlideScore. Lipo: lipophilic contact plus phobic
attractive term in the GlideScore. hbond: hydrogen-bonding term in the GlideScore. Rewards: various reward or penalty terms. evdw: Van der Waals energy. ecoul: Coulomb
energy. erotb: penalty for freezing rotatable bonds in the GlideScore
Compd.
R
Glide score
lipo
hbond
Rewards
evdw
ecoul
erotb
Protein
(R)-7
(S)-7
(R)-8
(S)-8
Cyclohexylmethyl
Cyclohexylmethyl
Phenethyl
À12.39
À11.85
À12.48
À12.60
À7.50
À6.85
À7.08
À7.81
À0.29
À0.42
À0.38
À0.28
À0.78
À0.56
À0.77
À0.84
À57.16
À54.56
À56.80
À56.32
À10.10
À12.25
À13.22
À9.59
0.55
0.55
0.59
0.59
3AN3
3AN4
3AN3
3AN3
Phenethyl
(A)
(B)
(C)
H323
S289
R288
(S)-7
H449
(S)-7
(R )-7
L465
(R)-7
L453
Q286
(R)-7
(S)-7
Figure 7. Docking structures of each enantiomer of 7 and 8, complexed with our hPPAR
c
LBD structure obtained from 6-hPPARc complex (PDB: 3AN3A). (a) A Connolly
molecular surface map showing the binding poses of (R)-7 and (S)-7 complexed with hPPAR
group of 7.
c
; (b) and (c) Zoomed view of the binding modes around the cyclohexylmethyl
(S)-8
(R)-8
rmsd=2.46A
rmsd=1.04A
Figure 8. Left: superposition of the bound structure of (R)-8 and the energy-minimized structure of (R)-8. Right: superposition of the bound structure of (S)-8 and the energy-
minimized structure of (S)-8. The rmsd differences of the bound ligands are indicated.
poured into water and extracted with ethyl acetate. The organic
layer was washed with brine, dried over anhydrous magnesium sul-
fate and concentrated. The residue was purified by silica gel column
chromatography (eluant; n-hexane/ethyl acetate = 3:1 v/v) to af-
ford 3.90 g (91%) of the title compound as a pale yellow oil. 1H
NMR (400 MHz, CDCl3) d 9.90 (s, 1H), 8.33 (d, J = 2.0 Hz, 1H), 7.99
(dd, J = 8.6, 2.2 Hz, 1H), 7.46 (dd, J = 8.0, 1.2 Hz, 2H), 7.41–7.31
(m, 3H), 7.07 (d, J = 8.8 Hz, 1H), 5.37 (s, 2H), 4.08 (t, J = 6.4 Hz,
2H), 1.89–1.80 (m, 2H), 1.01 (t, J = 7.4 Hz, 3H); MS (FAB) 299
(M+H)+.

6382
M. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 6375–6383
3.2.2. Benzyl 5-hydroxymethyl-2-propoxybenzoate (11)
To a solution of 10 (3.90 g, 13.0 mmol) and 30 mL of ethanol
was added sodium borohydride (0.50 g, 13.0 mmol) portionwise
at 0 °C. The reaction mixture was stirred for 2 h at room tempera-
ture. Excess ethanol was evaporated, the residue was poured into
water, and the whole was extracted with ethyl acetate. The extract
was washed with water and brine, dried over anhydrous magne-
sium sulfate and concentrated. The residue was purified by silica
gel column chromatography (eluant; n-hexane/ethyl acetate = 1:1
v/v) to afford 3.58 g (92%) of the title compound as a colorless
oil. 1H NMR (400 MHz, CDCl3) d 7.79 (d, J = 2.4 Hz, 1H), 7.46–7.44
(m, 3H), 7.39–7.31 (m, 3H), 6.94 (d, J = 8.4 Hz, 1H), 5.37 (s, 2H),
4.62 (s, 2H), 3.98 (t, J = 6.4 Hz, 2H), 1.85–1.76 (m, 2H), 0.99 (t,
J = 7.4 Hz, 3H); MS (FAB) 301 (M+H)+.
3.2.6. 5-((R)-3-((S)-4-Benzyl-2-oxo-oxazolidin-3-yl)-2-
(cyclohexylmethyl)-3-oxopropyl)-2-propoxybenzaldehyde (15)
To a solution of 14 (0.48 g, 0.95 mmol) and 30 mL of dehydrated
tetrahydrofuran was added dropwise a 1 mol/L solution of borane-
tetrahydrofuran complex (1.89 mL, 1.89 mmol). After completion
of the addition, the mixture was stirred for 2 h at 0 °C and then
overnight at room temperature. A saturated aqueous solution of
ammonium chloride was added to the reaction mixture, and the
whole was extracted with ethyl acetate. The organic layer was
washed with water and brine, dried over anhydrous magnesium
sulfate and concentrated. The residue was purified by silica gel col-
umn chromatography (eluant; n-hexane/ethyl acetate = 3:1 v/v) to
afford 0.41 g (88%) of the intermediate hydroxymethyl derivative
as a colorless oil. A mixture of the hydroxymethyl derivative
(0.41 g, 0.83 mmol), pyridinium dichromate (0.47 g, 1.25 mmol),
and 20 mL of dehydrated dichloromethane was stirred for 12 h at
room temperature. The catalyst was removed by filtration, and
the filtrate was washed with ether and concentrated. The residue
was purified by silica gel column chromatography (eluant; n-hex-
ane/ethyl acetate = 4:1 v/v) to afford 0.25 g (61%) of the title com-
pound as a white oil. 1H NMR (400 MHz, CDCl3) d 10.47 (s, 1H),
7.67 (d, J = 2.0 Hz, 1H), 7.50 (dd, J = 8.6, 2,2 Hz, 1H), 7.30–7.23 (m,
3H), 7.09 (dd, J = 7.8, 1.4 Hz, 2H), 6.91 (d, J = 8.8 Hz, 1H), 4.69–
4.62 (m, 1H), 4.26–4.07 (m, 3H), 4.04–3.97 (m, 2H), 3.12 (dd,
J = 13.2, 3.3 Hz, 1H), 2.99 (dd, J = 13.6, 7.6 Hz, 1H), 2.72 (dd,
J = 13.4, 7.0 Hz, 1H), 2.49 (dd, J = 13.2, 10.0 Hz, 1H), 1.92–1.81 (m,
2H), 1.76–1.53 (m, 6H), 1.33–1.10 (m, 5H), 1.05 (t, J = 7.5 Hz, 3H),
0.96–0.75 (m, 2H); MS (FAB) 492 (M+H+).
3.2.3. Benzyl 5-bromomethyl-2-propoxybenzoate (12)
A mixture of 11 (3.58 g, 12.0 mmol), phosphorus tribromide
(1.13 ml, 12.0 mmol) and 30 mL of dehydrated ether was stirred
for 2 h at 0 °C, then poured into ice-water, and the whole was ex-
tracted with ether. The extract was washed with water and brine,
dried over anhydrous magnesium sulfate and concentrated to af-
ford 4.16 g (95%) of a colorless oil, which was used in the next step
without further purification. 1H NMR (400 MHz, CDCl3) d 7.84 (d,
J = 2.4 Hz, 1H), 7.49–7.44 (m, 3H), 7.40–7.31 (m, 3H), 6.92 (d,
J = 8.4 Hz, 1H), 5.35 (s, 2H), 4.47 (s, 2H), 3.98 (t, J = 6.4 Hz, 2H),
1.84–1.76 (m, 2H), 0.98 (t, J = 7.4 Hz, 3H).
3.2.4. Benzyl 5-((R)-2-benzyl-3-((S)-4-benzyl-2-oxo-oxazolidin-
3-yl)-3-oxopropyl)-2-propoxybenzoate (13)
3.2.7. 4-(1-Adamantyl)-N-((5-((R)-3-((S)-4-benzyl-2-oxo-
oxazolidin-3-yl)-2-(cyclohexylmethyl)-3-oxopropyl)-2-
propoxyphenyl)methyl)benzamide (16)
(S)-4-Benzyl-3-(3-cyclohexylpropanoyl)oxazolidin-2-one
(0.75 g, 2.38 mmol) and 10 mL of dehydrated tetrahydrofuran
were mixed under an argon atmosphere, and cooled to À50 °C.
A
mixture of 15 (0.25 g, 0.51 mmol), 4-phenylbenzamide
Under stirring,
a 1 mol/L solution of sodium bis(trimethyl-
(0.39 g, 1.53 mmol), triethylsilane (0.24 mL, 1.53 mmol), trifluoro-
acetic acid (0.11 mL, 1.53 mmol) and 30 mL of dehydrated toluene
was refluxed for 48 h under an argon atmosphere. The reaction
mixture was poured into water, and the whole was extracted with
ethyl acetate. The extract was washed with water and brine, dried
over anhydrous magnesium sulfate and concentrated. The residue
was purified by silica gel column chromatography (eluant; n-hex-
ane/ethyl acetate = 3:1 v/v) to afford 0.28 g (76%) of the title com-
pound as a pale yellow solid. 1H NMR (400 MHz, CDCl3) d 7.66 (d,
J = 8.8 Hz 2H), 7.36 (d, J = 8.4 Hz, 2H), 7.23–7.18 (m, 4H), 7.15 (d,
J = 8.0 Hz, 1H), 6.98 (d, J = 7.6 Hz, 2H), 6.78 (d, J = 8.4 Hz, 1H),
6.59 (t, J = 6.0 Hz, 1H), 4.67–4.57 (m, 3H), 4.33–4.26 (m, 1H), 4.11
(t, J = 7.2 Hz, 1H), 4.03 (d, J = 6.4 Hz, 1H), 3.98–3.89 (m, 2H), 3.02-
2.89 (m, 2H), 2.73 (dd, J = 13.2, 6.6 Hz, 1H), 2.39 (dd, J = 13.5,
9.8 Hz, 1H), 2.10 (s, 3H), 1.90–1.53 (m, 20H), 1.37–1.10 (m, 5H),
1.04 (t, J = 7.35 Hz, 3H), 0.94–0.76 (m, 2H); MS (FAB) 731 (M+H+).
silyl)amide in dehydrated tetrahydrofuran (7.20 mL, 7.20 mmol)
was added dropwise. After completion of the addition, the mixture
was stirred for 1 h at À15 °C and recooled to À50 °C. Then a solu-
tion of 12 (1.63 g, 4.49 mmol) in dehydrated tetrahydrofuran
(15 mL) was added dropwise to it. After completion of the addi-
tion, the mixture was further stirred for 1 h while being gradually
heated to room temperature. A saturated aqueous solution of
ammonium chloride was added to the reaction mixture, and the
whole was extracted with ethyl acetate. The extract was washed
with water and brine, dried over anhydrous magnesium sulfate
and concentrated. The residue was purified by silica gel column
chromatography (eluant; n-hexane/ethyl acetate = 15:2 v/v) to af-
ford 0.82 g (58%) of the title compound as a pale yellow oil; MS
(FAB) 598 (M+H)+.
3.2.5. 5-((R)-3-((S)-4-Benzyl-2-oxo-oxazolidin-3-yl)-2-
(cyclohexylmethyl)-3-oxopropyl)-2-propoxybenzoic acid (14)
Compound 13 (0.82 g, 1.37 mmol), 10% palladium on carbon
(0.17 g) and 30 mL of ethyl acetate were mixed and catalytic
hydrogenation was carried out at an initial hydrogen pressure of
200–300 kPa. After completion of the reaction, the catalyst was re-
moved by filtration and the filtrate was concentrated. The residue
was purified by silica gel column chromatography (eluant; n-hex-
ane/ethyl acetate = 4:1 v/v) to afford 0.48 g (69%) of the title com-
pound as a white solid. 1H NMR (400 MHz, CDCl3) d 10.99 (s, 1H),
8.03 (dd, J = 2.0 Hz, 1H), 7.54 (dd, J = 8.4, 2.4 Hz, 1H), 7.30–7.22 (m,
3H), 7.10 (dd, J = 8.0, 2.0 Hz, 2H), 6.97 (d, J = 8.4 Hz, 2H), 4.69–4.62
(m, 1H), 4.23–4.15 (m, 5H), 3.16 (dd, J = 13.2, 3.2 Hz, 1H), 3.03 (dd,
J = 13.2, 7.2 Hz, 1H), 2.74 (dd, J = 13.4, 7.0 Hz, 1H), 2.55 (dd, J = 13.6,
9.6 Hz, 1H), 1.97–1.88 (m, 2H), 1.76–1.54 (m, 6H), 1.30–1.12 (m,
5H), 1.08 (t, J = 7.4 Hz, 3H), 0.93–0.72 (m, 2H); MS (FAB) 508
(M+H+).
3.2.8. (R)-2-((3-(((4-(1-Adamantyl)benzoyl)amino)methyl)-4-
propoxyphenyl)methyl)-3-cyclohexylpropanoic acid ((R)-7)
Compound 16 (170 mg, 0.38 mmol) was dissolved in 24 mL of
tetrahydrofuran and 5 mL of water under an argon atmosphere
with ice-cooling. To this solution was added 30% aqueous hydro-
gen peroxide (0.43 mL, 3.80 mmol). Then a solution of lithium
hydroxide monohydrate (64.0 mg, 1.53 mmol) in water (1 mL)
was added to it, and the mixture was stirred for 2.5 h at 0 °C and
for 3 h at room temperature. An aqueous solution of sodium sulfite
(1.00 g/6 mL) was added to the mixture and the whole was stirred
further for 15 min. The reaction mixture was acidified with 10%
HCl, and then extracted with ethyl acetate. The extract was washed
with brine, dried over anhydrous magnesium sulfate and concen-
trated. The residue was purified by silica gel column chromatogra-
phy (eluant; n-hexane/ethyl acetate = 3:1 v/v) to afford 110 mg
(83%) of the title compound as a white crystalline solid; mp

M. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 6375–6383
6383
177–178 °C. 1H NMR (300 MHz, CDCl3) d 7.70 (d, J = 8.4 Hz, 2H),
7.39 (d, J = 8.4 Hz, 2H), 7.14 (s, 1H), 7.04 (dd, J = 8.4, 2.1 Hz, 1H),
6.78–6.70 (m, 2H), 4.65–4.53 (m, 2H), 3.95 (t, J = 6.5 Hz, 2H),
2.87–2.64 (m, 3H), 2.10 (s, 3H), 1.90–1.54 (m, 20H), 1.34–1.12
(m, 5H), 1.06 (t, J = 7.4 Hz, 3H), 0.92–0.73 (m, 2H); HRMS (FAB,
GlideScore, which represents the ligand-receptor interaction
energy, was calculated for each generated pose. The pose with
the best GlideScore was selected as the binding structure of 7 (or
8) to hPPAR
docking procedure using the complexed structures of TIPP703,
(S)-6 or (R)-6 with the hPPAR LBDs (PDB code 2ZNO, 3AN3 and
c LBD. In prior to the study, we validated the above
MH+) calcd for C37H50NO4 572,3740, found 572.3719; [
a]
À5.5°
c
D
1
(c 0.50, CHCl3); Anal. (C37H50NO4À /4H2O) C, H, N.
3AN4) determined by X-ray crystallography. In the validation step,
we also checked the orientation of the side chain of Phe363 of the
3.2.9. (S)-2-((3-(((4-(1-Adamantyl)benzoyl)amino)methyl)-4-
propoxyphenyl)methyl)-3-cyclohexylpropanoic acid ((S)-7)
This compound was prepared by means of a procedure similar
to that used for (R)-7; mp 177–178 °C; 1H NMR (300 MHz, CDCl3)
d 7.70 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.4 Hz, 2H), 7.14 (s, 1H), 7.04
(dd, J = 8.4, 2.1 Hz, 1H), 6.78–6.70 (m, 2H), 4.65–4.53 (m, 2H),
3.95 (t, J = 6.5 Hz, 2H), 2.87–2.64 (m, 3H), 2.10 (s, 3H), 1.90–1.54
(m, 20H), 1.34–1.12 (m, 5H), 1.06 (t, J = 7.4 Hz, 3H), 0.92–0.73 (m,
2H); HRMS (FAB, MH+) calcd for C37H50NO4 572,3740, found
hPPARc LBDs because Phe363 interacts with the R group of the li-
gand and the orientation of the side chain of Phe363 is different be-
tween the X-ray structures bound ligands. In results, we succeeded
in reproducing the correct docking structures (to less than 2 Å of
the X-ray structures) of TIPP703, (S)-6 and (R)-6 and reproducing
the orientation of side chain of Phe363 of the hPPARc LBDs.
Acknowledgment
572.3735; [
a
]
+5.5° (c 0.50, CHCl3); Anal. (C37H50NO4) C, H, N.
This work was supported in part by the Targeted Proteins Re-
search Program of the Japan Science and Technology Corporation
(JST), the Uehara Memorial Foundation, the Tokyo Biochemical Re-
search Foundation (TBRF), and the Okayama Foundation for Sci-
ence and Technology (OFST).
D
3.2.10. (R)-2-((3-(((4-(1-Adamantyl)benzoyl)amino)methyl)-4-
propoxyphenyl)methyl)-4-phenylbutanoic acid ((R)-8)
This compound was prepared by means of a procedure similar
to that used for (R)-7; mp 140–141 °C; 1H NMR (300 MHz, CDCl3)
d 7.69 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.1 Hz, 2H), 7.26–7.13 (m,
6H), 7.02 (dd, J = 8.4, 2.1 Hz, 1H), 6.76–6.69 (m, 2H), 4.59 (d,
J = 4.5 Hz, 2H), 3.93 (t, J = 6.5 Hz, 2H), 2.91 (dd, J = 13.1, 7.7 Hz,
1H), 2.77–2.53 (m, 4H), 2.10 (s, 3H), 2.04–1.72 (m, 17H), 1.05 (t,
J = 7.4 Hz, 3H); HRMS (FAB, MH+) calcd for C37H50NO4 580,3427,
References and notes
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found 580.3432; [a]D+4° (c 0.25, CHCl3).
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K.; Miyachi, H. J. Med. Chem. 2003, 46, 3581.
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15, 5177.
3.2.11. (S)-2-((3-(((4-(1-Adamantyl)benzoyl)amino)methyl)-4-
propoxyphenyl)methyl)-4-phenylbutanoic acid ((S)-8)
This compound was prepared by means of a procedure similar
to that used for (R)-7; mp 140–141 °C; 1H NMR (300 MHz, CDCl3)
d 7.69 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.1 Hz, 2H), 7.26–7.13 (m,
6H), 7.02 (dd, J = 8.4, 2.1 Hz, 1H), 6.76-6.69 (m, 2H), 4.59 (d,
J = 4.5 Hz, 2H), 3.93 (t, J = 6.5 Hz, 2H), 2.91 (dd, J = 13.1, 7.7 Hz,
1H), 2.77–2.53 (m, 4H), 2.10 (s, 3H), 2.04–1.72 (m, 17H), 1.05 (t,
J = 7.4 Hz, 3H); HRMS (FAB, MH+) calcd for C37H50NO4 580,3427,
found 580.3432; [
a
]DÀ4° (c 0.25, CHCl3).
3.3. Reporter gene assay
Human embryonic kidney HEK293 cells were cultured in DMEM
containing 5% fetal bovine serum and antibiotic-antimycotic mix-
ture (Nacalai) at 37 °C in a humidified atmosphere of 5% CO2 in
air. Transfections were performed by calcium phosphate coprecip-
itation. Eight hours after transfection, ligands were added. Cells
were harvested approximately 16–20 h after the treatment, and
luciferase and b-galactosidase activities were assayed using a lumi-
nometer and a microplate reader. DNA cotransfection experiments
included 50 ng of reporter plasmid, 20 ng pCMX-b-galactosidase,
15 ng each receptor expression plasmid, and pGEM carrier DNA
to make a total of 150 ng of DNA per well in a 96-well plate. Lucif-
erase data were normalized to an internal b-galactosidase control
and reported values are means of triplicate assays.
19. Kasuga, J.; Oyama, T.; Nakagome, I.; Makishima, M.; Hirono, S.; Morikawa, K.;
Hashimoto, Y.; Miyachi, H. ChemMedChem 2008, 3, 1662.
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T.; Hashimoto, Y.; Miyachi, H. Bioorg. Med. Chem. Lett. 2008, 18, 1110.
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J. P.; Collins, J. L.; Dezube, M.; Henke, B. R.; Hull-Ryde, E. A.; Lake, D. H.;
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24. Evans, D. A.; Ennis, M. D.; Mathre, D. J. J. Am. Chem. Soc. 1982, 104, 1737.
25. The enantiomeric excess was determined with a CHIRALPAK AD-H column,
with hexane/i-PrOH/trifluoroacetic acid = 90:10:0.1 v/v/v as the mobile phase,
at a flow rate of 0.5 mL/min, with detection at 254 nm.
26. Computer-graphic work and measurement of hydrogen bonding distances
were performed using MOE (Molecular Operating Environment) (Ryoka
Systems Inc.).
3.4. Computational ligand docking
27. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J. J. Med. Chem.
2004, 47, 1739.
28. Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.; Greenwood, J. R. J. Med.
Chem. 2006, 49, 6177.
Optically active 7 and 8 were docked into the hPPAR
binding domains (LBDs) (PDB code: 3AN3 and 3AN4) with standard
precision mode for Glide 5.5 in the Schrödinger Suite. The
c ligand