Improvement of water-solubility of biarylcarboxylic acid peroxisome proliferator-activated receptor (PPAR) δ-selective partial agonists by disruption of molecular planarity/symmetry

Article abstract of DOI:10.1016/j.bmc.2010.08.041

To elucidate the molecular basis of peroxisome proliferator-activated receptor (PPAR) δ partial agonism, X-ray crystal structures of complexes of the PPARδ ligand-binding site with partial agonists are required. Unfortunately, reported PPARδ partial agoni

Full text of DOI:10.1016/j.bmc.2010.08.041

Bioorganic & Medicinal Chemistry 18 (2010) 7164–7173
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Improvement of water-solubility of biarylcarboxylic acid peroxisome
proliferator-activated receptor (PPAR) d-selective partial agonists by
disruption of molecular planarity/symmetry
Jun-ichi Kasuga a, Minoru Ishikawa a, , Mitsuhiro Yonehara a, Makoto Makishima b, Yuichi Hashimoto a,
⇑
Hiroyuki Miyachi c,
⇑
a Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
b Division of Biochemistry, Department of Biomedical Sciences, Nihon University School of Medicine, Itabashi-ku, Tokyo 173-8610, Japan
c Division of Pharmaceutical Sciences, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 1-1-1, Tsushima-Naka, Kita-ku,
Okayama 700-8530, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
To elucidate the molecular basis of peroxisome proliferator-activated receptor (PPAR) d partial agonism,
X-ray crystal structures of complexes of the PPARd ligand-binding site with partial agonists are required.
Unfortunately, reported PPARd partial agonists, biphenylcarboxylic acids 1 and 2, possess insufficient
aqueous solubility to allow such crystals to be obtained. To improve the aqueous solubility of 1 and 2,
substituents were introduced at the 2-position of the biaryl moiety, focusing on disruption of molecular
planarity and symmetry. All 2-substituted biphenyl analogs examined showed more potent PPARd ago-
nistic activity with greater aqueous solubility than 1 or 2. Among these biphenyls, 25 showed potent and
selective PPARd partial agonistic activity (EC50: 5.7 nM), with adequate solubility in phosphate buffer
(0.022 mg/mL). The 2-substituted pyridyl analog 27 showed weaker PPARd partial agonistic activity
(EC50: 76 nM) with excellent solubility in phosphate buffer (2.7 mg/mL; at least 2700 times more soluble
than 2). Our results indicate that two strategies to improve aqueous solubility, that is, introduction of
substituent(s) to modify the dihedral angle and to disrupt molecular symmetry, may be generally appli-
cable to bicyclic molecules. Combination of these approaches with the traditional approach of reducing
the molecular hydrophobicity may be particularly effective.
Received 3 August 2010
Accepted 19 August 2010
Available online 24 August 2010
Keywords:
PPAR agonist
Aqueous solubility
Molecular design
Nuclear receptor
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
ligands.5,6 PPAR
c plays a critical role in the differentiation of pre-
adipocytes to adipocytes, promotes lipid storage, and enhances
glucose disposal into peripheral tissues.7,8 A group of clinically
used insulin sensitizers called the thiazolidinediones are agonists
Peroxisome proliferator-activated receptors (PPARs) are ligand-
activated transcription factors belonging to the nuclear receptor
superfamily.1 In the presence of a ligand, PPARs heterodimerize
with retinoid X receptor (RXR), and the heterodimers modulate
transcription of target genes by binding to PPAR response elements
in the promoter region of target genes. Three different PPAR genes
for PPARc 9,10
PPARc agonists enhance insulin sensitivity in target
.
tissues and lower glucose and fatty acid levels in type 2 diabetic
patients. However, despite their proven benefits, clinical applica-
tion of these drugs has been plagued by certain adverse effects,
such as weight gain, increased rate of bone fractures, fluid accumu-
lation, and pulmonary edema, leading to increased frequency of
congestive heart failure.11–13 Therefore, partial agonists have been
developed with the aim of retaining the beneficial effects while
diminishing the adverse effects of full agonists. Metaglidasen, a
(a, b/d referred to as d, and c) have been identified so far. Each
PPAR isotype displays a distinct pattern of tissue distribution and
a distinct pharmacological profile, suggesting that each has unique
functions in different cell types.2
PPARa is highly expressed in tissues with high fatty acid oxida-
tion and, upon stimulation, increases high-density lipoprotein
cholesterol synthesis, promotes reverse cholesterol transport, in-
duces cellular uptake of fatty acids, and reduces triglycerides.3,4
PPARc partial agonist, is the most advanced insulin sensitizer,
and is currently in phase III clinical trials. The results of the phase
II clinical trials showed that this compound, which is a prodrug
ester that is rapidly and completely hydrolyzed in vivo to the
circulating free acid form, significantly improved metabolic param-
eters without the side effects of fluid retention/edema or weight
The fibrate class of hypolipidemic drugs are synthetic PPAR
a
⇑
Corresponding authors. Tel.: +81 3 5841 7849; fax: +81 3 5841 8495 (M.I.).
E-mail addresses: m-ishikawa@iam.u-tokyo.ac.jp (M. Ishikawa), miyachi@phar
gain.12 It has been proposed that the molecular basis of PPAR
c
par-
tial agonism is stabilization of helix 3 in PPAR
c, based on X-ray
m.okayama-u.ac.jp (H. Miyachi).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.041

J. Kasuga et al. / Bioorg. Med. Chem. 18 (2010) 7164–7173
7165
crystallographic and docking studies.14,15 Furthermore, it has been
reported that the reason for the differences in gene expression pro-
or fluorine atom(s) at the 2-position of the biaryl moiety of 1 and
2 might be effective, and we considered that this approach might
be useful if the traditional methodology of introducing hydrophilic
substituents into the PPARd agonists resulted in a decrease of their
activity. We also planned to analyze the relationship between
aqueous solubility and molecular symmetry, focusing on the posi-
tion of the carboxyl group.
files induced by various PPARc partial agonists is differences in
conformational change of the receptor associated with selective
cofactor recruitment.16 In contrast, there was initially limited
interest in PPARd, probably due to its ubiquitous distribution, or
the lack of selective agonists. However, the availability of PPARd-
knockout animals and selective agonists, especially GW501516
(Fig. 1) has revealed that PPARd is involved in fatty acid metabo-
lism, insulin resistance, reverse cholesterol transport and other
biological pathways.17–19 Recently, a PPARd partial agonist showed
full efficacy on free fatty acid oxidation in vitro and corrected the
plasma lipid parameters and insulin sensitivity in vivo.20
So far, we have discovered several PPARd-selective agonists,
including TIPP-204.21 We have also elucidated the X-ray crystal
structure of the complex of TIPP-204 and human PPARd ligand-
binding domain.22 Based on the X-ray crystal structure, biphenyl-
carboxylic acids 1 and 2 were designed and synthesized as
PPARd-selective partial agonists/antagonists (Fig. 1).23 Compound
1 shows antagonistic activity with EC50 of 170 nM and 8% maxi-
mum efficacy, whereas 2 shows partial agonistic activity with
EC50 of 29 nM and 48% maximum efficacy. The molecular mecha-
nism of PPARd partial agonism remains unclear. We thought that
it might be clarified by comparing the X-ray crystal structures of
PPARd-partial agonist complexes with those with PPARd-full ago-
nist and PPARd-antagonist complexes. Unfortunately, the reported
PPARd partial agonists, biphenylcarboxylic acids 1 and 2, possess
insufficient aqueous solubility to allow the preparation of suitable
crystals. Herein, we describe the design and synthesis of novel
PPARd partial agonists with sufficient aqueous solubility for this
purpose. To improve the aqueous solubility, we focused on the
dihedral angle of the biaryl moiety and introduced substituent(s)
to disrupt the molecular planarity and symmetry.
2.2. Chemistry
Compounds 12–14, in which an oxygen atom is introduced into
the n-butyl group of 1, were synthesized as shown in Scheme 1.
Introduction of two methyl groups into 40-hydroxybiphenyl-4-car-
boxylic acid (3) gave compound 4. Compound 4 was formylated,
followed by reductive amide alkylation to afford 6. Simultaneous
deprotection of the carboxyl and the hydroxyl group of 6 followed
by selective benzylation of the carboxyl group30 afforded 8. Intro-
duction of hydrophilic side chains into 8 gave 9–11. Compounds
9–11 were deprotected to afford 12–14.
Compounds in which the biphenyl moiety was modified were
synthesized as outlined in Scheme 2. Briefly, Suzuki coupling reac-
tion of bromide 1523 with appropriate boronic acids or pinacol bo-
rate afforded 1, 2, 16, and 17. Compound 16 was hydrolyzed to
afford 19, while 17 was oxidized to afford 23. As an alternative
route, pinacol borate 18 was prepared from 15. Then, borate 18
was coupled with the appropriate aryl bromides by means of Suzu-
ki coupling reaction to afford 20–22 and 24–27.
2.3. Biological activity and structure–activity relationships
To investigate the cell-level PPARd-agonistic activity of the
biaryl series, we utilized a PPARd-responsive reporter gene assay
with CMX-GAL4N-hPPARd LBD as the recombinant receptor gene,
TK-MH100x4-LUC as the reporter gene, and the CMX b-galactosi-
dase gene for normalization, as previously reported.31 After
incubation, cells were assayed for luciferase reporter gene and
b-galactosidase activities. None of the compounds evaluated in
our experiments reduced b-galactosidase activity in the concentra-
tion range investigated. Percent efficacy of PPARd partial agonists is
estimated at the maximal stimulatory response in relation to the
2. Results and discussion
2.1. Molecular design
In general, the aqueous solubility of drugs depends on their
hydrophobicity (Log P).24,25 Thus, the strategy of introducing
hydrophilic group(s) into the molecule is widely used for increas-
ing aqueous solubility. Therefore, as the first approach for increas-
ing the aqueous solubility of 1 and 2, we tried (i) introduction of an
oxygen atom into the n-butyl group, and (ii) replacement of the
phenyl ring with hetero rings. On the other hand, we also focused
on the relationship between solubility and molecular planarity
and/or symmetry. We hypothesized that introduction of substitu-
ent(s) into bicyclic structures with the aim of modifying the
dihedral angle would result in disruption of the planarity and sym-
metry, leading to decreased crystal packing energy and lower melt-
ing point, and so in turn increasing the solubility. Following this
hypothesis, we have previously increased the aqueous solubility
of integrin antagonists26–28 and aryl hydrocarbon receptor
agonists.29 We anticipated that introduction of methyl group(s)
maximal activity of GW501516. Similar PPAR
agonist assay systems were also utilized, with fenofibric acid as the
positive control for PPAR and ciglitazone for PPAR
a and PPARc receptor
a
c.
Initially, designed analogs possessing hydrophilic side chains
were evaluated (Table 1). Introduction of an oxygen atom into
the n-butyl group (12–14) abrogated the PPARd partial agonistic
activity. This result indicated that introduction of hydrophilic sub-
stituents into the n-butyl group blocks the PPARd–drug interaction.
Next, the effect of replacement of the phenyl group in the
biphenyl moiety with hetero rings was investigated (Table 2).
2-Pyridyl derivative 22 and 2-furanyl derivative 23 lacked PPARd
affinity. 3-Pyridyl derivative 21 showed about two times weaker
PPARd agonistic activity than 1, and its efficacy was similar to that
of 1. The other 3-pyridyl derivative 26 showed about eight times
Figure 1. Chemical structures of PPARd ligands.

7166
J. Kasuga et al. / Bioorg. Med. Chem. 18 (2010) 7164–7173
Scheme 1. Reagents and conditions: (a) MeI, K2CO3, DMF, rt, 98%; (b) TiCl4, CHCl2OCH3, DCM, ꢀ78 °C to rt, 86%; (c) 2-F-4-CF3-benzamide, Et3SiH, TFA, toluene, reflux, 73%; (d)
BBr3, DCM, ꢀ78 °C to rt, 82%; (e) BnBr, CsF–Celite, CH3CN, reflux, 74%, (f) CH3OCH2CH2Cl, K2CO3, DMF, 80 °C, 91% (for 9); (g) HOCH2CH2CH2Br, K2CO3, DMF, 100 °C, 63% (for
10); (h) butyryl chloride, Et3N, DCM, 0 °C, 99% (for 11); (i) 10% Pd/C, H2, AcOEt, rt, 82–90%.
weaker PPARd agonistic activity than 2. Overall, these approaches
to decrease the hydrophobicity of the molecules led to decreased
PPARd partial agonistic activity.
PPARd over PPAR
of these compounds possess weak PPAR
All these results (Table 3) indicate that introduction of substituents
into the biaryl moiety enhanced PPARd agonistic activity with
broadly similar levels of maximum efficacy and selectivity.
a
and PPAR
c
(at least 55 times), although some
full agonistic activity.
a
Next, we examined an alternative approach to increase aqueous
solubility, that is, disruption of molecular planarity and/or symme-
try (Table 3). Introduction of a methyl group (19) or fluorine atom
(20) at the 2-position of 1 led to improved PPARd partial agonistic
activity with similar efficacy. In particular, methyl analog 19
showed 15 times stronger PPARd partial agonistic activity than 1.
The effect of substitution with a meta-carboxyl group (2, 24–27)
was similar to that in the case of a para-carboxyl group (1, 19, and
20). Methyl analog 24 and difluoro analog 25 showed eighteen and
five times stronger activity than 2, respectively. These results indi-
cate that hydrophobic biaryl structures are preferable for PPARd
partial agonistic activity as compared with hydrophilic biaryl
structures.
Pyridyl analog 26 showed increased aqueous solubility (vide in-
fra), although the PPARd agonistic activity was weak. We hypothe-
sized that introduction of methyl group(s) into the biaryl moiety in
the pyridyl analog 26 would improve both the activity and aqueous
solubility. As expected, dimethylpyridyl analog 27 showed three
times stronger activity than 26, and comparable activity to 2.
Interestingly, the PPAR activity profile was highly dependent on
the position of the carboxyl group. The meta-carboxylic acid ana-
logs (2, 24–27) exhibited an efficacy of 40–68% compared to the
full agonist GW501516. The para-carboxylic acid analogs (1, 19,
and 20) exhibited very weak efficacy of around 10%.
2.4. Physico-chemical properties
Thermodynamic aqueous solubility (solubility of a compound as
asaturatedsolutioninequilibrium)ofpotentpartialagonists1, 2, 19,
20, and 24–27 was evaluated according to Avdeef and Testa.32 The
aqueous solubility of 1 and 2 in 1:15 M phosphate buffer (pH 7.4)
was quite low (<0.001 mg/mL). So, a mixture of an equal volume of
1:15 M phosphate buffer (pH 7.4) and EtOH was also used as an
aqueous medium for the evaluation of thermodynamic solubility.
All compounds shown in Table 4 had higher solubility than the
parent compounds. In the para-carboxyl series 1, 19, and 20, intro-
duction of a methyl group (19) or fluorine atom (20) into the
biphenyl moiety resulted in better solubility than 1, as expected.
The fluoro analog 20 was nine times more soluble (3.22 mg/mL)
than 1 in aqueous EtOH. Although compounds 19 and 20 were
more soluble in aqueous EtOH than 1, they were still essentially
insoluble in phosphate buffer. In the case of the meta-carboxyl ser-
ies 2 and 24–27, introduction of a methyl group (24) or two fluo-
rine atoms (25) resulted in seven times greater solubility than 2
in aqueous EtOH. Furthermore, 25 showed moderate solubility in
phosphate buffer (0.0217 mg/mL) for the first time among these
PPAR ligands. Pyridyl analog 26 showed better solubility in both
50% EtOH and phosphate buffer than the parent compound 2. Dim-
ethylpyridyl analog 27 showed two times better solubility than 26
in aqueous EtOH. It is noteworthy that 27 also has greatly
improved solubility in phosphate buffer (2.70 mg/mL), that is, it
Finally, all compounds in Table 3 showed improved selectivity
for PPARd over PPARa and PPARc. para-Carboxylic acids 1, 19,
and 20 proved to be PPARd modulators with complete selectivity
at the tested concentrations. The meta-carboxylic acids 2 and 24–
27 showed agonistic activity with sufficiently high selectivity for

J. Kasuga et al. / Bioorg. Med. Chem. 18 (2010) 7164–7173
7167
Scheme 2. Reagents and conditions: (a) ArB(OH)2 or aryl boronic acid pinacol ester, (PPh3)2PdCl2, Na2CO3, DME, EtOH, H2O, 60 °C (for 1, 2, 16, 17); (b) 6 M HCl, AcOH, 100 °C,
96%; (c) 2-methyl-2-butene, NaClO2, NaH2PO4, t-BuOH, H2O, 80 °C, 84%; (d) bis(pinacolate)diborane, (PPh3)2PdCl2, AcOK, 1,4-dioxane, 100 °C, 87%; (e) ArBr, (PPh3)2PdCl2,
Na2CO3, DME, EtOH, H2O, 60 °C (for 20–22, 24–27).
Table 1
X-ray crystallographic study. Difluoro analog 25 was five times
more potent and eight times more soluble than 2 in aqueous EtOH,
and was soluble in phosphate buffer (0.0217 mg/mL). Dimethyl
PPARd transactivation activities of biphenyl compounds modified at the butoxy side
chain
pyridyl analog 27 had comparable activity to 2 and was highly sol-
uble in phosphate buffer (2.70 mg/mL).
CO2H
F
O
To confirm the mechanism of the improved solubility of the
biaryl analogs, physico-chemical parameters, that is, melting point,
calculated dihedral angle, C Log P and retention time on reversed-
phase HPLC, were evaluated (Table 4). Dihedral angles in optimized
structures of simplified models 28 (Fig. 2) were obtained by means
N
H
RO
F3C
1, 12-14
33
*
of density functional theory (DFT) calculations (B3LYP/6-31G ).
Concerning the compounds with methyl group(s) introduced at
the 2-position of the biaryl moiety, all compounds (19, 24, and 27)
possess increased hydrophobicity, larger dihedral angle and lower
melting point than those of the parent compounds 1, 2, and 26,
respectively. The most soluble analog 27 possesses the lowest
melting point and the largest dihedral angle in this series. The only
one exception was the smaller C Log P of 24 compared with 2,
although the other hydrophobicity parameter, retention time,
was larger than that of 2. These results suggested that introduction
of methyl group(s) into biaryl molecules results in disruption of the
planarity, increasing the dihedral angle and leading in turn to de-
creased crystal packing energy and lower melting point, with a
consequent increase of solubility. Our strategy to improve solubil-
ity by focusing on dihedral angle, as presented in this paper, is
quite distinct from the general/classical strategy based on decreas-
ing the hydrophobicity of the molecule.
R
PPARd
a
EC50 (nM)
% Efficacy
1
12
13
14
n-Bu
CH3O(CH2)2
HO(CH2)3
170
8
N.A.b
N.A.b
N.A.b
—
—
—
CH3(CH2)2CO
a
EC50 values and % efficacy are given relative to the positive control, GW501516,
for PPARd.
b
N.A. means not active at 1 lM as an agonist.
is at least 2700 and 350 times more soluble than 2 and 26,
respectively.
Among the synthesized compounds, 19 and 20 were more po-
tent and more soluble PPARd-selective partial agonists/antagonists
than 1. In the meta-carboxyl series, 24 and 25 were more potent
PPARd-selective partial agonists than 2, with improved solubility.
Two PPAR partial agonists, 25 and 27, showed excellent overall
profiles and appeared to be suitable for preparing crystals for the
When 27 was compared with 2, two kinds of modifications
(replacement of the phenyl group with pyridine ring and introduc-

7168
J. Kasuga et al. / Bioorg. Med. Chem. 18 (2010) 7164–7173
Table 2
PPARs transactivation activities of heterocyclic compounds
Ar
PPARd
PPAR
a
PPARc
a
a
a
EC50 (nM)
% Efficacy
EC50 (nM)
% Efficacy
EC50 (nM)
% Efficacy
1
21
22
23
2
4-CO2H–C6H4
170
400
N.A.b
N.A.b
29
8
9
—
—
48
62
N.A.b
N.A.b
N.A.b
N.A.b
790
—
—
—
—
100
—
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
—
—
—
—
—
—
6-CO2H-pyridin-3-yl
5-CO2H-pyridin-2-yl
5-CO2H-furan-2-yl
3-CO2H–C6H4
26
5-CO2H-pyridin-3-yl
220
4000
a
EC50 values and % efficacy are given relative to the positive controls, GW501516 for PPARd, fenofibric acid for PPAR
N.A. means not active at 1 lM as an agonist.
a
, and ciglitazone for PPARc.
b
Table 3
PPAR transactivation activities of compounds possessing substituents at the 2-position
R2
CO2H
R2
X
F
O
F
O
N
H
N
H
CO2H
R1
R1
F3C
O
F3C
O
Me
Me
1, 19, 20
2, 24-27
R1
R2
X
PPARd
PPAR
a
PPARc
a
a
a
EC50 (nM)
% Efficacy
EC50 (nM)
% Efficacy
EC50 (nM)
% Efficacy
1
19
20
2
24
25
26
27
H
Me
F
H
Me
F
H
H
H
H
H
F
—
—
—
CH
CH
CH
N
170
11
53
8
9
N.A.b
N.A.b
N.A.b
790
—
—
—
100
100
—
—
100
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
—
—
—
—
—
—
—
—
11
48
40
68
62
55
29
1.6
5.7
220
76
130
N.A.b
4000
4200
H
Me
H
Me
N
a
EC50 values and % efficacy are given relative to the positive controls, GW501516 for PPARd, fenofibric acid for PPARa, and ciglitazone for PPARc.
N.A. means not active at 1 lM as an agonist.
b
tion of methyl groups) led more than 2700 times higher solubility
in phosphate buffer. We consider that this improvement of solubil-
ity can be ascribed to two factors, that is, decrease of hydrophobic-
ity by introduction of the pyridine ring (2 vs 26) and disruption of
molecular planarity by introduction of the methyl groups (26 vs
27). Thus, these results indicated that a combination of strategies
for improving aqueous solubility is an effective approach.
In the case of compounds containing fluorine atom(s), the rea-
son for the improvement of aqueous solubility is not entirely clear.
Compound 20 possessed a smaller calculated dihedral angle, lower
melting point and higher hydrophobicity than 1. A possible expla-
nation of the small dihedral angle would be interaction between
the fluorine lone pair and hydrogen at the 20-position. Lack of
molecular symmetry of 20 might lead to a lower melting point
and greater solubility, or the changes of electron density arising
from the introduction of fluorine might have resulted in increased
solubility. Compound 25 possessed the same melting point as 2,
but had a larger calculated dihedral angle and higher hydrophobic-
ity. We have reported that fluorination of b-naphthoflavone leads
to improved solubility, a relatively small calculated dihedral angle
and relatively high hydrophobicity.29 Taken together, these results
indicate that introduction of fluorine atom(s) might improve aque-
ous solubility not only by disrupting molecular planarity, but also
via other mechanism(s). A recent report noted that a larger differ-
ence between the highest occupied and lowest unoccupied molec-
ular orbitals (HOMO and LUMO, respectively), or larger molecular
polarizability, is associated with lower solubility.34 Among the
compounds reported here, however, the aqueous solubility was
not related to the difference between calculated HOMO and LUMO,
or calculated dipole moment (data not shown).
On the other hand, we have previously reported examples
where disruption of symmetry led to increased aqueous solubil-
ity.27,28 Thus, we analyzed the relationship between aqueous solu-
bility and molecular symmetry, focusing on the position of the
carboxyl group. When the position of the carboxyl group was chan-
ged from para to meta, the solubility increased (four times: 1 vs 2,
and 10 times: 19 vs 24). The melting points of meta-carboxylic
acids 2 and 24 were lower than those of the corresponding para-
derivatives 1 and 19, respectively. These results suggest that dis-
ruption of symmetry results in decreased crystal packing energy

J. Kasuga et al. / Bioorg. Med. Chem. 18 (2010) 7164–7173
7169
Table 4
Physico-chemical data of PPARd modulators
R2
CO2H
R2
X
F
O
F
O
N
H
N
H
CO2H
R1
R1
F3C
O
F3C
O
Me
Me
1, 19, 20
2, 24-27
R1
R2
X
Solubility (mg/mL)
Melting point (°C)
Calculated dihedral angleb (°)
C Log Pc
HPLC retention timed (min)
50% EtOHa
Phosphate buffer (pH7.4)
1
19
20
2
24
25
26
27
H
Me
F
H
Me
F
H
H
H
H
H
F
—
—
—
CH
CH
CH
N
0.375
0.985
3.22
1.35
9.95
10.4
9.03
17.7
<0.001
<0.001
<0.001
<0.001
<0.001
0.0217
0.00762
2.70
259–262
241–243
221–223
177–178
146–149
177
43.5
52.5
36.1
36.9
57.5
46.2
37.4
78.1
7.05
7.25
7.29
7.05
6.95
7.10
6.02
6.12
7.98
9.46
8.72
7.79
8.65
7.42
3.61
4.36
H
Me
H
Me
152
104–106
N
a
b
c
Solubility in a mixture of equal volumes of EtOH and 1:15 M phosphate buffer (pH 7.4).
Calculated dihedral angles of simplified models 28 (Fig. 2) were estimated by using GAUSSIAN 03.
C Log P values were estimated with ChemDraw Ultra version 10.0.
d
Waters
lBondapak reversed-phase column (3.9 mm ꢁ 150 mm).
these substituents disrupt the molecular planarity, increasing
the dihedral angle and leading in turn to decreased crystal pack-
ing energy and lower melting point, thereby increasing the
solubility. We suggest that the strategy to improve aqueous solu-
bility by introduction of substituent(s) that modify the dihedral
angle of bicyclic molecules could have general applicability. We
also suggest that the use of a combination of strategies for
improving aqueous solubility, that is, both reduction of hydropho-
bicity and disruption of molecular planarity, is effective. We also
found examples in which disruption of molecular symmetry led
to increased aqueous solubility. Further studies on the mecha-
nisms involved are needed.
Figure 2. Simplified models of PPARd modulators for calculation of dihedral angle.
and lower melting point, leading to increased solubility. Determi-
nation of the crystal structure of these molecules will be needed
to take this discussion further.
3. Conclusion
4. Experimental
To elucidate the molecular basis of PPARd partial agonism,
PPARd partial agonists possessing sufficient aqueous solubility to
permit the preparation of crystals suitable for X-ray structure anal-
ysis are required. Focusing on disruption of molecular planarity
and symmetry of PPARd partial agonists 1 and 2, substituents were
introduced at the 2-position of the biaryl moiety. Structure–activ-
ity relationship study revealed that (1) a hydrophobic biaryl moi-
ety enhances PPARd agonistic activity, (2) substituents at the 2-
position of the biaryl moiety improve aqueous solubility, (3) PPARd
efficacy was highly dependent on the position of the carboxyl
group, and (4) the substituted PPARd modulators possess high
selectivity over other PPARs. As a result of structure development,
we obtained PPARd-selective partial agonists 25 and 27 which are
sufficiently soluble in phosphate buffer. We are currently investi-
gating the X-ray crystal structure of the complexes of these PPARd
selective partial agonists with the PPARd ligand-binding domain. In
addition, more potent PPARd selective partial agonists/antagonists
19 and 20 with improved solubility were obtained.
4.1. General methods
1H NMR spectra were recorded on a JEOL JNMGX500 (500 MHz)
spectrometer. Chemical shifts are expressed in parts per million
relative to tetramethylsilane. Mass spectra were recorded on a
JEOL JMS-DX303 spectrometer. Melting points were determined
by using a Yanagimoto hot-stage melting point apparatus and are
uncorrected. Analytical thin-layer chromatography (TLC) was per-
formed on Merck 60 F254 pre-coated silica gel plates. Flash chro-
matography was performed on a column of Kanto Silica Gel N 60.
HPLC analyses were performed on an analytical column (Waters
lBondapak reversed-phase column (C18, 125 Å, 10 lm,
3.9 mm ꢁ 150 mm) eluted with a mobile phase consisting of 0.1%
TFA in 55% CH3CN at a flow rate of 1.0 mL/min, with UV monitoring
at 262–295 nm, at 40 °C.
4.2. Chemistry
The reasons why introduction of substituents to the 2-position
of the biaryl moiety led to improve aqueous solubility were dis-
cussed. Analogs in which a methyl group(s) was introduced at
the 2-position of the biaryl moiety showed increased hydropho-
bicity, a larger dihedral angle and a lower melting point than
those of the parent compounds. These results suggested that
4.2.1. Methyl 40-methoxybiphenyl-4-carboxylate (4)
A solution of 40-hydroxy-4-biphenylcarboxylic acid (3, 1.02 g,
4.76 mmol) in DMF (30 mL) was treated with MeI (3.0 g) followed
by K2CO3 (2.0 g) at rt, and the mixture was stirred at rt for 16 h. The
reaction was quenched with aqueous NH4Cl, and the whole was
extracted with DCM. The organic layer was dried over MgSO4

7170
J. Kasuga et al. / Bioorg. Med. Chem. 18 (2010) 7164–7173
and concentrated in vacuo, and the residue was purified by silica
4.2.6. Benzyl 30-{[2-fluoro-4-(trifluoromethyl)phenylamido]
methyl}-40-(2-methoxyethoxy)biphenyl-4-carboxylate (9)
gel chromatography (hexane/EtOAc = 10:1) to give
4.66 mmol, 98%) as a white solid.
4 (1.13 g,
A solution of 8 (30.2 mg, 57.7 lmol) and CH3OCH2CH2Cl (20 mg,
1H NMR (500 MHz, CDCl3) d 8.08 (d, J = 8.5 Hz, 2H), 7.62 (d,
J = 8.5 Hz, 2H), 7.57 (d, J = 8.5 Hz, 2H), 7.00 (d, J = 8.5 Hz, 2H),
3.93 (s, 3H), 3.86 (s, 3H). MS (FAB) 242 (M)+.
0.21 mmol) in DMF (1 mL) was treated with K2CO3 (10 mg) and the
mixture was stirred at 80 °C for 2 h. The reaction was quenched with
brine, and the whole was extracted with DCM. The organic layer was
dried over MgSO4 and concentrated in vacuo, and the residue was
purified by silica gel chromatography (hexane/EtOAc = 5:1 to 2:1)
4.2.2. Methyl 30-formyl-40-methoxybiphenyl-4-carboxylate (5)
A solution of 4 (1.13 g, 4.66 mmol) in DCM (100 mL) was cooled
to ꢀ78 °C, then treated with TiCl4 (3.0 mL) followed by CHCl2OCH3
(1.5 mL), and the mixture was stirred for 30 min at that tempera-
ture, warmed to rt, and stirred for 6 h. The reaction was quenched
with 2 M aqueous HCl, and the whole was extracted with DCM and
AcOEt. The organic layer was dried over MgSO4 and concentrated
in vacuo, and the residue was purified by silica gel chromatography
(hexane/EtOAc = 10:1 to 3:1) to give 5 (1.08 g, 4.00 mmol, 86%) as a
white solid.
to give 9 (30.5 mg, 52.4 lmol, 91%) as a white solid.
1H NMR (500 MHz, CDCl3) d 8.15 (t, J = 7.9 Hz, 1H), 8.11 (d,
J = 8.5 Hz, 2H), 7.65 (d, J = 2.4 Hz, 1H), 7.62 (d, J = 8.5 Hz, 2H),
7.55–7.34 (m, 9H), 6.99 (d, J = 8.5 Hz, 1H), 5.38 (s, 2H), 4.75 (d,
J = 4.9 Hz, 2H), 4.24 (t, J = 4.7 Hz, 2H), 3.79 (t, J = 4.7 Hz, 2H), 3.35
(s, 3H). MS (FAB) 582 (M+H)+.
4.2.7. Benzyl 30-{[2-fluoro-4-(trifluoromethyl)phenylamido]
methyl}-40-(3-hydroxypropoxy)biphenyl-4-carboxylate (10)
1H NMR (500 MHz, CDCl3) d 10.53 (s, 1H), 8.12 (d, J = 2.4 Hz,
1H), 8.10 (d, J = 8.2 Hz, 2H), 7.84 (dd, J = 8.8, 2.4 Hz, 1H), 7.65 (d,
J = 8.2 Hz, 2H), 7.11 (d, J = 8.8 Hz, 1H), 4.00 (s, 3H), 3.94 (s, 3H).
MS (FAB) 271 (M+H)+.
A solution of 8 (58.1 mg, 111 lmol) and 3-bromo-1-propanol
(28 mg, 0.20 mmol) in DMF (3 mL) was treated with K2CO3
(26 mg) and the mixture was stirred at 100 °C for 2 h. The reaction
was quenched with aqueous NH4Cl, and the whole was extracted
with DCM and AcOEt. The organic layer was dried over MgSO4
and concentrated in vacuo, and the residue was purified by silica
gel chromatography (hexane/EtOAc = 1:1) to give 10 (40.6 mg,
4.2.3. Methyl 30-{[2-fluoro-4-(trifluoromethyl)phenylamido]
methyl}-40-methoxybiphenyl-4-carboxylate (6)
A solution of 5 (1.08 g, 4.00 mmol) and 2-fluoro-4-trifluorom-
ethylbenzamide (1.66 g, 8.01 mmol) in toluene (30 mL) was trea-
ted with TFA (0.5 mL) followed by Et3SiH (3.0 mL) and the
mixture was refluxed for 24 h, then concentrated in vacuo. The
residue was purified by silica gel chromatography (hexane/
EtOAc = 5:1 to 3:1) to give 6 (1.35 g, 2.93 mmol, 73%) as a white
solid.
69.8 lmol, 63%) as a white solid.
1H NMR (500 MHz, CDCl3) d 8.22 (t, J = 7.9 Hz, 1H), 8.11 (d,
J = 8.5 Hz, 2H), 7.62–7.33 (m, 12H), 7.01 (d, J = 8.5 Hz, 1H), 5.38
(s, 2H), 4.74 (d, J = 4.9 Hz, 2H), 4.25 (t, J = 6.1 Hz, 2H), 3.91 (t,
J = 6.1 Hz, 2H), 2.13 (m, 2H). MS (FAB) 582 (M+H)+.
4.2.8. Benzyl 40-(butanoyloxy)-30-{[2-fluoro-4-(trifluoromethyl)
phenylamido]methyl}biphenyl-4-carboxylate (11)
1H NMR (500 MHz, CDCl3) d 8.25 (t, J = 7.9 Hz, 1H), 8.07 (d,
J = 7.9 Hz, 2H), 7.64 (d, J = 1.8 Hz, 1H), 7.62 (d, J = 7.9 Hz, 2H),
7.57 (dd, J = 8.5, 1.8 Hz, 1H), 7.52 (d, J = 7.9 Hz, 1H), 7.42 (m, 1H),
7.39 (d, J = 11.6 Hz, 1H), 7.00 (d, J = 8.5 Hz, 1H), 4.74 (d, J = 5.5 Hz,
2H), 3.96 (s, 3H), 3.93 (s, 3H). MS (FAB) 461 (M)+.
A solution of 8 (44.3 mg, 84.6 lmol) in DCM (5 mL) was cooled
to 0 °C, then treated with butyryl chloride (24 mg) followed by
Et3N (0.1 mL), and the mixture was stirred at rt for 1 h. The reac-
tion was quenched with aqueous NaHCO3, and the whole was ex-
tracted with DCM. The organic layer was dried over MgSO4 and
concentrated in vacuo, and the residue was purified by silica gel
chromatography (hexane/EtOAc = 5:1) to give 11 (49.6 mg,
4.2.4. 30-{[2-Fluoro-4-(trifluoromethyl)phenylamido]methyl}-
40-hydroxybiphenyl-4-carboxylic acid (7)
A solution of 6 (1.15 g, 2.49 mmol) in DCM (80 mL) was cooled
to ꢀ78 °C, then treated with BBr3 (10 mmol, 1.0 M solution in
DCM) at the same temperature, and the mixture was warmed to
rt, and stirred for 3 h. The reaction was quenched with brine,
and the whole was extracted with DCM and AcOEt. The organic
layer was dried over MgSO4 and concentrated in vacuo, and the
residue was purified by silica gel chromatography (hexane/
EtOAc = 2:1 to 0:1) to give 7 (880 mg, 2.03 mmol, 82%) as a white
solid.
83.6 lmol, 99%) as a white solid.
1H NMR (500 MHz, CDCl3) d 8.23 (t, J = 7.7 Hz, 1H), 8.13 (d,
J = 8.5 Hz, 2H), 7.68 (d, J = 2.2 Hz, 1H), 7.62 (d, J = 8.5 Hz, 2H),
7.58 (dd, J = 8.4, 2.2 Hz, 1H), 7.53 (d, J = 7.7 Hz, 1H), 7.47–7.34
(m, 6H), 7.19 (d, J = 8.4 Hz, 1H), 7.06 (m, 1H), 5.38 (s, 2H), 4.68
(d, J = 4.9 Hz, 2H), 2.63 (t, J = 7.3 Hz, 2H), 1.80 (m, 2H), 1.05 (t,
J = 7.3 Hz, 3H). MS (FAB) 594 (M+H)+.
4.2.9. 30-{[2-Fluoro-4-(trifluoromethyl)phenylamido]methyl}-
40-(2-methoxyethoxy)biphenyl-4-carboxylic acid (12)
A solution of 9 (30.5 mg, 52.4 lmol) and 10% Pd-C (20 mg) in
AcOEt (5 mL) under an H2 atmosphere was stirred for 1 h, then fil-
1H NMR (500 MHz, DMSO) d 9.96 (s, 1H), 8.99 (t, J = 5.5 Hz, 1H),
7.96 (d, J = 8.5 Hz, 2H), 7.84–7.81 (m, 2H), 7.68–7.66 (m, 3H), 7.59
(d, J = 2.4 Hz, 1H), 7.50 (dd, J = 8.5, 2.4 Hz, 1H), 6.94 (d, J = 8.5 Hz,
1H), 4.49 (d, J = 5.5 Hz, 2H). MS (FAB) 434 (M+H)+.
tered through Celite. The filtrate was concentrated in vacuo to give
12 (21.0 mg, 42.7 lmol, 82%) as a white solid.
4.2.5. Benzyl 30-{[2-fluoro-4-(trifluoromethyl)phenylamido]
methyl}-40-hydroxybiphenyl-4-carboxylate (8)
A solution of 7 (396 mg, 0.914 mmol) in CH3CN (80 mL) was
treated with CsF–Celite (0.36 g) followed by benzyl bromide
(0.66 g, 3.9 mmol) and the mixture was refluxed for 5 h, then con-
centrated in vacuo. The residue was purified by silica gel chroma-
1H NMR (500 MHz, DMSO) d 2.92 (s, 1H), 8.94 (t, J = 5.5 Hz, 1H),
7.99 (d, J = 7.9 Hz, 2H), 7.86–7.81 (m, 2H), 7.73–7.64 (m, 5H), 7.14
(d, J = 8.5 Hz, 1H), 4.53 (d, J = 5.5 Hz, 2H), 4.21 (t, J = 4.0 Hz, 2H),
3.72 (t, J = 4.0 Hz, 2H), 3.31 (s, 3H). MS (FAB) 492 (M+H)+.
4.2.10. 30-{[2-Fluoro-4-(trifluoromethyl)phenylamido]methyl}-
40-(3-hydroxypropoxy)biphenyl-4-carboxylic acid (13)
tography (hexane/EtOAc = 5:1 to 2:1) to give
0.674 mmol, 74%) as a white solid.
8 (353 mg,
A solution of 10 (40.6 mg, 69.8 lmol) and 10% Pd–C (10 mg) in
1H NMR (500 MHz, CDCl3) d 9.25 (s, 1H), 8.29 (t, J = 8.2 Hz, 1H),
8.11 (d, J = 8.2 Hz, 2H), 7.64 (m, 1H), 7.60 (d, J = 8.2 Hz, 2H), 7.56 (d,
J = 8.2 Hz, 1H), 7.52 (dd, J = 8.5, 2.4 Hz, 1H), 7.47–7.34 (m, 7H), 7.06
(d, J = 8.5 Hz, 1H), 5.38 (s, 2H), 4.67 (d, J = 6.1 Hz, 2H). MS (FAB) 524
(M+H)+.
AcOEt (10 mL) under H2 atmosphere was stirred for 1 h. The reac-
tion mixture was filtered through Celite, the filtrate was concen-
trated in vacuo, and the residue was purified by silica gel
chromatography (hexane/EtOAc = 1:1 to 0:1) to give 13 (29.4 mg,
59.8 lmol, 86%) as a white solid.

J. Kasuga et al. / Bioorg. Med. Chem. 18 (2010) 7164–7173
7171
1H NMR (500 MHz, DMSO) d 2.92 (s, 1H), 8.98 (t, J = 5.7 Hz, 1H),
7.98 (d, J = 8.5 Hz, 2H), 7.85–7.80 (m, 2H), 7.71 (d, J = 8.5 Hz, 2H),
7.68 (d, J = 9.2 Hz, 1H), 7.65–7.63 (m, 2H), 7.13 (d, J = 9.2 Hz, 1H),
4.57 (t, J = 5.2 Hz, 1H), 4.52 (d, J = 5.7 Hz, 2H), 4.14 (t, J = 6.1 Hz,
2H), 3.61 (m, 2H), 1.91 (m, 2H).
1H NMR (500 MHz, CDCl3) d 9.60 (s, 1H), 8.25 (t, J = 7.3 Hz, 1H),
7.78–7.77 (m, 2H), 7.52 (d, J = 8.5 Hz, 1H), 7.40 (d, J = 11.0 Hz, 1H),
7.37 (m, 1H), 7.29 (d, J = 3.7 Hz, 1H), 6.95 (d, J = 9.2 Hz, 1H), 6.73 (d,
J = 3.7 Hz, 1H), 4.71 (d, J = 5.5 Hz, 2H), 4.09 (t, J = 6.4 Hz, 2H), 1.85
(m, 2H), 1.53 (m, 2H), 1.00 (t, J = 7.3 Hz, 3H). MS (FAB) 464 (M+H)+.
4.2.11. 40-(Butanoyloxy)-30-{[2-fluoro-4-(trifluoromethyl)pheny-
lamido]methyl}biphenyl-4-carboxylic acid (14)
4.2.16. 40-Butoxy-30-{[2-fluoro-4-(trifluoromethyl)phenylami-
do]methyl}-2-methylbiphenyl-4-carboxylic acid (19)
A solution of intermediate 16 (32.1 mg, 62.0 lmol) in 6 M aque-
ous HCl (3 mL) and AcOH (8 mL) was stirred at 100 °C for 2 h. The
reaction mixture was poured into water, and the precipitated solid
was collected by filtration to give 19 (30.1 mg, 59.8 lmol, 96%) as a
white solid.
Compound 14 (37.9 mg, 75.3
white solid from 11 (49.6 mg, 83.6
l
mol, 90%) was synthesized as a
mol) following the general
l
procedure for 12.
1H NMR (500 MHz, DMSO) d 13.01 (s, 1H), 9.06 (t, J = 5.7 Hz,
1H), 8.03 (d, J = 8.5 Hz, 2H), 7.84 (d, J = 9.2 Hz, 1H), 7.79–7.67
(m, 6H), 7.26 (d, J = 7.9 Hz, 1H), 4.47 (d, J = 5.7 Hz, 2H), 2.65
(t, J = 7.3 Hz, 2H), 1.68 (m, 2H), 0.98 (t, J = 7.3 Hz, 3H).
Mp 241–243 °C. 1H NMR (500 MHz, DMSO) d 12.88 (s, 1H),
8.94 (t, J = 5.9 Hz, 1H), 7.84–7.77 (m, 4H), 7.66 (d, J = 7.9 Hz,
1H), 7.28–7.25 (m, 3H), 7.08 (m, 1H), 4.50 (d, J = 5.9 Hz, 2H), 4.07
(t, J = 6.1 Hz, 2H), 2.28 (s, 3H), 1.76 (m, 2H), 1.50 (m, 2H), 0.95 (t,
J = 7.6 Hz, 3H). MS (FAB) 504 (M+H)+. UV monitoring of HPLC at
280 nm.
4.2.12. 40-Butoxy-30-{[2-fluoro-4-(trifluoromethyl)phenylami-
do]methyl}biphenyl-4-carboxylic acid (1)
To a solution of 15 (60 mg, 0.13 mmol) and 4-carboxyphenylbo-
ronic acid (50 mg, 0.30 mmol) in 6 mL of DME, 4 mL of ethanol and
2 M aq Na2CO3 were added (Ph3P)2PdCl2 (10 mg, 0.014 mmol). The
reaction mixture was stirred at 50 °C for 5 h. The reaction was
quenched by the addition of aq NH4Cl, and the whole was ex-
tracted with AcOEt. The organic layer was dried over MgSO4 and
concentrated, and the residue was purified by silica gel chromatog-
raphy (hexane/ethyl acetate = 3:1) to give 1 (30 mg, 0.061 mmol,
47%) as a white solid.
4.2.17. 5-(4-Butoxy-3-{[2-fluoro-4-(trifluoromethyl)phenylami-
do]methyl}phenyl)furan-2-carboxylic acid (23)
A solution of 17 (58.4 mg, 0.126 mmol) and NaH2PO4 (18 mg,
0.15 mmol) in t-BuOH (6 mL) and H2O (2 mL) was treated with
2-methyl-2-butene (1 mL) followed by sodium chlorite (45 mg,
0.50 mmol), and the mixture was stirred at 80 °C for 5 h. The reac-
tion was quenched with aqueous HCl and the whole was extracted
with DCM. The organic layer was dried over MgSO4 and concen-
trated in vacuo, and the residue was purified by silica gel chroma-
tography (hexane/AcOEt = 2:1 to 0:1) to give 23 (51.0 mg,
0.106 mmol, 84%) as a yellow solid.
Mp 259–262 °C. 1H NMR (500 MHz, DMSO) d 8.96 (t, J = 5.7 Hz,
1H), 7.98 (d, J = 8.5 Hz, 2H), 7.83 (m, 2H), 7.71 (d, J = 8.5 Hz, 2H),
7.69–7.62 (m, 3H), 7.12 (d, J = 9.2 Hz, 1H), 4.52 (d, J = 5.7 Hz, 2H),
4.08 (t, J = 6.1 Hz, 2H), 1.75 (m, 2H), 1.49 (m, 2H), 0.95 (t,
J = 7.6 Hz, 3H). MS (FAB) 490 (M+H)+. UV monitoring of HPLC at
295 nm.
1H NMR (500 MHz, CDCl3) d 8.26 (t, J = 7.6 Hz, 1H), 7.77 (d,
J = 2.4 Hz, 1H), 7.75 (dd, J = 8.5, 2.4 Hz, 1H), 7.53 (d, J = 8.5 Hz,
1H), 7.41–7.36 (m, 3H), 6.94 (d, J = 8.5 Hz, 1H), 6.67 (d, J = 3.7 Hz,
1H), 4.72 (d, J = 5.5 Hz, 2H), 4.09 (t, J = 6.7 Hz, 2H), 1.85 (m, 2H),
1.53 (m, 2H), 1.00 (t, J = 7.3 Hz, 3H). MS (FAB) 479 (M)+.
4.2.13. 40-Butoxy-30-{[2-fluoro-4-(trifluoromethyl)phenylami-
do]methyl}biphenyl-3-carboxylic acid (2)
Compound 2 (29 mg, 59 lmol, 67%) was synthesized as a white
solid from 15 and 3-carboxyphenyl boronic acid following the gen-
eral procedure for 1.
4.2.18. N-[2-Butoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)benzyl]-2-fluoro-4-(trifluoromethyl)benzamide (18)
A mixture of 15 (145 mg, 0.323 mmol), bis(pinacolate)diboron
(120 mg, 0.473 mmol), (Ph3P)2PdCl2 (18 mg, 0.039 mmol), and
KOAc (140 mg, 1.43 mmol) was dissolved in 1,4-dioxane (3 mL)
and the whole was stirred at 100 °C for 3 h, then diluted with
AcOEt and H2O, and extracted with AcOEt. The organic layer was
dried over MgSO4 and concentrated in vacuo, and the residue
was purified by silica gel chromatography (hexane/EtOAc = 10:1)
to give 18 (139 mg, 0.281 mmol, 87%) as a colorless oil.
1H NMR (500 MHz, CDCl3) d 8.24 (t, J = 7.9 Hz, 1H), 7.78 (d,
J = 1.4 Hz, 1H), 7.74 (dd, J = 8.0, 1.4 Hz, 1H), 7.51 (d, J = 8.5 Hz,
1H), 7.37 (d, J = 11.6 Hz, 1H), 7.31 (m, 1H), 6.89 (d, J = 8.0 Hz,
1H), 4.69 (d, J = 4.9 Hz, 2H), 4.06 (t, J = 6.7 Hz, 2H), 1.82 (m, 2H),
1.50 (m, 2H), 1.32 (s, 12H), 0.97 (t, J = 7.6 Hz, 3H). MS (FAB)
495 (M)+.
Mp 177–178 °C. 1H NMR (500 MHz, DMSO) d 8.28 (t, J = 1.8 Hz,
1H), 8.26 (t, J = 7.9 Hz, 1H), 8.03 (d, J = 7.9 Hz, 1H), 7.79 (d,
J = 7.9 Hz, 1H), 7.63 (d, J = 1.8 Hz, 1H), 7.56–7.50 (m, 3H), 7.42
(m, 1H), 7.40 (d, J = 11.6 Hz, 1H), 6.99 (d, J = 7.9 Hz, 1H), 4.76 (d,
J = 5.5 Hz, 2H), 4.11 (t, J = 6.7 Hz, 2H), 1.87 (m, 2H), 1.55 (m, 2H),
1.01 (t, J = 7.6 Hz, 3H). MS (FAB) 490 (M+H)+. UV monitoring of
HPLC at 270 nm.
4.2.14. 40-Methyl butoxy-30-{[2-fluoro-4-(trifluoromethyl)
phenylamido]methyl}-2-methylbiphenyl-4-carboxylate (16)
Compound 16 (32.1 mg, 0.0620 mmol, 60%) was synthesized as
a white solid from 15 and methyl 3-methyl-4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)benzoate following the general procedure
for 1.
1H NMR (500 MHz, CDCl3) d 8.25 (t, J = 7.6 Hz, 1H), 7.93 (s, 1H),
7.86 (dd, J = 7.9, 1.8 Hz, 1H), 7.52 (d, J = 7.9 Hz, 1H), 7.42 (m, 1H),
7.39 (d, J = 11.6 Hz, 1H), 7.31 (d, J = 2.2 Hz, 1H), 7.27 (m, 1H),
7.23 (dd, J = 8.3, 2.2 Hz, 1H), 6.95 (d, J = 8.3 Hz, 1H), 4.73 (d,
J = 5.5 Hz, 2H), 4.10 (t, J = 6.7 Hz, 2H), 3.92 (s, 3H), 2.32 (s, 3H),
1.86 (m, 2H), 1.55 (m, 2H), 1.01 (t, J = 7.6 Hz, 3H). MS (FAB) 517
(M)+.
4.2.19. 40-Butoxy-2-fluoro-30-{[2-fluoro-4-(trifluoromethyl)
phenylamido]methyl}biphenyl-4-carboxylic acid (20)
A solution of 18 (30.8 mg, 62.2
lmol) and 4-bromo-3-fluoro-
benzoic acid (19.5 mg, 89.0 mol) in DME (3 mL), ethanol (2 mL),
l
and 2 M aqueous Na2CO3 (3 mL) was treated with (Ph3P)2PdCl2
(10 mg, 0.014 mmol) and the mixture was stirred at 60 °C for 3 h,
then diluted with AcOEt. The reaction was quenched with brine,
and the whole was extracted with AcOEt. The organic layer was
dried over MgSO4 and concentrated in vacuo, and the residue
was purified by silica gel chromatography (hexane/AcOEt = 3:1)
4.2.15. N-[2-Butoxy-5-(5-formylfuran-2-yl)benzyl]-2-fluoro-4-
(trifluoromethyl)benzamide (17)
Compound 17 (58.4 mg, 0.126 mmol, 93%) was synthesized as a
yellow oil from 15 and 5-formyl-2-furanboronic acid following the
general procedure for 1.
to give 20 (22.4 mg, 44.1 lmol, 71%) as a white solid.

7172
J. Kasuga et al. / Bioorg. Med. Chem. 18 (2010) 7164–7173
Mp 221–223 °C. 1H NMR (500 MHz, DMSO) d 13.25 (s, 1H), 8.96
4.2.25. 5-(4-Butoxy-3-{[2-fluoro-4-(trifluoromethyl)phenylam-
ido]methyl}phenyl)-4,6-dimethylpyridine-3-carboxylic acid
(27)
(t, J = 5.7 Hz, 1H), 7.83–7.78 (m, 3H), 7.72 (dd, J = 11.3, 1.5 Hz, 1H),
7.68 (d, J = 7.9 Hz, 1H), 7.60 (t, J = 7.9 Hz, 1H), 7.52–7.49 (m, 2H),
7.14 (d, J = 9.2 Hz, 1H), 4.51 (d, J = 5.7 Hz, 2H), 4.09 (t, J = 6.1 Hz,
2H), 1.76 (m, 2H), 1.49 (m, 2H), 0.95 (t, J = 7.3 Hz, 3H). MS (FAB)
508 (M+H)+. UV monitoring of HPLC at 290 nm.
Compound 27 (78 mg, 0.15 mmol, 71%) was synthesized as a
white solid from 18 and 5-bromo-4,6-dimethylpyridine-3-carbox-
ylic acid36 following the general procedure for 20.
Mp 104–106 °C. 1H NMR (500 MHz, DMSO) d 8.89 (br s, 2H),
7.82–7.75 (m, 2H), 7.65 (d, J = 7.9 Hz, 1H), 7.16–7.08 (m, 3H),
4.51 (dd, J = 5.5, 2.4 Hz, 2H), 4.08 (t, J = 6.1 Hz, 2H), 2.29 (s, 6H),
1.80–1.74 (m, 2H), 1.51 (tq, J = 7.3, 7.3 Hz, 2H), 0.96 (t, J = 7.3 Hz,
3H). MS (FAB) 519 (M+H)+. UV monitoring of HPLC at 262 nm.
4.2.20. 5-(4-Butoxy-3-{[2-fluoro-4-(trifluoromethyl)phenylam-
ido]methyl}phenyl)pyridine-2-carboxylic acid (21)
Compound 21 (10.9 mg, 0.0222 mmol, 28%) was synthesized as
a white solid from 18 and 5-bromopicolinic acid following the gen-
eral procedure for 20.
1H NMR (500 MHz, DMSO) d 8.98–8.94 (m, 2H), 8.16 (dd, J = 7.9,
2.4 Hz, 1H), 8.09 (d, J = 7.9 Hz, 1H), 7.86–7.81 (m, 2H), 7.73–7.68
(m, 3H), 7.17 (d, J = 8.5 Hz, 1H), 4.53 (d, J = 6.1 Hz, 2H),4.10 (t,
J = 6.4 Hz, 2H), 1.76 (m, 2H), 1.49 (m, 2H), 0.95 (t, J = 7.3 Hz, 3H).
MS (FAB) 491 (M+H)+.
4.3. Reporter gene assay
Transfections of CMX-GAL4N-hPPARa, CMX-GAL4N-hPPARc,
CMX-GAL4N-hPPARd and CMX-b-GAL into HEK 293 cells were per-
formed by the calcium phosphate coprecipitation method.
GW501516 (100 nM) for PPARd, fenofibric acid (100 nM) for
4.2.21. 6-(4-Butoxy-3-{[2-fluoro-4-(trifluoromethyl)pheny-
lamido]methyl}phenyl)pyridine-3-carboxylic acid (22)
Compound 22 (26.2 mg, 0.0534 mmol, 69%) was synthesized as
a white solid from 18 and 6-bromonicotinic acid following the gen-
eral procedure for 20.
PPARa, and ciglitazone (100 nM) for PPARc (positive controls)
and test compounds were added 6 h later. After overnight incuba-
tion, luciferin was added and luminescence was measured on an
ARVOTM SX microplate reader. b-Galactosidase was added and
the absorbance was measured on the microplate reader with emis-
sion detection at 405 nm.
1H NMR (500 MHz, DMSO) d 13.24 (s, 1H), 9.08 (d, J = 2.4 Hz,
1H), 8.97 (t, J = 5.5 Hz, 1H), 8.27 (dd, J = 8.5, 2.4 Hz, 1H), 8.12 (d,
J = 2.4 Hz, 1H), 8.07 (dd, J = 8.5, 2.4 Hz, 1H), 7.98 (d, J = 8.5 Hz,
1H), 7.85–7.81 (m, 2H), 7.70 (d, J = 7.3 Hz, 1H), 7.14 (d,
J = 8.5 Hz, 1H), 4.52 (d, J = 5.5 Hz, 2H), 4.10 (t, J = 6.1 Hz, 2H),
1.76 (m, 2H), 1.49 (m, 2H), 0.94 (t, J = 7.3 Hz, 3H). MS (FAB) 491
(M+H)+.
4.4. Thermodynamic aqueous solubility
The thermodynamic solubility determination was based on the
method of Avdeef and Testa.32 Briefly, about 1 mg of compound
was ground with an agate mortar and taken up in 1.0 mL of an
equal volume of a mixture of 1:15 M phosphate buffer (pH 7.4)
and EtOH, or 1:15 M phosphate buffer (pH 7.4). The suspension
was shaken for 48 h at 25 °C. An aliquot was filtered through a Mil-
4.2.22. 40-Butoxy-30-{[2-fluoro-4-(trifluoromethyl)phenylami-
do]methyl}-2-methylbiphenyl-3-carboxylic acid (24)
Compound 24 (17.5 mg, 0.0348 mmol, 56%) was synthesized as
a white solid from 18 and 3-bromo-2-methylbenzoic acid follow-
ing the general procedure for 20.
lipore DIMEX-13 (0.22 lm). The filtrate was diluted in DMF and in-
jected into an HPLC with UV detection; peak areas were recorded
at 262–295 nm. The concentration of the sample solution was cal-
culated using a previously determined calibration curve, corrected
for the dilution factor of the sample.
Mp 146–149 °C. 1H NMR (500 MHz, CDCl3) d 8.26 (t, J = 7.9 Hz,
1H), 7.94 (dd, J = 7.9, 1.2 Hz, 1H), 7.53 (d, J = 8.5 Hz, 1H), 7.47–
7.27 (m, 5H), 7.19 (dd, J = 8.5, 2.4 Hz, 1H), 6.95 (d, J = 8.5 Hz, 1H),
4.73 (d, J = 5.5 Hz, 2H), 4.10 (t, J = 6.4 Hz, 2H), 2.47 (s, 3H), 1.87
(m, 2H), 1.55 (m, 2H), 1.01 (t, J = 7.3 Hz, 3H). MS (FAB) 504
(M+H)+. UV monitoring of HPLC at 263 nm.
4.5. DFT Calculations
All calculations were performed at the DFT level, using the hy-
brid Becke3LYP (B3LYP) function as implemented in Gaussian
*
4.2.23. 40-Butoxy-2,6-difluoro-30-{[2-fluoro-4-(trifluoromethyl)
phenylamido]methyl}biphenyl-3-carboxylic acid (25)
Compound 25 (33.1 mg, 0.0630 mmol, 53%) was synthesized as
a white solid from 18 and 3-bromo-2,4-difluorobenzoic acid35 fol-
lowing the general procedure for 20.
2003. The 6-31G basis set was used for H, C, N, O and F atoms.
Geometry optimization and vibrational analysis were performed
at the same level. All stationary points were optimized without
any symmetry assumptions and characterized by normal coordi-
nate analysis at the same level of the theory (number of imaginary
frequencies, Nimag, 0).
Mp 177 °C. 1H NMR (500 MHz, CDCl3) d 8.25 (t, J = 7.9 Hz, 1H),
8.02–7.97 (m, 1H), 7.52 (d, J = 7.9 Hz, 1H), 7.46 (s, 1H), 7.44–7.37
(m, 3H), 7.04 (t, J = 8.5 Hz, 1H), 6.99 (d, J = 8.5 Hz, 1H), 4.74 (d,
J = 5.5, Hz, 2H), 4.10 (t, J = 6.1 Hz, 2H), 1.89–1.83 (m, 2H), 1.54
(tq, J = 7.9, 7.3 Hz, 2H), 1.01 (t, J = 7.3 Hz, 3H). MS (FAB) 526
(M+H)+. UV monitoring of HPLC at 262 nm.
Acknowledgment
The work described in this paper was partially supported by
Grants-in-Aid for Scientific Research from The Ministry of Educa-
tion, Culture, Sports, Science and Technology, Japan, and the Japan
Society for the Promotion of Science.
4.2.24. 5-(4-Butoxy-3-{[2-fluoro-4-(trifluoromethyl)phenylami-
do]methyl}phenyl)pyridine-3-carboxylic acid (26)
Compound 26 (55 mg, 0.11 mmol, 54%) was synthesized as a
white solid from 18 and 5-bromonicotinic acid following the gen-
eral procedure for 20.
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