Design, synthesis, and biological activity of potent and orally available g protein-coupled receptor 40 agonists

Article abstract of DOI:10.1021/jm101405t

G protein-coupled receptor 40 (GPR40) is being recently considered to be a new potential drug target for the treatment of type 2 diabetes because of its role in the enhancement of free fatty acid-regulated glucose-stimulated insulin secretion in pancreatic β-cells. We initially identified benzyloxyphenylpropanoic acid (1b) (EC₅₀ = 510 nM), which was designed based on the structure of free fatty acids, as a promising lead compound with GPR40 agonist activity. Chemical modification of compound 1b led to the discovery of 3-{4-[(2′,6′-dimethylbiphenyl-3-yl)methoxy]-2- fluorophenyl}propanoic acid (4p) as a potent GPR40 agonist (EC₅₀ = 5.7 nM). Compound 4p exhibited acceptable pharmacokinetic profiles and significant glucose-lowering effects during an oral glucose tolerance test in diabetic rats. Moreover, no hypoglycemic event was observed even after administration of a high dose of compound 4p to normal fasted rats. These pharmacological results suggest that GPR40 agonists might be novel glucose-dependent insulin secretagogues with little or no risk of hypoglycemia.

Full text of DOI:10.1021/jm101405t

ARTICLE
pubs.acs.org/jmc
Design, Synthesis, and Biological Activity of Potent and Orally
Available G Protein-Coupled Receptor 40 Agonists
Shinobu Sasaki,* Shuji Kitamura, Nobuyuki Negoro, Masami Suzuki, Yoshiyuki Tsujihata, Nobuhiro Suzuki,
Takashi Santou, Naoyuki Kanzaki, Masataka Harada, Yasuhiro Tanaka, Makoto Kobayashi, Norio Tada,
Miyuki Funami, Toshimasa Tanaka, Yoshio Yamamoto, Kohji Fukatsu, Tsuneo Yasuma, and Yu Momose
Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 17-85, Jusohonmachi 2-chome, Yodogawa-ku,
Osaka 532-8686, Japan
ABSTRACT: G protein-coupled receptor 40 (GPR40) is being
recently considered to be a new potential drug target for the
treatment of type 2 diabetes because of its role in the enhance-
ment of free fatty acid-regulated glucose-stimulated insulin secre-
tion in pancreatic β-cells. We initially identified benzyloxy-
phenylpropanoic acid (1b) (EC50 = 510 nM), which was designed
based on the structure of free fatty acids, as a promising lead
compound with GPR40 agonist activity. Chemical modification
of compound 1b led to the discovery of 3-{4-[(20,60-dimethylbi-
phenyl-3-yl)methoxy]-2-fluorophenyl}propanoic acid (4p) as a
potent GPR40 agonist (EC50 = 5.7 nM). Compound 4p exhibited
acceptable pharmacokinetic profiles and significant glucose-low-
ering effects during an oral glucose tolerance test in diabetic rats.
Moreover, no hypoglycemic event was observed even after administration of a high dose of compound 4p to normal fasted rats.
These pharmacological results suggest that GPR40 agonists might be novel glucose-dependent insulin secretagogues with little or no
risk of hypoglycemia.
’ INTRODUCTION
and glinides, are commonly used for its treatment.14 However,
these drugs promote insulin secretion independent of blood
glucose levels, thereby leading to the risk of hypoglycemia and
β-cell dysfunction.14,15 The above-mentioned findings suggest
that activation of GPR40 may lead to the enhancement of insulin
secretion in a glucose-dependent manner and that small mole-
cule agonists of GPR40 may serve as novel insulin secretagogues
with little or no risk of hypoglycemia.
Recently, several studies have reported GPR40 agonists that
contain acidic moieties such as a carboxylic acid or thiazolidine-
dione.16,17 However, in 2002, we independently identified a range
of synthetic agonists by ligand-based drug design methods.18,19
We had previously reported that saturated and unsaturated long-
chain FFAs have GPR40 agonist activity. Among them, docosa-
hexaenoic acid (DHA), a polyunsaturated fatty acid, showed the
most potent activity (EC50 = 1.1 μM), whereas the methyl ester
of linoleic acid showed no activity (EC50 > 300 μM).9 On the
basis of these results, we speculated that both a hydrogen bond
interaction of carboxylate and a π-π interaction of olefin might
have a great impact on the receptor-ligand interaction. We
selected several commercially available arylalkanoic acids and
screened them for their activity by using a fluorometric imag-
ing plate reader (FLIPR) assay. Fortunately, we found that
Free fatty acids (FFAs) are not only an important energy
source but they play key roles in the regulation of a range of
physiological responses, including insulin secretion.1-7 Acute
administration of FFAs promotes glucose-stimulated insulin
secretion (GSIS),1,2 whereas chronic exposure to high levels of
FFAs impairs β-cell function by induction of secretory failure and
β-cell apoptosis.3,4 Several possible mechanisms for FFA-ampli-
fied GSIS have been proposed, for example, that FFAs might
simply provide energy to β-cells or that the intracellular meta-
bolites of FFAs (fatty acyl-coenzyme A molecules) act to stim-
ulate insulin secretion.5-7 In the past decade, by using the G
protein-coupled receptor (GPCR) deorphanizing strategy,8 sev-
eral studies have reported that a number of FFAs act as ligands for
GPCRs, including GPR40, GPR41, GPR43, and GPR120.9-13
GPR41 and GPR43 are activated by short-chain FFAs,12 whereas
GPR40 and GPR120 are activated by medium- or long-chain
FFAs.9-11,13 GPR40 is highly expressed in mouse, rat, and
human pancreatic β-cells and is also found in several areas of
the human brain.9-11 GPR40 couples mainly with a G protein
R-subunit of the Gq family (GRq) and has been shown to
amplify GSIS from pancreatic β-cells only under high-glucose
concentration.9
Impaired insulin secretion is one of the major causes of type 2
diabetes, and several insulin secretagogues, such as sulfonylureas
Received: October 29, 2010
Published: February 14, 2011
r
2011 American Chemical Society
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Figure 1. Optimization of GPR40 agonist.
Scheme 1a
a Reagents and conditions: (a) R4C6H4CH2OH, ADDP, P(n-Bu)3, THF, rt; (b) R4C6H4CH2Br, NaH, DMF, 60 °C; (c) 2 M NaOH aq or LiOH aq,
MeOH or EtOH, THF, rt, 5-85% (2 steps).
3-phenylpropanoic acid had modest agonist activity at a con-
centration of 100 μM. On the basis of this result, we synthesized
or selected several phenylpropanoic acid derivatives having a
phenylalkoxy side chain for an additional π-π interaction with
GPR40 and identified benzyloxyphenylpropanoic acid (1b)
(EC50 = 510 nM) as a promising lead series (Figure 1). The
optimization of compound 1b led to the discovery of com-
pound 4p as a potent and orally bioavailable GPR40 agonist.
In this paper, we describe our early efforts with regard to
the synthesis, structure-activity relationship (SAR) study
data, and pharmacological effects of phenylpropanoic acid
derivatives.
reaction of 3-bromobenzyl alcohol with methyl 3-(4-hydroxy-
phenyl)propanoate (2a). Suzuki-Miyaura cross-coupling of
bromide 5 with appropriate arylboronic acids followed by basic
hydrolysis provided the biaryl analogues 6a-l. Stille coupling of
compound 5 with 2-(trimethylstannyl)pyridine followed by basic
hydrolysis afforded pyridyl derivative 7. Coupling of compound 5
with pinacolborane gave boronic ester 8. Suzuki coupling of
compound 8 with 2-bromothiazole followed by basic hydrolysis
provided the thiazolyl derivative 9.20
The synthesis of the 4-aminophenylpropanoic acid derivatives
11 and 12 is depicted in Scheme 3. Reductive amination of 20,60-
dimethylbiphenyl-3-carbaldehyde (22) with methyl 3-(4-amino-
phenyl)propanoate (2b) gave intermediate 10. Basic hydrolysis
of intermediate 10 afforded the corresponding carboxylic acid 11.
Compound 12 was synthesized from intermediate 10 by methy-
lation followed by basic hydrolysis.
Compound 17 was synthesized as shown in Scheme 4. 3-
(Bromophenyl)acetic acid (13) was converted to the corre-
sponding acyl chloride and then treated with ethyl 3-phenyl-
propanoate under Friedel-Crafts condition to give ketone 14.
Suzuki coupling of compound 14 with 2,6-dimethylphenylboro-
nic acid followed by basic hydrolysis provided compound 15.
Reduction of the carbonyl group of compound 15 with sodium
borohydride followed by dehydration under acidic conditions
gave compound 16. Hydrogenation of the double bond of
compound 16 afforded compound 17.
’ CHEMISTRY
The synthesis of phenylpropanoic acid derivatives having
an ether or thioether linker 4a-s is summarized in Scheme 1.
The phenoxy or phenylthio derivatives 2a-j was condensed
with appropriate alcohols using Mitsunobu reaction or
with appropriate alkyl bromides in the presence of a base
to give the intermediates 3a-s. Subsequent basic hydrolysis
of the intermediates 3a-s afforded the desired carboxylic
acids 4a-s.
The biaryl analogues 6a-l, 7, and 9 were synthesized by
standard palladium-catalyzed cross-coupling reactions as shown
in Scheme 2. The key intermediate 5 was prepared by Mitsunobu
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Scheme 2a
a Reagents and conditions: (a) 3-bromobenzyl alcohol, ADDP, P(n-Bu)3, THF, rt, 68%; (b) (i) R4B(OH)2, Pd(PPh3)4, Na2CO3, toluene, MeOH, H2O,
reflux, (ii) LiOH H2O, MeOH, THF, H2O, rt, 3-76% (2 steps); (c) (i) 2-(trimethylstannyl)pyridine, PdCl2(PPh3)2, DMF, reflux, (ii) LiOH H2O,
3
3
MeOH, THF, H2O, rt, 19% (2 steps); (d) pinacolborane, PdCl2(dppf), triethylamine, toluene, 100 °C; (e) (i) 2-bromothiazole, Pd(PPh3)4, Na2CO3,
toluene, MeOH, H2O, reflux, (ii) LiOH H2O, MeOH, THF, H2O, rt, 31% (3 steps from 5).
3
Scheme 3a
a Reagents and conditions: (a) 20,60-dimethylbiphenyl-3-carbaldehyde 22, 4 Å molecular sieves, toluene, then NaBH3CN, AcOH, THF, rt, 61%; (b) 2 M
NaOH aq, MeOH, THF, rt, 57-70%; (c) MeI, K2CO3, acetone, reflux, 48%.
Scheme 5 illustrates the synthesis of benzyl alcohols 20,
23, and 25a-d. Suzuki coupling of bromide 18 with 2,
4-dimethylphenylboronic acid followed by reduction of the ester
group provided alcohol 20. Compound 23 was synthesized from
bromide 21 and 2,6-dimethylphenylboronic acid in the similar
procedure. Ullmann coupling of bromide 21 with appropriate
phenols provided the aldehydes 24a-d, which were then
reduced with NaBH4 to give the alcohols 25a-d.
The synthesis of the esters 2b-j is outlined in Scheme 6.
Esterification of acids 26a-b with MeOH under acidic condi-
tions afforded esters 2b and 2j. Heck coupling of bromophenol
27 with methyl acrylate followed by hydrogenation of the double
bond provided compound 2c. Horner-Emmons reaction of the
protected benzaldehydes 28a-c, 28e, and ketone 29 using
phosphonoacetate reagents followed by catalytic hydrogenation
gave the corresponding phenylpropanoates. The protecting
groups were, in case of necessity, subsequently removed21 to
afford the esters 2d-i.
’ RESULTS AND DISCUSSION
The agonist activities of all synthesized compounds were
measured by a FLIPR assay in Chinese hamster ovary (CHO)
cells stably expressing human GPR40 in the presence of 0.1%
bovine serum albumin (BSA).
As the first step of our chemical modification, we introduced a
phenyl group or phenoxy group into the lead compound 1b to
potentiate the agonist activity due to an additional aromatic
interactions (e.g., π-π, CH-π, and cation-π interaction) with
GPR40 (Table 1). Among the modifications, introduction of a
substituent in the C3 position was more effective than that in the
C2 or C4 position (6a vs 4a and 4c; 4e vs 4b and 4d). Extending
the length of the linker reduced the potency (4e vs 4f and 4g).
These results indicated that the distance between the terminal
benzene ring and the central benzene ring is important for the
potent agonist activity. Furthermore, we replaced the terminal
phenyl group of compound 6a with a variety of heteroaromatic
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Scheme 4a
a Reagents and conditions: (a) (i) (COCl)2, DMF, THF, rt, (ii) ethyl 3-phenylpropanoate, AlCl3, nitromethane, rt, 61% (2 steps); (b) 2,6-dimethyl-
phenylboronic acid, Na2CO3, Pd(PPh3)4, toluene, EtOH, H2O, reflux; (c) 1 M NaOH aq, THF, EtOH, rt, 79% (2 steps); (d) NaBH4, THF, EtOH,
0 °C; (e) p-TsOH H2O, toluene, reflux, 87% (2 steps); (f) H2, Pd/C, THF, rt, 77%.
3
Scheme 5a
a Reagents and conditions: (a) 2,4-dimethylphenylboronic acid or 2,6-dimethylphenylboronic acid, Pd(PPh3)4, Cs2CO3 or 1 M Na2CO3 aq, toluene,
EtOH, 70-80 °C, 97-100%; (b) LiAlH4, THF, rt, 96%; (c) NaBH4, DME, THF, 0 °C-rt, 24-84%; (d) RPhOH, CuO, K2CO3, pyridine, quinoline,
170 °C, 72-83%.
groups. Although replacement of the phenyl group with a
2-thienyl group maintained activity (6a vs 6l), replacement
with a 2-pyridyl group or a 2-thiazolyl group which has a lower
Log D value was not tolerated (6a vs 7 and 9). These results
led us to speculate that the terminal aromatic ring contributed to
the aromatic interactions and/or lipophilic interactions with
GPR40.
In the next step of our chemical modification, we investigated
the influence of substituents on the terminal benzene rings of
derivative 4e and 6a (Table 2). Introduction of methyl group(s)
at different positions of the terminal benzene ring of the phenoxy
derivative 4e hardly affected the agonist activity (4e vs 4h-k). In
contrast, introduction of a 2-methyl group in the phenyl deriva-
tive 6a drastically increased the potency compared with a
3-methyl or a 4-methyl group (6d vs 6b-c). Encouraged by
the result of the 2-methyl derivative 6d, we focused on the
investigation of ortho-substituents in the terminal benzene ring
of derivative 6a. Introduction of a slightly smaller 2-fluoro group
(6f) decreased the activity compared with a 2-methyl derivative
6d, while incorporation of a bulkier substitution such as a
2-methoxy (6e), a 2-chloro (6g), or a 2-isopropyl (6h) group
maintained the potent activity. In addition, introduction of a
2,6-dimethyl (6i), a 2,3-dimethyl (6j), or a 2,4-dimethyl (6k)
group maintained the potent activity.
In general, most fatty acids bind to serum albumin under
physiological conditions.23 Owing to the structural similarity to
fatty acids, our GPR40 agonists readily bound to serum albumin.
For that reason, we measured the agonist activity of selected
compounds in both the absence (0%) and the presence (0.5%) of
BSA to investigate the influence of binding to serum albumin on
the pharmacological activity. As the result, the serum shift of the
nonsubstituted biphenyl derivative (6a 282-fold) was signifi-
cantly higher than these of the ortho-substituted biphenyl
analogues (6d 38-fold and 6i 36-fold) and the phenoxybenzene
analogues (4e, 4j, and 4k 27-47-fold). Moreover, these effects
of the serum shift were comparably correlated with the activity
of these compounds (6a vs 4e, 4j, 4k, 6d, and 6i) in the
presence of 0.1% BSA. These results led us to hypothesize that
a large dihedral angle of the 2,6-dimethylbiphenyl or the
phenoxybenzene core was favorable for binding to the
GPR40 receptor but on the other hand was unfavorable for
binding to serum albumin. Conformational analyses of simple
fragments (biphenyl, 2,6-dimethylbiphenyl, and phenoxyben-
zene) were performed using the MNDO-PM3 (MOPAC
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Scheme 6a
a Reagents and conditions: (a) SOCl2, MeOH, rt, 99% (for 2b) or H2SO4, MeOH, reflux, 86% (for 2j); (b) methyl acrylate, Pd(OAc)2, Bu4NCl, DMF,
100 °C, 24-34%; (c) H2, Pd/C, THF, MeOH or EtOH, 71-100% (for 2c-i); (d) (method A) (EtO)2P(O)CH2CO2Et (for 2d-e and 2h-i) or
(EtO)2P(O)CH(Me)CO2Et (for 2f), NaH, THF, (method B) (EtO)2P(O)CHFCO2Et (for 2g), n-BuLi, THF, rt, 52-100%; (e) AlCl3, CH3(CH2)7SH,
CH2Cl2, rt, 83-91%; (f) MOMCl, NaH, DMF, 87%; (g) 3 M HCl aq, EtOH, reflux, 44%.
Table 1. Effect of Substituents in the Benzyl Moiety on
GPR40 Agonist Activity
Table 2. Effect of Substituents in the Terminal Benzene Ring
on GPR40 Agonist Activity
compd
4e PhO
R4
EC50 (nM)a compd
R4
EC50 (nM)a
EC50
EC50
(nM)a Log Db
compd R4 (nM)a Log Db compd
R4
34
40
45
38
47
6e 2-MeO-C6H4
6f 2-F-C6H4
82
120
20
4h 4-Me-C6H4O
4i 3-Me-C6H4O
4j 2-Me-C6H4O
4k 2,6-diMe-C6H3O
1b
4a
4b
4c
4d
6a
4e
H
510
300
1.96
3.41
3.26
3.25
3.32
3.37
3.26
4f
4g
7
3-PhCH2O
300
3.27
3.63
1.94
3.22
2.11
6g 2-Cl-C6H4
6h 2-iPr-C6H4
4-Ph
3-Ph(CH2)2O 260
30
4-PhO 310
2-Ph 3600
2-PhO 1100
3-(2-pyridyl)
3-(2-thienyl)
2700
530
6i 2,6-diMe-C6H3
6j 2,3-diMe-C6H3
6k 2,4-diMe-C6H3
8.8
19
6l
9
3-(2-thiazolyl) 2700
6a Ph
260
120
240
27
9.9
3-Ph
260
6b 4-Me-C6H4
6c 3-Me-C6H4
6d 2-Me-C6H4
3-PhO
34
a All values are averages of n = 3 in the presence of 0.1% BSA. b The Log
D values were determined at pH 7.4 according to the reported method.22
a All values are averages of n = 3 in the presence of 0.1% BSA.
version 7.01) method in MOE.24 The global minimum con-
formations are shown in Figure 2. The dihedral angle of
biphenyl (orange) was almost planar, while that of 2,6-
dimethylbiphenyl (green) and phenoxybenzene (blue) were
orthogonal. These results were consistent with our hypothesis
mentioned above.
Next, we examined the effects of a linker atom (X) between
the biphenylylmethyl and the phenyl propanoic acid moiety
(Table 3). Replacement of the oxygen atom (6i) with a nitrogen
atom (11) maintained potent activity, whereas replacement with
a carbon atom (17) and with a sulfur atom (4l) markedly reduced
the agonist activity. These results suggest that the length of the
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linker region (-CH2X-) is very important for the potent
agonist activity. Moreover, introduction of a methyl group
(12) on the nitrogen atom of compound 11 reduced the potency,
implying that the substituent on the nitrogen atom may intro-
duce an unfavorable steric interaction due to the limited space in
the binding pocket.
While the compounds 6i and 11 exhibited potent activities,
unfortunately, these compounds showed poor PK profiles with
high clearance (CLtotal) (6i 4816 mL/h/kg; 11 1691 mL/h/kg)
in rats. In addition, a β-oxidation product (6i-1) and its taurine
conjugate (6i-2) were presumed to be major metabolites in the
rat plasma and liver after oral administration of compound 6i (10
mg/kg) as shown in Figure 3.
Therefore, to block β-oxidation of compound 6i, we focused
on chemical modifications of the phenylpropanoic acid moiety
(Table 4). Introduction of a fluoro group in the R-position of the
carboxylic acid moiety remarkably reduced the activity (6i vs
4m), indicating that an increasing acidity is unfavorable
(calculated pKa: 6i = 4.7; 4m = 2.6). Furthermore, introduction
of a methyl group in either the R- or β-position reduced the
activity (6i vs 4n and 4o). Next, we turned our attention to the
effect of the substituent in the benzene ring of the phenylpro-
panoic acid moiety. Introduction of a fluoro group in the
2-position of the benzene ring maintained the potency (6i vs
4p). Moreover, an additional incorporation of a fluoro group
in the 6-position of compound 4p reduced the activity (4p vs
4s). Our hypothesis is that the benzene ring of the phenyl-
propanoic acid moiety plays a key role in the π-π interaction
with the GPR40 receptor. This interaction most likely con-
tributes to the potent activity seen for this series. Therefore,
we hypothesize that the drop in potency seen with compound
4s is due to a decreased electron density of the benzene ring.
However, introduction of a bulkier substituent in this position
reduced the activity (4p vs 4q-r). One possible explanation
is that incorporation of a bulkier substituent moves the
propanoic acid moiety into a different and less favorable
conformation.
On the other hand, these steric or electronic effects of the
substituents drastically improved the PK profiles with reduced
Table 4. Effect of Substituents in the Phenylpropanoic Acid
Moiety on GPR40 Agonist Activity and Pharmacokinetic
Profilea
Figure 2. Overlap of minimized structures: 2,6-dimethylbiphenyl (carbon
atoms colored green), biphenyl (orange), and phenoxybenzene (blue).
Table 3. Effect of a Linker Atom (X) on GPR40 Agonist
Activity
EC50
CLtotal
Cmax
AUC0-8h
F
compd R1 R2
R3 (nM)b (mL/h/kg) (ng/mL) (ng h/mL) (%)
3
6i
H
F
H
H
H
H
H
8.8
170
4816
528
1136
914
900
187
42
5.3
300.9
268.7
256.2
86.0
2.0
0.9
4m
1264.1 61.2
848.4 94.0
1188.8 87.2
249.0 21.5
4637.9 85.2
10275.3 40.3
2618.8 54.2
compd
X
EC50 (nM)a
4n Me H
170
32
4o
4p
4q
4r
H
H
H
H
H
Me H
6i
O
8.8
6.3
H
H
H
H
2-F
2-Me
5.7
30
11
12
17
4l
NH
NMe
CH2
S
939.5
1593.7
472.8
520
2-MeO 30
2,6-diF 35
320
4s
207
1800
a Rat cassette dosing at 0.1 mg/kg, iv and 1 mg/kg, po. Average of 3 rats.
a All values are averages of n = 3 in the presence of 0.1% BSA.
b All values are averages of n = 3 in the presence of 0.1% BSA.
Figure 3. Presumed structures of metabolites in the plasma and liver after oral administration of compound 6i at a dose of 10 mg/kg to rats.
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plasma clearance compared to the original compound 6i (6i vs
4m-s). These results demonstrate the utility of our strategy to
improve PK profiles by introduction of substituents in the
phenylpropanoic acid moiety. One of the speculations for the
improved PK profiles of these compounds is that they might be
resistant to β-oxidation, as we hypothesized above. Among them,
the most potent compound (4p) (EC50 = 5.7 nM) showed
moderate bioavailability (F = 21.5%). To assess the utility of
compound 4p as a tool compound for in vivo studies, we
examined a direct insulinotropic effect of compound 4p. Com-
pound 4p enhanced glucose-stimulated insulin secretion (GSIS)
in rat insulinoma INS-1 833/15 cells (EC50 = 0.38 μM) and rat
isolated pancreatic islets (at 3 μM) (data not shown). Encour-
aged by these results, compound 4p was selected to test the
antidiabetic efficacy in rats.
In vivo efficacy of compound 4p was evaluated by the oral
glucose tolerance test (OGTT) in male N-STZ-1.5 rats, a model
for nonobese type 2 diabetes with impaired insulin secretion.25
As a result, compound 4p apparently reduced glucose excursion
at doses above 3 mg/kg (Figure 4A) in parallel with increased
insulin levels in the early phase (Figure 4B) when administered
0.5 h before an oral glucose challenge (1 g/kg). Similarly,
outstanding efficacy of compound 4p (3 mg/kg) was observed
in female Wistar fatty rats, a model that develops obesity and
obesity-related features such as impaired glucose tolerance,
hyperinsulinemia, and hyperlipidemia26 (data not shown). In
addition, the risk of hypoglycemia was assessed in fasted healthy
Sprague-Dawley rats by oral administration of compound 4p
(30 mg/kg) and nateglinide (50 mg/kg), a well-known non-
sulfonylurea rapid insulin secretagogue which acts on the ATP-
sensitive potassium channel in pancreatic β-cells.27 As expected,
with the glucose-dependent insulinotropic action of the GPR40
agonists,9,28 no significant differences were observed in the
normal fasting glucose levels between compound 4p-treated
and control groups despite the remarkable hypoglycemic effects
seen in the nateglinide-treated group (Figure 5A). Nateglinide
also potently enhanced plasma insulin levels in these rats, while
compound 4p showed a slight increase in plasma insulin levels
which did not affect plasma glucose levels (Figure 5B).
These results indicate that compound 4p may be a novel
glucose-dependent insulin secretagogue with little or no risk of
hypoglycemia.
Figure 4. Effects of compound 4p on plasma glucose and insulin levels
during an OGTT in male N-STZ-1.5 rats. (A) and (B) show time-
dependent changes of plasma glucose and plasma insulin levels after oral
administration of compound 4p followed by 1 g/kg oral glucose
challenge, respectively. Values are mean ( SD (n = 6). *:p e 0.025
versus control by one-tailed Williams’ test. *:p e 0.025 versus control by
Shirley-Williams’ test.
of the phenylpropanoic acid moiety of compound 4p were
performed, which was published recently.29
’ EXPERIMENTAL SECTION
::
Melting points were determined on a BUCHI B-545 melting point
apparatus and were uncorrected. Proton nuclear magnetic resonance
(1H NMR) spectra were recorded on Bruker Ultra Shield-300 (300
MHz) instruments. Chemical shifts are given in parts per million (ppm)
with tetramethylsilane as an internal standard. Abbreviations are used as
follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet,
dd = doublets of doublet, br = broad. Coupling constants (J values) are
given in hertz (Hz). Elemental analyses were carried out by Takeda
Analytical Laboratories, Ltd. and were within 0.4% of the theoretical
values unless otherwise noted. Low-resolution mass spectra (MS) were
determined on a Waters liquid chromatography-mass spectrometer
system (MS), using a CAPCELL PAK UG-120 ODS (Shiseido Co.,
Ltd.) column (2.0 mm i.d. ꢀ 50 mm) with aqueous CH3CN (10-95%)
containing 0.05% trifluoroacetic acid (TFA) and an HP-1100 (Agilent
Technologies) apparatus for monitoring at 220 nm. All MS experiments
were performed using electrospray ionization (ESI) in positive ion
mode. Analytical HPLC was performed on a Shimadzu LC-VP instru-
ment, equipped with CAPCELL PAK C18 UG120 S-3 μm, 2.0 mm ꢀ
50 mm column with a 4 min linear gradient from 90/10 to 5/95 and
subsequently with a 1.5 min isocratic elution 5/95 A/B, where A =
H2O-0.1%TFA, B = CH3CN-0.1%TFA, at a flow rate of 0.5 μL/min,
with UV detection at 220, at column temperature of 25 °C. Reaction
’ CONCLUSION
Optimization of the lead compound 1b, which was identified
from FFAs by ligand-based drug design, led to the discovery of
the phenylpropanoic acid derivative 4p as a potent and orally
bioavailable GPR40 agonist. The important aspects that led to
the discovery of compound 4p were as follows: (1) introduction
of ortho-substituents in the terminal benzene ring of compound
6a increased the agonist activities and reduced serum shifts, and
(2) introduction of a substituent in the phenylpropanoic acid
moiety dramatically improved PK profiles with reduced plasma
clearance. Compound 4p showed a significant glucose-lowering
effect during OGTT in diabetic rats. In addition, oral adminis-
tration of compound 4p in healthy rats neither increased insulin
secretion nor changed the normal fasting blood glucose levels,
even at a high dose. These results indicated that GPR40 agonists
may be safe insulin secretagogues with little or no risk of
hypoglycemia. To improve the PK profiles, further optimizations
1371
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Journal of Medicinal Chemistry
ARTICLE
(m, 4H). Anal. Calcd for C22H20O3 0.5 H2O: C, 77.40; H, 6.20. Found:
3
C, 77.25; H, 6.17.
3-{4-[(4-Phenoxybenzyl)oxy]phenyl}propanoic Acid (4b).
Step 1: To a solution of (4-phenoxyphenyl)methanol (300 mg, 1.50
mmol) and 2a (324 mg, 1.80 mmol) in THF (20 mL) were added
tributylphosphine (607 mg, 3.00 mmol) and 1,10-(azodicarbonyl)-
dipiperidine (757 mg, 3.00 mmol). After stirring overnight, the mixture
was concentrated and diluted with diisopropyl ether (i-Pr2O). The
precipitate was filtered off, washed with i-Pr2O, and the filtrate was
concentrated. The residue was purified by silica gel column chromatog-
raphy (hexane-EtOAc = 12/1) to give 3b (500 mg, 92%) as a white
powder. Step 2: A mixture of 3b (490 mg, 1.35 mmol) and litium
hydroxide monohydrate (LiOH H2O) (170 mg, 4.06 mmol) in THF
3
(12 mL), MeOH (8 mL), and water (8 mL) was stirred at room
temperature for 3 h. The mixture was diluted with water, neutralized
with aqueous HCl, and extracted with EtOAc. The extract was washed
with brine, dried over MgSO4, and concentrated. The residue was
recrystallized from EtOAc-hexane to give 4b (242 mg, 51%) as color-
less crystals; mp 144-145 °C (from EtOAc-hexane). 1H NMR
(CDCl3) δ 2.65 (t, J = 7.9 Hz, 2H), 2.91 (t, J = 7.9 Hz, 2H), 5.00 (s,
2H), 6.91 (d, J = 8.6 Hz, 2H), 7.00-7.03 (m, 4H), 7.08-7.15 (m, 3H),
7.34 (t, J = 8.3 Hz, 2H), 7.39 (d, J = 8.6 Hz, 2H). Anal. Calcd for
C22H20O4: C, 75.84; H, 5.79. Found: C, 75.79; H, 5.73.
3-[4-(Biphenyl-2-ylmethoxy)phenyl]propanoic Acid (4c).
Compound 4c was prepared in a manner similar to that described for
4a in 23% yield as colorless crystals; mp 103-104 °C (from EtOAc-
hexane). 1H NMR (CDCl3) δ 2.63 (t, J = 7.9 Hz, 2H), 2.88 (t, J = 7.9 Hz,
2H), 4.91 (s, 2H), 6.79 (d, J = 8.6 Hz, 2H), 7.08 (d, J = 8.6 Hz, 2H),
7.33-7.50 (m, 8H), 7.60-7.70 (m, 1H). Anal. Calcd for C22H20O3
0.1H2O: C, 79.07; H, 6.09. Found: C, 79.00; H, 6.11.
3
The following compounds 4d-s were also prepared as described for
4b from the appropriate alcohols and 2a-i as colorless crystals.
3-{4-[(2-Phenoxybenzyl)oxy]phenyl}propanoic Acid (4d).
Yield 43%; mp 114-115 °C (from EtOAc-hexane). 1H NMR (CDCl3)
δ 2.63 (t, J = 7.9 Hz, 2H), 2.89 (t, J = 7.9 Hz, 2H), 5.13 (s, 2H),
6.86-6.92 (m, 3H), 6.95-7.00 (m, 2H), 7.06-7.12 (m, 3H), 7.16 (dd,
J = 7.5 Hz, 1.0 Hz, 1H), 7.24-7.36 (m, 3H), 7.58 (dd, J = 7.5 Hz, 1.4 Hz,
1H). Anal. Calcd for C22H20O4: C, 75.84; H, 5.79. Found: C, 75.67;
H, 5.86.
3-{4-[(3-Phenoxybenzyl)oxy]phenyl}propanoic Acid (4e).
Yield 33%; mp 94-95 °C (from EtOAc-hexane). 1H NMR (CDCl3)
δ 2.64 (t, J = 7.9 Hz, 2H), 2.90 (t, J = 7.9 Hz, 2H), 5.01 (s, 2H),
6.86-6.90 (m, 2H), 6.88-6.98 (m, 1H), 7.00-7.03 (m, 2H),
7.08-7.17 (m, 5H), 7.30-7.36 (m, 3H). Anal. Calcd for C22H20O4:
C, 75.84; H, 5.79. Found: C, 75.68; H, 5.77.
Figure 5. Effects of compound 4p (30 mg/kg, po) and nateglinide (50
mg/kg, po) on fasting plasma glucose and insulin levels in healthy
Sprague-Dawley rats. (A) and (B) show time-dependent changes of
plasma glucose and plasma insulin level after oral administration of
compound 4p or nateglinide, respectively. Values are mean ( SD
(n = 6). *: p e 0.05 and **: p e 0.01 versus control by Dunnett’s test.
#: p e 0.05 and ##: p e 0.01 versus control by Steel test.
progress was determined by thin layer chromatography (TLC) analysis
on Merck Kieselgel 60 F254 plates or Fuji Silysia NH plates. Chromato-
graphic purification was carried out on silica gel columns [(Merck
Kieselgel 60, 70-230 mesh or 230-400 mesh, Merck) or (Chroma-
torex NH-DM 1020, 100-200 mesh)] or on Purif-Pack (SI 60 μm or
NH 60 μm, Fuji Silysia Chemical, Ltd.). The purities of all compounds
tested in biological systems were assessed as being >95% using analytical
HPLC or elemental analyses.
3-(4-{[3-(Benzyloxy)benzyl]oxy}phenyl)propanoic Acid
1
(4f). Yield 67%; mp 107-108 °C (from EtOAc-hexane). H NMR
(CDCl3) δ 2.65 (t, J = 7.9 Hz, 2H), 2.90 (t, J = 7.9 Hz, 2H), 5.01 (s, 2H),
5.07 (s, 2H), 6.87-6.94 (m, 3H), 7.01 (d, J = 7.6 Hz, 1H), 7.06-7.13
(m, 3H), 7.26-7.45 (m, 6H). Anal. Calcd for C23H22O4: C, 76.22; H,
6.12. Found: C, 76.20; H, 6.21.
3-[4-(Biphenyl-4-ylmethoxy)phenyl]propanoic Acid (4a).
Step 1: To a solution of methyl 3-(4-hydroxyphenyl)propanoate (2a)
(500 mg, 2.06 mmol) in DMF (10 mL) was added sodium hydride
(NaH) (60% in oil, 144 mg, 3.61 mmol). After stirring for 0.5 h,
4-(bromomethyl)biphenyl (753 mg, 3.05 mmol) was added to the
solution. After stirring at 60 °C for 5 h, the reaction mixture was poured
into water. The organic materials were extracted with THF and EtOAc.
The extract was washed with water and brine, dried over magnesium
sulfate (MgSO4), and concentrated to give 3a as a colorless oil. Step 2:
A mixture of 3a and 2 M NaOH (10 mL, 20 mmol) in MeOH (30 mL)
was stirred for 6 h under refluxed condition. After concentration, the
mixture was diluted with water, acidified with aqueous HCl, and
extracted with EtOAc. The extract was dried over MgSO4 and concen-
trated. The residue was recrystallized from THF-hexane to give 4a
(60 mg, 11%) as colorless crystals; mp 187-189 °C. 1H NMR (CDCl3)
δ 2.66 (t, J = 7.7 Hz, 2H), 2.91 (t, J = 7.7 Hz, 2H), 5.08 (s, 2H), 6.93 (d,
J = 8.4 Hz, 2H), 7.14 (d, J = 8.4 Hz, 2H), 7.30-7.50 (m, 5H), 7.50-7.60
3-(4-{[3-(2-Phenylethoxy)benzyl]oxy}phenyl)propanoic
1
Acid (4g). Yield 41%; mp 96-97 °C (from EtOAc-hexane). H
NMR (CDCl3) δ 2.64 (t, J = 8.0 Hz, 2H), 2.90 (t, J = 8.0 Hz, 2H), 3.09
(t, J = 7.1 Hz, 2H), 4.18 (t, J = 7.1 Hz, 2H), 5.00 (s, 2H), 6.83-6.92 (m,
3H), 6.97-7.00 (m, 2H), 7.12 (d, J = 8.6 Hz, 2H), 7.21-7.35 (m, 6H).
Anal. Calcd for C24H24O4: C, 76.57; H, 6.43. Found: C, 76.30;
H, 6.26.
3-(4-{[3-(4-Methylphenoxy)benzyl]oxy}phenyl)propanoic
Acid (4h). Yield 50%; mp 133-134 °C (from EtOAc-hexane).
1H NMR (CDCl3) δ 2.34 (s, 3H), 2.65 (t, J = 7.7 Hz, 2H), 2.90
(t, J = 7.7 Hz, 2H), 4.99 (s, 2H), 6.86-6.93 (m, 5H), 7.05 (s, 1H),
7.10-7.15 (m, 5H), 7.31 (t, J = 7.8 Hz, 1H). Anal. Calcd for C23H22O4:
C, 76.22; H, 6.12. Found: C, 76.18; H, 6.24.
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Journal of Medicinal Chemistry
ARTICLE
3-(4-{[3-(3-Methylphenoxy)benzyl]oxy}phenyl)propano-
ic Acid (4i). Yield 32%; mp 94-95 °C (from EtOAc-hexane). H
NMR (CDCl3) δ 2.32 (s, 3H), 2.65 (t, J = 7.7 Hz, 2H), 2.90 (t, J = 7.7
Hz, 2H), 5.01 (s, 2H), 6.79-6.96 (m, 6H), 7.06-7.24 (m, 5H), 7.33 (t,
J = 7.8 Hz, 1H). Anal. Calcd for C23H22O4: C, 76.22; H, 6.12. Found: C,
76.31; H, 6.23.
3-(4-{[3-(2-Methylphenoxy)benzyl]oxy}phenyl)propano-
ic Acid (4j). Yield 15%; mp 105-106 °C (from EtOAc-hexane). 1H
NMR (CDCl3) δ 2.22 (s, 3H), 2.65 (t, J = 7.8 Hz, 2H), 2.90 (t, J = 7.8
Hz, 2H), 4.99 (s, 2H), 6.79-6.94 (m, 4H), 6.98 (m, 1H), 7.03-7.21 (m,
5H), 7.21-7.33 (m, 2H). Anal. Calcd for C23H22O4: C, 76.22; H, 6.12.
Found: C, 75.92; H, 6.21.
2H), 2.93 (t, J = 7.8 Hz, 2H), 5.06 (s, 2H), 6.50 (d, J = 9.6 Hz, 2H),
7.08-7.48 (m, 7H). Anal. Calcd for C24H22F2O3: C, 72.71; H, 5.59.
Found: C, 72.54; H, 5.64.
Methyl 3-{4-[(3-Bromobenzyl)oxy]phenyl}propanoate (5).
Compound 5 was prepared in a manner similar to that described for 4b
(step 1) in 68% yield as a colorless powder. 1H NMR (CDCl3) δ 2.60 (t,
J =8.0 Hz, 2H), 2.90 (t, J =8.0 Hz, 2H), 3.66 (s, 3H), 5.00 (s, 2H), 6.88(d,
J = 8.6 Hz, 2H), 7.12 (d, J = 8.6 Hz, 2H), 7.21-7.27 (m, 1H), 7.34 (d, J =
7.5 Hz, 1H), 7.45 (d, J = 7.8 Hz, 1H), 7.59 (s, 1H).
3-[4-(Biphenyl-3-ylmethoxy)phenyl]propanoic Acid (6a).
Step 1: To a mixture of 5 (600 mg, 1.72 mmol), sodium carbonate
(547 mg, 5.16 mol), and phenylboronic acid (251 mg, 2.06 mmol) in
toluene (25 mL), MeOH (5 mL), and water (5 mL) was added tetra-
kis(triphenylphosphine)palladium (Pd(PPh3)4) (99 mg, 0.086 mmol)
at room temperature. The resulting mixture was refluxed with stirring
overnight under argon atmosphere. Then the reaction mixture was
concentrated under reduced pressure, diluted with water, and extracted
with EtOAc. The extract was washed with brine, dried over MgSO4, and
concentrated. The residue was purified by silica gel column chro-
matography (hexane-EtOAc = 18/1) to give methyl 3-[4-(biphenyl-
3-ylmethoxy)phenyl]propanoate (547 mg, 92%) as a white powder. 1H
NMR (CDCl3) δ 2.60 (t, J = 8.0 Hz, 2H), 2.90 (t, J = 8.0 Hz, 2H), 3.66
(s, 3H), 5.10 (s, 2H), 6.92 (d, J = 8.5 Hz, 2H), 7.12 (d, J = 8.5 Hz, 2H),
7.35-7.47 (m, 5H), 7.54-7.65 (m, 4H). Step 2: A mixture of 3--
[4-(biphenyl-3-ylmethoxy)phenyl]propanoate (547 mg, 1.58 mmol)
1
3-(4-{[3-(2,6-Dimethylphenoxy)benzyl]oxy}phenyl)pro-
panoic Acid (4k). Yield 32%; mp 132-133 °C (from EtOAc-
1
hexane). H NMR (CDCl3) δ 2.11 (s, 6H), 2.65 (t, J = 7.8 Hz, 2H),
2.90 (t, J = 7.8 Hz, 2H), 4.97 (s, 2H), 6.67 (dd, J = 2.1, 7.8 Hz, 1H),
6.81-6.91 (m, 3H), 7.00-7.15 (m, 6H), 7.24 (t, J = 7.8 Hz, 1H). Anal.
Calcd for C24H24O4 0.1H2O: C, 75.84; H, 6.39. Found: C, 75.73;
3
H, 6.43.
3-(4-{[(20,60-Dimethylbiphenyl-3-yl)methyl]sulfanyl}phenyl)-
propanoic Acid (4l). Yield 58%; mp 100-101 °C (from EtOAc-
hexane). 1H NMR (CDCl3) δ 1.95 (s, 6H), 2.62 (t, J = 7.7 Hz, 2H), 2.89
(t, J = 7.7 Hz, 2H), 4.09 (s, 2H), 6.97-7.01 (m, 2H), 7.04-7.09 (m, 4H),
7.11-7.17 (m, 1H), 7.20-7.29 (m, 3H), 7.31-7.36 (m, 1H). Anal. Calcd
for C24H24O2S 0.2H2O: C, 75.83; H, 6.47. Found: C, 75.84; H, 6.44.
30
3-{4-[(20,6 -Dimethylbiphenyl-3-yl)methoxy]phenyl}-2-
fluoropropanoic Acid (4m). Yield 34%; mp 143-144 °C (from i-
Pr2O-hexane). 1H NMR (CDCl3) δ 2.01 (s, 6H), 2.98-3.37 (m, 2H),
4.97-5.26 (m, 3H), 6.93 (d, J = 8.5 Hz, 2H), 7.05-7.22 (m, 7H),
7.34-7.50 (m, 2H). Anal. Calcd for C24H23FO3: C, 76.17; H, 6.13.
Found: C, 76.01; H, 6.11.
and LiOH H2O (199 mg, 4.74 mmol) in THF (9 mL), MeOH (6 mL),
3
and water (6 mL) was stirred at room temperature for 3 h. The reaction
mixture was diluted with water, neutralized with aqueous HCl, and extracted
with EtOAc. The extract was washed with water and brine, dried over
MgSO4, and concentrated. The residue was recrystallized from EtOAc-
hexane to give 6a (253 mg, 48%) as colorless crystals; mp 125-126 °C. 1H
NMR (CDCl3) δ 2.65 (t, J = 7.9 Hz, 2H), 2.91 (t, J = 7.9 Hz, 2H), 5.10 (s,
2H), 6.93 (d, J = 8.6 Hz, 2H), 7.13 (d, J = 8.6 Hz, 2H), 7.30-7.47 (m, 5H),
3-{4-[(20,60-Dimethylbiphenyl-3-yl)methoxy]phenyl}-2-
methylpropanoic Acid (4n). Yield 30%; mp 95-96 °C (from
i-Pr2O-hexane). 1H NMR (CDCl3) δ 1.16 (d, J = 6.6 Hz, 3H), 2.01
(s, 6H), 2.58-2.76 (m, 2H), 3.00 (dd, J = 13.2, 6.3 Hz, 1H), 5.09 (s,
2H), 6.87-6.92 (m, 2H), 7.07-7.20 (m, 7H), 7.38-7.47 (m, 2H).
Anal. Calcd for C25H26O3: C, 80.18; H, 7.00. Found: C, 80.21; H, 6.94.
3-{4-[(20,60-Dimethylbiphenyl-3-yl)methoxy]phenyl}butanoic
7.50-7.61 (m, 3H), 7.65 (s, 1H). Anal. Calcd for C22H20O3 0.3H2O: C,
3
78.22; H, 6.15. Found: C, 77.90; H, 5.88.
The following compounds 6b-l were also prepared as described for
6a from the appropriate arylboronic acid as colorless crystals.
3-{4-[(40-Methylbiphenyl-3-yl)methoxy]phenyl}propanoic
1
1
Acid (4o). Yield 45%; mp 143-144 °C (from i-Pr2O-hexane). H
Acid (6b). Yield 64%; mp 150-151 °C (from EtOAc-hexane). H
NMR (CDCl3) δ 1.28 (d, J = 6.9 Hz, 3H), 2.00 (s, 6H), 2.49-2.66 (m,
2H), 3.15-3.29 (m, 1H), 5.09 (s, 2H), 6.88-6.93 (m, 2H), 7.08-7.19
(m, 7H), 7.38-7.46 (m, 2H). Anal. Calcd for C25H26O3: C, 80.18; H,
7.00. Found: C, 79.98; H, 7.04.
NMR (CDCl3) δ 2.39 (s, 3H), 2.65 (t, J = 7.9 Hz, 2H), 2.90 (t, J = 7.9 Hz,
2H), 5.09 (s, 2H), 6.85-6.96 (m, 2H), 7.13 (d, J = 8.7 Hz, 2H), 7.21-
7.29 (m, 2H), 7.35-7.57 (m, 5H), 7.63 (s, 1H). Anal. Calcd for C23-
H22O3: C, 79.74; H, 6.49. Found: C, 79.58; H, 6.53.
3-{4-[(20,60-Dimethylbiphenyl-3-yl)methoxy]-2-fluoro-
phenyl}propanoic Acid (4p). Yield 29%; mp 107-107.5 °C
(from i-Pr2O-hexane). 1H NMR (CDCl3) δ 2.00 (s, 6H), 2.63 (t,
J = 7.6 Hz, 2H), 2.90 (t, J = 7.6 Hz, 2H), 5.06 (s, 2H), 6.63-6.70 (m,
2H), 7.06-7.18 (m, 6H), 7.36-7.46 (m, 2H). Anal. Calcd for
3-{4-[(30-Methylbiphenyl-3-yl)methoxy]phenyl}propanoic
1
Acid (6c). Yield 57%; mp 102-103 °C (from EtOAc-hexane). H
NMR (CDCl3) δ 2.42 (s, 3H), 2.65 (t, J = 8.0 Hz, 2H), 2.91 (t, J = 8.0 Hz,
2H), 5.10 (s, 2H), 6.93 (d, J = 8.6 Hz, 2H), 7.12-7.18 (m, 3H),
7.30-7.47 (m, 5H), 7.54 (dt, J = 7.3 Hz, 1.6 Hz, 1H), 7.64 (s, 1H).
Anal. Calcd for C23H22O3: C, 79.74; H, 6.49. Found: C, 79.53; H, 6.35.
3-{4-[(20-Methylbiphenyl-3-yl)methoxy]phenyl}propanic
C
24H23FO3: C, 76.17; H, 6.13. Found: C, 76.07; H, 6.09.
3-{4-[(20,60-Dimethylbiphenyl-3-yl)methoxy]-2-methyl-
1
phenyl}propanoic Acid (4q). Yield 38%; mp 79-80 °C (from
i-Pr2O-hexane). 1H NMR (CDCl3) δ 2.01 (s, 6H), 2.28 (s, 3H), 2.60
(t, J = 7.8 Hz, 2H), 2.89 (t, J = 7.8 Hz, 2H), 5.08 (s, 2H), 6.73-6.80 (m,
2H), 7.04-7.20 (m, 6H), 7.38-7.46 (m, 2H). Anal. Calcd for C25H26-
O3: C, 80.18; H, 7.00. Found: C, 80.35; H, 6.75.
Acid (6d). Yield 41%; mp 135-136 °C (from EtOAc-hexane). H
NMR (CDCl3) δ 2.25 (s, 3H), 2.65 (t, J = 7.9 Hz, 2H), 2.91 (t, J = 7.9
Hz, 2H), 5.09 (s, 2H), 6.92 (d, J = 8.5 Hz, 2H), 7.13 (d, J = 8.5 Hz, 2H),
7.23-7.31 (m, 5H), 7.39-7.45 (m, 3H). Anal. Calcd for C23H22O3: C,
79.74; H, 6.40. Found: C, 79.67; H, 6.57.
3-{4-[(20,60-Dimethylbiphenyl-3-yl)methoxy]-2-methoxy-
phenyl}propanoic Acid. (4r). Yield 5%; mp 118-119 °C (from
i-Pr2O-hexane). 1H NMR (CDCl3) δ 2.01 (s, 6H), 2.62 (t, J = 7.6 Hz,
2H), 2.87 (t, J = 7.6 Hz, 2H), 3.77 (s, 3H), 5.08 (s, 2H), 6.45-6.52 (m,
3-{4-[(20-Methoxybiphenyl-3-yl)methoxy]phenyl}propanoic
Acid(6e). Yield 45%; mp 128-129 °C(fromEtOAc-hexane). 1HNMR
(CDCl3) δ 2.65 (t, J = 7.9 Hz, 2H), 2.91 (t, J = 7.9 Hz, 2H), 3.79 (s, 3H),
5.08 (s, 2H), 6.90-7.05 (m, 4H), 7.13 (d, J = 8.6 Hz, 2H), 7.29-7.50 (m,
2H), 7.02-7.21 (m, 6H), 7.38-7.47 (m, 2H). Anal. Calcd for C25H26O4
5H), 7.58 (s, 1H). Anal. Calcd for C23H22O4 0.25H2O: C, 75.29; H, 6.18.
3
3
0.2H2O: C, 76.20; H, 6.75. Found: C, 76.18; H, 6.75.
Found: C, 75.38; H, 6.28.
3-{4-[(20,60-Dimethylbiphenyl-3-yl)methoxy]-2,6-difluoro-
phenyl}propanoic Acid (4s). Yield 85%; mp 112-113 °C (from
Et2O-hexane). 1H NMR (CDCl3) δ 2.01 (s, 6H), 2.61 (t, J = 7.8 Hz,
3-{4-[(20-Fluorobiphenyl-3-yl)methoxy]phenyl}propanoic
Acid (6f). Yield 58%; mp 112-113 °C (from EtOAc-hexane). H
NMR (CDCl3) δ 2.66 (t, J = 7.9 Hz, 2H), 2.91 (t, J = 7.9 Hz, 2H), 5.10 (s,
1
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ARTICLE
2H), 6.93 (d, J = 8.6 Hz, 2H), 7.12-7.24 (m, 4H), 7.29-7.36 (m, 1H),
7.42-7.54 (m, 4H), 7.61 (s, 1H). Anal. Calcd for C22H19FO3: C, 75.41;
H, 5.47. Found: C, 74.33; H, 5.33.
ladium(II) (PdCl2(dppf)) (0.056 g, 0.069 mmol). The mixture was
stirred at 100 °C under argon atmosphere overnight. After cooling to
room temperature, the reaction mixture was diluted with aqueous
ammonium chloride and extracted with EtOAc. The extract was
washed with brine, dried over MgSO4, and concentrated. The residue
was diluted with toluene (25 mL), MeOH (5 mL), and water (6 mL),
and added 2-bromothiazole (0.49 g, 3.0 mmol), sodium carbonate
(0.73 g, 6.8 mmol), and Pd(PPh3)4 (0.13 g, 0.12 mmol) at room
temperature. The resulting mixture was refluxed with stirring over-
night under argon atmosphere. Then the reaction mixture was diluted
with water and extracted with EtOAc. The extract was washed with
brine, dried over MgSO4, and concentrated. The residue was purified
by silica gel column chromatography (hexane-EtOAc = 3/1) to give
methyl 3-(4-{[3-(1,3-thiazol-2-yl)benzyl]oxy}phenyl)propanonate
3-{4-[(20-Chlorobiphenyl-3-yl)methoxy]phenyl}propanoic
1
Acid (6g). Yield 24%; mp 127-128 °C (from EtOAc-hexane). H
NMR (CDCl3) δ 2.65 (t, J = 7.8 Hz, 2H), 2.91 (t, J = 7.8 Hz, 2H), 5.10 (s,
2H), 6.93 (d, J = 8.5 Hz, 2H), 7.13 (d, J = 8.5 Hz, 2H), 7.28-7.50 (m,
8H). Anal. Calcd forC22H19 ClO3: C, 72.03;H, 5.22. Found: C, 71.92; H,
5.09.
3-(4-{[20-(1-Methylethyl)biphenyl-3-yl]methoxy}phenyl)-
propanoic Acid (6h). Yield 3%; mp 120-121 °C (from Et2O-
hexane). 1H NMR (CDCl3) δ 1.13 (d, J = 6.9 Hz, 6H), 2.65 (t, J = 8.0
Hz, 2H), 2.91 (t, J = 8.0 Hz, 2H), 2.97-3.06 (m, 1H), 5.09 (s, 2H), 6.91
(d, J = 8.6 Hz, 2H), 7.11-7.26 (m, 5H), 7.31-7.45 (m, 5H). Anal. Calcd
for C25H26O3 0.1H2O: C, 79.80; H, 7.02. Found: C, 79.87; H, 7.01.
(310 mg, 38%) as a colorless oil. A mixture of this oil and LiOH H2O
30
3
3-{4-[(20,6 -Dimethylbiphenyl-3-yl)methoxy]phenyl}pro-
panoic Acid (6i). Yield 45%; mp 136-137 °C (from EtOAc-
(110 mg, 2.63 mmol) in a mixture of THF (12 mL), MeOH (9 mL),
and water (9 mL) was stirred at room temperature for 3 h. The
reaction mixture was diluted with water, neutralized with aqueous
HCl, and extracted with EtOAc. The extract was washed with water
and brine, dried over MgSO4, and concentrated. The residue was
recrystallized from EtOAc-hexane to give 9 (241 mg, 81%) as
colorless crystals; mp 126-127 °C (from EtOAc-hexane). 1H
NMR (CDCl3) δ 2.65 (t, J = 8.0 Hz, 2H), 2.91 (t, J = 8.0 Hz, 2H),
5.10 (s, 2H), 6.93 (d, J = 8.6 Hz, 2H), 7.14 (d, J = 8.6 Hz, 2H), 7.35 (d,
J = 3.3 Hz, 1H), 7.43-7.52 (m, 2H), 7.88-7.92 (m, 2H), 8.04 (s, 1H).
Anal. Calcd for C19H17NO3S: C, 67.24; H, 5.05; N, 4.13. Found: C,
67.18; H, 5.22; N, 3.85.
1
hexane). H NMR (CDCl3) δ 2.00 (s, 6H), 2.64 (t, J = 8.0 Hz, 2H),
2.90 (t, J = 8.0 Hz, 2H), 5.09 (s, 2H), 6.90 (d, J = 8.6 Hz, 2H), 7.08-7.25
(m, 7H), 7.35-7.50 (m, 2H). Anal. Calcd for C24H24O3: C, 79.97; H,
6.71. Found: C, 79.88; H, 6.74.
3-{4-[(20,30-Dimethylbiphenyl-3-yl)methoxy]phenyl}pro-
panoic Acid (6j). Yield 43%; mp 146-147 °C (from EtOAc-
hexane). 1H NMR (CDCl3) δ 2.13 (s, 3H), 2.33 (s, 3H), 2.65 (t, J =
8.0 Hz, 2H), 2.90 (t, J = 8.0 Hz, 2H), 5.08 (s, 2H), 6.90-6.93 (m, 2H),
7.09-7.16 (m, 5H), 7.24-7.27 (m, 1H), 7.36-7.42 (m, 3H). Anal.
Calcd for C24H24O3: C, 79.97; H, 6.71. Found: C, 79.83; H, 6.73.
3-{4-[(20,40-Dimethylbiphenyl-3-yl)methoxy]phenyl}pro-
panoic Acid (6k). Yield 76%; mp 104-105 °C (from Et2O-hexane)
1H NMR (CDCl3) δ 2.22 (s, 3H), 2.36 (s, 3H), 2.65 (t, J = 7.6 Hz, 2H),
2.91 (t, J = 7.6 Hz, 2H), 5.08 (s, 2H), 6.91 (d, J = 8.4 Hz, 2H), 7.00-7.46
(m, 9H). Anal. Calcd for C24H24O3: C, 79.97; H, 6.71. Found: C, 79.99;
H, 6.78.
Methyl 3-(4-{[(20,60-Dimethylbiphenyl-3-yl)methyl]amino}-
phenyl)propanoate (10). To a solution of 2j (3.33 g, 18.6 mmol)
and 22 (3.91 g, 18.6 mmol) in toluene (40 mL) were added molecular
sieves (0.4 nm, beads, 7.2 g), and the mixture was stirred at room
temperature for 55 h. The reaction mixture was filtered through Celite,
and the filtrate was concentrated under reduced pressure. The obtained
residue was dissolved in THF (100 mL). Sodium cyanoborohydride
(2.53 g, 40.3 mmol) and acetic acid (2.31 mL, 40.3 mmol) were
successively added, and the mixture was stirred under a nitrogen
atmosphere at room temperature for 3 h. The reaction mixture was
weakly acidified with 10% aqueous citric acid solution and extracted with
EtOAc. The extract was washed with brine, dried over sodium sulfate
(Na2SO4), and concentrated. The residue was purified by silica gel
column chromatography (hexane-EtOAc = 9/1-3/2) to give 10 (4.24
g, 61%) as a colorless oil. 1H NMR (CDCl3) δ 1.99 (s, 6H), 2.56 (t, J =
7.8 Hz, 2H), 2.83 (t, J = 7.8 Hz, 2H), 3.66 (s, 3H), 4.01 (s, 1H), 4.35 (s,
2H), 6.57 (d, J = 8.4 Hz, 2H), 6.99 (d, J = 8.4 Hz, 2H), 7.03-7.17 (m,
5H), 7.31-7.34 (m, 1H), 7.39 (t, J = 7.5 Hz, 1H).
3-(4-{[3-(Thiophen-2-yl)benzyl]oxy}phenyl)propanoic
1
Acid (6l). Yield 17%; mp 127-128 °C (from EtOAc-hexane). H
NMR (CDCl3) δ 2.65 (t, J = 8.0 Hz, 2H), 2.91 (t, J = 8.0 Hz, 2H), 5.07
(s, 2H), 6.93 (d, J = 8.6 Hz, 2H), 7.08 (dd, J = 4.5 Hz, 3.5 Hz, 1H), 7.14
(d, J = 8.6 Hz, 2H), 7.27-7.41 (m, 4H), 7.57 (dt, J = 7.4 Hz, 1.6 Hz, 1H),
7.66 (s, 1H). Anal. Calcd for C20H18O3S: C, 70.98; H, 5.36. Found: C,
70.79; H, 5.26.
3-{4-[(3-Pyridin-2-ylbenzyl)oxy]phenyl}propanoic Acid (7).
To a solution of 5 (700 mg, 2.00 mmol) and 2-(trimethylstannyl)pyridine
(598 mg, 2.40 mmol) in DMF (20 mL) was added dichlorobis(triphenyl-
phosphane)palladium(II) (70 mg, 0.1 mmol). The mixture was refluxed
with stirring overnight under argon atmosphere. After cooling to room
temperature, the reaction mixture was diluted with water and extracted
with EtOAc. The extract was washed with brine, dried over MgSO4, and
concentrated. The residue was purified by silica gel column chromatogra-
phy (hexane-EtOAc = 5/1) to give a colorless oil. A mixture of this oil and
3-(4-{[(20,60-Dimethylbiphenyl-3-yl)methyl]amino}phenyl)-
propanoic Acid (11). To a solution of 10 (0.486 g, 1.30 mmol) in
a mixture of MeOH (6 mL) and THF (6 mL) was added 2 M NaOH
(2 mL), and the mixture was stirred at room temperature for 21 h. Water
was added to the reaction mixture, and the mixture was weakly acidified
with 10% aqueous citric acid solution and then extracted with EtOAc. The
extract was washed with brine, dried over Na2SO4, and concentrated. The
residue was purified by silica gel column chromatography (hexane-EtOAc
= 70/30-0/100) and recrystallized from EtOAc-hexane to give 11 (0.266
g, 57%) as colorless needle-like crystals; mp 87-88 °C. 1H NMR (CDCl3)
δ 1.99 (s, 6H), 2.61 (t, J = 7.7 Hz, 2H), 2.84 (t, J = 7.7 Hz, 2H), 4.35 (s,
2H), 6.57 (d, J = 8.5 Hz, 2H), 6.98-7.17 (m, 7H), 7.30-7.35 (m, 1H),
7.39 (t, J = 7.5 Hz, 1H). Anal. Calcd for C24H25NO2: C, 80.19; H, 7.01; N,
3.90. Found: C, 80.03; H, 7.20; N, 3.88.
LiOH H2O (87 mg, 2.01 mmol) in a mixture of THF (9 mL), MeOH
3
(6 mL), and water (6 mL) was stirred at room temperature for 3 h. The
reactionmixture was dilutedwithwater, neutralizedwith aqueous HCl, and
extracted with EtOAc. The extract was washed with water and brine, dried
over MgSO4, and concentrated. The residue was recrystallized from
EtOAc-hexane to give 7 (130 mg, 19% in 2 steps) as colorless crystals;
mp 160-161 °C. 1H NMR (CDCl3) δ 2.63 (t, J = 8.0 Hz, 2H), 2.90 (t, J =
8.0 Hz, 2H), 5.12 (s, 2H), 6.93 (d, J = 8.6 Hz, 2H), 7.12 (d, J = 8.6 Hz, 2H),
7.24-7.29 (m, 1H), 7.46-7.52 (m, 2H), 7.71-7.81 (m, 2H), 7.87-7.91
(m, 1H), 8.05 (s, 1H), 8.72-8.75 (m, 1H). Anal. Calcd for C21H19NO3:
C, 75.66; H, 5.74; N, 4.20. Found: C, 75.67; H, 5.85; N, 4.16.
3-{4-[[(20,60-Dimethylbiphenyl-3-yl)methyl](methyl)amino]-
phenyl}propanoic Acid (12). To a solution of 10 (0.598 g, 1.60
mmol) and iodomethane (0.498 mL, 8.00 mmol) in acetone (10 mL) was
added potassium carbonate (0.332 g, 2.40 mmol), and the mixture was
heated under reflux under a nitrogen atmosphere for 6 h. After cooling, the
3-(4-{[3-(1,3-Thiazol-2-yl)benzyl]oxy}phenyl)propanoic
Acid (9). To a solution of 5 (0.80 g, 2.3 mmol), pinacolborane (0.50
mL, 3.4 mmol), and triethylamine (0.70 g, 6.9 mmol) in toluene (20
mL) was added [1,10-bis(diphenylphosphino)ferrocene]dichloropal-
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ARTICLE
reaction mixture was concentrated under reduced pressure. Water was
added to the obtained residue, and the mixture was extracted with EtOAc.
The extract was washed with brine, dried over Na2SO4, and concentrated.
The residue was purified by silica gel column chromatography (hexane-
EtOAc = 100/0-75/25) to give a yellow oil. To a solution of this oil in
a mixture of MeOH (4 mL) and THF (4 mL) was added 2 M NaOH
(1.2 mL), and the mixture was stirred at room temperature for 3 days.
Water was added to the reaction mixture, and the mixture was weakly
acidified with 10% aqueous citric acid solution and extracted with EtOAc.
The extract was washed with brine, dried over Na2SO4, and concentrated.
The residue was purified by HPLC to give 12 (0.198 g, 28% in 2 steps) as a
brown viscous oil. 1H NMR (CDCl3) δ 1.98 (s, 6H), 2.61 (t, J = 7.7 Hz,
2H), 2.85 (t, J = 7.7 Hz, 2H), 2.99 (s, 3H), 4.53 (s, 2H), 6.68 (d, J = 8.7 Hz,
2H), 6.98-7.16 (m, 7H), 7.20 (d, J = 7.7 Hz, 1H), 7.36 (t, J = 7.5 Hz, 1H).
MS m/z 374 (M þ H)þ. HPLC (220 nm) 96.4%.
acid (3.0 g, 20.0 mmol), and cesium carbonate (9.8 g, 30.0 mmol) were
dissolved in a mixture of EtOH (20 mL) and toluene (80 mL). After
argon substitution, Pd(PPh3)4 (0.30 g, 0.26 mmol) was added. The
reaction mixture was stirred under an argon atmosphere at 70 °C for 18
h. The reaction mixture was cooled, and insoluble material was filtered
off through Celite. The filtrate was concentrated under reduced
pressure, and the residue was purified by silica gel column chromatog-
raphy (EtOAc-hexane = 1/10) to give 19 (5.0 g, 100%) as a colorless
oil. 1H NMR (CDCl3) δ 1.39 (t, J = 7.0 Hz, 3H), 2.23 (s, 3H), 2.37 (s,
3H), 4.38 (q, J = 7.0 Hz, 2H), 7.02-7.54 (m, 5H), 8.00-8.05 (m, 2H).
(20,40-Dimethylbiphenyl-3-yl)methanol (20). To a solution
of 19 (5.0 g, 19.7 mmol) in anhydrous THF (50 mL) was added lithium
aluminum hydride (0.91 g, 24.0 mmol) under ice-cooling, and the
mixture was stirred at room temperature for 3 h. The reaction solution
was ice-cooled, and sodium sulfate decahydrate (8.0 g, 24.8 mmol) was
added. The mixture was stirred at room temperature for 1 h. The
precipitated insoluble material was filtered off through Celite, and the
filtrate was concentrated under reduced pressure to give 20 as a colorless
oil (4.16 g, 96%). 1H NMR (CDCl3) δ 2.24 (s, 3H), 2.36 (s, 3H), 4.73
(d, J = 6.0 Hz, 2H), 7.00-7. 45 (m, 7H).
Ethyl 3-{4-[(3-Bromophenyl)acetyl]phenyl}propanoate (14).
(3-Bromophenyl)acetic acid (13) (4.00 g, 18.6 mmol) in THF (30 mL)
containing a catalytic amount of DMF was treated with oxalyl chloride (2.83
g, 22.3 mmol) at room temperature. After evolution of gas ceased, the
reaction mixture was evaporated to dryness, and the residue was dried under
vacuum. To a stirred solution of the residue and aluminum chloride (5.46 g,
40.9 mmol) in nitromethane (30 mL), ethyl 3-phenylpropanoate (3.32 g,
18.6 mmol) was added dropwise and the mixture was stirred at room
temperature for 4 h. The reaction mixture was quenched with ice-cold 2 M
HCl and extracted with EtOAc. The extract was washed successively with
water, saturated aqueous sodium bicarbonate (NaHCO3) and brine, dried
over MgSO4, and concentrated. The residue was purified by silica gel column
chromatography (EtOAc-hexane = 1/5) to give 14 (4.25 g, 61%) as
colorless crystals; mp 69-70 °C. 1H NMR (CDCl3) δ 1.23 (t, J = 7.2 Hz,
3H), 2.64 (t, J = 8.0 Hz, 2H), 3.01 (t, J = 8.0 Hz, 2H), 4.12 (q, J = 7.2 Hz,
2H), 4.22 (s2H,), 7.18-7.41 (m, 6H), 7.93 (d, J = 8.2 Hz, 1H). Anal. Calcd
for C19H19BrO3: C, 60.81; H, 5.10. Found: C, 60.67; H, 5.05.
20,60-Dimethylbiphenyl-3-carbaldehyde (22). 3-Bromoben-
zaldehyde (21) (18.5 g, 100 mmol) and (2,6-dimethylphenyl)boronic
acid (21.0 g, 140 mmol) were dissolved in a mixture of 1 M aqueous
sodium carbonate solution (200 mL), EtOH (100 mL), and toluene
(200 mL). After argon substitution, Pd(PPh3)4 (5.78 g, 5.00 mmol) was
added. The reaction mixture was stirred under argon atmosphere at
80 °C for 20 h. The reaction mixture was cooled, and water was added to
the reaction mixture. The mixture was diluted with EtOAc, and the
insoluble material was filtered through Celite. The organic layer of the
filtrate was washed with brine, dried over MgSO4, and concentrated. The
residue was purified by silica gel column chromatography (EtOAc-
hexane = 0/100-10/90) to give 22 (20.4 g, 97%) as a colorless oil. 1H
NMR (CDCl3) δ 2.02 (s, 6H), 7.11-7.23 (m, 3H), 7.42-7.46 (m, 1H),
7.61 (t, J = 7.6 Hz, 1H), 7.68-7.69 (m, 1H), 7.86-7.90 (m, 1H), 10.06
(s, 1H).
3-{4-[(20,60-Dimethylbiphenyl-3-yl)acetyl]phenyl}propanoic
Acid (15). Compound 15 was prepared in a manner similar to that des-
cribed for 6a in 79% yield as colorless crystals; mp 143-144 °C (EtOAc-
hexane). 1H NMR (CDCl3) δ 1.98 (s, 6H), 2.69 (t, J = 7.6 Hz, 2H), 3.00 (t,
J = 7.6 Hz, 2H), 4.28 (s, 2H), 7.01-7.40 (m, 9H), 7.93 (d, J = 8.4 Hz, 1H).
3-{4-[(E)-2-(20,60-Dimethylbiphenyl-3-yl)ethenyl]phenyl}-
propanoic Acid (16). To a solution of 15 (2.00 g, 5.37 mmol) in
EtOH (50 mL) was added sodium borohydride (50 mg, 1.32 mmol) at
0 °C. The mixture was stirred at room temperature for 1 h. The reaction
mixture was poured into water, neutralized with 2 M HCl, and extracted
with EtOAc. The extract was washed with brine, dried over MgSO4, and
concentrated to give a white powder. A mixture of the powder, p-
toluenesulfonic acid monohydrate (0.285 g, 1.90 mmol), and toluene
(30 mL) was heated under reflux for 0.5 h. The reaction mixture was
concentrated, and the residue was recrystallized from EtOAc-hexane to
give 16 as colorless crystals (0.97 g, 87%); mp 178-179 °C. 1H NMR
(CDCl3) δ 2.07 (s, 6H), 2.69 (t, J = 8.0 Hz, 2H), 2.97 (t, J = 8.0 Hz, 2H),
7.02-7.46 (m, 13H). Anal. Calcd for C25H24O2: C, 84.24; H, 6.79.
Found: C, 84.08; H, 7.00.
(20,60-Dimethylbiphenyl-3-yl)methanol (23). Compound 22
(18.5 g, 88.0 mmol) was dissolved in a mixture of DME (100 mL) and
THF (100 mL), and sodium borohydride (1.66 g, 44.0 mmol) was added
at 0 °C. The mixture was stirred at 0 °C for 3 h and further stirred at room
temperature for 3 h. The reaction mixture was quenched with aqueous
HCl and extracted with EtOAc. The extract was washed with brine, dried
over MgSO4, and concentrated. The residue was purified by silica gel
column chromatography (EtOAc-hexane = 0/100-50/90) to give 23
(15.6 g, 83%) as a colorless oil. 1H NMR (CDCl3) δ 1.66 (t, J = 5.9 Hz,
1H), 2.03 (s, 6H), 4.74 (d, J = 5.9 Hz, 2H), 7.07-7.19 (m, 5H), 7.35 (d,
J = 7.5 Hz, 1H), 7.43 (t, J = 7.5 Hz, 1H).
[3-(4-Methylphenoxy)phenyl]methanol (25a). Step 1: To a
solution of 4-methylphenol (3.57 g, 33.0 mol) and 3-bromobenzalde-
hyde (21) (5.55 g, 30.0 mmol) in a mixture of pyridine (40 mL) and
quinoline (20 mL) under nitrogen were added potassium carbonate
(6.22 g, 45.0 mmol) and cupric oxide (3.58 g, 45.0 mol). The mixture
was heated at 170 °C with vigorous stirring for 24 h. After cooling,
pyridine was evaporated and the residue was diluted with EtOAc. The
insoluble material was removed by filtration, and the filtrate was washed
with 1 M HCl and brine, dried over MgSO4, and concentrated. The
residue was purified by silica gel column chromatography (EtOAc-
hexane = 0/100-10/90) to give 3-(4-methylphenoxy)benzaldehyde
24a as a yellow oil (5.29 g, 83%). 1H NMR (CDCl3) δ 2.36 (s, 3H), 6.94
(d, J = 8.5 Hz, 2H), 7.18 (d, J = 8.5 Hz, 2H), 7.24-7.28 (m, 1H),
7.41-7.43 (m, 1H), 7.48 (t, J = 7.6 Hz, 1H), 7.55-7.59 (m, 1H), 9.95 (s,
1H). Step 2: To a solution of 24a (5.29 g, 24.9 mmol) in THF (30 mL)
and DME (30 mL) was added portionwise sodium borohydride (473
mg, 12.5 mmol) at 0 °C, and the mixture was stirred at 0 °C for 1 h. The
mixture was poured into 1 M HCl, and extracted with EtOAc. The
3-{4-[2-(20,60-Dimethylbiphenyl-3-yl)ethyl]phenyl}pro-
panoic Acid (17). Compound 16 (0.40 g, 1.12 mmol) was hydro-
genated on 10% palladium on carbon (0.1 g, containing 50% water) in
THF (40 mL) under hydrogen atmosphere (balloon pressure) at room
temperature overnight. The catalyst was removed by filtration, and the
filtrate was concentrated. The residue was purified by silica gel column
chromatography (acetone-hexane = 1/3-1/2) to give 17 as colorless
crystals (0.31 g, 77%); mp 178-179 °C (from Et2O-hexane). 1H NMR
(CDCl3) δ 1.98 (s, 6H), 2.62-2.67 (m, 2H), 2.88-2.93 (m, 6H),
6.87-7.34 (m, 11H). Anal. Calcd for C25H26O2: C, 83.76; H, 7.31.
Found: C, 83.46; H, 7.56.
Ethyl 20,40-Dimethylbiphenyl-3-carboxylate (19). Ethyl
3-bromobenzoate (18) (4.3 g, 18.8 mmol), 2,4-dimethylphenylboronic
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extract was washed with brine, dried over MgSO4, and concentrated.
The residue was purified by silica gel column chromatography (EtOAc-
hexane = 10/90-50/50) to give 25a as a colorless oil (4.59 g, 86%). 1H
NMR (CDCl3) δ 1.62 (t, J = 6.1 Hz, 1H), 2.34 (s, 3H), 4.66 (d, J =
6.1 Hz, 2H), 6.88-6.94 (m, 3H), 6.98 (s, 1H), 7.06 (d, J = 7.7 Hz, 1H),
7.14 (d, J = 8.7 Hz, 2H), 7.30 (t, J = 7.7 Hz, 1H).
Ethyl 3-(2-Fluoro-4-hydroxyphenyl)propanoate (2d). Step 1:
To an ice-cooled solution of ethyl diethylphosphonoacetate (9.45 g, 42.1
mmol) in THF (50 mL) was added NaH (60% in mineral oil, 1.54 g, 38.5
mmol), and the mixture was stirred for 15 min. A solution of 2-fluoro-4-
methoxybenzaldehyde (28a) (5.00 g, 32.4 mmol) in THF (30 mL) was
added dropwise. The mixture was stirred at room temperature for 2 h, and
water was added. The mixture was extracted with EtOAc. The extract was
washed with brine, dried over MgSO4, and concentrated. The residue was
purified by silica gel column chromatography (hexane-EtOAc = 4/1) to
give a colorless oil. Step 2: A mixture of this oil, THF (50 mL), EtOH (5
mL), and platinum dioxide (300 mg) was stirred overnight under a
hydrogen atmosphere at room temperature. The catalyst was filtered off,
and the filtrate was concentrated. The residue was purified by silica gel
column chromatography (hexane-EtOAc = 4/1) to give ethyl 3-(2-fluoro-
4-methoxyphenyl)propanoate (5.97 g, 81% in 2 steps) as a colorless oil.
Step 3: To a solution of this oil (57.4 g, 254 mmol) and aluminum chloride
(101 g, 761 mmol) in dichloromethane (250 mL) was added dropwise
1-octanethiol (74.3 g, 508 mmol), and the mixture was stirred at room
temperature for 2 h. The reaction mixture was poured into ice water and the
mixture was stirred for 30 min. The organic layer was separated, washed
with brine, dried over MgSO4, and concentrated. The catalyst was filtered
off, and the filtrate was concentrated. The residue was purified by silica gel
column chromatography (hexane-EtOAc = 4/1) to give 2d (44.6 g, 83%)
as a colorless oil. 1H NMR (CDCl3) δ 1.23 (t, J = 7.2 Hz, 3H), 2.58 (t, J =
8.1 Hz, 2H), 2.89 (t, J = 8.1 Hz, 2H), 4.12 (q, J = 7.2 Hz, 2H), 6.51-6.56
(m, 2H), 7.01-7.06 (m, 1H).
Ethyl 3-(2,6-Difluoro-4-hydroxyphenyl)propanoate (2e).
Compound 2e was prepared from 28b by a similar to that described
for 2d in 25% yield (3 steps) as a colorless oil. 1H NMR (CDCl3) δ 1.24
(t, J = 7.2 Hz, 3H), 2.55 (t, J = 7.8 Hz, 2H), 2.92 (t, J = 7.8 Hz, 2H), 3.76
(s, 3H), 4.13 (q, J = 7.2 Hz, 2H), 6.42 (d, J = 9.6 Hz, 2H).
Ethyl 3-(4-Hydroxyphenyl)-2-methylpropanoate (2f).
Compound 2f was prepared from 28c by a similar to that described
for 2d (step 1 and step 2) in 81% yield (2 steps) as a colorless oil. 1H
NMR (CDCl3) δ 1.14 (d, J = 6.3 Hz, 3H), 1.19 (t, J = 7.2 Hz, 3H),
2.57-2.73 (m, 2H), 2.85-2.99 (m, 1H), 4.09 (q, J = 7.2 Hz, 2H), 6.74
(d, J = 8.4 Hz, 2H), 7.03 (d, J = 8.4 Hz, 2H).
Ethyl 2-Fluoro-3-(4-hydroxyphenyl)propanoate (2g).
Step 1: A solution of ethyl diethylphosphono-2-fluoroacetate (4.90 g,
20.2 mmol) in THF (40 mL) was stirred under a nitrogen atmosphere at
0 °C, and 1.6 M n-butyllithium/hexane solution (13.1 mL, 21.0 mmol)
was added dropwise. The reaction mixture was stirred at 0 °C for 30 min,
and a solution of 4-(benzyloxy)benzaldehyde (28c) (4.29 g, 20.2 mmol)
in THF (20 mL) was added dropwise. The mixture was stirred at room
temperature for 3 h, and ice-cooled aqueous ammonium chloride
solution was added. The mixture was extracted with EtOAc, and the
extract was washed with brine, dried over MgSO4, and concentrated.
The catalyst was filtered off, and the filtrate was concentrated. The
residue was purified by silica gel column chromatography (hexane-
EtOAc = 97/3-80/20) to give a colorless oil. Step 2: A mixture of this
oil, THF (30 mL), EtOH (30 mL), and 10% palladium on carbon (4.9 g,
containing 50% water) was stirred overnight under a hydrogen atmo-
sphere at room temperature. The catalyst was filtered off, and the filtrate
was concentrated. The residue was purified by silica gel column chro-
matography (hexane-EtOAc = 90/10-60/40) to give 2g (1.75 g, 50%
in 2 steps) as a colorless oil. 1H NMR (CDCl3) δ 1.22-1.30 (m, 3H),
3.00-3.25 (m, 2H), 4.17-4.27 (m, 2H), 4.76-4.78 (m, 1H), 4.92-
5.15 (m, 1H), 6.74-6.81 (m, 2H), 7.08-7.15 (m, 2H).
[3-(3-Methylphenoxy)phenyl]methanol (25b). Compound
25b was prepared in a manner similar to that described for 25a in 69%
yield as a pale-yellow oil. 1H NMR (CDCl3) δ 1.66 (t, J = 6.0 Hz, 1H),
2.33 (s, 3H), 4.67 (d, J = 6.0 Hz, 2H), 6.79-6.83 (m, 2H), 6.90-6.94
(m, 2H), 7.01 (s, 1H), 7.09 (d, J = 7.5 Hz, 1H), 7.19-7.34 (m, 2H).
[3-(2-Methylphenoxy)phenyl]methanol (25c). Compound
25c was prepared in a manner similar to that described for 25a in
43% yield as a colorless oil. 1H NMR (CDCl3) δ 1.62 (t, J = 6.0 Hz, 1H),
2.24 (s, 3H), 4.66 (d, J = 6.0 Hz, 2H), 6.82 (dd, J = 2.2, 8.0 Hz, 1H),
6.87-6.97 (m, 2H), 7.03-7.22 (m, 3H), 7.22-7.32 (m, 2H).
[3-(2,6-Dimethylphenoxy)phenyl]methanol (25d). Com-
pound 25d was prepared in a manner similar to that described for 25a
in 23% yield as yellow crystals. 1H NMR (CDCl3) δ 1.60 (t, J = 6.0 Hz,
1H), 2.12 (s, 6H), 4.64 (d, J = 6.0 Hz, 2H), 6.65 (dd, J = 2,7 Hz, 8.1 Hz,
1H), 6.80 (s, 1H), 6.97 (d, J = 7.5 Hz, 1H) 7.02-7.13 (m, 3H), 7.22 (d,
J = 7.5 Hz, 1H).
Methyl 3-(4-Aminophenyl)propanoate (2b). Under ice-
cooling, thionyl chloride (15 mL, 206 mmol) was added dropwise to
MeOH (60 mL), and the mixture was stirred for 10 min. 3-(4-
Aminophenyl)propanoic acid (26a) (10.1 g, 61.1 mmol) was added to
the reaction mixture, and the mixture was stirred at room temperature
for 18 h. The solvent and excess thionyl chloride was evaporated under
reduced pressure, water and saturated aqueous NaHCO3 were added,
and the mixture was extracted with EtOAc. The extract was washed with
brine, dried over Na2SO4, and concentrated. The obtained solid was
washed with hexane to give 2b (10.9 g, 99%) as pale-brown prism
crystals. 1H NMR (CDCl3) δ 2.57 (t, J = 7.8 Hz, 2H) 2.84 (t, J = 7.8 Hz,
2H) 3.56 (br s, 2H) 3.66 (s, 3H) 6.62 (d, J = 8.5 Hz, 2H) 6.99 (d, J = 8.5
Hz, 2H).
Methyl 3-(4-Sulfanylphenyl)propanoate (2j). To a solution
of 3-(4-sulfanylphenyl)propanoic acid (26b) (2.19 g, 12.0 mmol) in
MeOH (10 mL) was added concentrated sulfuric acid (1.2 mL, 22.5
mmol) and refluxed for 3 h. After concentration, the mixture was diluted
with EtOAc, washed successively with water, saturated aqueous NaH-
CO3, and brine, dried over MgSO4, and concentrated. The residue was
purified by silica gel column chromatography (EtOAc-hexane = 0/
1
100-20/80) to give 2j as colorless crystals (2.02 g, 86%). H NMR
(CDCl3) δ 2.60 (t, J = 7.7 Hz, 2H), 2.90 (t, J = 7.7 Hz, 2H), 3.40 (s, 1H),
3.66 (s, 3H), 7.07 (d, J = 8.3 Hz, 2H), 7.21 (d, J = 8.3 Hz, 2H).
Methyl 3-(4-Hydroxy-2-methylphenyl)propanoate (2c). To
a solution of 4-bromo-3-methylphenol (27a) (10.4 g, 55.6 mmol) in DMF
(200 mL) was added methyl acrylate (7.18 g, 83.4 mmol), NaHCO3 (11.7
g, 139 mmol), and tetrabutylammonium chloride (30.9 g, 111 mmol), and
the solution was degassed and filled with argon. To this mixture was added
palladium acetate(II) (375 mg, 1.67 mmol) and then heated under argon
atmosphere at 100 °C for 24 h. The reaction mixture was cooled to room
temperature and filtered through Celite. The filtrate was diluted with
EtOAc, washed with water and brine, dried over MgSO4, and concentrated.
The residue was purified by silica gel column chromatography (hexane-
EtOAc = 1/10-1/4) to give yellow crystals. These crystals were dissolved
inMeOH(20mL) andTHF(30mL) andhydrogenatedon10%palladium
on carbon (127 mg, containing 50% water) under hydrogen atmosphere
(balloon pressure) at room temperature for 16 h. The catalyst was removed
by filtration, and the filtrate was concentrated. The residue was purified by
silica gel column chromatography (hexane-EtOAc = 1/15-1/8) to give
2c (2.25 g, 21% in 2 steps) as a colorless oil. 1H NMR (CDCl3) δ 2.27 (s,
3H), 2.55 (t, J = 8.4 Hz, 2H), 2.87 (t, J = 8.4 Hz, 2H), 3.68 (s, 3H), 4.69 (s,
1H), 6.59-6.65 (2H, m), 6.99 (d, J = 8.1 Hz, 1H).
2-Methoxy-4-(methoxymethoxy)benzaldehyde (28e). To
a solution of 4-hydroxy-2-methoxybenzaldehyde (28d) (5.00 g, 32.9
mmol) in DMF (100 mL) was added NaH (60% in mineral oil, 1.45 g,
36.2 mmol) at room temperature. After stirring for 0.5 h, chloromethyl
methyl ether (3.97 g, 49.4 mmol) was added and the mixture was stirred
at room temperature for 16 h. The reaction mixture was poured into
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ARTICLE
water. The organic materials were extracted with EtOAc. The extract was
washed with brine, dried over MgSO4, and concentrated. The residue
was purified by silica gel column chromatography (hexane-EtOAc =
6/1) to give 28e (5.58 g, 87%) as a colorless powder. 1H NMR (CDCl3)
δ 3.50 (s, 3H), 3.91 (s, 3H), 5.24 (s, 2H), 6.61 (d, J = 2.2 Hz, 1H),
6.64-6.70 (m, 1H), 7.80 (d, J = 8.6 Hz, 1H), 10.31 (s, 1H).
chow CE-2 (CLEA, Japan) and tap water ad libitum and were housed in
cages in a room with controlled temperature (23 ( 1 °C), humidity (55
( 5%), and lighting (lights on from 07:30 to 19:30). The care and use of
the animals and the experimental protocols used in this research were
approved by the Experimental Animal Care and Use Committee of
Takeda Pharmaceutical Company (Osaka, Japan).
Oral Glucose Tolerance Test in Diabetic Rats. Twenty-week-
old male N-STZ-1.5 rats were fasted overnight, and blood samples were
collected from tail vein, followed by measuring plasma glucose levels
(referred to as time point pre) by the automatic analyzer Hitachi 7080
(Hitachi, Japan). Rats were divided into four groups based on body weight
and plasma glucose levels (n=6). Compound4p (1, 3, or 5 mg/kg) or 0.5%
methylcellulose was orally administered 30 min before oral glucose load (1
g/kg). Blood samples were collected from the tail vein 0 (just before glucose
load), 10, 30, 60, and 120 min after the glucose load. Plasma glucose levels
were measured as described above, and plasma insulin levels were measured
with insulin RIA kit (SHIONOGI & Co., Ltd., Japan) according to the
manufacturer’s instruction. Statistical differences versus control were ana-
lyzed with one-tailed Williams’ test or Shirley-Williams’ test.
Effects on Normal Fasting Plasma Glucose and Insulin
Levels in SD Rats. Eight-week-old male SD rats were fasted overnight,
and blood was collected from tail vein, followed by measuring plasma
glucose levels (referred to as time point 0) by the automatic analyzer
Hitachi 7080 (Hitachi, Japan). Rats were divided into three groups based
on body weight and plasma glucose levels (n = 6). Compound4p (30 mg/
kg), nateglinide (50 mg/kg), or vehicle (0.5% methylcellulose) was orally
administered, and blood was collected from tail vein at time points 30, 60,
and 120 min after the administrations. Plasma glucose and insulin levels
were measured as described above. Statistical differences versus control
were analyzed with Dunnett’s test or Steel test.
Rotamer Analyses. Conformational analyses of biphenyl, 2,6-
dimethylbiphenyl, biphenyl, and phenoxyphenyl were performed using
MNDO-PM3 (MOPAC version 7.01) method in MOE. Each focused
bond was fixed and the other part of each compound was fully optimized.
Energy curves around each focused bond were obtained by plotting
calculated total energies against an axis of a dihedral angle.
Ethyl 3-(4-Hydroxy-2-methoxyphenyl)propanoate (2h). To
an ice-cooled solution of ethyl diethylphosphonoacetate (8.28 g, 36.9
mmol) in THF (50 mL) was added NaH (60% in mineral oil, 1.37 g, 34.1
mmol), and the mixture was stirred for 30 min. A solution of 2-methoxy-
4-(methoxymethoxy)benzaldehyde 28e (5.58 g, 28.4 mmol) in THF (30
mL) was added dropwise. The mixture was stirred at room temperature for
45 min. The reaction mixture was poured into water and extracted with
EtOAc. The extract was washed with brine, dried over MgSO4, and
concentrated. The residue was purified by silica gel column chromatogra-
phy (hexane-EtOAc = 7/1) to give a colorless oil. A mixture of this oil,
THF (100 mL), EtOH (100 mL), and 10% palladium on carbon (742 mg,
containing 50% water) was stirred overnight under a hydrogen atmosphere
at room temperature. The catalyst was filtered off, and the filtrate was
concentrated to give an oil. To a solution of this oil in EtOH (100 mL) was
added 40 mL of 3 M HCl. The resulting mixture was refluxed after 30 min
and then poured into cold waterand extracted with EtOAc. The extract was
washed with brine, dried over MgSO4, and concentrated. The residue was
purified by silica gel column chromatography (hexane-EtOAc = 4/1) to
give 2h (2.76 g, 44%) as a colorless oil. 1H NMR (CDCl3) δ 1.24 (t, J = 7.2
Hz, 3H), 2.55 (t, J = 8.0 Hz, 2H), 2.85 (t, J = 8.0 Hz, 2H), 3.78 (s, 3H), 4.12
(q, J = 7.2 Hz, 2H), 4.80 (s, 1H), 6.31 (dd, J = 8.1 Hz, 2.4 Hz, 1H), 6.38 (d,
J = 2.4 Hz, 1H), 6.97 (d, J = 8.1 Hz, 1H).
Ethyl 3-(4-Hydroxyphenyl)butanoate (2i). Compound 2i
was prepared from 29 by a similar to that described for 2d (step 1 and
step 2) in 89% yield (2 steps) as a colorless oil. 1H NMR (CDCl3) δ 1.18
(t, J = 7.2 Hz, 3H), 1.61 (d, J = 3.4 Hz, 2H), 2.46-2.60 (m, 2H),
3.16-3.28 (m, 1H), 4.07 (q, J = 7.2 Hz, 2H), 4.94 (br s, 1H), 6.73-6.77
(m, 2H), 7.06-7.10 (m, 2H).
Ca Influx Activity of CHO Cells Expressing Human GPR40
(FLIPR Assay). CHO dhfr cells stably expressing human GPR40
(accession no. NM_005303) were plated and incubated overnight in 5%
CO2 at 37 °C. Then, cells were incubated in loading buffer (recording
medium containing 2.5 μg/mL fluorescent calcium indicator Fluo 4-AM
(Molecular Devices), 2.5 mmol/L probenecid (Dojindo), and 0.1% fatty
acid-free BSA (Sigma)) for 60 min at 37 °C. Various concentrations of test
compounds or γ-linolenic acid (Sigma) were added into the cells, and
increase of the intracellular Ca2þ concentration after addition was mon-
itored by FLIPR Tetra system (Molecular Devices) for 90 s. The
agonistic activities of test compounds and γ-linolenic acid on human
GPR40 were expressed as [(A - B)/(C - B)] ꢀ 100 (increase of the
intracellular Ca2þ concentration (A) in test compounds-treated cells,
(B) in vehicle-treated cells, and (C) in 10 μM γ-linolenic acid-treated
cells). EC50 value of each compound was obtained with Prism 5
software (GraphPad).
’ AUTHOR INFORMATION
Corresponding Author
*Phone: þ81-6-6300-6838. Fax: þ81-6-6300-6306. E-mail:
Sasaki_Shinobu@takeda.co.jp.
’ ACKNOWLEDGMENT
The authors thank Toshio Tanaka and Yoji Hayase for helpful
discussions, and Tomoko Asakawa, Takeshi Miyazaki, Tomoko
Kaisho, and Shiro Takekawa for in vitro and in vivo experiments.
’ ABBREVIATIONS USED
Pharmacokinetic Analyses in Rat Cassette Dosing. Test
compounds were administered as a cassette dosing to nonfasted rats.
After oral and intravenous administration, blood samples were collected.
The blood samples were centrifuged to obtain the plasma fraction. The
plasma samples were deproteinized with acetonitrile containing an
internal standard. After centrifugation, the supernatant was diluted
and centrifuged again. The compound concentrations in the supernatant
were measured by LC/MS/MS.
GPCR, G-protein coupled receptor; GSIS, glucose-stimulated
insulin secretion; FFAs, free fatty acids; OGTT, oral glucose
tolerance test; DHA, docosahexaenoic acid; FLIPR, fluorometric
imaging plate reader; PK, pharmacokinetic; BSA, bovine serum
albumin; CHO, Chinese hamster ovary
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subcutaneous injection of 120 mg/kg of streptozotocin (STZ) into
male Wistar Kyoto rats 1-2 days after birth. All rats were fed regular
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