Optimization of (2,3-dihydro-1-benzofuran-3-yl)acetic acids: Discovery of a non-free fatty acid-like, highly bioavailable G protein-coupled receptor 40/free fatty acid receptor 1 agonist as a glucose-dependent insulinotropic agent

Article abstract of DOI:10.1021/jm300170m

G protein-coupled receptor 40 (GPR40)/free fatty acid receptor 1 (FFA1) is a free fatty acid (FFA) receptor that mediates FFA-amplified glucose-stimulated insulin secretion in pancreatic β-cells. We previously identified (2,3-dihydro-1-benzofuran-3-yl)acetic acid derivative 2 as a candidate, but it had relatively high lipophilicity. Adding a polar functional group on 2 yielded several compounds with lower lipophilicity and little effect on caspase-3/7 activity at 30 μM (a marker of toxicity in human HepG2 hepatocytes). Three optimized compounds showed promising pharmacokinetic profiles with good in vivo effects. Of these, compound 16 had the lowest lipophilicity. Metabolic analysis of 16 showed a long-acting PK profile due to high resistance to β-oxidation. Oral administration of 16 significantly reduced plasma glucose excursion and increased insulin secretion during an OGTT in type 2 diabetic rats. Compound 16 (TAK-875) is being evaluated in human clinical trials for the treatment of type 2 diabetes.

Full text of DOI:10.1021/jm300170m

Article
pubs.acs.org/jmc
Optimization of (2,3-Dihydro-1-benzofuran-3-yl)acetic Acids:
Discovery of a Non-Free Fatty Acid-Like, Highly Bioavailable G
Protein-Coupled Receptor 40/Free Fatty Acid Receptor 1 Agonist as a
Glucose-Dependent Insulinotropic Agent
Nobuyuki Negoro,† Shinobu Sasaki,† Satoshi Mikami,† Masahiro Ito,† Yoshiyuki Tsujihata,† Ryo Ito,†
Masami Suzuki,† Koji Takeuchi,† Nobuhiro Suzuki,‡ Junichi Miyazaki,‡ Takashi Santou,†
Tomoyuki Odani,† Naoyuki Kanzaki,† Miyuki Funami,‡ Akio Morohashi,† Masami Nonaka,†
Shinichiro Matsunaga,† Tsuneo Yasuma,*,‡ and Yu Momose†
†Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1 Muraoka-Higashi 2-chome, Fujisawa, Kanagawa
251-8555, Japan
‡Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 17-85 Jusohonmachi 2-chome, Yodogawa-ku, Osaka
532-8686, Japan
S
* Supporting Information
ABSTRACT: G protein-coupled receptor 40 (GPR40)/free
fatty acid receptor 1 (FFA1) is a free fatty acid (FFA) receptor
that mediates FFA-amplified glucose-stimulated insulin
secretion in pancreatic β-cells. We previously identified (2,3-
dihydro-1-benzofuran-3-yl)acetic acid derivative 2 as a
candidate, but it had relatively high lipophilicity. Adding a
polar functional group on 2 yielded several compounds with
lower lipophilicity and little effect on caspase-3/7 activity at 30
μM (a marker of toxicity in human HepG2 hepatocytes).
Three optimized compounds showed promising pharmacokinetic profiles with good in vivo effects. Of these, compound 16 had
the lowest lipophilicity. Metabolic analysis of 16 showed a long-acting PK profile due to high resistance to β-oxidation. Oral
administration of 16 significantly reduced plasma glucose excursion and increased insulin secretion during an OGTT in type 2
diabetic rats. Compound 16 (TAK-875) is being evaluated in human clinical trials for the treatment of type 2 diabetes.
drug that has a low risk of hypoglycemia and potent antidiabetic
effects would be advantageous.
INTRODUCTION
■
According to the International Diabetes Federation, approx-
imately 285 million people worldwide suffer from diabetes as of
2010. Its incidence is increasing rapidly, and the number of
patients will reach 438 million by the year 2030.1 Type 2
diabetes accounts for almost 90% of total diabetes and is
characterized by reduced insulin sensitivity combined with
impaired insulin secretion. Persistent or uncontrolled hyper-
glycemia may cause a significantly increased risk of macro-
vascular and microvascular complications, including athero-
sclerosis, coronary heart disease, nephropathy, neuropathy, and
retinopathy. Therefore, therapeutic control of glucose homeo-
stasis is crucially important in the clinical management and
treatment of diabetes. Drugs that enhance insulin secretion
such as sulfonylureas and meglitinides are commonly used for
the treatment of type 2 diabetes. However, these drugs enhance
insulin secretion by directly closing KATP channel independent
of blood glucose levels, leading to excess blood glucose-
lowering, called hypoglycemia.2 Furthermore, chronic exposure
of them may induce β-cell apoptosis and reduction of the
therapeutic effect, called secondary failure.3,4 Hence, a novel
G protein-coupled receptor 40 (GPR40)/free fatty acid
receptor 1 (FFA1) was identified as an orphan G protein-
coupled receptor (GPCR),5 and then deorphanized as a
receptor for medium- to long-chain free fatty acids
(FFAs).6−8 Several reports have shown that GPR40/FFA1 is
predominantly expressed in pancreatic β-cells, and the receptor
mediates FFA-amplified insulin secretion.6 The insulinotropic
effects via GPR40/FFA1 are dependent on glucose concen-
tration,6,9,10 indicating that a selective agonist has a low risk of
hypoglycemia and some supporting evidence has already been
reported.11,12 GPR40/FFA1 mainly couples with the Gαq
protein, which activates phospholipase C, resulting in the
production of inositol triphosphate and mobilization of
intracellular Ca2+ from the endoplasmic reticulum.9,10 The
activation of GPR40/FFA1 also stimulates Ca2+ influx through
voltage-gated Ca2+ channels,13 and the resulting increase in
Received: February 7, 2012
Published: April 10, 2012
© 2012 American Chemical Society
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Figure 1. Design of (2,3-dihydro-1-benzofuran-3-yl)acetic acids.
a
Scheme 1
a
Reagents and conditions: (a) CHIRALPAK AD HPLC separation, hexane/EtOH (88/12, v/v).
a
Scheme 2
a
Reagents and conditions: (a) 4a−c (condition A) or R1OH (condition D), ADDP, P(n-Bu)3, toluene, rt, 60−95%; (b) 2 M NaOH aq, MeOH,
THF, 50 °C, 32−94% except for 18, 20, 21, 16−42% (from 17a in 2 steps); 25, 15% (from 43c in 3 steps); 19 as a HCl-salt; (c) m-CPBA, AcOEt, 0
°C, 79%; (d) oxone, MeOH, H2O, 0 °C to rt, 73%; (e) TBAF, THF, rt, 88−94%; (f) 30b, K3PO4, DMF, 90 °C, 88%; (g) piperidine-4-ol, DEAD,
PPh3, toluene, rt, 63%.
intracellular Ca2+ concentrations enhances glucose-stimulated
insulin secretion (GSIS).
We independently identified several types of synthetic
agonists and recently reported phenylpropanoic acid derivatives
as represented by compound 1 with potent GPR40/FFA1
agonist activity and glucose-lowering effect during an oral
glucose tolerance test (OGTT) in diabetic animal models.18
However, this compound showed low sustainability in plasma
due to its proposed vulnerability to β-oxidation at the
propanoic acid moiety. Therefore, we intended to improve
metabolic stability against β-oxidation by constructing a fused
structure, leading to the identification of (2,3-dihydro-1-
A variety of synthetic GPR40/FFA1 agonists have been
reported.14 Importantly, some of them were designed based on
FFAs as the endogenous ligands, so they inherit the feature of
FFAs with the lipotoxic potential.15,16 We postulated that
reducing lipophilicity of compounds would be important to
eliminate the undesirable off-target activities derived from
highly lipophilic nature of FFAs.17
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a
Scheme 3
a
Reagents and conditions: (a) p-TsCl, pyridine, 0 °C, 70%; (b) p-TsCl, N,N,N′,N′-tetramethyl-1,6-hexanediamine, Et3N, toluene, 0 °C, 94%; (c)
oxone, MeOH, H2O, 0 °C to rt, 96%; (d) NaBH4, MeOH, THF, 0 °C to rt, 92−97%; (e) R1Cl (for 5a and 5e), R1OTs (for 5b and 5f), or 1-oxa-6-
thiaspiro[2.5]octane (for 5d), K2CO3, (KI), DMF, 70−100 °C, 47−98%; (f) TBSCl, imidazole, DMF, rt, 77%; (g) tetrahydro-4H-thiopyran-4-ol,
DEAD, PPh3, THF, rt, 86%; (h) (i) 1.6 M n-BuLi in hexanes, THF, −78 °C, (ii) B(i-PrO)3, −78 °C to rt, (iii) 2 M HCl aq, rt, 71% (from 34 in 2
steps); (i) methyl 3-bromobenzoate, Pd(PPh3)4, 2 M Cs2CO3 aq, DME, reflux, 86%; (j) m-CPBA, AcOEt, 0 °C, 85%; (k) LiAlH4, THF, 10 °C to rt,
93%.
benzofuran-3-yl)acetic acid derivative 2 with potent agonist
activity for human GPR40/FFA1 and superior pharmacokinetic
(PK) profile in rats.19 Furthermore, 2 exhibited in vivo efficacy
at a dose of 10 mg/kg in rats despite the inferior potency
against rat GPR40/FFA1. On the other hand, the lipophilicity
of 2 was still high (see Log D value: 3.83),17 so we decided to
explore the introduction of a polar substituent with the goal of
reducing lipophilicity and assessed their toxic profiles in human
hepatocyte HepG2 cells in which FFAs show their toxicity.20
From our previous work on other chemical series, it was
revealed that incorporation of the various functionalities into
the 4′-position of the biphenyl ring was tolerated.21 With these
observations in mind, we devised the following strategies to
identify new chemical entities with potent and durable glucose-
lowering effect and favorable safety profiles suitable for clinical
development (Figure 1). First, in an attempt to decrease the
lipophilicity, we conducted an extensive evaluation of the 4′-
substituent on the biphenyl group. Second, to determine the
importance of the stereochemistry, we separated the
enantiomers. Finally, we expected to improve in vitro activities
and PK profiles by appending substituents on the biphenyl
core. Herein, we describe the identification and development of
(2,3-dihydro-1-benzofuran-3-yl)acetic acid derivatives leading
to the discovery of TAK-875, the hemihydrate of [(3S)-6-
({2′,6′-dimethyl-4′-[3-(methylsulfonyl)propoxy]biphenyl-3-yl}-
methoxy)-2,3-dihydro-1-benzofuran-3-yl]acetic acid (16), as a
promising anti-diabetic drug candidate.22
Chemistry. The racemic dihydrobenzofuran intermediate
4a was synthesized by the reported method in our previous
publications.19,22 Preparative chiral HPLC separation of 4a
using a CHIRALPAK AD column afforded enantiomers 4b and
4c (Scheme 1). The absolute stereochemistry was determined
by X-ray crystallography of eutomer 16 derived from 4b as
described hereinafter.
The target GPR40/FFA1 agonists were prepared as outlined
in Scheme 2 (for R groups: see Tables 1−3). Phenols 4a−c
were condensed with biphenylylmethanols 5a−f and 5i−n
(vide infra) by Mitsunobu reaction or with the corresponding
mesylates by alkylation, followed by ester hydrolysis of the
coupling products to afford the carboxylic acids 6−16, 22−26,
and 28, including oxidation of sulfide to sulfone for 14 and 15.
Conversion of 4′-alkoxy substituents on the biphenyl scaffold
was also performed in an alternative method. Alkylation of the
key intermediates 17a and 17b, derived from 5g and 5h, with
alcohols or tosylates followed by hydrolysis of the ester,
furnished the desired compounds 18−21 and 27.
Biphenylylmethanols 5a−g were synthesized as depicted in
Scheme 3. Tosylate 30a was prepared by a conventional
method. Meanwhile, tosylation of (3-methylthio)-1-propanol
(29b) was performed by Tanabe’s procedure23 with a catalytic
amount of diamine to avoid the self-cyclization reaction,
followed by oxidation to give the tosylate 30b. Intermediate
3119 was converted to alcohols 5a, 5b, and 5d−g in two
different approaches. Reduction of 31 with NaBH4 to alcohol
32, followed by alkylation, afforded 5a and 5e (Route A).
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a
Scheme 4
a
Reagents and conditions: (a) AlCl3, 115 °C, 78%; (b) n-Bu4NBr3, MeOH, rt, 72%; (c) dichloromethyl methyl ether, TiCl4, CH2Cl2, 0 °C, 40%; (d)
H2, Pd/C, MeOH, toluene, rt, 97%; (e) Br2, AcOH, rt, 83%; (f) 1-fluoropyridinium triflate, 1,2-dichloroethane, reflux, 36%; (g) 3-
formylphenylboronic acid, PdCl2(dppf)·CH2Cl2, K3PO4, THF, 80 °C, 49−79%; (h) NCS, DMF, rt, 60−65%; (i) NaBH4, MeOH, THF, 0 °C, 65−
98%; (j) TBSCl, imidazole, DMF, rt, 88%; (k) R1OTs, K2CO3, (KI), DMF, 90−95 °C, 53−95%.
Figure 2. Stereoscopic molecular view of compound 16.
Alternatively, 31 was alkylated with 30a, 1-oxa-6-thiaspiro[2.5]-
octane, or 30b, or silylated with tert-butyldimethylchlorosilane,
followed by reduction to provide alcohols 5b, 5d, 5f, and 5g.
Synthesis of alcohol 5c with 1,1-dioxidotetrahydrothiopyranyl
group was accomplished using another way. Phenol 34 was
alkylated under Mitsunobu condition to give tetrahydrothio-
pyranyl ether, which was converted to boronic acid 35. Suzuki
cross-coupling of 35 with methyl 3-bromobenzoate, followed
by oxidation of the thioether, gave sulfone, which was treated
with lithium aluminum hydride to give the desired alcohol 5c.
The 3′- and/or 5′-substituted biphenylylmethanols 5h−n
were prepared following the routes illustrated in Scheme 4.
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Table 1. In Vitro Activities of (2,3-Dihydro-1-benzofuran-3-yl)acetic Acids
a
All values are average of n = 3 in the presence of 0.1% BSA. EC50 values and 95% confidence intervals of each compound were obtained with Prism
b
5 software (GraphPad). Efficacies of compounds at 10 μM were 103−113% of γ-linolenic acid at 10 μM. All values are average of n = 2 in the
c
d
presence of 0.2% BSA. Percent of activation was compared to maximal activity of staurosporine as a reference compound. The Log D values were
determined at pH 7.4 according to the reported method.28
According to the procedure of Baddeley,24 4-ethylphenol (36)
was converted to 3,5-diethylphenol (37), which underwent
selective bromination at the para-position with n-Bu4NBr3 to
yield 38a. Formylation of 2,3,5-trimethylphenol (39) with
TiCl4 and dichloromethyl methyl ether25 afforded a mixture of
desired o-formylated product 40 and p-formylated product as a
regioisomer (ca. 2:1). Subsequent catalytic hydrogenation
proceeded smoothly to give phenol 41, which was then treated
with bromine to afford bromide 38b. Monofluorination of 34
with 1-fluoropyridinium triflate gave 38c in moderate yield. The
obtained bromophenols 38a−c were subjected to Suzuki
coupling condition to afford biphenyls 42a−c. Mono- and
dichlorinated biphenyls 42d and 42e were synthesized by
treatment of 31 with NCS. Biphenyls 42a−e were converted to
the corresponding alcohols 5h−n by the same reaction
sequence as described in Scheme 3.
Determination of the absolute stereochemistry in compound
16 was performed by X-ray diffraction analysis.26 As shown in
Figure 2, the absolute configuration of 16 was determined to be
(S). Because 16 was derived from the key intermediate 4b, the
stereochemistry of 4b was assigned as (S) and the stereo-
chemistry of enantiomer 4c assigned as (R) as described in
Scheme 1.
reader (FLIPR) system in the presence of 0.1% bovine serum
albumin (BSA), and binding affinities for human and rat
receptors were measured using the cell membranes in the
presence of 0.2% BSA.22 Compounds were evaluated for their
apoptotic potential by measuring caspase-3/7 activity, which is
a marker of apoptosis.27 We utilized HepG2 cells in which
FFAs, the endogenous ligands of GPR40/FFA1, have been
reported to enhance the enzymatic activity.20 The Log D values
were measured at pH 7.4 with relative retention time over
standard compounds of HPLC analysis.28
First, we briefly verified the tolerability of the substituent at
the 4′-position of the biphenyl scaffold by use of other two
functional groups (Table 1). (3-Methyloxetan-3-yl)methoxy
derivative 8, the cyclized analogue of 2-ethoxyethoxy derivative
2, exhibited potent agonist activity; moreover, more bulky and
polar (1,1-dioxidotetrahydro-2H-thiopyran-4-yl)oxy derivative
11 still retained agonist activity and human/rat binding
affinities. These results imply that the binding pocket
encompassing the tail substituent can tolerate a wide range of
groups including polar functionality. In parallel, we focused on
the effect of stereochemistry at the 3-position of the
dihydrobenzofuran ring. GPCRs are cell surface proteins
known to dynamically change their conformations by ligand
activation. As for GPR40/FFA1, a variety of long-chain fatty
acids have been reported as natural ligands, suggesting that the
ligand recognition site of this protein is not strict for binding to
the lipophilic alkyl chain part. However, the binding pocket for
RESULTS AND DISCUSSION
■
Agonist activities of the prepared compounds were measured
by monitoring Ca2+ influx using the fluorometric imaging plate
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Table 2. In Vitro Activities of (2,3-Dihydro-1-benzofuran-3-yl)acetic Acids
a
All values are average of n = 3 in the presence of 0.1% BSA. EC50 values and 95% confidence intervals of each compound were obtained with Prism
b
5 software (GraphPad). Efficacies of compounds at 10 μM were 107−113% of γ-linolenic acid at 10 μM. All values are average of n = 2 or 3 in the
c
d
presence of 0.2% BSA. The activity was measured with anhydrous 16. Percent of activation was compared to maximal activity of staurosporine as a
reference compound. The Log D values were determined at pH 7.4 according to the reported method.28 The activities were measured as
e
f
hydrochloride.
the carboxylic acid moiety of the ligand is presumably restricted
because this part is essential for signal transduction. Actually,
previous SAR studies revealed that the electrostatic interaction
of the carboxylic group and π−π interaction of the olefin group
or the phenyl group are important for extension of GPR40/
FFA1 activity.6,18 Accordingly, we speculated that the absolute
configuration of the chiral center neighboring the carboxylic
acid in dihydrobenzofuran derivatives may be critical for both
binding and signaling. It turned out that the (S)-enantiomer 6
showed more potent agonist activity to the human GPR40/
FFA1 than the corresponding (R)-enantiomer 7. Similarly, 6
had higher binding affinities against human and rat GPR40/
FFA1 than 7. Preference for (S)-stereochemistry over (R) was
also observed in other 4′-alkoxybiphenyl series (9 vs 10 and 12
vs 13). These results indicate that the direction of the acetic
acid moiety attached to the dihydrobenzofuran ring makes a
significant impact on GPR40/FFA1 activity, namely, the
relative position between the aromatic ring of the dihydro-
benzofuran and the carboxylic acid moiety would affect the
interaction with the GPR40/FFA1 receptor as expected.
Having identified the preferable stereochemistry on the (2,3-
dihydro-1-benzofuran-3-yl)acetic acid moiety, we employed the
(S)-isomer for subsequent investigations.
As we expected, some polar functionalities such as ether or
sulfone were tolerated at the 4′-position of the terminal
biphenyl ring on the (2,3-dihydro-1-benzofuran-3-yl)acetic acid
series. We then pursued replacement of the substituent at this
position with the goal of optimizing lipophilicity of compounds
(Table 2). Cyclic or linear sulfone analogues 14−16 with low
lipophilicity (see Log D values: 2.43−2.73) exhibited potent
agonist activity and binding affinities. Lactam analogue 18 was
also tolerated in terms of agonist activity and binding affinities.
Replacement of 1,1-dioxidotetrahydro-2H-thiopyran-4-yl group
on 12 with 1-methylpiperidin-4-yl group (19) increased
binding affinities in human and rats. This effect might be
derived from the enhanced interaction between the positive
charge at the protonated piperidine ring and a polar
functionality such as Ser8 (TM1).22 Heteroaryl analogues
such as thiazole 20 and imidazopyridine 21 showed potent
agonist activity and affinities. However, heteroaryl analogues 20
and 21 showed caspase-3/7 activities, probably owing to their
high lipophilicities (see Log D values: 4.27 and 3.80,
respectively).
Various polar substituents such as sulfone, amide, amine, and
heteroaromatics at the 4′-position of the biphenyl ring were
tolerated for agonist activity and binding affinities regardless of
their polarities, suggesting that human and rat GPR40/FFA1
receptors would have a large cavity in the ligand binding pocket
located around the 4′-position of the biphenyl moiety. Thus,
this position was thought to be suitable for modulating
absorption, distribution, metabolism, excretion, and toxicology
(ADME-Tox) properties. As can be seen, analogues 14−16, 18,
Regarding caspase-3/7 activity, (1,1-dioxidotetrahydro-2H-
thiopyran-4-yl)oxy derivatives (11−13) with low Log D value
(ca. 2.8) tend to be lower compared to the ether series (2 and
7−10). Among these compounds, 12 was selected for further
evaluation.
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Table 3. In Vitro Activities of (2,3-Dihydro-1-benzofuran-3-yl)acetic Acids
a
All values are average of n = 3 in the presence of 0.1% BSA. EC50 values and 95% confidence intervals of each compound were obtained with Prism
b
5 software (GraphPad). Efficacies of compounds at 10 μM were 104−114% of γ-linolenic acid at 10 μM. All values are average of n = 2 in the
c
d
presence of 0.2% BSA. Percent of activation was compared to maximal activity of staurosporine as a reference compound. The Log D values were
determined at pH 7.4 according to the reported method.28
and 19 with polar groups have good potency and minimum
caspase activity, so they were selected for further investigation.
Last, we turned our attention to further increasing potency
and modulating ADME-Tox profiles through introduction of
hydrophobic substituent(s) such as a methyl group or a
halogeno group on the biphenyl ring (Table 3) because most of
the ligand binding pocket was thought to consist of
hydrophobic amino acids.22 Introduction of one or more
small hydrophobic substituents (22−28) resulted in almost the
same agonist activities and binding affinities or slight improve-
ment in both. In this series, 2′,6′-diethyl analogue 22 exhibited
marginally increased affinities in human and rat binding assays
compared to the parent compound 16.
In terms of caspase-3/7 activity, 1,1-dioxidotetrahydrothio-
pyran derivatives as with 2′,3′,5′,6′-tetramethyl analogue 23 and
monofluoro analogue 25 tend to have a weak potential of
activating caspase-3/7. Among 3-(methylsulfonyl)propoxy
derivatives, 2′,3′,5′,6′-tetramethyl analogue 24 and monofluoro
analogue 26 did not show any activities, whereas introduction
of chloro group(s) (27 and 28) increased caspase-3/7 activity
as the lipophilicity is increased (Log D value: 3.07 and 3.53,
respectively). Thus, all compounds possessed comparable
GPR40/FFA1 activity, but some of them slightly induced
caspase-3/7 activities with increasing lipophilicity. The thresh-
old of Log D value for apoptosis induction would be estimated
as 2.9−3.2 according to these results. Consequently, we
selected two compounds (24 and 26) in Table 3 for rat PK
study.
efficacy in mind, plasma concentrations at 1 and 4 h (C1h and
C4h) after oral administration are shown in Table 4 in addition
Table 4. Pharmacokinetic Profiles for (2,3-Dihydro-1-
a
benzofuran-3-yl)acetic Acids
Cmax
(ng/mL)
Tmax
(h)
AUC0−8h
(ng·h/mL)
C1h
(ng/mL)
C4h
(ng/mL)
compd
12
14
15
16
18
19
24
26
2667.7
1082.4
1941.3
1883.5
626.4
0.7
13007.6
5210.4
9621.7
11840.4
3474.9
4372.3
3963.9
8036.1
2614.2
1082.4
1737.7
1855.6
566.8
1621.0
638.8
1240.0
1601.7
477.3
574.5
340.5
966.6
1.0
1.17
2.00
1.83
1.58
0.67
0.50
743.6
578.0
1275.2
2033.3
1152.2
1541.1
a
Rat cassette dosing at 1 mg/kg, po (fasted). Test compounds were
suspended in 0.5% methylcellulose aqueous solution. All values are
averages of three rats.
to the PK parameters (Cmax, Tmax, AUC0−8h). The 1,1-
dioxidotetrahydrothiopyran derivative 12 presented a highly
desirable PK profile, namely, rapid absorption (Tmax = 0.7 h),
high maximum concentration (Cmax = 2667.7 ng/mL), and
good plasma duration (C4h = 1621.0 ng/mL), leading to a high
plasma exposure (AUC0−8h = 13007.6 ng·h/mL). The more
polar analogue 14 with tertiary alcohol functionality showed
lower Cmax and AUC0−8h compared to 12. One of the reasons
for this decrease was probably due to its decline in membrane
permeability (data not shown). The ring-opened analogue 15,
with the flexible side chain, exhibited a good PK profile, but it
showed somewhat lower plasma concentration relative to 12. In
Potent compounds without caspase-3/7 activity were further
evaluated for oral PK profiles through cassette dosing
experiments in fasted rats. With in vivo evaluation of drug
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the study on the analogous compound, we have confirmed that
the 2-(ethylsulfonyl)ethoxy group was degraded by β-
elimination under basic conditions. It remains possible that
similar decomposition could occur on 15 in vivo. In the
meantime, the 3-(methylsulfonyl)propoxy analogue 16, whose
sulfonyl group was shifted to terminal side for one carbon,
eliminates the possible concern for β-elimination and possesses
an excellent PK profile as expected. Although the AUC0−8h of
lactam 18 and cyclic amine 19 were lower compared to the
sulfone derivatives, their C1h and C4h were found to be at the
same level near Cmax, indicating favorable duration in plasma.
Compared to the corresponding unsubstituted analogue,
2′,3′,5′,6′-tetramethyl derivative 24 exhibited lowered
AUC0−8h, likely due to relatively high clearance with increased
lipophilicity. The 3′-Fluoro analogue 26 showed good
absorption (high Cmax and rapid Tmax) but relatively low
AUC0−8h and C4h, indicating that its clearance from plasma
might be faster than that of the parent compound 16.
As a whole, sulfonyl derivatives possessed favorable PK
profiles, and introduction of lipophilic substituents at the 3′-
and/or 5′-position on the biphenyl scaffold tended to decrease
the plasma concentration. On the basis of the above results,
compounds (12, 14−16, 19, and 26) possessing favorable PK
profile, notably long duration in plasma, were selected for in
vivo pharmacological study.
In vivo efficacies of the selected compounds were evaluated
by OGTT in female Wistar fatty rats, where compounds were
orally administered 1 h or 4 h prior to glucose challenge (these
tests were referred to 1H−OGTT or 4H−OGTT, respec-
tively). The 1H−OGTT assessed intrinsic efficacy of
compounds at 1 mg/kg dose, while the 4H−OGTT assessed
their durability at 3 mg/kg dose. Among the tested compounds,
12, 16, and 26, with especially desirable PK profiles, produced
significantly improved glucose intolerance in both 1H−OGTT
and 4H−OGTT (Table 5). Tertiary alcohol derivative 14 with
prominent PK profiles. Among three compounds (12, 16, and
26) with pronounced in vivo efficacies, 16 possessed the lowest
log D value (2.58). On the basis of the above information (i.e.,
in vitro and in vivo potencies, ADME-Tox profiles, and PK
parameters), including drug-likeness (Log D), 16 was selected
as a candidate for further investigation.
As reported in the previous communication,22 16 showed
excellent PK profiles in rats and dogs. In an attempt to
rationalize its PK profile, 16 was further characterized by
performing additional PK studies using [14C]-labeled 16 in rats
and dogs. The observed metabolic pathways of 16 are
summarized in Figure 3. The major component in plasma
was unchanged 16 in rats and dogs, and a small amount of
biphenylylcarboxylic acid 45 was detected in rats. In addition,
taurine conjugate 46 and acyl glucuronide 47 were detected in
rat bile, and these three metabolites (45−47) were also
detected in feces of both species. Thus, the β-oxidation
metabolite of 16 was not observed in rats and dogs. These
results demonstrate that our design to introduce a fused
structure into the phenylpropanoic acid indeed decreased β-
oxidation, leading to an improved PK profile. The favorable
metabolic process of 16 would contribute to a desirable PK
profile suitable for once-daily dosing in humans.29,30 The more
detailed metabolic analysis of 16 will be presented elsewhere.
To assess the pharmacological effects of 16 in detail, we
performed an OGTT in male Goto-Kakizaki (GK) rats, which
are a spontaneous type 2 diabetic model with impaired insulin
secretion in response to glucose. While insulin secretion after
glucose challenge rapidly occurred in Wistar Kyoto rats as
healthy control, the early phase insulin secretion was impaired
in GK rats (Figure 4C) and the impaired insulin secretion was
reflected in the increased glucose excursion (Figure 4A). Oral
administration of 16 (1−10 mg/kg) 1 h prior to oral glucose
challenge dose-dependently augmented insulin secretion
(Figure 4C) and suppressed plasma glucose excursion (Figure
4A) in GK rats. The areas under the curves of plasma glucose
(AUC0−120min) (Figure 4B) and plasma insulin (AUC0−120min
)
Table 5. Effects of Selected Compounds during an OGTT in
Female Wistar Fatty Rats
a
(Figure 4D) showed that the minimum effective dose for
glucose-lowering and for increasing insulin secretion were 3 and
10 mg/kg, respectively. Moreover, the 16-treated group had
already augmented insulin secretion at glucose charge (0 min)
as the GK rats have high level of fasting plasma glucose. We
have already reported that 16, even at an oral dose of 30 mg/kg,
had no impact on fasting plasma glucose levels in Sprague−
Dawley (SD) rats with normal glucose homeostasis and did not
significantly promote insulin secretion.12 Our present and
previous results indicate that the insulinotropic action of 16 is
strictly dependent on blood glucose levels. From the unique
features of 16, we believe that the compound may pose a low
risk of hypoglycemia, while showing potent glucose-lowering
effects in diabetic pathology caused by the dysfunction of
pancreatic β-cells.
b
ED (mg/kg)
compd
1H−OGTT
4H−OGTT
12
14
15
16
19
26
1
3
3
3
3
c
NE
d
NT
1
d
c
NT
NE
1
3
a
Effects of compounds during an OGTT in female Wistar fatty rats (n
b
= 6). See Experimental Section for details. Effective dose (ED) was
determined by statistical significance on AUC of plasma glucose. NE:
not effective. NT: not tested.
c
d
relatively lower plasma concentration showed efficacy only in
the 4H−OGTT and did not exhibit significant activity in the
1H−OGTT. Although ring-opened analogue 15 significantly
reduced plasma glucose excursions in the 4H−OGTT, the
potencies of these compounds were relatively lower compared
to that of the parent compound 12. The reason for insufficient
efficacy of 1-methylpiperidine derivative 19 would be its
relatively lower plasma concentration.
CONCLUSION
■
We have made considerable efforts to identify novel GPR40/
FFA1 agonists with reduced undesirable hydrophobic and toxic
profiles of FFAs as endogenous ligands while exhibiting potent
GPR40/FFA1 agonist activity. Optimization of (2,3-dihydro-1-
benzofuran-3-yl)acetic acid lead compound 2 culminated in the
discovery of 16. Introduction of various polar substituents at
the 4′-position of the biphenyl ring resulted in: (1) maintained
GPR40/FFA1 agonist activity, (2) reduced toxic profile (as
measured by caspase-3/7 activity) accompanied by decreased
Despite their lower affinities for the rat receptor as previously
mentioned, a series of dihydrobenzofuran derivatives exhibited
rapid and long-lasting in vivo efficacy by virtue of their
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Figure 3. Presumed metabolic pathways of 16.
Figure 4. Effects of 16 during an OGTT in male GK rats. (A) and (C) show time-dependent changes of plasma glucose (PG) and plasma insulin
after oral administration of 16, followed by 1 g/kg oral glucose challenge, respectively. Data in (B) and (D) represent AUC0−120min of PG levels and
AUC0−120min of plasma insulin levels shown in (A) and (C), respectively. Values are mean SD (n = 6). # P ≤ 0.025 compared to vehicle-treated
GK rats by one-tailed Shirley−Williams test. $ P ≤ 0.025 compared to vehicle-treated GK rats by one-tailed Williams’ test. ++ P ≤ 0.01 compared to
vehicle-treated GK rats by Aspin−Welch test. * P ≤ 0.05 compared to vehicle-treated GK rats by Student’s t test.
Log D values, and (3) improved PK profile, in particular, the
sulfonyl group is the best as for long-lasting plasma
concentration. Metabolism studies of 16 showed that the
compound was highly resistant to β-oxidation as we expected.
Encouraged by the results of the safety studies in rats and dogs,
16 was selected as a candidate for human clinical trials.
EXPERIMENTAL SECTION
■
Chemistry. Reagents and solvents were obtained from commercial
sources and used without further purification. Reaction progress was
determined by thin layer chromatography (TLC) analysis on Merck
Kieselgel 60 F254 plates or Fuji Silysia NH plates. Chromatographic
purification was carried out on silica gel columns [(Merck Kieselgel 60,
70−230 mesh or 230−400 mesh Merck) or (Chromatorex NH-DM
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1020, 100−200 mesh)] or on Purif-Pack (SI: 60 μM or NH: 60 μM,
added 2 M NaOH aqueous solution (0.750 mL, 1.50 mmol) at room
temperature, and the mixture was stirred at 50 °C for 2 h. The mixture
was diluted with water, acidified with 10% citric acid aqueous solution,
and extracted with AcOEt. The organic layer was washed with brine,
dried over anhydrous MgSO4, and concentrated. The residue was
purified by silica gel column chromatography (AcOEt/hexane =
30:70−80:20) to give an oil, which was triturated with hexane to give
crystals. Recrystallization from hexane/AcOEt gave 6 (0.315 g, 72%)
as colorless crystals; mp 58−59 °C. [α]D +6.5° (c 0.30, CH3CN).
99.7% ee [column, CHIRALPAK AD, 4.6 mm i.d. × 250 mmL; mobile
phase, hexane/IPA/TFA (85:15:0.1) (v/v/v) by isocratic elution; flow
rate, 0.5 mL/min; detection, UV 220 nm; temperature, room
Fuji Silysia Chemical, Ltd.). 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). 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% trifluoro-
acetic 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 instrument, 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 mL/min, with UV detection
at 220 nm, at column temperature of 25 °C. Elemental analyses were
carried out by Takeda Analytical Laboratories Limited and were within
0.4% of the theoretical values unless otherwise noted. The purity of
compounds was assessed by elemental analysis or analytical HPLC
(>95%). Optical rotations were determined on a JASCO P-1030
polarimeter. Preparative purifications were performed using a Gilson
pumping system in conjunction with a photodiode array detector
(Hewlett-Packard 1100 series) and a Gilson 215 auto sampler.
Separations were achieved using the following method, which utilized
a YMC packed column (CombiPrep ODS-A, 5 μm, 20 mm i.d. × 50
mm) with a 1 min isocratic elution 10/90, a 3.7 linear gradient from
10/90 to 0/100, and then a 2.7 min isocratic elution 0/100 A/B at a
flow rate of 25 mL/min.
1
temperature]. H NMR (CDCl3) δ 1.25 (t, J = 7.0 Hz, 3H), 1.98
(s, 6H), 2.55−2.66 (m, 1H), 2.76−2.85 (m, 1H), 3.62 (q, J = 7.0 Hz,
2H), 3.75−3.86 (m, 3H), 4.14 (t, J = 5.0 Hz, 2H), 4.28 (dd, J = 9.1, 6.0
Hz, 1H), 4.75 (t, J = 9.1 Hz, 1H), 5.05 (s, 2H), 6.44−6.52 (m, 2H),
6.68 (s, 2H), 7.02−7.10 (m, 2H), 7.16 (s, 1H), 7.34−7.45 (m, 2H).
MS m/z 477 (M + H)+. HPLC purity (220 nm) 100.0%. Anal. Calcd
for C29H32O6: C, 73.09; H, 6.77. Found: C, 73.02; H, 6.73.
[(3S)-6-({4′-[(4-Hydroxy-1,1-dioxidotetrahydro-2H-thiopyr-
an-4-yl)methoxy]-2′,6′-dimethylbiphenyl-3-yl}methoxy)-2,3-
dihydro-1-benzofuran-3-yl]acetic Acid (14). The title compound
was prepared from 4b and 5d by a method similar to that described for
6 except for step 2. Step 1: Methyl [(3S)-6-({4′-[(4-hydroxytetrahy-
dro-2H-thiopyran-4-yl)methoxy]-2′,6′-dimethylbiphenyl-3-yl}-
methoxy)-2,3-dihydro-1-benzofuran-3-yl]acetate in 90% yield as a
1
yellow oil. H NMR (CDCl3) δ 1.77−1.89 (m, 2H), 1.99 (s, 6H),
2.06−2.15 (m, 2H), 2.18 (s, 1H), 2.42−2.60 (m, 3H), 2.70−2.79 (m,
1H), 3.04−3.17 (m, 2H), 3.72 (s, 3H), 3.74−3.86 (m, 3H), 4.26 (dd, J
= 9.1, 6.0 Hz, 1H), 4.75 (t, J = 9.1 Hz, 1H), 5.05 (s, 2H), 6.44−6.51
(m, 2H), 6.67 (s, 2H), 7.01 (d, J = 7.9 Hz, 1H), 7.04−7.09 (m, 1H),
7.15 (s, 1H), 7.35−7.45 (m, 2H). MS m/z 531 (M − 18 + H)+. Step 2:
To a solution of the obtained oil (0.586 g, 1.07 mmol) in AcOEt (5
mL) was added gradually m-CPBA (0.568 g, 2.14 mmol) at 0 °C, and
the mixture was stirred at 0 °C for 2 h. The mixture was quenched
with Na2S2O3 aqueous solution and saturated NaHCO3 aqueous
solution and extracted with AcOEt. The organic layer was washed with
brine, dried over anhydrous MgSO4, and concentrated. The residue
was purified by silica gel column chromatography (AcOEt/hexane =
40:60−80:20) to give methyl [(3S)-6-({4′-[(4-hydroxy-1,1-dioxidote-
trahydro-2H-thiopyran-4-yl)methoxy]-2′,6′-dimethylbiphenyl-3-yl}-
methoxy)-2,3-dihydro-1-benzofuran-3-yl]acetate (0.489 g, 79%) as a
Methyl [(3S)-6-Hydroxy-2,3-dihydro-1-benzofuran-3-yl]-
acetate (4b) and Methyl [(3R)-6-Hydroxy-2,3-dihydro-1-ben-
zofuran-3-yl]acetate (4c). Methyl (6-hydroxy-2,3-dihydro-1-benzo-
furan-3-yl)acetate (4a) was optically resolved using normal phase
preparative HPLC [column, CHIRALPAK AD, 50 mm i.d. × 500
mmL; mobile phase, hexane/EtOH (88/12) (v/v) by isocratic elution;
flow rate, 60 mL/min; detection, UV 220 nm; temperature, 30 °C].
Retention times of the two enantiomers were 16.3 and 18.7 min. The
(S)-isomer 4b (retention time 16.3 min) was thus obtained with a
99.7% ee [column, CHIRALPAK AD-H, 4.6 mm i.d. × 250 mmL;
mobile phase, hexane/IPA (80/20) (v/v) by isocratic elution; flow
rate, 0.5 mL/min; detection, UV 220 nm; temperature, 30 °C]. [α]D
+5.4° (c 0.92, CHCl3). The (R)-isomer 4c (retention time 18.7 min)
was thus obtained with a 99.1% ee [column, CHIRALPAK AD-H, 4.6
mm i.d. × 250 mmL; mobile phase, hexane/IPA (80/20) (v/v) by
isocratic elution; flow rate, 0.5 mL/min; detection, UV 220 nm;
temperature: 30 °C]. [α]D −6.4° (c 0.94, CHCl3).
1
colorless oil. H NMR (CDCl3) δ 2.00 (s, 6H), 2.16−2.33 (m, 4H),
2.46 (s, 1H), 2.50−2.61 (m, 1H), 2.69−2.80 (m, 1H), 2.90−3.01 (m,
2H), 3.43−3.57 (m, 2H), 3.72 (s, 3H), 3.74−3.86 (m, 1H), 3.88 (s,
2H), 4.26 (dd, J = 9.1, 6.1 Hz, 1H), 4.75 (t, J = 9.1 Hz, 1H), 5.06 (s,
2H), 6.43−6.51 (m, 2H), 6.67 (s, 2H), 6.99−7.10 (m, 2H), 7.15 (s,
1H), 7.35−7.47 (m, 2H). MS m/z 581 (M + H)+. Step 3: 14 in 76%
yield as colorless crystals (hexane/AcOEt); mp 198−201 °C. [α]D
+5.1° (c 0.30, CH3CN). 1H NMR (CDCl3) δ 2.00 (s, 6H), 2.17−2.33
(m, 4H), 2.56−2.67 (m, 1H), 2.76−2.85 (m, 1H), 2.90−3.01 (m, 2H),
3.43−3.56 (m, 2H), 3.75−3.86 (m, 1H), 3.88 (s, 2H), 4.29 (dd, J =
9.1, 6.0 Hz, 1H), 4.76 (t, J = 9.1 Hz, 1H), 5.06 (s, 2H), 6.44−6.52 (m,
2H), 6.67 (s, 2H), 7.02−7.09 (m, 2H), 7.15 (s, 1H), 7.35−7.46 (m,
2H). MS m/z 567 (M + H)+. HPLC purity (220 nm) 99.4%. Anal.
Calcd for C31H34O8S: C, 65.71; H, 6.05. Found: C, 65.69; H, 6.03.
[(3S)-6-({4′-[2-(Ethylsulfonyl)ethoxy]-2′,6′-dimethylbiphen-
yl-3-yl}methoxy)-2,3-dihydro-1-benzofuran-3-yl]acetic Acid
(15). The title compound was prepared from 4b and 5e by a method
similar to that described for 6 except for step 3. Step 1: Methyl [(3S)-
6-({4′-[2-(ethylsulfanyl)ethoxy]-2′,6′-dimethylbiphenyl-3-yl}-
methoxy)-2,3-dihydro-1-benzofuran-3-yl]acetate in 60% yield as a
pale-yellow oil. 1H NMR (CDCl3) δ 1.31 (t, J = 7.4 Hz, 3H), 1.99 (s,
6H), 2.50−2.79 (m, 4H), 2.92 (t, J = 7.0 Hz, 2H), 3.71 (s, 3H), 3.74−
3.86 (m, 1H), 4.15 (t, J = 7.0 Hz, 2H), 4.26 (dd, J = 9.1, 6.1 Hz, 1H),
4.75 (t, J = 9.1 Hz, 1H), 5.05 (s, 2H), 6.44−6.51 (m, 2H), 6.66 (s,
2H), 7.01 (d, J = 7.9 Hz, 1H), 7.05−7.10 (m, 1H), 7.16 (s, 1H), 7.34−
7.45 (m, 2H). MS m/z 507 (M + H)+. Step 2: [(3S)-6-({4′-[2-
(Ethylsulfanyl)ethoxy]-2′,6′-dimethylbiphenyl-3-yl}methoxy)-2,3-dihy-
[(3S)-6-{[4′-(2-Ethoxyethoxy)-2′,6′-dimethylbiphenyl-3-yl]-
methoxy}-2,3-dihydro-1-benzofuran-3-yl]acetic Acid (6). Step
1: To a mixture of 4b (0.250 g, 1.20 mmol), 5a (0.360 g, 1.20 mmol),
and P(n-Bu)3 (0.388 g, 1.92 mmol) in toluene (20 mL) was added
gradually ADDP (0.484 g, 1.92 mmol), and the mixture was stirred
under nitrogen atmosphere at room temperature for 20 h. Hexane (10
mL) was added, and the insoluble material was removed by filtration.
The filtrate was concentrated, and the residue was purified by silica gel
column chromatography (AcOEt/hexane = 5:95−40:60) to give
methyl [(3S)-(6-{[4′-(2-ethoxyethoxy)-2′,6′-dimethylbiphenyl-3-yl]-
methoxy}-2,3-dihydro-1-benzofuran-3-yl)acetate (0.456 g, 77%) as a
1
colorless oil. H NMR (CDCl3) δ 1.25 (t, J = 7.0 Hz, 3H), 1.98 (s,
6H), 2.50−2.61 (m, 1H), 2.70−2.79 (m, 1H), 3.62 (q, J = 7.0 Hz,
2H), 3.71 (s, 3H), 3.75−3.86 (m, 3H), 4.11−4.16 (m, 2H), 4.26 (dd, J
= 9.1, 6.1 Hz, 1H), 4.75 (t, J = 9.1 Hz, 1H), 5.05 (s, 2H), 6.44−6.51
(m, 2H), 6.69 (s, 2H), 7.01 (d, J = 8.1 Hz, 1H), 7.08 (dt, J = 7.1, 1.5
Hz, 1H), 7.16 (s, 1H), 7.34−7.45 (m, 2H). MS m/z 491 (M + H)+.
HPLC purity (220 nm) 100.0%. Step 2: To a solution of the obtained
ester (0.451 g, 0.919 mmol) in MeOH (2 mL) and THF (4 mL) was
1
dro-1-benzofuran-3-yl]acetic acid in 89% yield as a colorless oil. H
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NMR (CDCl3) δ 1.31 (t, J = 7.4 Hz, 3H), 1.99 (s, 6H), 2.56−2.71 (m,
3H), 2.76−2.86 (m, 1H), 2.92 (t, J = 7.0 Hz, 2H), 3.75−3.87 (m, 1H),
4.15 (t, J = 6.8 Hz, 2H), 4.28 (dd, J = 9.1, 6.1 Hz, 1H), 4.76 (t, J = 9.1
Hz, 1H), 5.05 (s, 2H), 6.44−6.52 (m, 2H), 6.66 (s, 2H), 7.02−7.10
(m, 2H), 7.16 (s, 1H), 7.34−7.45 (m, 2H). MS m/z 493 (M + H)+.
Step 3: To a solution of the obtained oil (0.304 g, 0.617 mmol) in
MeOH (10 mL) was added dropwise a solution of oxone (0.569 g,
0.926 mmol) in water (5 mL) at 0 °C, and the mixture was stirred at 0
°C to room temperature for 12 h. MeOH was evaporated. The residue
was diluted with water and extracted with AcOEt. The extract was
washed with brine, dried over anhydrous MgSO4, and concentrated.
The residue was purified by preparative HPLC to give crystals.
Recrystallization from heptane/AcOEt gave 15 (0.237 g, 73%) as
solution of the obtained solid (2.27 g, 4.26 mmol) in THF (25 mL)
was added 1 M TBAF in THF (4.7 mL, 4.7 mmol) at room
temperature, and the mixture was stirred under nitrogen atmosphere at
room temperature for 1 h. The mixture was concentrated, and the
residue was partitioned between water and AcOEt. The organic layer
was separated, washed with brine, dried over anhydrous MgSO4, and
concentrated. The residue was purified by silica gel column
chromatography (AcOEt/hexane = 20:80−60:40) to give 17a (1.67
g, 94%) as a colorless oil. 1H NMR (CDCl3) δ 1.97 (s, 6H), 2.55 (dd, J
= 16.5, 9.8 Hz, 1H), 2.75 (dd, J = 16.5, 4.8 Hz, 1H), 3.72 (s, 3H),
3.74−3.86 (m, 1H), 4.26 (dd, J = 9.0, 6.2 Hz, 1H), 4.63 (s, 1H), 4.75
(t, J = 9.0 Hz, 1H), 5.05 (s, 2H), 6.43−6.50 (m, 2H), 6.59 (s, 2H),
7.01 (d, J = 8.1 Hz, 1H), 7.04−7.11 (m, 1H), 7.16 (s, 1H), 7.34−7.46
(m, 2H). MS m/z 419 (M + H)+.
1
colorless crystals; mp 130−131 °C. [α]D +6.6° (c 0.30, CH3CN). H
NMR (CDCl3) δ 1.47 (t, J = 7.5 Hz, 3H), 1.99 (s, 6H), 2.55−2.67 (m,
1H), 2.75−2.86 (m, 1H), 3.19 (q, J = 7.5 Hz, 2H), 3.42 (t, J = 5.4 Hz,
2H), 3.75−3.87 (m, 1H), 4.29 (dd, J = 9.1, 6.0 Hz, 1H), 4.44 (t, J = 9.1
Hz, 2H), 4.76 (t, J = 5.4 Hz, 1H), 5.06 (s, 2H), 6.44−6.52 (m, 2H),
6.64 (s, 2H), 7.02−7.09 (m, 2H), 7.15 (s, 1H), 7.35−7.46 (m, 2H).
MS m/z 525 (M + H)+. HPLC purity (220 nm) 99.8%. Anal. Calcd for
C29H32O7S: C, 66.39; H, 6.15. Found: C, 66.35; H, 6.15.
[(3S)-6-({2′,6′-Dimethyl-4′-[3-(2-oxopyrrolidin-1-yl)-
propoxy]biphenyl-3-yl}methoxy)-2,3-dihydro-1-benzofuran-3-
yl]acetic Acid (18). Step 1: To a mixture of 17a (0.325 g, 0.777
mmol), 1-(3-hydroxypropyl)pyrrolidin-2-one (0.167 g, 1.16 mmol),
and P(n-Bu)3 (0.314 g, 1.55 mmol) in toluene (15 mL) was added
ADDP (0.392 g, 1.55 mmol), and the mixture was stirred at room
temperature for 15 h. Hexane (15 mL) was added, and the insoluble
material was removed by filtration. The filtrate was concentrated, and
the residue was purified by silica gel column chromatography (AcOEt/
hexane = 30:70−100:0) to afford methyl [(3S)-6-({2′,6′-dimethyl-4′-
[3-(2-oxopyrrolidin-1-yl)propoxy]biphenyl-3-yl}methoxy)-2,3-dihy-
dro-1-benzofuran-3-yl]acetate (0.420 g, crude) as a colorless oil. The
crude product was used for the next reaction without further
purification. MS m/z 544 (M + H)+. Step 2: Compound 18 was
prepared from the obtained oil by a method similar to that described
for 6, step 2 in 42% yield (from 17a) as colorless crystals (heptane/
MeCN); mp 90−92 °C. [α]D +5.2° (c 0.32, CH3CN). 1H NMR
(CDCl3) δ 1.94−1.99 (m, 6H), 1.99−2.10 (m, 4H), 2.41 (t, J = 8.1
Hz, 2H), 2.59 (dd, J = 16.5, 9.3 Hz, 1H), 2.78 (dd, J = 16.5, 5.4 Hz,
1H), 3.42−3.52 (m, 4H), 3.73−3.85 (m, 1H), 4.00 (t, J = 6.3 Hz, 2H),
4.28 (dd, J = 9.2, 5.8 Hz, 1H), 4.74 (t, J = 9.2 Hz, 1H), 5.06 (s, 2H),
6.43−6.50 (m, 2H), 6.64 (s, 2H), 7.02−7.09 (m, 2H), 7.13 (s, 1H),
7.33−7.44 (m, 2H). MS m/z 530 (M + H)+. HPLC purity (220 nm)
99.7%. Anal. Calcd for C32H35NO6·0.5H2O: C, 71.36; H, 6.74; N, 2.60.
Found: C, 71.39; H, 6.60; N, 2.61.
[(3S)-6-({2′,6′-Dimethyl-4′-[3-(methylsulfonyl)propoxy]-
biphenyl-3-yl}methoxy)-2,3-dihydro-1-benzofuran-3-yl]acetic
Acid Hemihydrate (16). Step 1: To a mixture of 4b (0.208 g, 1.00
mmol), 5f (0.348 g, 1.00 mmol), and P(n-Bu)3 (0.324 g, 1.60 mmol)
in toluene (15 mL) was added portionwise ADDP (0.404 g, 1.60
mmol), and the mixture was stirred under nitrogen atmosphere at
room temperature for 1.5 h. Hexane (8 mL) was added, and the
insoluble material was removed by filtration. The filtrate was
concentrated, and the residue was purified by silica gel column
chromatography (AcOEt/hexane = 40:60−80:20) to give methyl
[(3S)-6-({2′,6′-dimethyl-4′-[3-(methylsulfonyl)propoxy]biphenyl-3-
yl}methoxy)-2,3-dihydro-1-benzofuran-3-yl]acetate (0.442 g, 82%) as
a colorless oil. 1H NMR (CDCl3) δ 1.99 (s, 6H), 2.30−2.41 (m, 2H),
2.49−2.61 (m, 1H), 2.69−2.79 (m, 1H), 2.97 (s, 3H), 3.23−3.31 (m,
2H), 3.71 (s, 3H), 3.74−3.86 (m, 1H), 4.08−4.13 (m, 2H), 4.26 (dd, J
= 9.1, 6.1 Hz, 1H), 4.75 (t, J = 9.1 Hz, 1H), 5.05 (s, 2H), 6.43−6.51
(m, 2H), 6.64 (s, 2H), 7.01 (d, J = 8.0 Hz, 1H), 7.07 (dt, J = 7.1, 1.6
Hz, 1H), 7.15 (s, 1H), 7.34−7.46 (m, 2H). MS m/z 539 (M + H)+.
Step 2: To a solution of the obtained oil (11.2 g, 20.8 mmol) in
MeOH (40 mL) and THF (80 mL) was added 2 M NaOH aqueous
solution (20.0 mL, 40.0 mmol), and the mixture was stirred at 50 °C
for 2 h. The mixture was concentrated, diluted with water, acidified
with 1 M hydrochloric acid aqueous solution, and extracted with
AcOEt. The organic layer was washed with brine, dried over MgSO4,
and concentrated to give crystals, which were washed with heptane/
AcOEt. Recrystallization from EtOH/H2O gave 16 (9.31 g, 85%) as
colorless crystals; mp 127−129 °C. [α]D +5.3° (c 0.3085, CH3CN).
99.6% ee [column, CHRALPAK AD-3 (NC002), 4.6 mm i.d. × 250
mmL; mobile phase, hexane/2-propanol/TFA = 500:500:1 (v/v/v) by
isocratic elution; flow rate, 0.5 mL/min; detection, UV 220 nm;
column temperature, 30 °C]. 1H NMR (CDCl3) δ 1.99 (s, 6H), 2.29−
2.41 (m, 2H), 2.61 (dd, J = 16.9, 9.2 Hz, 1H), 2.81 (dd, J = 16.9, 5.5
Hz, 1H), 2.97 (s, 3H), 3.23−3.31 (m, 2H), 3.75−3.87 (m, 1H), 4.13
(t, J = 5.8 Hz, 2H), 4.28 (dd, J = 9.1, 6.0 Hz, 1H), 4.76 (t, J = 9.1 Hz,
1H), 5.06 (s, 2H), 6.44−6.52 (m, 2H), 6.64 (s, 2H), 7.02−7.10 (m,
2H), 7.16 (s, 1H), 7.35−7.46 (m, 2H). MS m/z 525 (M + H)+. HPLC
purity (220 nm) 100.0%. Anal. Calcd for C29H32O7S·0.5H2O: C,
65.27; H, 6.23. Found: C, 65.23; H, 6.15.
[(3S)-6-({2′,6′-Dimethyl-4′-[(1-methylpiperidin-4-yl)oxy]-
biphenyl-3-yl}methoxy)-2,3-dihydro-1-benzofuran-3-yl]acetic
Acid Hydrochloride (19). Step 1: To a mixture of 17a (0.350 g,
0.836 mmol), 1-methylpiperidin-4-ol (0.143 g, 1.25 mmol), and PPh3
(0.351 g, 1.34 mmol) in toluene (15 mL) was added 40% DEAD in
toluene (0.582 g, 1.34 mmol), and the mixture was stirred at room
temperature for 15 h. Then, 1-methylpiperidin-4-ol (0.098 g, 0.851
mmol), PPh3 (0.197 g, 1.05 mmol), and 40% DEAD in toluene (0.328
g, 0.750 mmol) were added to the mixture. After stirring at room
temperature for 8 h, hexane (15 mL) was added to the mixture, and
the insoluble material was removed by filtration. The filtrate was
concentrated, and the residue was purified by basic silica gel column
chromatography (AcOEt/hexane = 20:80−50:50) to afford methyl
[(3S)-6-({2′,6′-dimethyl-4′-[(1-methylpiperidin-4-yl)oxy]biphenyl-3-
yl}methoxy)-2,3-dihydro-1-benzofuran-3-yl]acetate (0.270 g, 63%) as
1
a colorless oil. H NMR (CDCl3) δ 1.80−1.93 (m, 2H), 1.95−2.10
(m, 8H), 2.24−2.36 (m, 5H), 2.55 (dd, J = 16.5, 9.3 Hz, 1H), 2.66−
2.80 (m, 3H), 3.71 (s, 3H), 3.74−3.87 (m, 1H), 4.21−4.39 (m, 2H),
4.75 (t, J = 9.0 Hz, 1H), 5.05 (s, 2H), 6.44−6.51 (m, 2H), 6.66 (s,
2H), 7.01 (d, J = 7.9 Hz, 1H), 7.06−7.11 (m, 1H), 7.17 (s, 1H), 7.33−
7.45 (m, 2H). MS m/z 516 (M + H)+. Step 2: To a stirred solution of
the obtained oil (0.270 g, 0.52 mmol) in MeOH (4 mL) and THF (8
mL) was added 2 M NaOH aqueous solution (2.0 mL, 4.0 mmol).
The mixture was stirred at room temperature for 15 h. Then the
mixture was neutralized with saturated NH4Cl aqueous solution. To
the mixture was added sodium chloride, and the mixture was extracted
with AcOEt/THF/CH2Cl2. The extract was dried over anhydrous
MgSO4 and concentrated. The resultant residue was treated with 4 M
HCl in AcOEt (5 mL, 20 mmol). Then hexane (20 mL) was added to
the mixture, and the resulting crystals were collected by filtration to
afford 19 (91 mg, 32%) as colorless crystals; mp 165−167 °C. MS m/z
Methyl {(3S)-6-[(4′-Hydroxy-2′,6′-dimethylbiphenyl-3-yl)-
methoxy]-2,3-dihydro-1-benzofuran-3-yl}acetate (17a). Step 1:
Methyl {(3S)-6-[(4′-{[tert-butyl(dimethyl)silyl]oxy}-2′,6′-dimethylbi-
phenyl-3-yl)methoxy]-2,3-dihydro-1-benzofuran-3-yl}acetate was pre-
pared from 4b and 5g by a method similar to that described for 6, step
1
1 in 85% yield as a colorless solid. H NMR (CDCl3) δ 0.23 (s, 6H),
1.00 (s, 9H), 1.95 (s, 6H), 2.55 (dd, J = 16.5, 9.3 Hz, 1H), 2.75 (dd, J
= 16.5, 5.5 Hz, 1H), 3.71 (s, 3H), 3.74−3.88 (m, 1H), 4.26 (dd, J =
9.2, 6.0 Hz, 1H), 4.75 (t, J = 9.2 Hz, 1H), 5.05 (s, 2H), 6.44−6.51 (m,
2H), 6.57 (s, 2H), 7.01 (d, J = 7.9 Hz, 1H), 7.06−7.10 (m, 1H), 7.17
(s, 1H), 7.33−7.44 (m, 2H). MS m/z 533 (M + H)+. Step 2: To a
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1
502 (M + H)+ as a free form. H NMR (DMSO-d6) δ 1.82−2.30 (m,
3′-(Hydroxymethyl)-2,6-dimethylbiphenyl-4-ol (32). To a
solution of 4′-hydroxy-2′,6′-dimethylbiphenyl-3-carbaldehyde (31)
(6.95 g, 30.7 mmol) in MeOH (30 mL) and THF (60 mL) was
added gradually NaBH4 (1.29 g, 30.7 mmol) at 0 °C, and the mixture
was stirred under nitrogen atmosphere at 0 °C to room temperature
for 20 h. The mixture was concentrated, quenched with water and 1 M
HCl aqueous solution, and extracted with AcOEt. The organic layer
was washed with brine, dried over anhydrous MgSO4, and
concentrated to give crystals. Recrystallization from heptane/AcOEt
4H), 1.92 (s, 6H), 2.40−2.56 (m, 1H), 2.61−2.84 (m, 4H), 3.03−3.46
(m, 5H), 3.59−3.72 (m, 1H), 4.18 (dd, J = 9.1, 6.9 Hz, 1H), 4.67 (t, J
= 9.1 Hz, 1H), 5.10 (s, 2H), 6.40−6.53 (m, 2H), 6.77 (s, 2H), 7.02−
7.15 (m, 3H), 7.35−7.49 (m, 2H). HPLC purity (220 nm) 99.0%.
[(3S)-6-({3′-Chloro-2′,6′-dimethyl-4′-[3-(methylsulfonyl)-
propoxy]biphenyl-3-yl}methoxy)-2,3-dihydro-1-benzofuran-3-
yl]acetic Acid (27). Step 1: A mixture of 17b (0.616 g, 1.36 mmol),
30b (0.517 g, 1.77 mmol), and K3PO4 (0.433 g, 2.04 mmol) in DMF
(2 mL) was stirred under nitrogen atmosphere at 90 °C for 2.5 h. The
mixture was diluted with water and extracted with AcOEt. The organic
layer was washed with brine, dried over anhydrous MgSO4, and
concentrated. The residue was purified by silica gel column
chromatography (AcOEt/hexane = 30:70−80:20) to give methyl
[(3S)-6-({3′-chloro-2′,6′-dimethyl-4′-[3-(methylsulfonyl)propoxy]-
biphenyl-3-yl}methoxy)-2,3-dihydro-1-benzofuran-3-yl]acetate (0.681
1
gave 32 (6.56 g, 93%) as colorless crystals; mp 175 °C. H NMR
(CDCl3) δ 1.67 (t, J = 5.8 Hz, 1H), 1.98 (s, 6H), 4.65 (s, 1H), 4.74 (d,
J = 5.8 Hz, 2H), 6.59 (s, 2H), 7.06 (dt, J = 7.3, 1.5 Hz, 1H), 7.12 (s,
1H), 7.33 (dt, J = 7.5, 1.5 Hz, 1H), 7.41 (t, J = 7.5 Hz, 1H). MS m/z
211 (M − 18 + H)+. Anal. Calcd for C15H16O2: C, 78.92; H, 7.06.
Found: C, 78.76; H, 7.04.
[4′-(2-Ethoxyethoxy)-2′,6′-dimethylbiphenyl-3-yl]methanol
(5a). A mixture of 32 (4.57 g, 20.0 mmol), 2-chloroethyl ethyl ether
(3.29 mL, 30.0 mmol), K2CO3 (3.32 g, 24.0 mmol), and KI (0.332 g,
2.00 mmol) in DMF (30 mL) was stirred under nitrogen atmosphere
at 80 °C for 70 h. The mixture was diluted with water and extracted
with AcOEt. The organic layer was washed sequentially with 1 M
NaOH aqueous solution and brine, dried over anhydrous MgSO4, and
concentrated. The residue was purified by basic silica gel column
chromatography (AcOEt/hexane = 20:80−60:40) and crystallized
from heptane/AcOEt to give 5a (4.43 g, 74%) as colorless crystals; mp
1
g, 88%) as a colorless viscous oil. H NMR (CDCl3) δ 1.97 (s, 3H),
2.05 (s, 3H), 2.35−2.46 (m, 2H), 2.55 (dd, J = 16.5, 9.1 Hz, 1H), 2.75
(dd, J = 16.5, 5.7 Hz, 1H), 2.99 (s, 3H), 3.31−3.41 (m, 2H), 3.71 (s,
3H), 3.75−3.86 (m, 1H), 4.20 (t, J = 5.9 Hz, 2H), 4.26 (dd, J = 9.2, 6.2
Hz, 1H), 4.75 (t, J = 9.2 Hz, 1H), 5.06 (s, 2H), 6.43−6.51 (m, 2H),
6.69 (s, 1H), 6.99−7.07 (m, 2H), 7.13 (s, 1H), 7.37−7.47 (m, 2H).
MS m/z 573 (M + H)+. Step 2: Compound 27 was prepared by a
method similar to that described for 6, step 2 in 63% yield as colorless
crystals (Et2O−AcOEt); mp 127−128 °C. [α]D +5.6° (c 0.30,
1
1
CH3CN). H NMR (CDCl3) δ 1.97 (s, 3H), 2.05 (s, 3H), 2.35−
62−63 °C. H NMR (CDCl3) δ 1.25 (t, J = 7.1 Hz, 3H), 1.66 (t, J =
2.47 (m, 2H), 2.62 (dd, J = 16.8, 9.2 Hz, 1H), 2.81 (dd, J = 16.8, 5.5
Hz, 1H), 2.99 (s, 3H), 3.32−3.40 (m, 2H), 3.75−3.87 (m, 1H), 4.20
(t, J = 5.7 Hz, 2H), 4.29 (dd, J = 9.1, 6.0 Hz, 1H), 4.76 (t, J = 9.1 Hz,
1H), 5.06 (s, 2H), 6.44−6.52 (m, 2H), 6.69 (s, 1H), 7.02−7.08 (m,
2H), 7.13 (s, 1H), 7.37−7.47 (m, 2H). MS m/z 559 (M + H)+. HPLC
purity (220 nm) 99.63%. Anal. Calcd for C29H31ClO7S: C, 62.30; H,
5.59. Found: C, 62.03; H, 5.58.
5.9 Hz, 1H), 2.00 (s, 6H), 3.62 (q, J = 7.1 Hz, 2H), 3.80 (t, J = 5.1 Hz,
2H), 4.14 (t, J = 5.1 Hz, 2H), 4.73 (d, J = 5.9 Hz, 2H), 6.69 (s, 2H),
7.06 (d, J = 7.3 Hz, 1H), 7.12 (s, 1H), 7.33 (d, J = 7.3 Hz, 1H), 7.40 (t,
J = 7.3 Hz, 1H). MS m/z 301 (M + H)+. Anal. Calcd for C19H24O3: C,
75.97; H, 8.05. Found: C, 75.75; H, 8.10.
4′-{tert-Butyl(dimethyl)silyl}oxy}-2′,6′-dimethylbiphenyl-3-
carbaldehyde (33d). To a solution of 31 (9.0 g, 39.8 mmol) and
imidazole (2.98 g, 43.8 mmol) in DMF (100 mL) was added TBSCl
(6.6 g, 43.8 mmol) at room temperature, and the mixture was stirred at
room temperature for 4 h. The mixture was diluted with AcOEt,
washed sequentially with water and brine, dried over MgSO4, and
concentrated. The residue was purified by silica gel column
chromatography (AcOEt/hexane = 9:91−20:80) to give 33d (10.5
g, 77%) as a yellow oil. 1H NMR (CDCl3) δ 0.25 (s, 6H), 1.02 (s, 9H),
1.97 (s, 6H), 6.62 (s, 2H), 7.44 (dt, J = 7.5, 1.5 Hz, 1H), 7.59 (t, J =
7.5 Hz, 1H), 7.68 (t, J = 1.5 Hz, 1H), 7.86 (dt, J = 7.5, 1.5 Hz, 1H),
10.06 (s, 1H). MS m/z 341 (M + H)+.
(3-Methyloxetan-3-yl)methyl 4-Methylbenzenesulfonate
(30a). To a suspension of p-TsCl (14.3 g, 75.0 mmol) in pyridine
(60 mL) was added slowly 3-methyl-3-oxetanemethanol (29a) (5.11 g,
50.0 mmol) at 0 °C, and the mixture was stirred under nitrogen
atmosphere at 0 °C for 4 h. The mixture was added to ice water and
stirred for 1 h. The precipitate was collected by filtration, washed with
cold water, and dried to give 30a (8.97 g, 70%) as colorless crystals;
mp 60−61 °C. 1H NMR (CDCl3) δ 1.31 (s, 3H), 2.47 (s, 3H), 4.11 (s,
2H), 4.32−4.39 (m, 4H), 7.37 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.4 Hz,
2H). MS m/z 257 (M + H)+. HPLC purity (220 nm) 98.6%. Anal.
Calcd for C12H16O4S: C, 56.23; H, 6.29. Found: C, 56.21; H, 6.22.
3-(Methylsulfonyl)propyl 4-Methylbenzenesulfonate (30b).
Step 1: To a solution of 3-methylthio-1-propanol (29b) (5.30 g, 50.0
mmol), Et3N (10.5 mL, 75.0 mmol), and N,N,N′,N′-tetramethyl-1,6-
hexanediamine (0.861 g, 5.00 mmol) in toluene (50 mL) was added
dropwise p-TsCl (14.3 g, 75.0 mmol) in toluene (50 mL) at 0 °C, and
the mixture was stirred under nitrogen atmosphere at 0 °C for 3 h.
The mixture was quenched with water and extracted with AcOEt. The
extract was washed with brine, dried over anhydrous Na2SO4, and
concentrated. The residue was purified by silica gel column
chromatography (AcOEt/hexane = 10:90−40:60) to give 3-
(methylthio)propyl 4-methylbenzenesulfonate (12.2 g, 94%) as a
[2,6-Dimethyl-4-(tetrahydro-2H-thiopyran-4-yloxy)phenyl]-
boronic Acid (35). Step 1: 4-(4-Bromo-3,5-dimethylphenoxy)-
tetrahydro-2H-thiopyran was prepared from 3,5-dimethyl-4-bromo-
phenol (34) and tetrahydro-4H-thiopyran-4-ol by a method similar to
1
that described for 17, step 1 in 86% yield as a white solid. H NMR
(CDCl3) δ 1.93−2.07 (m, 2H), 2.10−2.23 (m, 2H), 2.37 (s, 6H),
2.49−2.62 (m, 2H), 2.85−2.98 (m, 2H), 4.25−4.36 (m, 1H), 6.65 (s,
2H). Step 2: To a solution of the obtained solid (3.01 g, 10.0 mmol) in
THF (50 mL) was added dropwise a solution of 1.6 M n-BuLi in
hexane (6.57 mL, 10.5 mmol) under argon atmosphere at −78 °C.
The mixture was stirred at −78 °C for 1.5 h, and then B(i-PrO)3 (6.92
mL, 30.0 mmol) was added at the same temperature. The mixture was
gradually warmed to room temperature and stirred for 16 h. After the
mixture was cooled to 0 °C, 2 M HCl aqueous solution (50 mL) was
added. The resulting mixture was stirred at 0 °C for 2.5 h. The phases
were separated, and the aqueous phase was extracted with AcOEt (pH
of the aqueous phase was adjusted to neutral with saturated NaHCO3
aqueous solution). The combined organic phase was dried over
anhydrous MgSO4, and concentrated to give a white solid. The
resulting solid was washed with cold hexane and dried to afford 35
1
colorless oil. H NMR (CDCl3) δ 1.87−1.98 (m, 2H), 2.04 (s, 3H),
2.45 (s, 3H), 2.51 (t, J = 7.1 Hz, 2H), 4.14 (t, J = 6.1 Hz, 2H), 7.35 (d,
J = 8.2 Hz, 2H), 7.80 (d, J = 8.2 Hz, 2H). MS m/z 261 (M + H)+. Step
2: To a solution of the obtained sulfonate (12.2 g, 46.9 mmol) in
MeOH (250 mL) was added dropwise a solution of oxone (57.7 g,
93.8 mmol) in water (250 mL) at 0 °C, and the mixture was stirred at
0 °C to room temperature for 20 h. After evaporation of the solvent,
the residue was diluted with water and extracted with AcOEt. The
extract was washed with brine, dried over anhydrous Na2SO4, and
concentrated. The resulting crystals were washed with heptane/AcOEt
1
(1.89 g, 71%) as a white solid. H NMR (CDCl3) δ 1.90−2.06 (m,
2H), 2.09−2.23 (m, 2H), 2.35 (s, 6H), 2.48−2.62 (m, 2H), 2.83−2.98
(m, 2H), 4.28−4.40 (m, 1H), 6.51 (s, 2H), 6.59 (s, 2H). MS m/z not
detected.
1
to give 30b (13.1 g, 96%) as colorless crystals; mp 88−89 °C. H
NMR (CDCl3) δ 2.17−2.28 (m, 2H), 2.46 (s, 3H), 2.92 (s, 3H),
3.07−3.15 (m, 2H), 4.18 (t, J = 5.9 Hz, 2H), 7.37 (d, J = 8.3 Hz, 2H),
7.79 (d, J = 8.3 Hz, 2H). MS m/z 293 (M + H)+. Anal. Calcd for
C11H16O5S2: C, 45.19; H, 5.52. Found: C, 44.96; H, 5.53.
{4′-[(1,1-Dioxidotetrahydro-2H-thiopyran-4-yl)oxy]-2′,6′-di-
methylbiphenyl-3-yl}methanol (5c). Step 1: A mixture of 35 (1.41
g, 5.30 mmol), methyl 3-bromobenzoate (1.14 g, 5.30 mmol), and
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Pd(PPh3)4 (0.306 g, 0.265 mmol) in 2 M Cs2CO3 (6.35 mL) and
DME (20 mL) was stirred under argon atmosphere at 95 °C for 24 h.
The mixture was diluted with water and extracted with AcOEt. The
combined organic layer was washed with brine, dried over anhydrous
MgSO4, and concentrated. The residue was purified by silica gel
column chromatography (AcOEt/hexane = 0:100−25:75) to give
crystals. Recrystallization from hexane−AcOEt gave methyl 2′,6′-
dimethyl-4′-(tetrahydro-2H-thiopyran-4-yloxy)biphenyl-3-carboxylate
(1.63 g, 86%) as colorless crystals; mp 69−71 °C. 1H NMR (CDCl3) δ
1.92−2.13 (m, 8H), 2.15−2.29 (m, 2H), 2.52−2.66 (m, 2H), 2.89−
3.03 (m, 2H), 3.91 (s, 3H), 4.33−4.44 (m, 1H), 6.66 (s, 2H), 7.34 (dt,
J = 7.8, 1.5 Hz, 1H), 7.49 (t, J = 7.8 Hz, 1H), 7.84 (t, J = 1.5 Hz, 1H),
8.01 (dt, J = 7.8, 1.5 Hz, 1H). MS m/z 379 (M + Na)+. HPLC purity
(220 nm) 99.7%. Anal. Calcd for C21H24O3S: C, 70.75; H, 6.79.
Found: C, 70.73; H, 6.80. Step 2: Methyl 4′-[(1,1-dioxidotetrahydro-
2H-thiopyran-4-yl)oxy]-2′,6′-dimethylbiphenyl-3-carboxylate was pre-
pared from the obtained crystals by a method similar to that described
for 14, step 2 in 85% yield as colorless crystals; mp 180 °C. 1H NMR
(CDCl3) δ 1.99 (s, 6H), 2.30−2.45 (m, 2H), 2.45−2.59 (m, 2H),
2.88−3.02 (m, 2H), 3.37−3.53 (m, 2H), 3.92 (s, 3H), 4.63−4.72 (m,
1H), 6.69 (s, 2H), 7.33 (dt, J = 7.6, 1.4 Hz, 1H), 7.50 (t, J = 7.6 Hz,
1H), 7.83 (t, J = 1.6 Hz, 1H), 8.02 (dt, J = 7.6, 1.4 Hz, 1H). MS m/z
389 (M + H)+. HPLC purity (220 nm) 98.6%. Step 3: To a solution of
the obtained crystals (0.128 g, 0.33 mmol) in THF (2 mL) was added
gradually LiAlH4 (80%, 15.7 mg, 0.33 mmol) at 0 °C. The mixture was
stirred for 1.5 h at 0 °C, followed by gradually addition of
Na2SO4·10H2O at the same temperature. After stirring at room
temperature for 1 h, the mixture was filtered through a pad of Celite.
The filtrate was concentrated to afford 5c (0.111 g, 93%) as a colorless
(CDCl3) δ 2.13 (s, 3H), 2.27 (s, 3H), 2.53 (s, 3H), 6.53 (s, 1H), 10.23
(s, 1H), 12.29 (s, 1H). MS m/z 165 (M + H)+.
2,3,5,6-Tetramethylphenol (41). Compound 40 (6.58 g, 40.1
mmol) was hydrogenated on 10% Pd/C (1.0 g) in MeOH (120 mL)
under hydrogen atmosphere (balloon pressure) at room temperature
for 22 h. The catalyst was removed by filtration, and the filtrate was
concentrated to give 41(5.83 g, 97%). An analytical sample was
1
obtained as colorless plates (MeOH). H NMR (CDCl3) δ 2.14 (s,
6H), 2.22 (s, 6H), 4.59 (s, 1H), 6.60 (s, 1H). MS m/z 151 (M + H)+.
Anal. Calcd for C10H14O: C, 79.96; H, 9.39. Found: C, 80.02; H, 9.42.
4-Bromo-2,3,5,6-tetramethylphenol (38b). To a suspension of
41 (5.10 g, 34.0 mmol) in AcOH (90 mL) was added dropwise a
solution of Br2 (1.98 mL, 38.6 mmol) in AcOH (30 mL) at room
temperature, and the mixture was stirred at room temperature for 5 h.
The mixture was concentrated, and the residue was diluted with
AcOEt, washed sequentially with Na2S2O3 aqueous solution and brine,
dried over anhydrous MgSO4, and concentrated to give 38b (6.48 g,
83%). An analytical sample was obtained as off-white crystals
1
(petroleum ether). H NMR (CDCl3) δ 2.23 (s, 6H), 2.40 (s, 6H),
4.59 (s, 1H). MS m/z not detected.
4-Bromo-2-fluoro-3,5-dimethylphenol (38c). A mixture of 34
(2.00 g, 9.95 mmol) and N-fluoropyridinium triflate (6.15 g, 24.9
mmol) in 1,2-dichloroethane (20 mL) was stirred at reflux for 7 h. The
mixture was quenched with 1 M Na2S2O3 aqueous solution and
extracted with AcOEt. The extract was washed sequentially with water
and brine, dried over anhydrous MgSO4, and concentrated. The
residue was purified by silica gel column chromatography (AcOEt/
hexane = 0:100−30:70) to afford 38c (0.790 g, 36%) as colorless
crystals. 1H NMR (CDCl3) δ 2.29−2.36 (m, 6H), 5.04 (d, J = 4.0 Hz,
1H), 6.79 (d, J = 9.0 Hz, 1H). MS m/z not detected.
1
solid. H NMR (CDCl3) δ 1.76 (t, J = 5.6 Hz, 1H), 2.00 (s, 6H),
3′-Chloro-4′-hydroxy-2′,6′-dimethylbiphenyl-3-carbalde-
hyde (42d). To a solution of 31 (11.3 g, 50.0 mmol) in DMF (50
mL) was added gradually NCS (6.68 g, 50.0 mmol) at 0 °C, and the
mixture was stirred at room temperature for 13 h. The mixture was
heated to 50 °C, and stirred for 3 h. To the mixture was added NCS
(1.34 g, 10.0 mmol), and the mixture was stirred at 50 °C for 3 h. To
the mixture was added NCS (0.668 g, 5.00 mmol), and the resulting
mixture was stirred at 50 °C for 1 h. The mixture was poured into
water and extracted with AcOEt. The extract was washed with brine,
dried over anhydrous MgSO4, and concentrated. The residue was
purified by silica gel column chromatography (AcOEt/hexane = 5:95−
40:60) to give crystals. Recrystallization from heptane/AcOEt gave
2.29−2.44 (m, 2H), 2.44−2.58 (m, 2H), 2.87−3.02 (m, 2H), 3.37−
3.53 (m, 2H), 4.63−4.70 (m, 1H), 4.74 (d, J = 5.6 Hz, 2H), 6.68 (s,
2H), 7.05 (dt, J = 7.4, 1.5 Hz, 1H), 7.12 (s, 1H), 7.31−7.38 (m, 1H),
7.42 (t, J = 7.4 Hz, 1H). MS m/z 343 (M − 18 + H)+. HPLC purity
(220 nm) 97.1%.
3,5-Diethylphenol (37). A mixture of 4-ethylphenol (36) (25.7 g,
210 mmol) and AlCl3 (62.5 g, 469 mmol) was stirred under nitrogen
atmosphere at 115 °C for 4 h. The reaction mixture was cooled to 60
°C, poured onto crushed ice, and extracted with AcOEt. The extract
was washed with brine, dried over anhydrous MgSO4, and
concentrated. The residue was purified by silica gel column
chromatography (AcOEt/hexane = 0:100−25:75) to give 37 (12.3
1
42d (8.47 g, 65%) as colorless crystals; mp 85−86 °C. H NMR
1
g, 78%) as a reddish-brown oil. H NMR (CDCl3) δ 1.21 (t, J = 7.7
(CDCl3) δ 1.95 (s, 3H), 2.05 (s, 3H), 5.61 (s, 1H), 6.84 (s, 1H),
7.36−7.42 (m, 1H), 7.57−7.66 (m, 2H), 7.85−7.91 (m, 1H), 10.06 (s,
1H). MS m/z 261 (M + H)+. Anal. Calcd for C15H13ClO2: C, 69.10;
H, 5.03. Found: C, 69.16; H, 4.97.
Hz, 6H), 2.58 (q, J = 7.7 Hz, 4H), 4.66 (s, 1H), 6.49−6.52 (m, 2H),
6.60−6.63 (m, 1H). MS m/z 151 (M + H)+.
4-Bromo-3,5-diethylphenol (38a). To a solution of 37 (3.00 g,
20.0 mmol) in MeOH (30 mL) was added n-Bu4NBr3 (9.64 g, 20.0
mmol) at room temperature, and the mixture was stirred for 1 h. After
evaporation of the solvent, the residue was diluted with water and
extracted with AcOEt. The extract was washed with brine, dried over
anhydrous MgSO4, and concentrated. The residue was purified by
silica gel column chromatography (AcOEt/hexane = 0:100−25:75) to
give 38a (3.28 g, 72%). An analytical sample was obtained as colorless
crystals (heptane). 1H NMR (CDCl3) δ 1.21 (t, J = 7.6 Hz, 6H), 2.73
(q, J = 7.6 Hz, 4H), 4.65 (s, 1H), 6.59 (s, 2H). MS m/z not detected.
Anal. Calcd for C10H13BrO: C, 52.42; H, 5.72. Found: C, 52.22; H,
5.66.
3′,5′-Dichloro-4′-hydroxy-2′,6′-dimethylbiphenyl-3-carbal-
dehyde (42e). To a solution of 31 (11.3 g, 50.0 mmol) in DMF (50
mL) was added gradually NCS (13.4 g, 100 mmol) at 0 °C, and the
mixture was stirred at room temperature for 14 h. The mixture was
heated to 50 °C, and the mixture was stirred for 2 h. The mixture was
poured into water and extracted with AcOEt. The extract was washed
with brine, dried over anhydrous MgSO4, and concentrated. The
resulting crystals were triturated with heptane/AcOEt to give 42e
(8.88 g, 60%) as colorless crystals; mp 116−117 °C. 1H NMR
(CDCl3) δ 2.03 (s, 6H), 6.00 (s, 1H), 7.35−7.40 (m, 1H), 7.60−7.66
(m, 2H), 7.88−7.94 (m, 1H), 10.06 (s, 1H). MS m/z 294 (M + H)+.
Anal. Calcd for C15H12Cl2O2: C, 61.04; H, 4.10. Found: C, 60.91; H,
3.98.
Ca Influx Activity of CHO Cells Expressing Human GPR40/
FFA1 (FLIPR Assay). CHO dhfr cells stably expressing human
GPR40/FFA1 (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 Ca2+
concentration after addition was monitored by FLIPR Tetra system
2-Hydroxy-3,4,6-trimethylbenzaldehyde (40). To a solution of
2,3,5-trimethylphenol (39) (13.6 g, 100 mmol) in CH2Cl2 (20 mL)
was added dropwise TiCl4 (41.7 g, 220 mmol) under nitrogen
atmosphere at 0 °C over 0.5 h, and the mixture was stirred at 0 °C for
1 h. To the mixture was added dropwise dichloromethyl methyl ether
(11.5 g, 100 mmol), and the mixture was stirred at 0 °C for 6 h. The
mixture was quenched with saturated NH4Cl aqueous solution and
extracted with CH2Cl2. The organic layer was washed sequentially with
diluted HCl aqueous solution, NaHCO3 aqueous solution and brine,
dried over anhydrous MgSO4, and concentrated. The residue was
purified by silica gel column chromatography (AcOEt/hexane = 5:95−
1
50:50) to give 40 (6.58 g, 40%) as pale-brown crystals. H NMR
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Journal of Medicinal Chemistry
Article
(Molecular Devices) for 90 s. The agonistic activities of test
compounds and γ-linolenic acid on human GPR40/FFA1 were
expressed as [(A − B)/(C − B)] × 100 (increase of the intracellular
Ca2+ concentration (A) in test compounds-treated cells, (B) in vehicle-
treated cells, and (C) in 10 μM γ-linolenic acid-treated cells). EC50
values and 95% confidence intervals of each compound were obtained
with Prism 5 software (GraphPad).
centrifugation, the supernatant was diluted and centrifuged again. The
compound concentrations in the supernatant were measured by LC/
MS/MS.
Oral Glucose Tolerance Test (OGTT). 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 Limited. Female Wistar fatty WF
rats and Male GK rats were obtained from Takeda Rabics Limited
(Hikari, Japan). They were fed the commercial diet CE-2 (Clea Japan
Co.) and tap water ad libitum. Female WF (12−17 weeks old) and
male GK (41 weeks old) rats were fasted overnight and orally given
vehicle (0.5% methylcellulose aqueous solution) or compounds
(suspended in vehicle). All animals received an oral glucose load (1
g/kg) 1 or 4 h after drug administration. Blood samples were collected
from tail vein before drug administration (pre) and just before glucose
load (time 0) and 10, 30, 60, and 120 min after glucose load. Plasma
glucose and plasma insulin levels were measured by Autoanalyzer 7080
(Hitachi, Japan) and radioimmunoassay (Millipore, USA), respec-
tively. Statistical differences were analyzed by the Student’s t test or the
Aspin−Welch test. In the dose-dependent study, statistical signifi-
cances versus vehicle control were assessed by the one-tailed Williams
test or the Shirley−Williams test.
Preparation of CHO Membrane for GPR40/FFA1 Receptor
Binding Assay. Cell lines stably expressing human GPR40/FFA1 and
rat GPR40/FFA1 were used for the experiments. Each cell was
cultured in Minimum Essential Medium Alpha (MEM-Alpha,
Invitrogen) supplemented with 10% dialyzed Fetal-Bovine-Serum
(dialyzed FBS, Thermo Trace Ltd.), 100 unit/mL penicillin, and 100
unit/mL streptomycin in 5% CO2/95% air atmosphere at 37 °C. Cells
were harvested at confluence in Dulbecco’s Phosphate-Buffered-Saline
(D-PBS, Invitrogen) containing 1 mM EDTA and centrifuged. Cells
were homogenized in ice-cold membrane preparation buffer (50 mM
Tris-HCl (pH 7.5), 5 mM EDTA, 0.5 mM PMSF (Wako), 20 μg/mL
leupeptin, 0.1 μg/mL pepstatin A, 100 μg/mL Phosphoramidon,
Peptide Institute, Inc.) and centrifuged (700g, 10 min, 4 °C). The
supernatant was filtered through 40 μm Cell Strainer (BD Falcon) and
ultracentrifuged (100000g, 1 H 4 °C) with Optima L-100 XP
Ultracentrifuge (Beckman Coulter). The precipitation was suspended
in the same buffer, and the protein concentration was determined with
the BCA Protein assay reagent (Pierce) following membrane
solubilization with 0.1% SDS and 0.1 M NaOH aqueous solution.
The membrane suspension was stored at −80 °C until receptor
binding assay.
GPR40/FFA1 Receptor Binding Assay. The frozen cell
membranes were resuspended in ice-cold assay buffer (25 mmol/L
Tris-HCl (pH7.5), 5 mmol/L EDTA, 0.5 mmol/L PMSF, 20 μg/mL
leupeptin, 0.1 μg/mL pepstatin A, 0.05% CHAPS (Wako), and 0.2%
fatty-acid-free BSA (Sigma)) and used for receptor binding assay. To
determine the Kd values of 3-[4-({2′,6′-dimethyl-6-[(4-[3H])-
phenylmethoxy]biphenyl-3-yl}methoxy)phenyl] propanoic acid
(Amersham Biosciences) for human and rat GPR40/FFA1, binding
assays were performed in the presence of various concentrations of the
labeled ligand. After incubation at room temperature for 90 min, the
membranes were harvested using GF/C filter plates (MILLIPORE)
and washed with ice-cold 50 mmol/L Tris-HCl (pH7.5) using a
FilterMate Harvester (PerkinElmer). The membrane-associated radio-
activities were counted using a TopCount liquid scintillation counter
(PerkinElmer). Nonspecific binding was defined as binding in the
presence of 10 μmol/L of the unlabeled ligand. To determine the
binding affinities of test compounds to human and rat GPR40/FFA1,
binding assays were performed in the presence of both various
concentrations of test compounds and 2 nmol/L or 6 nmol/L of the
labeled ligand. The 50% inhibitory concentrations (IC50 values) of test
compounds for the labeled ligand were calculated using nonlinear
regression analysis in GraphPad Prism 3.0 (GraphPad Software). Ki
values were converted as Ki = IC50/{1 + (the concentration of the
labeled ligand)/Kd}.
Caspase-3/7 Activity Assay. HepG2 cells were cultured at 37 °C,
5% CO2 in DMEM supplemented with 10% fetal bovine serum, 50
IU/mL penicillin, and 50 μg/mL streptomycin. Cells were seeded at 2
× 104cells/well in a 96-well white plate (Costar) and cultured with test
compounds in DMEM supplemented with 0.5% fetal bovine serum, 2
mM L-glutamine, 1 mM sodium pyruvate, 50 IU/mL penicillin, and 50
μg/mL streptomycin for 1 day. Caspase-3/7 activity was measured by
using Caspase-GloTM 3/7 assay Kit (Promega) according to the
manufacturer’s instruction. Caspase-3/7 activity was calculated (n = 3)
to the following. Caspase-3/7 activity (%) = (RLU of compound −
RLU of 1% DMSO)/(RLU of 30 μM staurosporine − RLU of 1%
DMSO) × 100.
Pharmacokinetic Analysis in Rat Cassette Dosing. Test
compounds were suspended in 0.5% methylcellulose aqueous solution
and administered as a cassette dosing to fasted rats. After oral
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
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic procedures and spectroscopic data of final com-
pounds and intermediates not described in the manuscript.
Methods and instrumentation used to obtain the X-ray crystal
structure of compound 16. This material is available free of
charge via the Internet at http://pubs.acs.org.
Accession Codes
CDCC code: CDCC 837682.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +81-6-6300-6553. Fax: +81-6-6300-6251. E-mail:
tsuneo.yasuma@takeda.com.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Shuji Kitamura, Osamu Ujikawa, Nobuyuki
Amano, Dr. Yoshihiko Tagawa, Dr. Hiroshi Miki, and Junichi
Sakamoto for helpful discussions, Keiko Higashikawa and
Yoichi Nagano for X-ray crystallographic analysis, Masayuki
Yamashita, Katsuhiko Miwa, Miki Ueda, Katsuaki Oda, Shozo
Yamamoto, and Mitsutaka Tanaka for preparations of the key
intermediate and analyses of enantiomeric excess, and Dr. Brian
G. Shearer, Dr. Lydia Hart, and Dr. Tsuyoshi Maekawa for
proofreading this manuscript.
ABBREVIATIONS USED
■
GPR40, G protein-coupled receptor 40; FFA1, free fatty acid
receptor 1; GPCR, G protein-coupled receptor; FFA, free fatty
acid; GSIS, glucose-stimulated insulin secretion; OGTT, oral
glucose tolerance test; PK, pharmacokinetic; FLIPR, fluoro-
metric imaging plate reader; BSA, bovine serum albumin;
ADME-Tox, absorption, distribution, metabolism, excretion,
and toxicology; C1h, plasma concentration at 1 h after
administration; C4h, plasma concentration at 4 h after
administration; Cmax, maximum plasma concentration; Tmax
,
time of maximum plasma concentration; AUC, area under
curve; GK, Goto-Kakizaki
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Journal of Medicinal Chemistry
Article
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