Discovery of phenylpropanoic acid derivatives containing polar functionalities as potent and orally bioavailable G protein-coupled receptor 40 agonists for the treatment of type 2 diabetes

Article

pubs.acs.org/jmc

Discovery of Phenylpropanoic Acid Derivatives Containing Polar

Functionalities as Potent and Orally Bioavailable G Protein-Coupled

Receptor 40 Agonists for the Treatment of Type 2 Diabetes

Satoshi Mikami,* Shuji Kitamura, Nobuyuki Negoro, Shinobu Sasaki, Masami Suzuki, Yoshiyuki Tsujihata,

Takeshi Miyazaki, Ryo Ito, Nobuhiro Suzuki, Junichi Miyazaki, Takashi Santou, Naoyuki Kanzaki,

Miyuki Funami, Toshimasa Tanaka, Tsuneo Yasuma, and Yu Momose

Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd., 26-1 Muraoka-higashi, 2-chome, Fujisawa, Kanagawa

251-8555, Japan

ABSTRACT: As part of a program to identify potent GPR40 agonists with drug-like properties suitable for clinical development,

the incorporation of polar substituents was explored with the intention of decreasing the lipophilicity of our recently disclosed

phenylpropanoic acid derivative 1. This incorporation would allow us to mitigate the cytotoxicity issues observed with compound

1 and enable us to move away from the multifunctional free fatty acid-like structure. Substitutions on the 2′,6′-dimethylbiphenyl

ring were initially undertaken, which revealed the feasibility of introducing polar functionalities at the biphenyl 4′-position.

Further optimization of this position and the linker led to the discovery of several 4′-alkoxybiphenyl derivatives, which showed

potent GPR40 agonist activities with the best balance in terms of improved cytotoxicity proﬁles and favorable pharmacokinetic

properties. Among them, 3-{2-ﬂuoro-4-[({4′-[(4-hydroxy-1,1-dioxidotetrahydro-2H-thiopyran-4-yl)methoxy]-2′,6′-dimethylbi-

phenyl-3-yl}methyl)amino]phenyl}propanoic acid (35) exhibited a robust plasma glucose-lowering eﬀect and insulinotropic

action during an oral glucose tolerance test in rats with impaired glucose tolerance.

glucagon-like peptide 1 analogues¹¹have been recently

developed.

Free fatty acids (FFAs) are well-known to amplify glucose-

stimulated insulin secretion (GSIS) from pancreatic β-cells.

INTRODUCTION

Type 2 diabetes mellitus (T2DM) is a disease characterized by

■

defects in insulin secretion from pancreatic β-cells and/or

insulin resistance in target tissues of insulin.¹⁻³Although the

Before the discovery of G protein-coupled receptor 40

relative importance of diminished insulin secretion and insulin

resistance varies among patients with T2DM, many studies

(GPR40), it was postulated that these eﬀects were mainly

mediated by the active metabolites of FFAs.¹²⁻¹⁵GPR40 is

indicate that impaired insulin secretion caused by β-cell

highly expressed in pancreatic β-cells and rodent insulin-

dysfunction is frequently present in patients.⁴In the clinical

secreting cell lines and is activated by naturally occurring

medium- to long-chain FFAs.¹⁶⁻¹⁸The mechanism of action of

management of T2DM, oral insulinotropic agents such as

sulfonylureas have been widely prescribed as a mono- or

GPR40 remains to be fully elucidated; however, several reports

combination therapy.^5,6However, because sulfonylureas act to

have shown that GPR40 is mainly coupled with the G protein

α-subunit of the Gq family (Gαq).^16,17,19Generally, the

stimulate continuous insulin release independently of blood

glucose levels, they often elicit hypoglycemia^7,8and possibly

activation of Gαq protein-coupled receptors results in increased

promote the deterioration of islet function, resulting in

attenuated eﬃcacy.^9,10To complement the major drawbacks

of sulfonylurea agents currently in use, research exploring novel

insulin secretagogues with distinct mechanism of action has

attracted a great deal of attention in the pharmaceutical

industry and in academia. As a result, novel drugs that

potentiate insulin secretion in a glucose-dependent manner,

such as dipeptidyl peptidase IV inhibitors and incretin hormone

phospholipase C (PLC) activity and, in turn, inositol 1,4,5-

triphosphate (IP₃)-mediated intracellular calcium mobilization

and protein kinase C (PKC) activation, which are related to

enhanced insulin secretion in pancreatic β-cells.^20,21In fact, it

has been reported that endogenous ligands and small molecule

Received: November 29, 2011

Published: March 19, 2012

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a

Figure 1. Structure of recently disclosed GPR40 agonist 1 and its proﬁles. Binding aﬃnities for human and rat GPR40 receptors. All values are

b

average of n = 2 or 3 measurements in the presence of 0.2% BSA. Fluorometric imaging plate reader (FLIPR) functional assay for human GPR40

receptor. The value is an average of n = 3 measurements in the presence of 0.1% BSA. The 95% conﬁdence intervals are shown in parentheses.

^cIntracellular ATP content (%) at 30 μM relative to untreated control. Lowered ATP content relative to untreated control (100%) indicates a

decrease in cell viability. ^dCaspase-3/7 activity (%) at 30 μM relative to the response of the reference compound (staurosporine), which was set to

100%. A larger value is interpreted as a higher risk of apoptotic cytotoxicity. ^eThe Log D value was determined at pH 7.4 as previously described.⁵⁵

a

Scheme 1. Synthesis of 2′,6′-Dimethylbiphenyl Derivatives 10−13

a

Reagents and conditions: (a) Pd(PPh₃)₄, Cs₂CO₃, toluene, EtOH or MeOH, reﬂux, 45−87%; (b) benzyl bromide, K₂CO₃, DMF, 70 °C, 46%; (c)

(i) (CF₃SO₂)₂O, i-Pr₂NEt, CH₂Cl₂, 0 °C, 87%, (ii) 2, Pd(PPh₃)₄, K₃PO₄, DME, 80 °C, 87%; (d) H₂(balloon pressure), 10% Pd/C, MeOH, THF,

rt, 91%; (e) MeI, K₂CO₃, DMF, 50 °C, 93%; (f) LAH, THF, 0 °C or rt, 43−99%; (g) NaBH₄, EtOH, 0 °C, 88%−quant; (h) methyl 3-(4-

hydroxyphenyl)propanoate, ADDP, n-Bu₃P, THF, rt, 71−87%; (i) ethyl 3-(2-ﬂuoro-4-hydroxyphenyl)propanoate, ADDP, n-Bu₃P, toluene, rt, 87%;

(j) KOH or NaOH aq, THF, MeOH or EtOH, rt, 91−98%.

GPR40 agonists increase intracellular calcium concentration via

GPR40 and lead to GSIS in pancreatic β-cells.^{16−18,22−27}In

addition, several reports have shown that synthetic GPR40

agonists stimulate insulin secretion in a glucose-dependent

manner and correct impaired glucose tolerance in a single dose

in rodents, suggesting that GPR40 agonists would be novel and

potential insulinotropic drugs.²⁶⁻³⁰

design, which culminated in the identiﬁcation of the potent

phenylpropanoic acid derivative 1 as a novel GPR40 agonist.³⁰

Although compound 1 displayed not only favorable agonist

activity (EC₅₀= 30 nM) in a ﬂuorometric imaging plate reader

(FLIPR)-based functional assay but also signiﬁcant glucose-

lowering action during an oral glucose tolerance test in diabetic

rat models, it exhibited cytotoxicity potential in human

hepatocellular carcinoma HepG2 cells, as evidenced by both

decreased intracellular ATP content and caspase-3/7 induction

activity as shown in Figure 1. It is generally recognized that

In our recent publications,^29,30we described our research

eﬀorts that began with a screening hit from our in-house library

of compounds selected on the basis of ligand-based drug

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a

Scheme 2. Synthesis of 4′-Alkoxybiphenyl Derivatives 22−35

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Scheme 2. continued

a

Reagents and conditions: (a) (3-formylphenyl)boronic acid, Na₂CO₃, Pd(PPh₃)₄, toluene, EtOH or DME, H₂O, 80 °C, 66−83%; (b)

EtOCH₂CH₂Cl, K₂CO₃, KI, DMF, 80 °C, 89%; (c) TBSCl, imidazole, rt, 77%; (d) (i) NaOH, MeOH, rt, (ii) Cu, 2-bromo-6-methylpyridine, 185

°C, 74% (2 steps); (e) NaH, 1-oxa-6-thiaspiro[2.5]octane 34, DMF, 0−80 °C, 44%; (f) 3,4-dihydro-2H-pyran, PPTS, CH₂Cl₂, rt, 77%; (g) NaBH₄,

THF, DME, or MeOH, 0 °C, 83%−quant; (h) ethyl 3-(2-ﬂuoro-4-hydroxyphenyl)propanoate or 38a or 38b, ADDP, n-Bu₃P, THF or toluene, rt,

75−94%; (i) methyl 3-(4-hydroxyphenyl)propanoate or 38b, DEAD, Ph₃P, THF, rt, 37%; (j) p-TsOH·H₂O, MeOH, rt, 88%; (k) TBAF, THF, rt,

79−99%; (l) ROH, DEAD or DIAD, Ph₃P, THF, rt, 51−100%; (m) ROH, ADDP, n-Bu₃P, THF, rt, 64−67%; (n) 1 M NaOH aq, THF, MeOH, rt,

49−91%; (o) m-CPBA, CH₂Cl₂or EtOAc, 0 °C to rt, 47−91%; (p) AcOH, concd H₂SO₄, H₂O, 90 °C, 61%; (q) (i) m-CPBA, EtOAc, 0 °C to rt, (ii)

HSCH₂CO₂H, LiOH·H₂O, DMF, rt, (iii) TFA, toluene, rt, (iv) MsOH, EtOAc, rt, 47−60% (4 steps); (r) HCl, EtOAc, rt, 83%; (s) HSCH₂CO₂H,

LiOH·H₂O, DMF, rt.

reducing the potential for cytotoxicity of drug candidates during

the lead optimization process is a prudent strategy to minimize

the potential risk for adverse eﬀects in animals and

humans.³¹⁻³⁴Therefore, we sought to minimize the cytotox-

icity liability observed for this compound series.

that less polar, more lipophilic compounds have an increased

likelihood of promiscuous binding to multiple targets, resulting

in adverse toxicological outcomes in vivo.⁴⁴⁻⁴⁶We postulated

that the cytotoxicity identiﬁed in compound 1 is attributable to

its highly lipophilic character. Therefore, our chemistry eﬀorts

were directed toward decreasing the lipophilicity of compound

1. This strategy would also allow structural diﬀerentiation

between GPR40 agonists and FFAs that are known to exert

pleiotropic physiological eﬀects and cellular toxicity⁴⁷⁻⁵⁴and

consequently lead to the generation of selective GPR40

agonists with more drug-like physicochemical properties.

Herein we describe our research eﬀorts toward identifying

positions that tolerate polar functionalities and the subsequent

optimization of the 4′-position on the biphenyl ring and the

linker that connects the biphenyl ring to the phenylpropanoic

acid moiety. These eﬀorts led us to discover several 4′-

alkoxylbiphenyl derivatives as potent and bioavailable GPR40

agonists, as represented by 3-{2-ﬂuoro-4-[({4′-[(4-hydroxy-1,1-

dioxidotetrahydro-2H-thiopyran-4-yl)methoxy]-2′,6′-dimethyl-

biphenyl-3-yl}methyl)amino]phenyl}propanoic acid (com-

pound 35; bold numbers in parentheses indicate speciﬁc

compounds illustrated in the schemes). In addition, this new

series successfully overcame the potential cytotoxicity issues

associated with compound 1 and demonstrated the compelling

pharmacological proﬁles that may help demonstrate that

GPR40 agonists might constitute an attractive therapeutic

approach to the treatment of T2DM.

To evaluate the potential cytotoxicity of the compounds, we

employed a combination of two assays that focus on diﬀerent

aspects of cell death. The two experiments measured diﬀerent

end points: intracellular ATP content as an indicator of viable

cells and caspase-3/7 induction activity as a marker for

apoptotic cells. In these assays, precultured HepG2 cells were

exposed to test compounds. After 24 h of incubation, the

intracellular ATP content and caspase-3/7 induction activity

were measured using ATPliteTM-M and Caspase-GloTM 3/7

assay kit, respectively (for more details, refer to the

Experimental Section). ATP assays were expressed as a

percentage of viable cells compared to untreated controls.

Caspase-3/7 induction activity was also expressed as a

percentage relative to the response of the reference compound

(staurosporine), which was set to 100%.

In the meantime, extensive searches for small molecule

GPR40 agonists have been recently pursued in pharmaceutical

companies and academia;³⁵⁻⁴³however, the synthetic GPR40

agonists reported in the literature to date have been often

plagued by high lipophilicity. This is likely due in part to the

fact that endogenous GPR40 ligands are FFAs; the ligand-

binding site in the receptor might therefore be hydrophobic in

nature. Indeed, compound 1, designed based on the structural

features of FFAs, has a high Log D value of 4.19. It is reported

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a

Scheme 3. Synthesis of 4-Aminophenylpropanoic Acid Moieties 38 and 39

a

Reagents and conditions: (a) ethyl acrylate or tert-butyl acrylate, Pd(OAc)₂, (o-tol)₃P, i-Pr₂NEt, DMF, 110 °C, 80−96%; (b) H₂, 10% Pd/C, EtOH,

rt, 79−98%; (c) NsCl, pyridine, rt, 76−77%.

a

Scheme 4. Synthesis of Compound 41

a

Reagents and conditions: (a) (i) 38a, MS4A, toluene, rt, (ii) H₂, 10% Pd/C, EtOH, rt, 83%; (b) 1 M NaOH aq, THF, EtOH, rt, 99%; (c) HCl,

AcOEt, rt, 97%.

Starting from a Suzuki−Miyaura cross-coupling reaction

between 4-bromo-3,5-dimethylphenol (14) and (3-

formylphenyl)boronic acid, phenol 15 was then alkylated

with 1-chloro-2-ethoxyethane or protected with a tert-

butyldimethylsilyl (TBS) group to provide 17a or 17e.

Alternatively, copper-mediated arylation of phenol 14 with 2-

bromo-6-methylpyridine, alkylation of 14 with 1-oxa-6-

thiaspiro[2.5]octane, or protection of phenol 14 as the

tetrahydropyranyl (THP) ether were carried out prior to

palladium-mediated cross-coupling reactions to aﬀord 17b, 17c,

and 17d. The resulting aldehydes 17b−d were then converted

to 19a−d and 19g in an analogous fashion to that described in

Scheme 1. Subsequently, 19d and 19e−f were deprotected with

tetrabutylammonium ﬂuoride (TBAF), or under acidic

conditions, to give phenols 20a and 20b−c, respectively,

which were then coupled with a range of alcohols under

Mitsunobu conditions. Finally, the desired compounds 22−35

were obtained from 19a−c, 19g, or 21a−i via the following sets

of conditions: methods A and C, simple hydrolysis of the esters

to carboxylic acids; method B, oxidation of sulﬁdes to the

corresponding sulfones prior to hydrolysis; method D,

sequential oxidation to the sulfone, removal of the 2-

nitrobenzenesulfonyl (nosyl = Ns) groups, hydrolysis to the

carboxylic acid, and acid salt formation; method E, hydrolysis to

the carboxylic acid, followed by HCl salt formation; method F,

sequential oxidation to the sulfone, removal of the nosyl group,

and hydrolysis to the carboxylic acid.

CHEMISTRY

■

2′,6′-Dimethylbiphenyl derivatives 10−13 with a substituent on

the central phenyl ring were synthesized following the route

illustrated in Scheme 1. The synthesis of biphenyl intermediates

8a−d was straightforward, but due to the limitations of

commercially available starting materials, three diﬀerent

approaches (route A−C) were required utilizing Suzuki−

Miyaura cross-coupling reactions of the appropriate phenyl

bromides 3 or 4 or the triﬂate of phenol 7 with the

corresponding boronic acids 2, 5a, or 5b as the key steps.

The preparation of the 5-substituted biphenyl intermediate 8c

began with an alkylation of phenol 6 with benzyl bromide

(route C). This alkylation provided a mixture of mono- and

disubstituted products, which was successfully separated by

silica gel chromatography to aﬀord phenol 7. Conversion of

phenol 7 to the corresponding triﬂate and the subsequent

Suzuki−Miyaura cross-coupling reaction with boronic acid 2

proceeded smoothly to yield the desired coupling product.

Elaboration into the intermediate 8c was accomplished in an

additional two-step sequence of reactions, involving depro-

tection of the benzyl group by catalytic hydrogenation followed

by methylation of the phenol. Next, the intermediates 8a−d

underwent reduction with lithium aluminum hydride (LAH) or

sodium borohydride (NaBH₄) to give alcohols 9a−d, which

were then treated with methyl 3-(4-hydroxyphenyl)propanoate

or ethyl 3-(2-ﬂuoro-4-hydroxyphenyl)propanoate³⁰under

Mitsunobu conditions followed by hydrolysis of the ester

moieties to deliver the ﬁnal products 10−13.

Preparation of 4-aminophenylpropanoic acid esters 38a−b

was accomplished by a Mizorogi−Heck reaction of bromo-

benzene 36 with ethyl acrylate followed by hydrogenation of

A series of 4′-alkoxybiphenyl derivatives 22−35 were

prepared following the procedures depicted in Scheme 2.

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Table 1. In Vitro Proﬁles of Compounds with Substitutions on 2′,6′-Dimethylbiphenyl Ring

a

binding

h-FLIPR

a

compd

X

R or R′

2-Me

h-K_i(nM)

r-K_i(nM)

EC₅₀(nM)

Log D

10

11

12

13

22

23

24

25

26

42

1

H

F

58

520

99

47

25

16

15

23

12

46

32

30

10000

120

87

40 (24−68)

4.22

4.04

4.16

3.38

3.83

4.61

5.08

3.89

4.14

4.04

4.19

b

4-MeO

ND

b

5-MeO

ND

H

F

6-MeO

20 (12−33)

12 (7.7−19)

15 (9.0−25)

14 (8.5−22)

13 (8.7−19)

4.5 (2.6−7.7)

22 (10−48)

30 (13−69)

4′-MeO

6.2

4′-c-PrCH₂O

4′-BnO

5.6

6.7

4′-EtOCH₂CH₂O

H

10

4.5

H

F

27

H

36

a

b

Refers to Figure 1. Not determined.

Table 2. In Vitro Activities and Cytotoxicity Proﬁles of 4′-Alkoxybiphenyl Derivatives with O Linker

a

b

c

Refers to Figure 1. Refers to Figure 1. Intracellular ATP content (%) at 30 μM relative to untreated control. Values in parentheses represent

d

intracellular ATP content (%) at 10 μM. Caspase-3/7 activity (%) at 30 μM relative to the response to the reference compound (staurosporine),

which was set to 100%. Values in parentheses represent caspase-3/7 activity (%) at 10 μM. Refers to Figure 1. HCl salt.

e

f

the oleﬁn moieties in 37a−b. Nosylation of the anilines 38a

and 38b aﬀorded 39a and 39b, respectively (Scheme 3).

Compound 41, possessing an amino linker, was readily

prepared in three steps from the intermediate 17a as illustrated

in Scheme 4. Aldehyde 17a was subjected to reductive

amination with 4-aminophenylpropanoic acid ethyl ester 38a

to furnish the coupled product 40, which was saponiﬁed and

then transformed into its hydrochloride salt 41 for crystal-

lization.

the 2′,6′-dimethylbiphenyl ring on potency. Synthesized

compounds were ﬁrst evaluated for in vitro receptor aﬃnities

in a competitive binding assay in the presence of 0.2% bovine

serum albumin (BSA) to characterize the ligand’s ability to

displace a synthetic radioligand (3-[4-({2′,6′-dimethyl-6-

[(4-³H)phenylmethoxy]biphenyl-3-yl}methoxy)phenyl]-

propanoic acid)²⁹bound to human and rat GPR40 receptors

stably expressed on Chinese hamster ovary (CHO) cells.

Compounds showing potency comparable to or better than

compounds 1 and 42³⁰in the binding assay were then

measured for agonist activity against human GPR40 receptor

expressed in CHO cells by a FLIPR assay in the presence of

0.1% BSA. Table 1 summarizes the in vitro proﬁles of

RESULTS AND DISCUSSION

■

In our search for sites where polar functionalities would be

tolerated, we initially investigated the eﬀects of substitutions in

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compounds bearing a substituent at various positions in the

2′,6′-dimethylbiphenyl ring. Incorporation of a methoxy group

into the 4- or 5-position (11 and 12, respectively) led to a

signiﬁcant loss in binding aﬃnities against both the human and

rat receptors when compared with 42 and 1, respectively. These

potency losses could be attributed to the limited space in the

binding pocket of the GPR40 receptor around the 4- and 5-

positions of the biphenyl ring. In contrast, although the 6-

methoxy derivative 13 displayed somewhat weaker activity

against the rat receptor (K_i= 87 nM), the 2-methyl derivative

10 and 13 maintained their potency against human receptors.

The observation that potency was preserved with the

introduction of a methyl group at the 2-position indicates

that this modiﬁcation has little impact on the overall binding

conformation of compound 42. On the other hand, methoxy

substitution at the 4′-position in compound 22 was shown to

improve binding aﬃnities for human and rat GPR40 receptors.

In light of the enhanced binding aﬃnity and the preserved

agonist activity of compound 22, we next directed our attention

to variations of substituents at the 4′-position. More lipophilic

and bulkier substituents such as cyclopropylmethoxy or

benzyloxy groups (23 and 24) were also well tolerated.

Notably, the incorporation of a 2-ethoxyethoxy group (25),

characterized as a moderately polar substituent, restored both

the binding aﬃnities and agonist activity in a FLIPR assay. Our

previous study demonstrated that the introduction of a ﬂuorine

atom at the 2-position of the right-hand phenyl ring remarkably

improved pharmacokinetic proﬁles in terms of maximum

plasma concentration (C_max) and area under the curve

(AUC), with no loss in potency, although it was accompanied

by a slight increase in the lipophilicity of the molecule (ΔLog D

∼0.15).³⁰On the basis of these observations, the 2-

ﬂuorophenylpropanoic acid derivative 26 with a 2-ethoxyethyl

group at the 4′-position of the biphenyl ring was prepared and

proﬁled for its GPR40 activity (Table 1) and rat pharmaco-

kinetics (Table 3). Consistent with previous ﬁndings, the oral

absorption of the ﬂuoro compound 26 turned out to be better

than that of the nonﬂuoro compound 25. Surprisingly, in

comparison, compound 26 exhibited more potent binding and

agonist activity. Thus, preliminary structure−activity relation-

ships of substitutions on the 2′,6′-dimethylbiphenyl ring

revealed that polar functionalities could potentially be tolerated

at the 4′-position.

compromising binding and agonist activities. Moreover,

compounds 30 and 31 exhibited a propensity toward improved

cytotoxicity proﬁles based on ATP content, especially at 10 μM,

although those at 30 μM were still not improved. These results

encouraged us to incorporate an additional polar moiety into

the sulfone substituent to further decrease the Log D value.

Compound 32, combining the sulfone moiety and a tertiary

alcohol group, had a Log D value of 2.78 and exhibited

considerable improvement in cytotoxicity even at the 30 μM

concentration. Thus, the examination of various substituents at

the 4′-position uncovered not only the promising attributes of

sulfone groups toward lowering the lipophilicity of the

molecules but also the possibility that lowering Log D to less

than 3 could alleviate concerns about cytotoxicity. Pharmaco-

kinetic evaluation of this series in rats also revealed additional

advantageous aspects of the sulfone substituents. As can be seen

in Table 3, sulfone derivatives (30−32) exhibited excellent

Table 3. Pharmacokinetic Proﬁles for 4′-Alkoxybiphenyl

a

Derivatives

b

c

C_max

AUC_{0−8 h}

F

(%)

HLM

RLM

compd (ng/mL) (ng·h/mL)

(μL/min/mg) (μL/min/mg)

25

26

27

28

29

30

31

32

9.4

32.2

35.4

120.4

206.9

142.7

232.3

780.7

758.8

448.8

13.9

24.6

0

8

0

67.4

36.9

0

25.3

27.8

0

53.3

84.8

7

5

184.1

221.4

185.2

145.1

132.9

53.3

6

10

0

3

d

ND

33

34

35

80.3

129.7

207.7

433.2

689.0

54.5

78.1

2

0

2

0

109.9

41

42

1

28.0

5.3

77.7

2.0

24.1

0.9

0

28

69

34

39

86.0

249.0

21.5

a

Rat cassette dosing at 0.1 mg/kg, iv and 1 mg/kg, po (nonfasted).

b

Average of 3 rats. Metabolic stability in the human hepatic

microsome. Metabolic stability in rat hepatic microsome. Not

determined.

c

d

We next focused on installing a range of polar functionalities

at the biphenyl 4′-position with the aim of decreasing

compound lipophilicity, as measured by Log D, to a more

druggable range (preferably less than ca. 3)⁴⁶and of improving

the cytotoxicity proﬁles. The resulting in vitro activities,

cytotoxicity proﬁles and Log D values of 4′-alkoxybiphenyl

derivatives are summarized in Table 2. A wide variety of polar

substituents, represented by ether (27), heteroaryl (28), amide

(29), and sulfone (30, 31, and 32) moieties, were tolerated

fairly well in terms of potency, and no signiﬁcant diﬀerence in

binding aﬃnities between human and rat was observed in this

series. Introduction of pyran or pyridine functionality (27 and

28, respectively) was found to have little impact on the overall

Log D value, and these compounds exhibited signiﬁcant

cytotoxicity at 30 μM in both the ATP and caspase assays. In

addition, compound 29 maintained comparable activity despite

having a lower Log D value; however, its cytotoxicity proﬁle

remained unacceptable. It should be noted that installation of

sulfone substituents, as seen in compounds 30−32, oﬀered the

advantage of dramatically reducing the Log D value without

pharmacokinetic proﬁles characterized by high C_maxand AUC

compared with the nonsulfone derivatives 25−29 and the 4′-

unsubstituted biphenyl derivative 1. Given the fact that most of

the compounds were generally quite stable toward oxidative

metabolism in both human and rat liver microsomes, which do

not allow the evaluation of β-oxidation or conjugation

metabolism of the phenylpropanoic acid moieties, the superior

pharmacokinetic proﬁles noted with sulfone derivatives may

result from less propensity to β-oxidation and/or conjugation

metabolism as a result of the decreased lipophilicity of these

molecules. The putative major metabolic pathways from the

metabolite studies of compound 42 are believed to be β-

oxidation and conjugation metabolism of the acids.³⁰Our

previous extensive studies of the in vivo eﬃcacy of compounds

in an oral glucose tolerance test (OGTT) using diabetic rat

models demonstrated that the magnitude of the plasma

concentration, coupled with the in vitro activity of tested

compounds, were crucial determinants of in vivo eﬃcacy in this

series. Accordingly, improved pharmacokinetic parameters with

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Figure 2. Docking model of 31 inside a GPR40 homology model.

higher C_maxand AUC, as seen with the sulfone derivatives, were

anticipated to translate well into in vivo activity.

In an eﬀort to rescue the poor cytotoxicity proﬁles of

compounds 30 and 31, and also to further examine the

Computational modeling served to provide some spec-

ulations about the tolerance of diﬀerent functional groups at the

4′-position of the biphenyl ring for a wide range of substituents.

A representative compound 31 was docked into the putative

binding pocket of the GPR40 homology model (Figure 2). The

results suggested that compound 31 binds in a fashion similar

to that of TAK-875, recently reported from our group.²⁹In this

model, the sulfone substituent of compound 31 extends toward

the relatively polar region sandwiched between Ser8 (TM1)

and Lys259 (TM7), and each oxygen atom of the sulfone group

makes hydrogen bonds with Ser8 and Lys259. The other polar

side chains listed in Table 3 also contain hydrogen-bond

acceptors that could potentially form hydrogen bonds with Ser8

and/or Lys259. In addition, lipophilic substituents, such as

cyclopropylmethoxy and benzyloxy groups, could interact with

the hydrophobic residues (−CH₂−) of Lys259 and/or Ser8.

Although compound 31 still possessed a risk of cytotoxicity,

this compound was considered valuable as an in vitro

pharmacological tool for validating the potential therapeutic

beneﬁts of GPR40 agonists for the treatment of diabetes.

Therefore, we ﬁrst examined in vitro insulinotropic eﬀects of

compound 31 in rat insulinoma, INS-1 cells (clone 833/15)

(Figure 3a). The INS-1 cells maintained glucose responsive-

ness, secreting 9.5-fold the amount of insulin at 11 mM glucose

concentration as that at 1 mM. Compound 31 augmented

insulin secretion in a dose-dependent manner in the presence

of 11 mM glucose and statistically signiﬁcant increases in

insulin secretion were observed at doses of above 0.1 μM of the

compound compared to that seen at 11 mM glucose alone.

Next, we examined the eﬀect of glucose concentration on the

insulinotropic action of 31. As shown in Figure 3b, compound

31, at a concentration of 1 μM, augmented insulin secretion in

the presence of >6 mM glucose, while it did not enhance

insulin secretion in the presence of either 1 or 4 mM glucose.

In the same experiment, nateglinide and glibenclamide, well-

known as sulfonylurea agents, potentiated insulin secretion

even at lower glucose concentrations. Moreover, the amount of

secreted insulin in the presence of high glucose levels (11 and

16 mM) was greater with 31 than with nateglinide (p ≤ 0.01

and p ≤ 0.01, respectively) or glibenclamide (p ≤ 0.05 and p ≤

0.01, respectively). These results indicate that GPR40 agonists

augment insulin secretion through a mechanism strictly

dependent on glucose concentration and, furthermore, that

they are more potent in enhancing insulin secretion than

sulfonylurea agents at high glucose concentrations.

Figure 3. Enhancement of GSIS by compound 31 in INS-1 833/15

cells. (a) INS-1 cells (clone 833/15) were stimulated with 11 mM

glucose in the presence or absence of 31 (0.01−10 μM), and the

amount of insulin secretion was quantiﬁed by enzyme immunoassay

(EIA). Data are expressed as the mean fold-increase over control (1

mM glucose stimulation alone) SD (n = 3). **p ≤ 0.01 by Aspin−

Welch test. #p ≤ 0.025 vs 11 mM glucose alone by one-tailed Williams

test. (b) INS-1 cells (clone 833/15) were stimulated with various

concentrations of glucose (1−16 mM), in the absence or presence of 1

μM of 31, 10 μM of nateglinide, or 30 μM of glibenclamide, and the

amount of insulin secreted was quantiﬁed by EIA. Data are expressed

as the mean fold-increase over control (1 mM glucose stimulation

alone) and represent as the mean values of six wells (each glucose

stimulation alone) SD (n = 3−6). *p ≤ 0.05 and **p ≤ 0.01 vs

control (stimulation by glucose alone at each concentration) by

Dunnett’s test.

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Table 4. In Vitro Activities and Cytotoxicity Proﬁles of 4′-Alkoxybiphenyl Derivatives with NH Linker

a

b

c

d

e

Refers to Figure 1. Refers to Figure 1. Refers to Table 2. Refers to Table 2. Refers to Figure 1.

relationship of cytotoxicity and compound lipophilicity, the O

linker in 4′-alkoxybiphenyl derivatives 30−32 was converted to

the corresponding NH linker (Table 4). We anticipated that

this modiﬁcation would lead to a signiﬁcant drop in the Log D

value and result in attenuation of cytotoxicity issues. As a result,

the binding aﬃnities and agonist activities of compounds 33−

35 were generally comparable to those of the O linker

counterparts 30−32, although compound 33 displayed some-

what weaker agonist activity than compound 30. Notably,

compounds 33−35 had signiﬁcantly lowered Log D values in

the range of 2.1−2.5 and exhibited substantially improved

cytotoxicity proﬁles, with the only exception of a somewhat

decreased ATP content at the 30 μM dose in the case of

compound 33. These results indicate that the ability to induce

cytotoxicity appears to be roughly correlated with the

molecule’s lipophilicity, and Log D values below 2.8 would

be desirable to minimize the potential cytotoxicity risk for this

series. It appears certain, however, that the NH linker itself

contributed to the mitigation of caspase-3/7 activation in a

distinct manner apart from just lowering the lipophilicity. This

is because the NH linker derivative 41, possessing a high Log D

value of 3.49, showed no signs of apoptotic cytotoxicity,

whereas the O linker derivative 29 with a similar Log D value of

3.51 did display signiﬁcant apoptotic cytotoxicity. In rat

pharmacokinetic studies (Table 3), oral plasma exposure of

compounds 33 and 34 turned out to be inferior to that of the

corresponding O linker-containing derivatives 30 and 31. In

contrast, compound 35 preserved the excellent oral absorption

noted with compound 32.

On the basis of its excellent in vitro potency, pharmacoki-

netic proﬁle, and minimal risk of cytotoxicity, compound 35

was selected for further in vivo pharmacological evaluation.

Compound 35 was assessed for its ability to improve glucose

tolerance in female Wistar fatty rats, a disease model that

develops obese and obesity-related features such as impaired

glucose tolerance, hyperinsulinemia, and hyperlipidemia.⁵⁶

Single oral doses of 35 robustly lowered the blood glucose

excursion (Figure 4a) and augmented insulin secretion (Figure

4b) during an oral glucose tolerance test in a dose-proportional

manner from 0.1 to 1 mg/kg when the compound was

administered 30 min before the oral glucose challenge. It

should be noted that compound 35 exhibited a signiﬁcant

Figure 4. Eﬀects of compound 35 on plasma insulin and glucose levels

during an oral glucose tolerance test in female Wistar fatty rats.

Overnight-fasted rats were orally given compound 35 (0.1, 0.3, 1 mg/

kg) 30 min before glucose load (1 g/kg), and time-dependent changes

in (a) plasma glucose and (b) plasma insulin were monitored. #p ≤

0.025 vs control by one-tailed Williams’ test. *p ≤ 0.025 vs control by

Shirley−Williams’ test. Data are means SD for six animals.

blood glucose-lowering eﬀect at a much lower dose (0.3 mg/

kg) in comparison to compound 1³⁰(3 mg/kg). These results

can be at least in part rationalized by the improved oral

exposure of 35. In addition, the risk of hypoglycemia was also

assessed in fasting normal Sprague−Dawley (SD) rats by oral

administration of a high dose of compound 35 in comparison

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with glibenclamide, a well-known sulfonylurea insulin secreta-

gogue that acts on ATP-sensitive potassium channels in

pancreatic β-cells. As shown in parts a and b of Figure 5,

reduced potential cytotoxicity may contribute to the clean

toxicological proﬁle of 35.

CONCLUSION

■

From the hypothesis that the cytotoxicity observed with

compound 1 would be ascribed to its highly lipophilic nature,

we envisioned that imparting polarity to compound 1 could

lead to the generation of GPR40 agonists with a minimized risk

of in vitro cellular toxicity as well as more druggable

physicochemical properties. In addition, we anticipated that

this strategy would also contribute to moving away from the

multifaceted FFA-like structure and result in the identiﬁcation

of the drug candidates that speciﬁcally activate the GPR40

receptor. Preliminary investigations of substituents on the 2′,6′-

dimethylbiphenyl ring indicated the potential for incorporating

polar side chains at the 4′-position. Through the subsequent

exploration of polar functionalities at the 4′-position and on the

linker, we discovered a series of 4′-alkoxybiphenyl derivatives

with a combination of excellent in vitro activities and

dramatically improved pharmacokinetic and cytotoxicity

proﬁles. Cytotoxicity issues were successfully overcome by

reducing the lipophilicity to a Log D value of less than 2.8.

Compound 35 displayed a superior blood glucose-lowering

eﬀect in female Wistar fatty rats over compound 1, and no

signiﬁcant adverse eﬀects were observed in a 4-week toxicity

study in rats. Potent and safe GPR40 agonists, such as

compound 35, are expected to provide a novel therapeutic

approach to enhance insulin secretion with a low risk of

hypoglycemia.

EXPERIMENTAL SECTION

General. 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). The acidic

protons of carboxylic acids, alcohols, or anilines were not frequently

Figure 5. Eﬀects of compound 35 (30 mg/kg, po) and glibenclamide

(10 mg/kg, po) on fasting plasma glucose and insulin levels in normal

Sprague−Dawley rats. (a) and (b) show time-dependent changes in

plasma glucose and plasma insulin level after oral administration of

compound 35 or glibenclamide, respectively. *p ≤ 0.05 and **p ≤

0.01 vs control by Student’s t test. ##p ≤ 0.01 vs control by Aspin−

Welch test. Data are means SD for six animals.

1

observed in H NMR spectra. Elemental analyses were carried out by

Takeda Analytical Laboratories, Ltd., and were within 0.3% 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 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 μL/min, with UV detection at 220 and 254 nm, at

column temperature of 25 °C, or performed on a Waters Quattro

micro API (Agilent HP1100, Gilson215) instrument, equipped with

CAPCELL PAK C18 UG120 S-3 μm, 1.5 mm × 35 mm column, by

gradient elution: 0.00 min (A/B = 100/0), 2.00 min (A/B = 0/100),

3.00 (A/B = 0/100), 3.01 (A/B = 100/0), 3.30 (A/B = 100/0) where

A = 2% CH₃CN/H₂O with 5 mM NH₄OAc, B = 95% CH₃CN/H₂O

with 5 mM NH₄OAc, at a ﬂow rate of 0.5 mL/min, with UV detection

at 220 nm, at column temperature of 40 °C. Purity of all tested

compounds was determined to be >97% by elemental analyses and/or

analytical HPLC using the aforementioned conditions. Reagents and

solvents were obtained from commercial sources and used without

glibenclamide (10 mg/kg) lowered plasma glucose levels below

normal fasting levels in SD rats by signiﬁcantly increasing

plasma insulin. In contrast, compound 35, even at the high dose

of 30 mg/kg, did not alter fasting glucose levels in SD rats with

normal glucose homeostasis (Figure 5a). In agreement with the

absence of hypoglycemic eﬀects, insulin secretion induced by

compound 35 was statistically signiﬁcant only at 30 min after

the administration, but the amount was much lower compared

to that by caused by administration of glibenclamide (Figure

5b). Thus, our results indicate that compound 35 not only

enhances GSIS and eﬀectively improves postprandial hyper-

glycemia in the diabetic state, but also it may present a low risk

of hypoglycemia, an adverse eﬀect common to sulfonylureas.

On the basis of the promising in vivo pharmacological proﬁle

of compound 35, a 4-week toxicity study was subsequently

carried out in normal rats. In this study, no particular adverse

eﬀects were observed at oral doses of 15, 50, and 150 mg/kg,

indicating the possibility that minimized oﬀ-target pharmacol-

ogy as a result of decreased lipophilicity (Log D = 2.14) and

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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 1020, 100−200 mesh)] or

on Purif-Pack (SI or NH, particle size: 60 μm, Fuji Silysia Chemical,

Ltd.). Abbreviations of the solvents are used as follows: EtOAc, ethyl

acetate; THF, tetrahydrofuran; EtOH, ethanol; DMF, N,N-dimethyl-

formamide; Et₂O, diethyl ether; MeOH, methanol; CH₃CN,

acetonitrile; DME, 1,2-dimethoxyethane; DMSO, dimethyl sulfoxide.

Methyl 3-(Benzyloxy)-5-hydroxybenzoate (7). To a mixture of

methyl 3,5-dihydroxybenzoate (6) (8.41 g, 50.0 mmol) and benzyl

bromide (3.57 mL, 300 mmol) in DMF (30 mL) was added potassium

carbonate (4.15 g, 30.0 mmol). The reaction mixture was stirred at 70

°C for 16 h under nitrogen, followed by solvent removal in vacuo. The

residue was partitioned between EtOAc and water. The phases were

separated, and the aqueous phase was extracted with EtOAc. The

combined organic phases were washed with saturated aqueous NaCl,

dried over MgSO₄, and concentrated in vacuo. The residue was

puriﬁed by column chromatography (silica gel, hexane/EtOAc, 19:1 to

7:3) and then crystallized from hexane/EtOAc to give 7 (3.56 g, 46%)

hexane/EtOAc to aﬀord methyl 5-hydroxy-2′,6′-dimethylbiphenyl-3-

carboxylate (1.64 g, 91%) as a white solid. ¹H NMR (300 MHz,

CDCl₃) δ 2.03 (6H, s), 3.91 (3H, s), 5.14 (1H, s), 6.86 (1H, dd, J =

2.5, 1.4 Hz), 7.07−7.21 (3H, m), 7.43 (1H, t, J = 1.4 Hz), 7.51 (1H,

dd, J = 2.5, 1.4 Hz). MS m/z 257 (M + H)⁺. Anal. Calcd for C₁₆H₁₆O₃:

C, 74.98; H, 6.29. Found: C, 74.79; H, 6.44. To a mixture of methyl 5-

hydroxy-2′,6′-dimethylbiphenyl-3-carboxylate (0.500 g, 1.95 mmol)

and iodomethane (0.243 mL, 3.90 mmol) in DMF (4 mL) was added

potassium carbonate (0.324 g, 2.34 mmol). The reaction mixture was

stirred at 50 °C for 14 h, diluted with water, and extracted with EtOAc.

The organic phase was washed with saturated aqueous NaCl, dried

over MgSO₄, and concentrated in vacuo. The residue was puriﬁed by

column chromatography (silica gel, hexane/EtOAc, 100:0 to 4:1) to

aﬀord 8c (0.490 g, 93%) as a colorless oil. ¹H NMR (300 MHz,

CDCl₃) δ 2.03 (6H, s), 3.87 (3H, s), 3.91 (3H, s), 6.91 (1H, dd, J =

2.6, 1.4 Hz), 7.08−7.13 (2H, m), 7.14−7.21 (1H, m), 7.45 (1H, t, J =

1.4 Hz), 7.55 (1H, dd, J = 1.4 Hz). MS m/z 271 (M + H)⁺.

6-Methoxy-2′,6′-dimethylbiphenyl-3-carbaldehyde (8d).

The title compound was prepared in 87% yield as a pale-yellow oil

from 4 and 5b using the procedure analogous to that described for the

synthesis of 8a, except that EtOH was employed as a solvent in place

of MeOH. ¹H NMR (300 MHz, CDCl₃) δ 1.99 (6H, s), 3.84 (3H, s),

6.98−7.22 (4H, m), 7.60 (1H, d, J = 2.2 Hz), 7.91 (1H, dd, J = 2.2, 8.8

Hz), 9.92 (1H, s). MS m/z 241 (M + H)⁺.

(2,2′,6′-Trimethylbiphenyl-3-yl)methanol (9a). To a solution

of 8a (1.30 g, 5.11 mmol) in THF (30 mL) at 0 °C was added

portionwise lithium aluminum hydride (220 mg, 5.80 mmol). The

reaction mixture was stirred 0 °C for 3 h, and Na₂SO₄·10H₂O (2.00 g,

3.83 mmol) was slowly added at the same temperature. After being

stirred at room temperature, the mixture was ﬁltered through a pad of

Celite using EtOAc and then the ﬁltrate was concentrated in vacuo.

The residue was puriﬁed by column chromatography (silica gel,

hexane/EtOAc, 10:1 to 5:1) to aﬀord 9a (500 mg, 43%) as a white

solid. ¹H NMR (300 MHz, CDCl₃) δ 1.94 (6H, s), 1.97 (3H, s), 4.69

(2H, d, J = 6.0 Hz), 7.01 (1H, s), 7.06−7.32 (5H, m).

1

as a white solid. H NMR (300 MHz, CDCl₃) δ 3.90 (3H, s), 5.07

(2H, s), 5.18 (1H, s), 6.68 (1H, t, J = 2.3 Hz), 7.14 (1H, dd, J = 2.3,

1.3 Hz), 7.24−7.28 (1H, m), 7.30−7.46 (5H, m). MS m/z 259 (M +

H)⁺.

Methyl 2,2′,6′-Trimethylbiphenyl-3-carboxylate (8a). A

mixture of methyl 3-bromo-2-methylbenzoate (3) (2.60 g, 11.4

mmol), (2,6-dimethylphenyl)boronic acid (2) (2.00 g, 13.3 mmol),

tetrakis(triphenylphosphine)palladium(0) (200 mg, 0.170 mmol), and

cesium carbonate (5.60 g, 17.2 mmol) in toluene (50 mL) and MeOH

(10 mL) was reﬂuxed for 18 h under argon. The insoluble materials

were ﬁltered oﬀ and washed with EtOAc. The ﬁltrate was concentrated

in vacuo to give a crude oil, which was puriﬁed by column

chromatography (silica gel, hexane/EtOAc, 10:1) to aﬀord 8a (1.30

g, 45%) as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 1.93 (6H, s),

2.03 (3H, s), 3.09 (3H, s), 7.07−7.96 (6H, m). MS m/z 255 (M +

H)⁺.

(4-Methoxy-2′,6′-dimethylbiphenyl-3-yl)methanol (9b). To a

solution of 8b (2.90 g, 12.1 mmol) in EtOH (40 mL) at 0 °C was

added portionwise sodium borohydride (460 mg, 12.2 mmol). The

reaction mixture was allowed to warm to room temperature and stirred

for 4 h. After being cooled to 0 °C, the reaction mixture was quenched

with citric acid aqueous solution and evaporated under reduced

pressure to remove EtOH. The residue was extracted with EtOAc,

washed with saturated aqueous NaCl, and then dried over MgSO₄.

The organic layer was concentrated in vacuo to aﬀord 9b (3.00 g,

quant.) as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 2.03 (6H, s),

2.37 (1H, t, J = 6.6 Hz), 3.92 (3H, s), 4.71 (2H, d, J = 6.6 Hz), 6.94

(1H, d, J = 8.8 Hz), 7.02−7.22 (5H, m).

4-Methoxy-2′,6′-dimethylbiphenyl-3-carbaldehyde (8b).

The title compound was prepared in 82% yield as a pale-yellow

solid from 4 and 5a using the procedure analogous to that described

for the synthesis of 8a, except that EtOH was employed as a solvent in

1

place of MeOH. H NMR (300 MHz, CDCl₃) δ 2.02 (6H, s), 3.99

(3H, s), 7.03−7.23 (4H, m), 7.35 (1H, dd, J = 2.6, 8.4 Hz), 7.63 (1H,

d, J = 2.2 Hz), 10.52 (1H, s). MS m/z 241 (M + H)⁺.

Methyl 5-Methoxy-2′,6′-dimethylbiphenyl-3-carboxylate

(8c). To a solution of 7 (3.10 g, 12.0 mmol) and N-ethyldiisopropyl-

amine (2.51 mL, 14.4 mmol) in dichloromethane (30 mL) at 0 °C was

added dropwise triﬂuoromethanesulfonic anhydride (2.22 mL, 13.2

mmol). The reaction mixture was stirred at the same temperature for 1

h and then washed with water, dried over MgSO₄, and concentrated in

vacuo to give methyl 3-(benzyloxy)-5-{[(triﬂuoromethyl)sulfonyl]-

oxy}benzoate as a brown oil. A mixture of the triﬂate obtained above,

(2,6-dimethylphenyl)boronic acid (2.25 g, 15.0 mmol), tetrakis-

(triphenylphosphine)palladium(0) (0.693 g, 0.600 mmol), and

potassium triphosphate (3.82 g, 18.0 mmol) in 1,2-dimethoxyethane

(40 mL), was stirred at 80 °C for 15 h under argon. After

concentration in vacuo, the residue was diluted with EtOAc, washed

sequentially with water and saturated aqueous NaCl, dried over

MgSO₄, and concentrated in vacuo. The residue was puriﬁed by

column chromatography (silica gel, hexane/EtOAc, 100:0 to 4:1) to

aﬀord methyl 5-(benzyloxy)-2′,6′-dimethylbiphenyl-3-carboxylate

(5-Methoxy-2′,6′-dimethylbiphenyl-3-yl)methanol (9c). To a

solution of 8c (0.485 g, 1.79 mmol) in THF (5 mL) at room

temperature was added portionwise lithium aluminum hydride (68.1

mg, 1.79 mmol). The reaction mixture was stirred at room

temperature for 15 h under nitrogen and then Na₂SO₄·10H₂O

(0.577 g, 1.79 mmol) was slowly added at the same temperature. After

being stirred for 1 h, the mixture was ﬁltered through a pad of Celite

using THF and the ﬁltrate was concentrated in vacuo to aﬀord 9c

(0.431 g, 99%) as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 1.67

(1H, t, J = 5.7 Hz), 2.05 (6H, s), 3.83 (3H, s), 4.71 (2H, d, J = 5.7

Hz), 6.63 (1H, dd, J = 2.2, 1.4 Hz), 6.72 (1H, s), 6.90−6.94 (1H, m),

7.06−7.19 (3H, m).

(6-Methoxy-2′,6′-dimethylbiphenyl-3-yl)methanol (9d). The

title compound was prepared in 88% yield as a colorless oil from 8d

using the procedure analogous to that described for the synthesis of

1

(3.60 g, 87%) as a pale-yellow oil. H NMR (300 MHz, CDCl₃) δ

1

2.00 (6H, s), 3.91 (3H, s), 5.13 (2H, s), 6.97 (1H, dd, J = 2.5, 1.4 Hz),

7.06−7.20 (3H, m), 7.29−7.48 (6H, m), 7.65 (1H, dd, J = 2.5, 1.4

Hz). MS m/z 347 (M + H)⁺. Methyl 5-(benzyloxy)-2′,6′-

dimethylbiphenyl-3-carboxylate was hydrogenated under atmospheric

hydrogen with 10% Pd on carbon (containing 50% water, 0.4 g) as a

catalyst in MeOH (20 mL) and THF (10 mL) at room temperature

for 24 h. The reaction mixture was ﬁltered by Millipore ﬁltration and

concentrated in vacuo. The resulting solid was recrystallized from

9b. H NMR (300 MHz, CDCl₃) δ 2.01 (6H, s), 3.74 (3H, s), 4.65

(2H, d, J = 5.2 Hz), 6.97 (1H, d, J = 8.4 Hz), 7.03 (1H, d, J = 2.2 Hz),

7.06−7.24 (3H, m), 7.35 (1H, dd, J = 8.4, 2.6 Hz).

General Procedure for the Synthesis of Compound 10−13.

To a mixture of 9a−d, methyl 3-(4-hydroxyphenyl)propanoate or

ethyl 3-(2-ﬂuoro-4-hydroxyphenyl)propanoate (1.06−1.18 equiv) and

tributylphosphine (1.28−2.00 equiv) in THF or toluene was added

portionwise 1,1′-(azodicarbonyl)dipiperidine (1.28−2.00 equiv). The

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reaction mixture was stirred at room temperature for 18−21 h. After

addition of hexane or Et₂O, the insoluble materials were removed by

ﬁltration and the ﬁltrate was concentrated in vacuo. The residue was

puriﬁed by silica gel column chromatography to aﬀord the coupled

ethyl esters. The above ethyl esters were dissolved in a 1:1 mixture of

THF and MeOH (or EtOH). To the solution were added potassium

hydroxide (2.20 equiv) aqueous solution or 2 M sodium hydroxide

aqueous solution (2.61 equiv). The reaction mixture was stirred at

room temperature for 7−72 h and then partitioned between EtOAc

and 10% citric acid aqueous solution. The phases were separated, and

the organic phase was washed with saturated aqueous NaCl, dried over

MgSO₄or Na₂SO₄, and concentrated in vacuo. The residue was

puriﬁed by silica gel column chromatography to aﬀord 10−13.

3-{4-[(2,2′,6′-Trimethylbiphenyl-3-yl)methoxy]phenyl}-

propanoic Acid (10). Starting from 9a and methyl 3-(4-

hydroxyphenyl)propanoate, the procedures summarized above pro-

vided the title compound in 76% yield as a white solid after

sodium salt was subsequently combined with 2-bromo-6-methlypyr-

idine (1.00 g, 5.81 mmol) and copper powder (11 mg, 0.17 mmol),

and the resulting reaction mixture was stirred at 185 °C for 1 h. After

cooling to room temperature, the reaction mixture was diluted with

EtOAc, washed sequentially with water and saturated aqueous NaCl,

dried over MgSO₄, and concentrated in vacuo. The residue was

puriﬁed by column chromatography (silica gel, hexane/EtOAc, 100:0

to 5:1) to aﬀord 16a (1.25 g, 74%) as a pale-yellow oil. ¹H NMR (300

MHz, CDCl₃) δ 2.40 (6H, s), 2.46 (3H, s), 6.57 (1H, d, J = 8.3 Hz),

6.83−6.92 (3H, m), 7.54 (1H, t, J = 8.3 Hz). MS m/z 292 (M + H)⁺.

4-[(4-Bromo-3,5-dimethylphenoxy)methyl]tetrahydro-2H-

thiopyran-4-ol (16b). To a solution of 14 (6.07 g, 30.2 mmol) in

DMF (60 mL) at 0 °C was added sodium hydride (60% dispersion in

mineral oil, 1.21 g, 30.2 mmol) in a single portion. After being stirred

for 1 h at room temperature, the reaction mixture was cooled to 0 °C

and a solution of 1-oxa-6-thiaspiro[2.5]octane (2.62 g, 20.1 mmol) in

DMF (20 mL) was slowly added. The reaction mixture was allowed to

warm to 80 °C and stirred overnight at the same temperature. After

concentration in vacuo to remove DMF, the residue was partitioned

between EtOAc and NaCl aqueous solution. The phases were

separated, and the aqueous phase was extracted with EtOAc. The

combined organic phases were washed sequentially with 2 M NaOH

aqueous solution and saturated aqueous NaCl, dried over Na₂SO₄, and

concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, hexane/EtOAc, 5:1) to aﬀord 16b (2.92

1

recrystallization from hexane/Et₂O. H NMR (300 MHz, CDCl₃) δ

1.92 (6H, s), 1.96 (3H, s), 2.63 (2H, t, J = 7.4 Hz), 2.89 (2H, t, J = 7.4

Hz), 5.04 (2H, s), 6.88 (2H, d, J = 8.4 Hz), 7.05−7.34 (8H, m). MS

m/z 375 (M + H)⁺; mp 106−107 °C. HPLC purity: 100%. Anal.

Calcd for C₂₅H₂₆O₃: C, 80.18; H, 7.00. Found: C, 80.04; H, 7.04.

3-{4-[(4-Methoxy-2′,6′-dimethylbiphenyl-3-yl)methoxy]-

phenyl}propanoic Acid (11). Starting from 9b and methyl 3-(4-

hydroxyphenyl)propanoate, the procedures summarized above pro-

vided the title compound in 79% yield as a white solid after

1

g, 44%) as a white solid. H NMR (300 MHz, CDCl₃) δ 1.73−1.88

(2H, m), 2.01−2.14 (3H, m), 2.34−2.52 (8H, m), 3.01−3.16 (2H, m),

3.73 (2H, s), 6.65 (2H, s).

1

recrystallization from hexane/Et₂O. H NMR (300 MHz, CDCl₃) δ

1.98 (6H, s), 2.63 (2H, t, J = 7.7 Hz), 2.89 (2H, t, J = 7.7 Hz), 3.90

(3H, s), 5.14 (2H, s), 6.84−6.99 (3H, m), 7.00−7.24 (7H, m). mp

130−131 °C. HPLC purity: 100%. Anal. Calcd for C₂₅H₂₆O₄: C,

76.90; H, 6.71. Found: C, 76.77; H, 6.61.

2-(4-Bromo-3,5-dimethylphenoxy)tetrahydro-2H-pyran

(16c). To a mixture of 14 (10.5 g, 52.2 mmol) and 3,4-dihydro-2H-

pyran (8.83 g, 105 mmol) in dichloromethane (160 mL) was added

pyridinium p-toluenesulfonate (2.64 g, 10.5 mmol). The reaction

mixture was stirred at room temperature for 80 h and then

concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, hexane/EtOAc, 4:1) to aﬀord 16c (11.5

3-{2-Fluoro-4-[(5-methoxy-2′,6′-dimethylbiphenyl-3-yl)-

methoxy]phenyl}propanoic Acid (12). Starting from 9c and ethyl

3-(2-ﬂuoro-4-hydroxyphenyl)propanoate, the procedures summarized

1

above provided the title compound in 86% yield as a colorless oil. H

1

NMR (300 MHz, CDCl₃) δ 2.03 (6H, s), 2.64 (2H, t, J = 7.6 Hz), 2.91

(2H, t, J = 7.6 Hz), 3.82 (3H, s), 5.05 (2H, s), 6.63−6.71 (3H, m),

6.76 (1H, s), 6.93 (1H, dd, J = 2.3, 1.5 Hz), 7.06−7.19 (4H, m). MS

m/z 409 (M + H)⁺. HPLC purity: 99.5%.

g, 77%) as a colorless oil. H NMR (300 MHz, CDCl₃) δ 1.56−1.75

(3H, m), 1.80−2.07 (3H, m), 2.37 (6H, s), 3.55−3.64 (1H, m), 3.83−

3.93 (1H, m), 5.37 (1H, t, J = 3.1 Hz), 6.80 (2H, s).

4′-(2-Ethoxyethoxy)-2′,6′-dimethylbiphenyl-3-carbaldehyde

(17a). To a mixture of 15 (8.52 g, 37.7 mmol) and 1-chloro-2-

ethoxyethane (6.15 g, 56.6 mmol) in DMF were added potassium

carbonate (6.25 g, 45.2 mmol) and potassium iodide (1.25 g, 7.54

mmol). The reaction mixture was stirred at 80 °C for 18 h and then

poured into water. The mixture was extracted with EtOAc, washed

with saturated aqueous NaCl, and then dried over MgSO₄. The

organic layer was concentrated in vacuo. The residue was puriﬁed by

column chromatography (silica gel, hexane/EtOAc, 3:1) to aﬀord 17a

3-{4-[(6-Methoxy-2′,6′-dimethylbiphenyl-3-yl)methoxy]-

phenyl}propanoic Acid (13). Starting from 9d and methyl 3-(4-

hydroxyphenyl)propanoate, the procedures summarized above pro-

vided the title compound in 67% yield as a white solid after

1

recrystallization from hexane/Et₂O. H NMR (300 MHz, CDCl₃) δ

2.00 (6H, s), 2.64 (2H, t, J = 7.8 Hz), 2.90 (2H, t, J = 7.8 Hz), 3.74

(3H, s), 5.00 (2H, s), 6.85−7.44 (10H, m). mp 103−104 °C. Anal.

Calcd for C₂₅H₂₆O₄·0.5H₂O: C, 75.16; H, 6.81. Found: C, 75.44; H,

6.64.

1

(10.0 g, 89%) as a white solid. H NMR (300 MHz, CDCl₃) δ 1.26

4′-Hydroxy-2′,6′-dimethylbiphenyl-3-carbaldehyde (15). A

ﬂask was charged with 14 (10.3 g, 51.0 mmol), (3-formylphenyl)-

boronic acid (7.67 g, 51.2 mmol), 1 M sodium carbonate aqueous

solution (150 mL), EtOH (50 mL), and toluene (150 mL). The

system was placed under argon by evacuation and ﬁlling with argon

ﬁve times. After tetrakis(triphenylphosphine)palladium(0) (2.95 g,

2.55 mmol) was added, the system was again placed under argon: the

above pump−ﬂush cycle was repeated three times. The reaction

mixture was stirred at 80 °C for 24 h under argon. After cooling to

room temperature, the reaction mixture was passed through a pad of

Celite. The ﬁltrate was washed with saturated aqueous NaCl, dried

over MgSO₄, and concentrated in vacuo. The residue was puriﬁed by

column chromatography (silica gel, hexane/EtOAc, 9:1 to 3:2) to

(3H, t, J = 7.0 Hz), 1.99 (6H, s), 3.62 (2H, q, J = 7.0 Hz), 3.81 (2H, t,

J = 4.9 Hz), 4.15 (2H, t, J = 4.9 Hz), 6.71 (2H, s), 7.42 (1H, dt, J = 7.5,

1.5 Hz), 7.58 (1H, t, J = 7.5 Hz), 7.66 (1H, t, J = 1.5 Hz), 7.86 (1H, dt,

J = 7.5, 1.5 Hz), 10.05 (1H, s). MS m/z 299 (M + H)⁺.

2′,6′-Dimethyl-4′-[(6-methylpyridin-2-yl)oxy]biphenyl-3-

carbaldehyde (17b). The title compound was prepared in 94% yield

as a colorless oil from 16a using the procedure analogous to that

described for the synthesis of 15. ¹H NMR (300 MHz, CDCl₃) δ 2.00

(6H, s), 2.50 (3H, s), 6.64 (1H, d, J = 8.1 Hz), 6.85−6.93 (3H, m),

7.46 (1H, dt, J = 7.6, 1.4 Hz), 7.53−7.66 (2H, m), 7.71 (1H, t, J = 1.4

Hz), 7.88 (1H, dt, J = 7.6, 1.4 Hz), 10.07 (1H, s). MS m/z 318 (M +

H)⁺.

4′-[(4-Hydroxytetrahydro-2H-thiopyran-4-yl)methoxy]-2′,6′-

dimethylbiphenyl-3-carbaldehyde (17c). The title compound was

prepared in 66% yield as a white solid from 16b using the procedure

analogous to that described for the synthesis of 15, except that 1,2-

1

aﬀord 15 (9.53 g, 83%) as a pale-yellow solid. H NMR (300 MHz,

CDCl₃) δ 1.97 (6H, s), 4.69 (1H, s), 6.62 (2H, s), 7.42 (1H, dt, J =

7.7, 1.4 Hz), 7.59 (1H, t, J = 7.6 Hz), 7.66 (1H, t, J = 1.7 Hz), 7.86

(1H, dt, J = 7.6, 1.5 Hz), 10.05 (1H, s). MS m/z 227 (M + H)⁺.

2-(4-Bromo-3,5-dimethylphenoxy)-6-methylpyridine (16a).

To a solution of sodium hydroxide (0.230 g, 5.81 mmol) in MeOH

(50 mL) was added 14 (1.17 g, 5.81 mmol). After being stirred at

room temperature for 10 min, the reaction mixture was concentrated

to dryness in vacuo to aﬀord sodium salt of 14 (1.30 g). The resultant

1

dimethoxyethane was employed as a solvent in place of EtOH. H

NMR (300 MHz, CDCl₃) δ 1.70 (1H, t, J = 5.8 Hz), 1.76−1.90 (2H,

m), 2.01 (6H, s), 2.05−2.16 (2H, m), 2.20 (1H, s), 2.40−2.53 (2H,

m), 3.03−3.18 (2H, m), 3.80 (2H, s), 4.73 (2H, d, J = 5.8 Hz), 6.67

(2H, s), 7.02−7.09 (1H, m), 7.12 (1H, s), 7.31−7.37 (1H, m), 7.41

(1H, t, J = 7.4 Hz).

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2′,6′-Dimethyl-4′-(tetrahydro-2H-pyran-2-yloxy)biphenyl-3-

carbaldehyde (17d). The title compound was prepared in 83% yield

as a yellow oil from 16c using the procedure analogous to that

18a (0.330 g, 1.10 mmol), ethyl 3-(2-ﬂuoro-4-hydroxyphenyl)-

propanoate (0.233 g, 1.10 mmol), and tributylphosphine (0.448 mL,

1.80 mmol) in toluene (20 mL) at 0 °C was added portionwise 1,1′-

(azodicarbonyl)dipiperidine (0.454 g, 1.80 mmol). The reaction

mixture was stirred at room temperature for 16 h. After addition of

hexane, the insoluble materials were removed by ﬁltration and the

ﬁltrate was concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, hexane/EtOAc, 19:1 to 3:1) to aﬀord 19a

(0.505 g, 93%) as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 1.20−

1.28 (6H, m), 1.98 (6H, s), 2.57 (2H, t, J = 7.7 Hz), 2.89 (2H, t, J =

7.7 Hz), 3.62 (2H, q, J = 7.0 Hz), 3.80 (2H, t, J = 7.0 Hz), 4.08−4.15

(4H, m), 5.06 (2H, s), 6.63−6.70 (4H, m), 7.05−7.11 (2H, m), 7.16

(1H, s), 7.34−7.35 (1H, m), 7.42 (1H, t, J = 7.4 Hz). MS m/z 495 (M

+ H)⁺.

1

described for the synthesis of 15. H NMR (300 MHz, CDCl₃) δ

1.57−1.78 (3H, m), 1.82−1.93 (2H, m), 1.99 (6H, s), 2.04 (1H, m),

3.65 (1H, m), 3.97 (1H, m), 5.47 (1H, t, J = 3.0 Hz), 6.84 (2H, s),

7.42 (1H, m), 7.58 (1H, t, J = 7.5 Hz), 7.67 (1H, s), 7.86 (1H, m),

10.05 (1H, s).

4′-{[tert-Butyl(dimethyl)silyl]oxy}-2′,6′-dimethylbiphenyl-3-

carbaldehyde (17e). To a mixture of 15 (9.00 g, 39.8 mmol) and

imidazole (2.98 g, 43.8 mmol) in DMF (100 mL) was added tert-

butyldimethylsilyl chloride (6.60 g, 43.8 mmol). After being stirred for

4 h, the reaction mixture was diluted with EtOAc and washed

sequentially with water and saturated aqueous NaCl. The organic layer

was dried over MgSO₄and concentrated in vacuo. The residue was

puriﬁed by column chromatography (silica gel, hexane/EtOAc, 10:1 to

Ethyl 3-[4-({2′,6′-Dimethyl-4′-[(6-methylpyridin-2-yl)oxy]-

biphenyl-3-yl}methoxy)-2-ﬂuorophenyl]propanoate (19b).

The title compound was prepared in 94% yield as a colorless oil

from 18b using the procedure analogous to that described for the

1

4:1) to aﬀord 17e (10.5 g, 77%) as a yellow oil. H NMR (300 MHz,

CDCl₃) δ 0.25 (6H, s), 1.02 (9H, s), 1.97 (6H, s), 6.62 (2H, s), 7.44

(1H, dt, J = 1.5, 7.5 Hz), 7.59 (1H, t, J = 7.5 Hz), 7.68 (1H, t, J = 1.5

Hz), 7.86 (1H, dt, J = 1.5, 7.5 Hz), 10.06 (1H, s).

1

synthesis of 19a. H NMR (300 MHz, CDCl₃) δ 1.23 (3H, t, J = 7.2

Hz), 1.99 (6H, m), 2.50 (3H, s), 2.58 (2H, t, J = 7.8 Hz), 2.90 (2H, t, J

= 7.8 Hz), 4.12 (2H, q, J = 7.2 Hz), 5.08 (2H, s), 6.60−6.73 (3H, m),

6.84−6.92 (3H, m), 7.05−7.16 (2H, m), 7.21 (1H, s), 7.36−7.49 (2H,

m), 7.56 (1H, t, J = 7.8 Hz). MS m/z 514 (M + H)⁺.

[4′-(2-Ethoxyethoxy)-2′,6′-dimethylbiphenyl-3-yl]methanol

(18a). To a solution of 17a (2.39 g, 9.70 mmol) in THF (20 mL) and

1,2-dimethoxyethane (20 mL) at 0 °C was added sodium borohydride

(0.227 g, 6.00 mmol). The reaction mixture was stirred at the same

temperature for 3 h and quenched with NH₄Cl aqueous solution. The

mixture was extracted with EtOAc, washed with saturated aqueous

NaCl, dried over MgSO₄, and then concentrated in vacuo. The residue

was puriﬁed by column chromatography (silica gel, hexane/EtOAc, 4:1

Ethyl 3-[2-Fluoro-4-({4′-[(4-hydroxytetrahydro-2H-thiopyr-

an-4-yl)methoxy]-2′,6′-dimethylbiphenyl-3-yl}methoxy)-

phenyl]propanoate (19c). The title compound was prepared in

89% yield as a colorless oil from 18c (0.900 g, 2.51 mmol) using the

procedure analogous to that described for the synthesis of 19a, except

1

to 1:1) to aﬀord 18a (3.55 g, 98%) as a white solid. H NMR (300

that THF was employed as a solvent in place of toluene. H NMR

MHz, CDCl₃) δ 1.25 (3H, t, J = 7.1 Hz), 1.66 (1H, t, J = 5.9 Hz), 2.00

(6H, s), 3.62 (2H, q, J = 7.1 Hz), 3.80 (2H, t, J = 5.1 Hz), 4.14 (2H, t,

J = 5.1 Hz), 4.73 (2H, d, J = 5.9 Hz), 6.69 (2H, s), 7.06 (1H, d, J = 7.3

Hz), 7.12 (1H, s), 7.33 (1H, d, J = 7.3 Hz), 7.40 (1H, t, J = 7.3 Hz).

MS m/z 301 (M + H)⁺.

(300 MHz, CDCl₃) δ 1.23 (3H, t, J = 7.2 Hz), 1.75−1.90 (2H, m),

1.99 (6H, s), 2.05−2.16 (2H, m), 2.19 (1H, s), 2.39−2.52 (2H, m),

2.57 (2H, t, J = 7.6 Hz), 2.89 (2H, t, J = 7.6 Hz), 3.03−3.19 (2H, m),

3.79 (2H, s), 4.12 (2H, q, J = 7.2 Hz), 5.06 (2H, s), 6.60−6.73 (4H,

m), 7.01−7.19 (3H, m), 7.33−7.48 (2H, m).

{2′,6′-Dimethyl-4′-[(6-methylpyridin-2-yl)oxy]biphenyl-3-

yl}methanol (18b). The title compound was prepared in 98% yield as

a colorless oil from 17b using the procedure analogous to that

Methyl 3-(4-{[2′,6′-Dimethyl-4′-(tetrahydro-2H-pyran-2-

yloxy)biphenyl-3-yl]methoxy}phenyl)propanoate (19d). To a

mixture of 18d (5.15 g, 16.5 mmol), methyl 3-(4-hydroxyphenyl)-

propanoate (3.28 g, 18.2 mmol), and triphenylphosphine (5.63 g, 21.5

mmol) in THF (100 mL) was added dropwise diethyl azodicarbox-

ylate (40 wt % in toluene, 9.70 mL, 24.8 mmol). The reaction mixture

was stirred at room temperature for 2 days. After concentration in

vacuo, the residue was puriﬁed by column chromatography (silica gel,

hexane/EtOAc, 10:1 to 3:1) to aﬀord 19d (2.92 g, 37%) as a pale-

1

described for the synthesis of 18a. H NMR (300 MHz, CDCl₃) δ

1.98−2.03 (6H, m), 2.47−2.53 (3H, m), 4.75 (2H, d, J = 5.1 Hz), 6.62

(1H, d, J = 8.3 Hz), 6.83−6.92 (3H, m), 7.07−7.13 (1H, m), 7.14−

7.19 (1H, m), 7.33−7.39 (1H, m), 7.43 (1H, t, J = 7.5 Hz), 7.56 (1H,

t, J = 7.7 Hz). MS m/z 320 (M + H)⁺.

4-({[3′-(Hydroxymethyl)-2,6-dimethylbiphenyl-4-yl]oxy}-

methyl)tetrahydro-2H-thiopyran-4-ol (18c). The title compound

was prepared in quantitative yield as a white solid from 17c using the

procedure analogous to that described for the synthesis of 18a, except

that MeOH was employed as a solvent in place of 1,2-dimethoxy-

ethane. ¹H NMR (300 MHz, CDCl₃) δ 1.70 (1H, t, J = 5.8 Hz), 1.76−

1.90 (2H, m), 2.01 (6H, s), 2.05−2.16 (2H, m), 2.20 (1H, s), 2.40−

2.53 (2H, m), 3.03−3.18 (2H, m), 3.80 (2H, s), 4.73 (2H, d, J = 5.8

Hz), 6.67 (2H, s), 7.02−7.09 (1H, m), 7.12 (1H, s), 7.31−7.37 (1H,

m), 7.41 (1H, t, J = 7.4 Hz).

[2′,6′-Dimethyl-4′-(tetrahydro-2H-pyran-2-yloxy)biphenyl-

3-yl]methanol (18d). The title compound was prepared in

quantitative yield as a colorless oil from 17d using the procedure

analogous to that described for the synthesis of 18a, except that

MeOH was employed as a solvent in place of 1,2-dimethoxyethane. ¹H

NMR (300 MHz, CDCl₃) δ 1.55−1.79 (4H, m), 1.80−1.93 (2H, m),

2.00 (6H, s), 2.03 (1H, m), 3.64 (1H, m), 3.97 (1H, m), 4.73 (2H, d, J

= 5.7 Hz), 5.45 (1H, t, J = 3.0 Hz), 6.81 (2H, s), 7.07 (1H, d, J = 7.5

Hz), 7.13 (1H, s), 7.33 (1H, d, J = 7.5 Hz), 7.40 (1H, t, J = 7.8 Hz).

(4′-{[tert-Butyl(dimethyl)silyl]oxy}-2′,6′-dimethylbiphenyl-3-

yl)methanol (18e). The title compound was prepared in quantitative

yield as a white solid from 17e using the procedure analogous to that

described for the synthesis of 18a, except that MeOH was employed as

a solvent in place of 1,2-dimethoxyethane. ¹H NMR (300 MHz,

CDCl₃) δ 0.23 (6H, s), 1.00 (9H, s), 1.67 (1H, t, J = 5.4 Hz), 1.96

(6H, s), 4.73 (2H, d, J = 5.4 Hz), 6.58 (2H, s), 7.07 (1H, d, J = 7.2

Hz), 7.13 (1H, s), 7.30−7.36 (1H, m), 7.39 (1H, t, J = 7.5 Hz).

Ethyl 3-(4-{[4′-(2-Ethoxyethoxy)-2′,6′-dimethylbiphenyl-3-

yl]methoxy}-2-ﬂuorophenyl)propanoate (19a). To a mixture of

1

yellow oil. H NMR (300 MHz, CDCl₃) δ 1.57−1.78 (3H, s), 1.82−

1.90 (2H, m), 1.98 (6H, s), 2.02 (1H, m), 2.59 (2H, t, J = 7.8 Hz),

2.89 (2H, t, J = 7.8 Hz), 3.62 (1H, m), 3.66 (3H, s), 3.97 (1H, m),

5.08 (2H, s), 5.45 (1H, t, J = 3.0 Hz), 6.81 (2H, s), 6.89 (2H, d, J = 8.4

Hz), 7.05−7.14 (3H, m), 7.18 (1H, s), 7.34−7.47 (2H, m).

Ethyl 3-{4-[(4′-{[tert-Butyl(dimethyl)silyl]oxy}-2′,6′-dimethyl-

biphenyl-3-yl)methoxy]-2-ﬂuorophenyl}propanoate (19e). The

title compound was prepared in 89% yield as a white solid from 18e

using the procedure analogous to that described for the synthesis of

19a, except that THF was employed as a solvent in place of toluene.

¹H NMR (300 MHz, CDCl₃) δ 0.23 (6H, s), 1.00 (9H, s), 1.23 (3H, t,

J = 7.2 Hz), 1.95 (6H, s), 2.57 (2H, t, J = 7.6 Hz), 2.90 (2H, t, J = 7.6

Hz), 4.12 (2H, q, J = 7.2 Hz), 5.06 (2H, s), 6.58 (2H, s), 6.62−6.73

(2H, m), 7.04−7.21 (3H, m), 7.32−7.48 (2H, m).

tert-Butyl 3-(4-{[(4′-{[tert-Butyl(dimethyl)silyl]oxy}-2′,6′-di-

methylbiphenyl-3-yl)methyl][(2-nitrophenyl)sulfonyl]amino}-

2-ﬂuorophenyl)propanoate (19f). The title compound was

prepared in 92% yield as a yellow oil from 18e and tert-butyl 3-(4-

amino-2-ﬂuorophenyl)propanoate (38b) using the procedure analo-

1

gous to that described for the synthesis of 19d. H NMR (300 MHz,

CDCl₃) δ 0.20−0.24 (6H, m), 0.96−1.02 (9H, m), 1.36−1.41 (9H,

m), 1.81 (6H, s), 2.45 (2H, t, J = 7.7 Hz), 2.82 (2H, t, J = 7.7 Hz),

4.92 (2H, s), 6.54 (2H, s), 6.71−6.80 (2H, m), 6.90−7.07 (3H, m),

7.19−7.33 (2H, m), 7.46−7.54 (1H, m), 7.56−7.61 (1H, m), 7.63−

7.72 (2H, m).

Ethyl 3-(2-Fluoro-4-{({4′-[(4-hydroxytetrahydro-2H-thiopyr-

an-4-yl)methoxy]-2′,6′-dimethylbiphenyl-3-yl}methyl)[(2-

nitrophenyl)sulfonyl]amino}phenyl)propanoate (19g). The title

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compound was prepared in 92% yield as a pale-yellow amorphous

solid from 18c and ethyl 3-(4-amino-2-ﬂuorophenyl)propanoate (38a)

using the procedure analogous to that described for the synthesis of

Ethyl 3-[4-({4′-[2-(Ethylsulfanyl)ethoxy]-2′,6′-dimethylbi-

phenyl-3-yl}methoxy)-2-ﬂuorophenyl]propanoate (21d). To a

mixture of 20b (1.67 g, 2.75 mmol), 2-(ethylsulfanyl)ethanol (0.325

mL, 3.03 mmol), and tributylphosphine (1.03 mL, 4.13 mmol) in THF

(33 mL) was added dropwise 1,1′-(azodicarbonyl)dipiperidine (1.04 g,

4.13 mmol). After the reaction mixture was stirred at room

temperature for 16 h, 2-(ethylsulfanyl)ethanol (0.325 mL, 3.03

mmol), tributylphosphine (1.03 mL, 4.13 mmol), and 1,1′-

(azodicarbonyl)dipiperidine (1.04 g, 4.13 mmol) were sequentially

added to the mixture, which was stirred for another 16 h. The

insoluble materials were removed by ﬁltration, and the ﬁltrate was

concentrated in vacuo. The residue was puriﬁed by silica gel column

1

19d. H NMR (300 MHz, CDCl₃) δ 0.22 (6H, s), 1.00 (9H, s), 1.21

(3H, t, J = 7.2 Hz), 1.81 (6H, s), 2.53 (2H, t, J = 7.8 Hz), 2.87 (2H, t, J

= 7.8 Hz), 4.10 (2H, q, J = 7.2 Hz), 4.92 (2H, s), 6.54 (2H, s), 6.71−

6.81 (2H, m), 6.90 (1H, s), 6.96−7.08 (2H, m), 7.22−7.36 (2H, m),

7.52 (1H, m), 7.60 (1H, m), 7.63−7.73 (2H, m).

Methyl 3-{4-[(4′-Hydroxy-2′,6′-dimethylbiphenyl-3-yl)-

methoxy]phenyl}propanoate (20a). A mixture of 19d (2.92 g,

6.15 mmol) and p-toluenesulfonic acid monohydrate (0.120 g, 0.620

mmol) in MeOH (60 mL) was stirred at room temperature for 2 h

and then concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, hexane/EtOAc, 10:1 to 1:2) to aﬀord 20a

1

chromatography to aﬀord 21d (1.22 g, 64%) as a pale-yellow oil. H

NMR (300 MHz, CDCl₃) δ 1.23 (3H, t, J = 7.2 Hz), 1.31 (3H, t, J =

7.2 Hz), 1.98 (6H, s), 2.59 (2H, t, J = 7.8 Hz), 2.67 (2H, q, J = 7.2

Hz), 2.85−2.97 (4H, m), 3.66 (3H, s), 4.15 (2H, t, J = 6.9 Hz), 5.08

(2H, s), 6.66 (2H, s), 6.89 (2H, d, J = 8.7 Hz), 7.04−7.14 (3H, m),

7.17 (1H, s), 7.35−7.47 (2H, m).

1

(2.12 g, 88%) as a red oil. H NMR (300 MHz, CDCl₃) δ 1.96 (6H,

s), 2.59 (2H, t, J = 7.8 Hz), 2.89 (2H, t, J = 7.8 Hz), 3.66 (3H, s), 4.63

(1H, s), 5.08 (2H, s), 6.59 (2H, s), 6.89 (2H, d, J = 8.7 Hz), 7.05−7.13

(3H, m), 7.17 (1H, s), 7.35−7.45 (2H, m).

Ethyl 3-{2-Fluoro-4-[(4′-hydroxy-2′,6′-dimethylbiphenyl-3-

yl)methoxy]phenyl}propanoate (20b). To a solution of 19e

(11.6 g, 20.8 mmol) in THF (110 mL) at 0 °C was added dropwise

tetrabutylammonium ﬂuoride (1 M solution in THF, 22.9 mmol, 22.9

mmol). The reaction mixture was stirred at room temperature

overnight, diluted with saturated aqueous NaCl, and extracted with

EtOAc. The organic layer was washed with saturated aqueous NaCl,

dried over MgSO₄, and then concentrated in vacuo. The residue was

puriﬁed by column chromatography (silica gel, hexane/EtOAc, 9:1 to

Ethyl 3-[4-({2′,6′-Dimethyl-4′-[3-(2-oxopyrrolidin-1-yl)-

propoxy]biphenyl-3-yl}methoxy)-2-ﬂuorophenyl]propanoate

(21e). To a mixture of 20b (0.400 g, 0.947 mmol), 1-(3-

hydroxypropyl)pyrrolidin-2-one (0.134 mL, 1.04 mmol), and tribu-

tylphosphine (0.354 mL, 1.42 mmol) in THF (5 mL) was added

dropwise 1,1′-(azodicarbonyl)dipiperidine (0.358 g, 1.42 mmol). After

the reaction mixture was stirred at room temperature for 16 h, the

insoluble materials were removed by ﬁltration and the ﬁltrate was

concentrated in vacuo. The residue was puriﬁed by silica gel column

1

chromatography to aﬀord 21e (0.350 g, 67%) as a colorless oil. H

2:1) to aﬀord 20b (8.70 g, 99%) as a colorless oil. H NMR (300

NMR (300 MHz, CDCl₃) δ 1.23 (3H, t, J = 7.2 Hz), 1.96−2.10 (4H,

m), 1.98 (6H, s), 2.40 (2H, t, J = 7.8 Hz), 2.57 (2H, t, J = 7.8 Hz),

2.90 (2H, t, J = 7.8 Hz), 3.38−3.53 (4H, m), 4.00 (2H, t, J = 6.0 Hz),

4.12 (2H, q, J = 7.2 Hz), 5.06 (2H, s), 6.62−6.71 (4H, m), 7.05−7.13

(2H, m), 7.16 (1H, s), 7.34−7.46 (2H, m). MS m/z 548 (M + H)⁺.

Ethyl 3-(4-{[2′,6′-Dimethyl-4′-(tetrahydro-2H-pyran-4-

yloxy)biphenyl-3-yl]methoxy}-2-ﬂuorophenyl)propanoate

(21f). The title compound was prepared in 92% yield as a colorless oil

from 20b and tetrahydro-2H-pyran-4-ol using the procedure analogous

to that described for the synthesis of 21d, except that diethyl

azodicarboxylate and triphenylphosphine were employed in place of

1,1′-(azodicarbonyl)dipiperidine and tributylphosphine, respectively.

¹H NMR (300 MHz, CDCl₃) δ 1.23 (3H, t, J = 7.1 Hz), 1.74−1.89

MHz, CDCl₃) δ 1.23 (3H, t, J = 7.2 Hz), 1.96 (6H, s), 2.58 (2H, t, J =

7.7 Hz), 2.90 (2H, t, J = 7.7 Hz), 4.12 (2H, q, J = 7.2 Hz), 4.78 (1H,

s), 5.06 (2H, s), 6.59 (2H, s), 6.61−6.72 (2H, m), 7.00−7.20 (3H, m),

7.31−7.47 (2H, m). MS m/z 423 (M + H)⁺.

tert-Butyl 3-(2-Fluoro-4-{[(4′-hydroxy-2′,6′-dimethylbiphen-

yl-3-yl)methyl][(2-nitrophenyl)sulfonyl]amino}phenyl)-

propanoate (20c). The title compound was prepared in 79% yield as

a yellow oil from 19f using the procedure analogous to that described

for the synthesis of 20b. ¹H NMR (300 MHz, CDCl₃) δ 1.21 (3H, t, J

= 7.2 Hz), 1.82 (6H, s), 2.53 (2H, t, J = 7.8 Hz), 2.87 (2H, t, J = 7.8

Hz), 4.10 (2H, q, J = 7.2 Hz), 4.58 (1H, s), 4.93 (2H, s), 6.55 (2H, s),

6.71−6.81 (2H, m), 6.88 (1H, s), 6.96−7.09 (2H, m), 7.23−7.37 (2H,

m), 7.52 (1H, m), 7.60 (1H, m), 7.64−7.73 (2H, m).

Methyl 3-(4-{[4′-(Cyclopropylmethoxy)-2′,6′-dimethylbi-

phenyl-3-yl]methoxy}phenyl)propanoate (21a). To a mixture

of 20a (0.200 g, 0.512 mmol), cyclopropymethanol (73.6 mg, 1.02

mmol), and triphenylphosphine (0.175 g, 0.666 mmol) in THF (4

mL) was added diethyl azodicarboxylate (40 wt % solution in toluene,

0.302 mL, 0.768 mmol). The reaction mixture was stirred for 12 h and

then concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, hexane/EtOAc, 9:1 to 2:1) to aﬀord 21a

(0.157 g, 69%) as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 0.31−

0.39 (2H, m), 0.60−0.69 (2H, m), 1.27 (1H, m), 1.98 (6H, s), 2.59

(2H, t, J = 7.8 Hz), 2.89 (2H, t, J = 7.8 Hz), 3.66 (3H, s), 3.81 (2H, d,

J = 6.9 Hz), 5.08 (2H, s), 6.66 (2H, s), 6.89 (2H, d, J = 8.7 Hz), 7.05−

7.13 (3H, m), 7.18 (1H, s), 7.35−7.45 (2H, m).

Methyl 3-(4-{[4′-(Benzyloxy)-2′,6′-dimethylbiphenyl-3-yl]-

methoxy}phenyl)propanoate (21b). The title compound was

prepared in 63% yield as a colorless oil from 20a and benzyl alcohol

using the procedure analogous to that described for the synthesis of

21a. ¹H NMR (300 MHz, CDCl₃) δ 1.99 (6H, s), 2.59 (2H, t, J = 7.8

Hz), 2.89 (2H, t, J = 7.8 Hz), 3.66 (3H, s), 5.07 (2H, s), 5.08 (2H, s),

6.75 (2H, s), 6.89 (2H, d, J = 8.7 Hz), 7.05−7.13 (3H, m), 7.18 (1H,

s), 7.30−7.49 (7H, m).

(2H, m), 1.93−2.10 (8H, m), 2.57 (2H, t, J = 7.6 Hz), 2.89 (2H, t, J =

7.6 Hz), 3.55−3.66 (2H, m), 3.95−4.06 (2H, m), 4.12 (2H, q, J = 7.2

Hz), 4.44−4.55 (1H, m), 5.06 (2H, s), 6.61−6.71 (4H, m), 7.04−7.13

(2H, m), 7.15−7.19 (1H, m), 7.34−7.46 (2H, m).

Ethyl 3-(4-{[2′,6′-Dimethyl-4′-(tetrahydro-2H-thiopyran-4-

yloxy)biphenyl-3-yl]methoxy}-2-ﬂuorophenyl)propanoate

(21g). The title compound was prepared in 91% yield as a yellow oil

from 20b and tetrahydro-2H-thiopyran-4-ol using the procedure

analogous to that described for the synthesis of 21d, except that

diethyl azodicarboxylate and triphenylphosphine were employed in

place of 1,1′-(azodicarbonyl)dipiperidine and tributylphosphine,

respectively. ¹H NMR (300 MHz, CDCl₃) δ 1.23 (3H, t, J = 7.1

Hz), 1.98 (6H, s), 1.99−2.11 (2H, m), 2.16−2.27 (2H, m), 2.53−2.64

(4H, m), 2.85−3.01 (4H, m), 4.07−4.17 (2H, m), 4.32−4.42 (1H, m),

5.06 (2H, s), 6.62−6.71 (4H, m), 7.05−7.12 (2H, m), 7.17 (1H, s),

7.34−7.46 (2H, m).

tert-Butyl 3-(4-{({4′-[2-(Ethylsulfanyl)ethoxy]-2′,6′-dimethyl-

biphenyl-3-yl}methyl)[(2-nitrophenyl)sulfonyl]amino}-2-

ﬂuorophenyl)propanoate (21h). The title compound was prepared

in 86% yield as a yellow oil from 20c and 2-(ethylsulfanyl)ethanol

using the procedure analogous to that described for the synthesis of

1

21d. H NMR (300 MHz, CDCl₃) δ 1.31 (3H, t, J = 7.5 Hz), 1.39

Methyl 3-(4-{[4′-(2-Ethoxyethoxy)-2′,6′-dimethylbiphenyl-3-

yl]methoxy}phenyl)propanoate (21c). The title compound was

prepared in 51% yield as a colorless oil from 20a and 2-

ethoxymethanol using the procedure analogous to that described for

(9H, s), 1.85 (6H, s), 2.45 (2H, t, J = 7.8 Hz), 2.67 (2H, q, J = 7.5

Hz), 2.82 (2H, t, J = 7.8 Hz), 2.91 (2H, t, J = 6.9 Hz), 4.14 (2H, t, J =

6.9 Hz), 4.93 (2H, s), 6.62 (2H, s), 6.70−6.81 (2H, m), 6.91 (1H, s),

6.96−7.07 (2H, m), 7.24 (1H, d, J = 7.8 Hz), 7.31 (1H, t, J = 7.5 Hz),

7.46−7.54 (1H, m), 7.58 (1H, d, J = 7.8 Hz), 7.64−7.72 (2H, m). MS

m/z 723 (M + H)⁺.

1

the synthesis of 21a. H NMR (300 MHz, CDCl₃) δ 1.25 (3H, t, J =

7.0 Hz), 1.98 (6H, s), 2.59 (2H, t, J = 7.7 Hz), 2.89 (2H, t, J = 7.7 Hz),

3.56−3.69 (5H, m), 3.80 (2H, t, J = 5.0 Hz), 4.14 (2H, t, J = 5.0 Hz),

5.08 (2H, s), 6.68 (2H, s), 6.85−6.93 (2H, m), 7.03−7.20 (4H, m),

7.33−7.46 (2H, m).

tert-Butyl 3-[4-({[2′,6′-Dimethyl-4′-(tetrahydro-2H-thiopyr-

an-4-yloxy)biphenyl-3-yl]methyl}[(2-nitrophenyl)sulfonyl]-

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amino)-2-ﬂuorophenyl]propanoate (21i). The title compound

was prepared in quantitative yield as a pale-yellow oil from 20c and 2-

(ethylsulfanyl)ethanol using the procedure analogous to that described

for the synthesis of 21d, except that diethyl azodicarboxylate and

triphenylphosphine were employed in place of 1,1′-(azodicarbonyl)-

3-(4-{[4′-(2-Ethoxyethoxy)-2′,6′-dimethylbiphenyl-3-yl]-

methoxy}-2-ﬂuorophenyl)propanoic Acid (26). The title com-

pound was prepared in 77% yield as a white solid from 19a using the

procedure analogous to that described for the synthesis of 23, except

that 10% citric acid aqueous solution was employed in place of 1 M

HCl aqueous solution. ¹H NMR (300 MHz, CDCl₃) δ 1.25 (3H, t, J =

7.0 Hz), 1.98 (6H, s), 2.64 (2H, t, J = 7.6 Hz), 2.91 (2H, t, J = 7.6 Hz),

3.62 (2H, q, J = 7.0 Hz), 3.80 (2H, t, J = 5.0 Hz), 4.14 (2H, t, J = 5.0

Hz), 5.06 (2H, s), 6.63−6.70 (4H, m), 7.06−7.13 (2H, m), 7.16 (1H,

s), 7.33−-7.38 (1H, m), 7.42 (1H, t, J = 7.4 Hz). MS m/z 467 (M +

H)⁺. HPLC purity: 99.8%.

1

dipiperidine and tributylphosphine, respectively. H NMR (300 MHz,

CDCl₃) δ 1.39 (9H, s), 1.85 (6H, s), 1.97−2.11 (2H, m), 2.14−2.28

(2H, m), 2.45 (2H, t, J = 7.7 Hz), 2.52−2.66 (2H, m), 2.82 (2H, t, J =

7.7 Hz), 2.89−3.02 (2H, m), 4.30−4.41 (1H, m), 4.93 (2H, s), 6.61

(2H, s), 6.76 (2H, t, J = 8.3 Hz), 6.88−7.08 (3H, m), 7.18−7.35 (2H,

m), 7.46−7.62 (2H, m), 7.63−7.73 (2H, m). MS m/z 735 (M + H)⁺.

3-{4-[(4′-Methoxy-2′,6′-dimethylbiphenyl-3-yl)methoxy]-

phenyl}propanoic Acid (22). To a mixture of 20a (0.200 g, 0.512

mmol), MeOH (0.0410 mL, 1.02 mmol), and triphenylphosphine

(0.175 g, 0.666 mmol) in THF (4 mL) at 0 °C was added dropwise

diethyl azodicarboxylate (40 wt % solution in toluene, 0.302 mL, 0.768

mmol). The reaction mixture was stirred for 12 h and then

concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, hexane/EtOAc, 10:1 to 3:1) to aﬀord

methyl 3-{4-[(4′-methoxy-2′,6′-dimethylbiphenyl-3-yl)methoxy]-

phenyl}propanoate (0.134 g, 65%) as a yellow oil. To a solution of

the above ester (0.134 g, 0.331 mmol) in THF (4 mL) and MeOH (2

mL) at room temperature was added 1 M NaOH aqueous solution

(0.662 mL). The reaction mixture was stirred at the same temperature

for 1 h, and then 1 M HCl aqueous solution was added until the

solution reached pH ∼ 3. The mixture was extracted with EtOAc,

washed sequentially with water and saturated aqueous NaCl, dried

over MgSO₄, and then concentrated in vacuo. The residue was puriﬁed

by silica gel column chromatography (silica gel, hexane/EtOAc, 4:1 to

1:2) to aﬀord 22 (0.115 g, 89%) as a white solid. ¹H NMR (300 MHz,

CDCl₃) δ 1.99 (6H, s), 2.64 (2H, t, J = 7.8 Hz), 2.90 (2H, t, J = 7.8

Hz), 3.81 (3H, s), 5.08 (2H, s), 6.66 (2H, s), 6.87−6.93 (2H, m),

7.06−7.15 (3H, m), 7.18 (1H, br), 7.35−7.45 (2H, m). mp 148−149

°C. HPLC purity: 99.4%. Anal. Calcd for C₂₅H₂₆O₄: C, 76.90; H, 6.71.

Found: C, 76.98; H, 6.76.

3-(4-{[4′-(Cyclopropylmethoxy)-2′,6′-dimethylbiphenyl-3-

yl]methoxy}phenyl)propanoic Acid (23). To a solution of 21a

(0.157 g, 0.353 mmol) in THF (5 mL) and MeOH (2.5 mL) at room

temperature was added 1 M NaOH aqueous solution (0.706 mL). The

reaction mixture was stirred at the same temperature for 4 h, and then

1 M HCl aqueous solution was added until the solution reached pH ∼

3. The mixture was extracted with EtOAc, washed sequentially with

water and saturated aqueous NaCl, dried over MgSO₄, and then

concentrated in vacuo. The residue was puriﬁed by silica gel column

chromatography (silica gel, hexane/EtOAc, 4:1 to 1:2) to aﬀord 23

(0.115 g, 76%) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 0.30−

0.40 (2H, m), 0.60−0.70 (2H, m), 1.20−1.36 (1H, m), 1.98 (6H, s),

2.64 (2H, t, J = 7.8 Hz), 2.90 (2H, t, J = 7.8 Hz), 3.81 (2H, d, J = 6.9

Hz), 5.08 (2H, s), 6.66 (2H, s), 6.90 (2H, d, J = 8.4 Hz), 7.05−7.19

(4H, m), 7.35−7.45 (2H, m). HPLC purity: 99.8%; mp 128−129 °C.

Anal. Calcd for C₂₈H₃₀O₄: C, 78.11; H, 7.02. Found: C, 78.21; H, 7.03.

3-(4-{[4′-(Benzyloxy)-2′,6′-dimethylbiphenyl-3-yl]methoxy}-

phenyl)propanoic Acid (24). The title compound was prepared in

91% yield as a white solid from 21b using the procedure analogous to

that described for the synthesis of 23. ¹H NMR (300 MHz, CDCl₃) δ

1.99 (6H, s), 2.64 (2H, t, J = 7.8 Hz), 2.90 (2H, t, J = 7.8 Hz), 5.07

(2H, s), 5.08 (2H, s), 6.75 (2H, s), 6.90 (2H, d, J = 8.4 Hz), 7.05−7.15

(3H, m), 7.18 (1H, s), 7.29−7.48 (7H, m). HPLC purity: 98.5%; mp

122−123 °C. Anal. Calcd for C₃₁H₃₀O₄: C, 79.80; H, 6.48. Found: C,

79.84; H, 6.50.

3-(4-{[2′,6′-Dimethyl-4′-(tetrahydro-2H-pyran-4-yloxy)-

biphenyl-3-yl]methoxy}-2-ﬂuorophenyl)propanoic Acid (27).

The title compound was prepared in 49% yield as a white solid

from 21g using the procedure analogous to that described for the

1

synthesis of 23. H NMR (300 MHz, CDCl₃) δ 1.74−1.91 (2H, m),

1.98 (6H, s), 1.98−2.11 (2H, m), 2.64 (2H, t, J = 7.8 Hz), 2.91 (2H, t,

J = 7.8 Hz), 3.54−3.66 (2H, m), 3.95−4.06 (2H, m), 4.50 (1H, m),

5.06 (2H, s), 6.62−6.72 (4H, m), 7.05−7.15 (2H, m), 7.17 (1H, s),

7.33−7.47 (2H, m). MS m/z 479 (M + H)⁺; mp 120−121 °C. HPLC

purity: 98.9%. Anal. Calcd for C₂₉H₃₁FO₅: C, 72.78; H, 6.53. Found:

C, 72.62; H, 6.59.

3-[4-({2′,6′-Dimethyl-4′-[(6-methylpyridin-2-yl)oxy]-

biphenyl-3-yl}methoxy)-2-ﬂuorophenyl]propanoic Acid Hy-

drochloride (28). To a mixture of 18b (0.920 g, 2.88 mmol), ethyl

3-(2-ﬂuoro-4-hydroxyphenyl)propanoate (0.672 g, 3.16 mmol), and

tributylphosphine (1.08 mL, 4.32 mmol) in THF (20 mL) was added

portionwise 1,1′-(azodicarbonyl)dipiperidine (1.09 g, 4.32 mmol). The

reaction mixture was stirred at room temperature for 70 h. After

removal of the solvent in vacuo, Et₂O was added to the residue. The

insoluble materials were removed by ﬁltration, and the ﬁltrate was

concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, hexane/EtOAc, 10:1 to 3:1) to aﬀord

ethyl 3-[4-({2′,6′-dimethyl-4′-[(6-methylpyridin-2-yl)oxy]biphenyl-3-

yl}methoxy)-2-ﬂuorophenyl]propanoate (1.39 g, 94%) as a colorless

oil. MS m/z 514 (M + H)⁺. To a solution of the above compound

(1.39 g, 2.71 mmol) in THF (20 mL) and MeOH (10 mL) at room

temperature was added 1 M NaOH aqueous solution (5.42 mL, 5.42

mmol). The reaction mixture was stirred at the same temperature for 2

h and then neutralized by addition of 1 M HCl aqueous solution. The

mixture was extracted with EtOAc, washed sequentially with water and

saturated aqueous NaCl, dried over MgSO₄, and then concentrated in

vacuo. The residue was puriﬁed by silica gel column chromatography

(silica gel, hexane/EtOAc, 2:1 to 1:2) to aﬀord the free base of 28

(0.820 g, 62%) as a colorless oil. MS m/z 486 (M + H)⁺. The obtained

oil (0.820 g, 1.69 mmol) was dissolved in EtOAc (8 mL), and then 4

M HCl solution in EtOAc (1.27 mL, 5.07 mmol) was added. After

removal of the solvent in vacuo, the residue was crystallized from

EtOAc/Et₂O to aﬀord 28 (0.730 g, 48% from 18b) as a white solid. ¹H

NMR (300 MHz, DMSO-d₆) δ 1.94 (6H, s), 2.38 (3H, s), 2.41−2.56

(2H, m), 2.68−2.84 (2H, m), 5.16 (2H, s), 6.68−6.94 (5H, m), 7.03

(1H, d, J = 7.2 Hz), 7.10−7.30 (3H, m), 7.38−7.56 (2H, m), 7.70−

7.85 (1H, m). MS m/z 486 (M + H)⁺(as a free base). HPLC purity:

99.4%. Anal. Calcd for C₃₀H₂₉ClFNO₄: C, 69.03; H, 5.60; N, 2.68.

Found: C, 68.86; H, 5.71; N, 2.66.

3-[4-({2′,6′-Dimethyl-4′-[3-(2-oxopyrrolidin-1-yl)propoxy]-

biphenyl-3-yl}methoxy)-2-ﬂuorophenyl]propanoic Acid (29).

The title compound was prepared in 88% yield as a white solid

from 21e using the procedure analogous to that described for the

synthesis of 23. ¹H NMR (300 MHz, CDCl₃) δ 1.90−2.12 (10H, m),

2.41 (2H, t, J = 8.1 Hz), 2.63 (2H, t, J = 7.4 Hz), 2.90 (2H, t, J = 7.4

Hz), 3.41−3.54 (4H, m), 3.99 (2H, t, J = 6.2 Hz), 5.08 (2H, s), 6.59−

6.73 (4H, m), 7.04−7.17 (3H, m), 7.31−7.47 (2H, m). MS m/z 520

(M + H)⁺; mp 138−139 °C. HPLC purity: 100%. Anal. Calcd for

C₃₁H₃₄FNO₅: C, 71.66; H, 6.60; N, 2.70. Found: C, 71.50; H, 6.49; N,

2.65.

3-(4-{[4′-(2-Ethoxyethoxy)-2′,6′-dimethylbiphenyl-3-yl]-

methoxy}phenyl)propanoic Acid (25). The title compound was

prepared in 75% yield as a white solid from 21c using the procedure

1

analogous to that described for the synthesis of 23. H NMR (300

MHz, CDCl₃) δ 1.25 (3H, t, J = 6.9 Hz), 1.98 (6H, s), 2.64 (2H, t, J =

7.8 Hz), 2.90 (2H, t, J = 7.8 Hz), 3.62 (2H, q, J = 6.9 Hz), 3.77−3.84

(2H, m), 4.10−4.17 (2H, m), 5.08 (2H, s), 6.68 (2H, s), 6.90 (2H, d, J

= 8.7 Hz), 7.05−7.20 (4H, m), 7.34−7.45 (2H, m). MS m/z 449 (M +

H)⁺. HPLC purity: 100%. Anal. Calcd for C₂₈H₃₂O₅: C, 74.97; H, 7.19.

Found: C, 74.82; H, 7.17.

3-[4-({4′-[2-(Ethylsulfonyl)ethoxy]-2′,6′-dimethylbiphenyl-3-

yl}methoxy)-2-ﬂuorophenyl]propanoic Acid (30). To a solution

of 21d (0.200 g, 0.392 mmol) in dichloromethane (4 mL) at 0 °C was

added portionwise m-chloroperbenzoic acid (70%, 0.212 g, 0.862

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Journal of Medicinal Chemistry

Article

mmol). The reaction mixture was stirred at the same temperature for 2

h. The mixture was washed sequentially with 1 M NaOH aqueous

solution and saturated aqueous NaCl, dried over MgSO₄, and

concentrated in vacuo. The residue was puriﬁed by silica gel column

chromatography (silica gel, hexane/EtOAc, 10:1 to 1:1) to aﬀord ethyl

3-[4-({4′-[2-(ethylsulfonyl)ethoxy]-2′,6′-dimethylbiphenyl-3-yl}-

methoxy)-2-ﬂuorophenyl]propanoate (0.190 g, 89%) as a colorless oil.

¹H NMR (300 MHz, CDCl₃) δ 1.23 (3H, t, J = 7.2 Hz), 1.47 (3H, t, J

= 7.5 Hz), 1.99 (6H, s), 2.57 (2H, t, J = 7.8 Hz), 2.90 (2H, t, J = 7.8

Hz), 3.19 (2H, q, J = 7.5 Hz), 3.42 (2H, t, J = 5.4 Hz), 4.12 (2H, q, J =

7.2 Hz), 4.44 (2H, t, J = 5.4 Hz), 5.07 (2H, s), 6.62−6.71 (4H, m),

7.05−7.13 (2H, m), 7.15 (1H, s), 7.35−7.48 (2H, m). MS m/z 543

(M + H)⁺. To a solution of the above ester (0.190 g, 0.350 mmol) in

AcOH (4.5 mL) and water (4 mL) was added concd H₂SO₄(0.65

mL). The reaction mixture was stirred at 90 °C for 4 h. After cooled to

room temperature, the mixture was diluted with EtOAc and washed

sequentially with water and saturated aqueous NaCl. The organic layer

was dried over MgSO₄and concentrated in vacuo. The residue was

puriﬁed by silica gel column chromatography (silica gel, hexane/

EtOAc, 4:1 to 0:100) to aﬀord 30 (0.124 g, 61%) as a white solid. ¹H

NMR (300 MHz, CDCl₃) δ 1.47 (3H, t, J = 7.5 Hz), 1.99 (6H, s), 2.64

(2H, t, J = 7.8 Hz), 2.91 (2H, t, J = 7.8 Hz), 3.19 (2H, q, J = 7.5 Hz),

3.42 (2H, t, J = 5.4 Hz), 4.44 (2H, t, J = 5.4 Hz), 5.07 (2H, s), 6.63−

6.72 (4H, m), 7.05−7.17 (3H, m), 7.35−7.47 (2H, m); mp 123−124

°C. HPLC purity: 99.8%. Anal. Calcd for C₂₈H₃₁FO₆S: C, 65.35; H,

6.07. Found: C, 65.14; H, 5.97.

3-{4-[({4′-[(1,1-Dioxidotetrahydro-2H-thiopyran-4-yl)oxy]-

2′,6′-dimethylbiphenyl-3-yl}methyl)amino]-2-ﬂuorophenyl}-

propanoic Acid Methanesulfonate (34). To a solution of 21i

(3.50 g, 4.76 mmol) in EtOAc (20 mL) at 0 °C was added portionwise

m-chloroperbenzoic acid (70%, 1.41 g, 5.72 mmol). The reaction

mixture was stirred at room temperature for 16 h and poured into 1 M

NaOH aqueous solution. The mixture was extracted with EtOAc, dried

over Na₂SO₄, and concentrated in vacuo. The residue was puriﬁed by

silica gel column chromatography (silica gel, hexane/EtOAc, 9:1 to

1:1) to aﬀord tert-butyl 3-(4-{({4′-[(1,1-dioxidotetrahydro-2H-thio-

pyran-4-yl)oxy]-2′,6′-dimethylbiphenyl-3-yl}methyl)[(2-nitrophenyl)-

sulfonyl]amino}-2-ﬂuorophenyl)propanoate (2.49 g, 68%) as a white

1

amorphous solid. H NMR (300 MHz, CDCl₃) δ 1.38 (9H, s), 1.87

(6H, s), 2.28−2.58 (6H, m), 2.83 (2H, t, J = 7.6 Hz), 2.88−3.02 (2H,

m), 3.36−3.53 (2H, m), 4.61−4.70 (1H, m), 4.94 (2H, s), 6.65 (2H,

s), 6.70−6.84 (2H, m), 6.93−7.10 (3H, m), 7.20 (1H, d, J = 7.7 Hz),

7.31 (1H, t, J = 7.8 Hz), 7.46−7.54 (1H, m), 7.54−7.62 (1H, m),

7.63−7.74 (2H, m). To a solution of the above compound (2.49 g,

3.25 mmol) in DMF (16 mL) were added 2-sulfanylacetic acid (0.678

mL, 9.75 mmol) and lithium hydroxide monohydrate (0.818 g, 19.5

mmol). The reaction mixture was stirred at room temperature for 16 h

and then concentrated in vacuo. The residue was partitioned between

EtOAc and NaCl aqueous solution. The phases were separated, and

the aqueous phase was extracted with EtOAc. The combined organic

phases were dried over Na₂SO₄and concentrated in vacuo. The

residue was puriﬁed by silica gel column chromatography (silica gel,

hexane/EtOAc, 9:1 to 1:1) to aﬀord tert-butyl 3-{4-[({4′-[(1,1-

dioxidotetrahydro-2H-thiopyran-4-yl)oxy]-2′,6′-dimethylbiphenyl-3-

yl}methyl)amino]-2-ﬂuorophenyl}propanoate (1.55 g, 82%) as a white

3-[4-({4′-[(1,1-Dioxidotetrahydro-2H-thiopyran-4-yl)oxy]-

2′,6′-dimethylbiphenyl-3-yl}methoxy)-2-ﬂuorophenyl]-

propanoic Acid (31). To a solution of 21g (0.53 g, 1.0 mmol) in

EtOAc (10 mL) at 0 °C was added portionwise m-chloroperbenzoic

acid (70%, 0.55 g, 2.2 mmol). The reaction mixture was stirred at the

same temperature for 2 h and diluted with EtOAc. The mixture was

washed sequentially with 1 M NaOH aqueous solution and saturated

aqueous NaCl, dried over MgSO₄, and concentrated in vacuo. The

residue was puriﬁed by silica gel column chromatography (silica gel,

hexane/EtOAc, 4:1 to 1:1) to aﬀord ethyl 3-[4-({4′-[(1,1-dioxidote-

trahydro-2H-thiopyran-4-yl)oxy]-2′,6′-dimethylbiphenyl-3-yl}-

methoxy)-2-ﬂuorophenyl]propanate (0.25 g, 49%) as a pale-yellow oil.

MS m/z 555 (M + H)⁺. Subsequently, the title compound was

prepared in 68% yield as a white solid from the above ester using the

1

amorphous solid. H NMR (300 MHz, CDCl₃) δ 1.41 (9H, s), 1.97

(6H, s), 2.28−2.61 (6H, m), 2.73−2.86 (2H, m), 2.88−3.03 (2H, m),

3.35−3.54 (2H, m), 4.04−4.22 (1H, m), 4.33 (2H, s), 4.60−4.73 (1H,

m), 6.21−6.41 (2H, m), 6.66 (2H, s), 6.88−7.16 (3H, m), 7.28−7.46

(2H, m). To a solution of the above compound (1.55 g, 2.66 mmol) in

toluene (15 mL) was added triﬂuoroacetic acid (15 mL). The reaction

mixture was stirred at room temperature for 2 h and then concentrated

in vacuo. The residue was partitioned between EtOAc and saturated

aqueous NaHCO₃, and the pH of the aqueous solution was adjusted to

ca. 6−7 with 1 M HCl aqueous solution. The phases were separated,

and the aqueous solution was extracted with EtOAc. The combined

organic phases were dried over Na₂SO₄and concentrated in vacuo.

The residue was puriﬁed by silica gel column chromatography (silica

gel, hexane/EtOAc, 9:1 to 1:1) to aﬀord 3-{4-[({4′-[(1,1-dioxidote-

trahydro-2H-thiopyran-4-yl)oxy]-2′,6′-dimethylbiphenyl-3-yl}methyl)-

amino]-2-ﬂuorophenyl}propanoic acid (1.30 g, 2.47 mmol) as a white

amorphous solid. This compound (1.30 g, 2.47 mmol) was then

dissolved in EtOAc (5 mL) and Et₂O (5 mL), and to this solution was

added methanesulfonic acid (0.160 mL, 2.47 mmol). The resulting

solid was collected by ﬁltration, rinsed with Et₂O/EtOAc (2:1), and

1

procedure analogous to that described for the synthesis of 23. H

NMR (300 MHz, CDCl₃) δ 1.98 (6H, s), 2.29−2.57 (4H, m), 2.65

(2H, t, J = 7.8 Hz), 2.85−3.01 (4H, m), 3.38−3.53 (2H, m), 4.67 (1H,

m), 5.07 (2H, s), 6.61−6.73 (4H, m), 7.04−7.18 (3H, m), 7.34−7.49

(2H, m). MS m/z 527 (M + H)⁺; mp 148−149 °C. HPLC purity:

100%. Anal. Calcd for C₂₉H₃₁FO₆S: C, 66.14; H, 5.93. Found: C,

65.92; H, 6.02.

3-[2-Fluoro-4-({4′-[(4-hydroxy-1,1-dioxidotetrahydro-2H-thi-

opyran-4-yl)methoxy]-2′,6′-dimethylbiphenyl-3-yl}methoxy)-

phenyl]propanoic Acid (32). The title compound was prepared in

56% yield as a white solid from 19c using the procedure analogous to

that described for the synthesis of 31. ¹H NMR (300 MHz, CDCl₃) δ

2.00 (6H, s), 2.17−2.38 (4H, m), 2.65 (2H, t, J = 7.5 Hz), 2.85−3.04

(4H, m), 3.41−3.60 (2H, m), 3.88 (2H, s), 5.08 (2H, s), 6.61−6.77

(4H, m), 7.03−7.21 (3H, m), 7.35−7.49 (2H, m). MS m/z 557 (M +

H)⁺; mp 198−199 °C. HPLC purity: 99.3%. Anal. Calcd for

C₃₀H₃₃FO₇S: C, 64.73; H, 5.98; S, 5.76; F, 3.41. Found: C, 64.57;

H, 5.94; S, 5.83; F, 3.48.

3-{4-[({4′-[2-(Ethylsulfonyl)ethoxy]-2′,6′-dimethylbiphenyl-

3-yl}methyl)amino]-2-ﬂuorophenyl}propanoic Acid Methane-

sulfonate (33). The title compound was prepared in 60% yield as a

white solid from 21h using the procedure analogous to that described

for the synthesis of 34. ¹H NMR (300 MHz, DMSO-d₆) δ 1.27 (3H, t,

J = 7.5 Hz), 1.88 (6H, s), 2.34 (3H, s), 2.39 (2H, t, J = 7.5 Hz), 2.65

(2H, t, J = 7.5 Hz), 3.18 (2H, q, J = 7.5 Hz), 3.58 (2H, t, J = 5.4 Hz),

4.28−4.37 (4H, m), 6.28−6.42 (2H, m), 6.72 (2H, s), 6.90−7.00 (2H,

m), 7.05 (1H, s), 7.31 (1H, d, J = 7.5 Hz), 7.39 (1H, t, J = 7.5 Hz). MS

m/z 514 (M + H)⁺(as a free base). HPLC purity: 99.0%. Anal. Calcd

for C₂₈H₃₂FNO₅S·CH₃SO₃H·0.4H₂O: C, 56.46; H, 6.01; N, 2.27.

Found: C, 56.39; H, 5.95; N, 2.12.

1

dried to aﬀord 34 (1.39 g, 47% from 21i) as a white solid. H NMR

(300 MHz, DMSO-d₆) δ 1.87 (6H, s), 2.10−2.30 (4H, m), 2.34−2.46

(5H, m), 2.67 (2H, t, J = 7.5 Hz), 3.06−3.28 (4H, m), 4.35 (2H, s),

4.63−4.77 (1H, m), 6.35−6.53 (2H, m), 6.78 (2H, s), 6.92−7.10 (3H,

m), 7.28−7.44 (2H, m). MS m/z 526 (M + H)⁺(as a free base).

H P L C p u r i t y : 9 9 . 9 % . A n a l . C a l c d f o r

C₂₉H₃₂FNO₅S·CH₃SO₃H·0.5H₂O: C, 57.13; H, 5.91; N, 2.22.

Found: C, 57.08; H, 5.85; N, 2.15.

3-{2-Fluoro-4-[({4′-[(4-hydroxy-1,1-dioxidotetrahydro-2H-

thiopyran-4-yl)methoxy]-2′,6′-dimethylbiphenyl-3-yl}methyl)-

amino]phenyl}propanoic Acid (35). To a solution of 19g (1.71 g,

2.32 mmol) in dichloromethane (20 mL) at 0 °C was added

portionwise m-chloroperbenzoic acid (70%, 1.26 g, 5.11 mmol). The

reaction mixture was stirred at room temperature for 3 h and

concentrated in vacuo. The residue was partitioned between EtOAc

and 1 M NaOH aqueous solution. The phases were separated, and the

aqueous phase was extracted with EtOAc. The combined organic

phases were dried over Na₂SO₄and concentrated in vacuo. The

residue was puriﬁed by silica gel column chromatography (silica gel,

hexane/EtOAc, 9:1 to 1:4) to aﬀord ethyl 3-(2-ﬂuoro-4-{({4′-[(4-

hydroxy-1,1-dioxidotetrahydro-2H-thiopyran-4-yl)methoxy]-2′,6′-di-

3771

dx.doi.org/10.1021/jm2016123 | J. Med. Chem. 2012, 55, 3756−3776

Journal of Medicinal Chemistry

Article

methylbiphenyl-3-yl}methyl)[(2-nitrophenyl)sulfonyl]amino}phenyl)-

propanoate (1.28 g, 72%) as a white amorphous solid. H NMR (300

3.66 (2H, s), 4.12 (2H, q, J = 7.2 Hz), 6.32−6.42 (2H, m), 6.96 (1H, t,

J = 8.4 Hz). MS m/z 212 (M + H)⁺.

1

tert-Butyl 3-(4-Amino-2-ﬂuorophenyl)propanoate (38b). The

title compound was prepared in 98% yield as a pale-yellow solid from

37b using the procedure analogous to that described for the synthesis

MHz, CDCl₃) δ 1.21 (3H, t, J = 7.3 Hz), 1.80−1.94 (6H, m), 2.15−

2.38 (4H, m), 2.45−2.64 (3H, m), 2.79−3.05 (4H, m), 3.40−3.61

(2H, m), 3.87 (2H, s), 4.10 (2H, q, J = 7.1 Hz), 4.93 (2H, s), 6.63

(2H, s), 6.71−6.86 (2H, m), 6.87−7.12 (3H, m), 7.19−7.41 (2H, m),

7.46−7.79 (4H, m). To a solution of the above compound (1.28 g,

1.67 mmol) in DMF (9 mL) were added sulfanylacetic acid (0.348

mL, 5.01 mmol) and lithium hydroxide monohydrate (0.420 g, 10.0

mmol). The reaction mixture was stirred at room temperature for 24 h

and then concentrated in vacuo. The residue was partitioned between

EtOAc and NaCl aqueous solution. The phases were separated, and

the aqueous phase was extracted with EtOAc. The combined organic

phases were dried over Na₂SO₄and concentrated in vacuo. The

residue was puriﬁed by silica gel column chromatography (silica gel,

hexane/EtOAc, 17:3 to 1:1) to aﬀord ethyl 3-{2-ﬂuoro-4-[({4′-[(4-

hydroxy-1,1-dioxidotetrahydro-2H-thiopyran-4-yl)methoxy]-2′,6′-di-

methylbiphenyl-3-yl}methyl)amino]phenyl}propanoate (0.820 g,

1

of 38a. H NMR (300 MHz, CDCl₃) δ 1.42 (9H, s), 2.47 (2H, t, J =

7.8 Hz), 2.81 (2H, t, J = 7.8 Hz), 3.65 (2H, s), 6.32−6.40 (2H, m),

6.95 (1H, t, J = 8.4 Hz). MS m/z 240 (M + H)⁺.

Ethyl 3-(2-Fluoro-4-{[(2-nitrophenyl)sulfonyl]amino}-

phenyl)propanoate (39a). To a solution of 38a in pyridine (70

mL) was added portionwise 2-nitrobenzenesulfonyl chloride (11.4 g,

51.5 mmol). After being stirred at room temperature for 70 h, the

reaction mixture was concentrated in vacuo. To the residue were

added water and EtOAc, and the resultant mixture was stirred at 80 °C

for 15 min and passed through a pad of Celite. The organic phase of

the ﬁltrate was washed with saturated aqueous NaCl, dried over

MgSO₄, and concentrated in vacuo. The residue was puriﬁed by silica

gel column chromatography (silica gel, hexane/EtOAc, 4:1 to 1:1) to

1

aﬀord 39a (14.2 g, 76%) as a pale-yellow solid. H NMR (300 MHz,

84%) as a white amorphous solid. H NMR (300 MHz, CDCl₃) δ

CDCl₃) δ 1.21 (3H, t, J = 7.2 Hz), 2.56 (2H, t, J = 7.6 Hz), 2.89 (2H,

t, J = 7.6 Hz), 4.10 (2H, q, J = 7.2 Hz), 6.86 (1H, dd, J = 8.3, 1.9 Hz),

6.99 (1H, dd, J = 10.7, 2.3 Hz), 7.10 (1H, t, J = 8.3 Hz), 7.24 (1H, s),

7.62 (1H, td, J = 7.7, 1.4 Hz), 7.72 (1H, td, J = 7.7, 1.4 Hz), 7.87 (1H,

dd, J = 3.4, 1.4 Hz), 7.89 (1H, dd, J = 3.4, 1.4 Hz). MS m/z 397 (M +

H)⁺.

1.23 (3H, t, J = 7.3 Hz), 1.98 (6H, s), 2.14−2.35 (4H, m), 2.44−2.61

(3H, m), 2.75−3.02 (4H, m), 3.37−3.59 (2H, m), 3.87 (2H, s), 4.11

(2H, q, J = 7.2 Hz), 4.33 (2H, s), 6.21−6.39 (2H, m), 6.66 (2H, s),

6.88−7.12 (3H, m), 7.27−7.44 (2H, m). To a solution of the above

compound (0.820 g, 1.40 mmol) in THF (3 mL) and MeOH (10 mL)

was added 1 M NaOH aqueous solution (4.20 mL, 4.20 mmol). After

being stirred at room temperature for 24 h, 10% citric acid aqueous

solution was added. The mixture was evaporated under reduced

pressure to remove MeOH, extracted with EtOAc, dried over Na₂SO₄,

and concentrated in vacuo. The residue was puriﬁed by silica gel

column chromatography (silica gel, hexane/EtOAc, 17:3 to 1:2). The

residue was recrystallized from EtOAc/hexane to aﬀord 35 (0.618 g,

48% from 19g) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 1.89−

2.07 (6H, m), 2.15−2.36 (4H, m), 2.60 (2H, t, J = 7.6 Hz), 2.84 (2H,

t, J = 7.6 Hz), 2.89−3.02 (2H, m), 3.38−3.59 (2H, m), 3.87 (2H, s),

4.34 (2H, s), 6.24−6.40 (2H, m), 6.65 (2H, s), 6.91−7.11 (3H, m),

7.28−7.45 (2H, m). MS m/z 556 (M + H)⁺; mp 180−181 °C. HPLC

purity: 97.8%. Anal. Calcd for C₃₀H₃₄FNO₆S: C, 64.85; H, 6.17; N,

2.52. Found: C, 64.55; H, 6.33; N, 2.41.

tert-Butyl 3-(2-Fluoro-4-{[(2-nitrophenyl)sulfonyl]amino}-

phenyl)propanoate (39b). The title compound was prepared in

77% yield as a beige solid from 38b using the procedure analogous to

that described for the synthesis of 39a. ¹H NMR (300 MHz, CDCl₃) δ

1.38 (9H, s), 2.48 (2H, t, J = 7.8 Hz), 2.85 (2H, t, J = 7.8 Hz), 6.86

(1H, dd, J = 2.1, 8.1 Hz), 6.98 (1H, dd, J = 2.1, 10.8 Hz), 7.10 (1H, t, J

= 8.1 Hz), 7.61 (1H, m), 7.71 (1H, m), 7.87 (2H, dd, J = 1.5, 7.8 Hz).

Ethyl 3-[4-({[4′-(2-Ethoxyethoxy)-2′,6′-dimethylbiphenyl-3-

yl]methyl}amino)-2-ﬂuorophenyl]propanoate (40). To a mixture

of 17a (1.48 g, 7.00 mmol) and 38a (2.09 g, 7.00 mmol) in toluene

(15 mL) were added molecular sieves (0.4 nm, beads, 3.5 g). After

being stirred at room temperature for 30 h, the reaction mixture was

passed through a pad of Celite and the ﬁltrate was concentrated in

vacuo to aﬀord the crude imine. The above imine was hydrogenated

under atmospheric hydrogen with 10% Pd on carbon (containing 50%

water, 0.5 g) as a catalyst in EtOH (20 mL) at room temperature for

12 h. The reaction mixture was ﬁltered by Millipore ﬁltration and

concentrated in vacuo. The residue was puriﬁed by silica gel column

chromatography (silica gel, hexane/EtOAc, 19:1 to 7:3) to aﬀord 40

(2.88 g, 83%) as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 1.19−

1.28 (6H, m), 1.97 (6H, s), 2.54 (2H, t, J = 7.7 Hz), 2.84 (2H, t, J =

7.7 Hz), 3.61 (2H, q, J = 7.0 Hz), 3.80 (2H, t, J = 5.0 Hz), 4.07−4.16

(5H, m), 4.33 (2H, s), 6.25−6.36 (2H, m), 6.68 (2H, s), 6.95 (1H, t, J

= 8.5 Hz), 7.04 (1H, dt, J = 7.3, 1.4 Hz), 7.10 (1H, s), 7.27−7.32 (1H,

m), 7.38 (1H, t, J = 7.5 Hz). MS m/z 494 (M + H)⁺.

3-[4-({[4′-(2-Ethoxyethoxy)-2′,6′-dimethylbiphenyl-3-yl]-

methyl}amino)-2-ﬂuorophenyl]propanoic Acid Hydrochloride

(41). To a solution of 40 (2.88 g, 5.83 mmol) in EtOH (90 mL) and

THF (90 mL) was added 2 M NaOH aqueous solution (30 mL). The

reaction mixture was stirred at room temperature for 16 h, and then

10% citric acid aqueous solution was added until the solution reached

pH ∼ 5. The mixture was extracted with EtOAc, washed with saturated

aqueous NaCl, dried over MgSO₄, and then concentrated in vacuo.

The residue was puriﬁed by silica gel column chromatography (silica

gel, hexane/EtOAc, 4:1 to 1:4) to give a free base of 41 (2.70 g, 5.80

mmol) as a pale-yellow oil. To a solution of the above carboxylic acid

(2.66 g, 5.72 mmol) in EtOAc (15 mL) was added 4 M HCl solution

in EtOAc (5 mL). The resulting precipitate was triturated with

EtOAc/Et₂O, collected by ﬁltration, rinsed with EtOAc/Et₂O, and

dried to aﬀord 41 (2.78 g, 96%) as a white solid. ¹H NMR (300 MHz,

CDCl₃) δ 1.24 (3H, t, J = 7.0 Hz), 1.83 (6H, s), 2.65 (2H, t, J = 6.5

Hz), 2.83 (2H, t, J = 6.5 Hz), 3.61 (2H, q, J = 7.0 Hz), 3.78 (2H, t, J =

4.8 Hz), 4.11 (2H, t, J = 4.8 Hz), 4.48 (2H, s), 6.63 (2H, s), 6.82 (1H,

d, J = 9.8 Hz), 6.89 (1H, s), 6.94−7.01 (1H, m), 7.02−7.12 (2H, m),

7.41 (1H, t, J = 7.6 Hz), 7.49−7.55 (1H, m). mp 122 °C. Anal. Calcd

Ethyl (2E)-3-(4-Amino-2-ﬂuorophenyl)prop-2-enoate (37a).

To a mixture of 36 (13.3 g, 70.0 mmol), ethyl acrylate (9.48 mL, 87.5

mmol), and tris(2-methylphenyl)phosphine (8.52 g, 28.0 mmol) in

N,N-diisopropylethylamine (50 mL) and DMF (50 mL) was added

palladium acetate(II) (0.786 g, 3.50 mmol). The reaction mixture was

stirred for 5 h at 110 °C under argon. After cooling to room

temperature, the reaction mixture was concentrated in vacuo. The

residue was diluted with water and EtOAc, and the insoluble materials

were removed by Celite ﬁltration. The organic phase of the ﬁltrate was

washed with saturated aqueous NaCl, dried over MgSO₄, and

concentrated in vacuo. The residue was puriﬁed by silica gel column

chromatography (silica gel, hexane/EtOAc, 4:1 to 2:3) to aﬀord 37a

1

(14.0 g, 96%) as a yellow solid. H NMR (300 MHz, CDCl₃) δ 1.32

(3H, t, J = 7.2 Hz), 4.03 (2H, s), 4.24 (2H, q, J = 7.2 Hz), 6.28−6.47

(3H, m), 7.32 (1H, t, J = 8.4 Hz), 7.71 (1H, d, J = 16.0 Hz). MS m/z

210 (M + H)⁺.

tert-Butyl (2E)-3-(4-Amino-2-ﬂuorophenyl)prop-2-enoate

(37b). The title compound was prepared in 80% yield as a yellow

solid from 36 and tert-butyl acrylate using the procedure analogous to

that described for the synthesis of 37a. ¹H NMR (300 MHz, CDCl₃) δ

1.52 (9H, s), 3.99 (2H, s), 6.26 (1H, d, J = 16.2 Hz), 6.31−6.45 (2H,

m), 7.30 (1H, t, J = 8.4 Hz), 7.62 (1H, d, J = 16.2 Hz).

Ethyl 3-(4-Amino-2-ﬂuorophenyl)propanoate (38a). Com-

pound 37a (12.4 g, 59.3 mmol) was hydrogenated under atmospheric

hydrogen with 10% Pd on carbon (containing 50% water, 4.0 g) as a

catalyst in EtOH (120 mL) at room temperature for 12 h. The

reaction mixture was ﬁltered by Millipore ﬁltration and concentrated

in vacuo. The residue was puriﬁed by silica gel column

chromatography (silica gel, hexane/EtOAc, 4:1 to 1:1) to aﬀord 38a

(9.89 g, 79%) as a pale-brown oil. ¹H NMR (300 MHz, CDCl₃) δ 1.23

(3H, t, J = 7.2 Hz), 2.55 (2H, t, J = 7.7 Hz), 2.85 (2H, t, J = 7.7 Hz),

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for C₂₈H₃₂FNO₄·HCl: C, 66.99; H, 6.63; N, 2.79. Found: C, 66.81; H,

6.68; N, 2.72.

manufacture’s instruction. ATP content was calculated (n = 3) as

follows. ATP content (% of control) = (RLU of compound/RLU of

1% DMSO) × 100. Caspase-3/7 activity was measured by using

Caspase-GloTM 3/7 assay kit (Promega) according to the

manufacture’s instruction. Caspase-3/7 activity was calculated (n =

3) as follows. 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 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 CH₃CN

containing an internal standard. After centrifugation, the supernatant

was diluted and centrifuged again. The compound concentrations in

the supernatant were measured by a high performance liquid

chromatography−tandem mass spectrometry.

Homology Modeling and Ligand Docking. A homology model

of GPR40 was constructed using the crystal structure of bovine

rhodopsin (PDB code 1GZM),⁵⁷which was obtained from the RCSB

Protein Data Bank as a structural template. An alignment of the amino

acid sequences between GPR40 and rhodopsin was created using

ClustalX (version 2.0.11)⁵⁸and manually revised. Procedures of

homology modeling were performed in MOE (version 2008.10).⁵⁹

The CL2 loop on the extra cellular domain was excluded except

Cys170 forming disulﬁde bond due to the diﬃculty of estimation. In

the previous step, compound 43 was docked into the obtained

receptor model using the program GOLD (version 4.1).⁶⁰Then, the

resultant docking models with receptor models, replacing compound

43 with 31, were subjected to energy minimization with MOE after

connecting each residual substituent. In the energy minimization

Preparation of CHO Membrane for GPR40 Receptor Binding

Assay. Cell lines stably expressing human GPR40 and rat GPR40

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) con-

taining 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 Receptor Binding Assay. The frozen cell membranes

were resuspended in ice-cold assay buﬀer (25 mmol/L Tris-HCl (pH

7.5), 5 mmol/L EDTA, 0.5 mmol/L PMSF, 20 μg/mL leupeptin, 0.1

μg/mL pepstatin A, 0.05% CHAPS (Wako), 0.2% fatty-acid-free BSA

(Sigma)), and used for receptor binding assay. To determine the K_d

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, 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 GF/C

ﬁlter plates (MILLIPORE) and washed with ice-cold 50 mmol/L Tris-

HCl (pH 7.5) using FilterMate Harvester (PerkinElmer). The

membrane-associated radioactivities 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, binding assays were performed in the presence

of both various concentrations of test compounds and 2 or 6 nmol/L

of the labeled ligand. The 50% inhibitory concentrations (IC₅₀values)

of test compounds for the labeled ligand were calculated using

nonlinear regression analysis in GraphPad Prism 3.0 (GraphPad

Software). K_ivalues were converted as K_i= IC₅₀/{1 + (the

concentration of the labeled ligand)/K_d}.

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% CO₂at 37 °C. Then cells were incubated in loading buﬀer

(recording medium containing 2.5 μg/mL ﬂuorescent calcium

indicator Fluo 4 a.m. (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 increases of the intracellular Ca²⁺

concentration after addition were monitored 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 Ca²⁺

concentration (A) in test compounds-treated cells, (B) in vehicle-

treated cells, and (C) in 10 μM γ-linolenic acid-treated cells). EC₅₀

value and 95% conﬁdence interval of each compound was obtained

with Prism 5.0 software (GraphPad).

Cytotoxicity Test. HepG2 cells were cultured at 37 °C, 5% CO₂in

DMEM supplemented with 10% fetal bovine serum, 50 IU/mL

penicillin, and 50 μg/mL streptomycin. Cells were seeded at 2 × 10⁴

cells/well in 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. The intracellular ATP content was

measured by using ATPliteTM-M (PerkinElmer) according to the

process, the MMFF94s force ﬁeld was used and the dielectric constant

was set to 2r, where r is the distance between two interacting atoms.

In Vitro Insulin Secretion Assay. Rat insulinoma INS-1 cell

clone, INS-1 833/15 cells were kindly donated by Duke University

Medical Center. INS-1 833/15 cells were maintained in RPMI 1640

(Invitorogen) containing 10% heat-inactivated fetal calf serum

(Thermo Trace), 10 mM Hepes, 0.05 mM β-mercaptoethanol,

penicillin/streptomycin, and 1 mM sodium pyruvate at 37 °C in a

humidiﬁed 5% CO₂/95% air. The cells were seeded into 96-well poly-

L-lysin coated plates (5 × 10⁴cells/well) and were subsequently

cultured for 2 days at 37 °C in a humidiﬁed 5% CO₂/95% air

environment. After the culture, media was removed and cells were

preincubated with Krebs−Ringer bicarbonate Hepes (KRBH) buﬀer

containing (116 mM NaCl, 4.7 mM KCl, 1.17 mM KH₂PO₄, 1.17 mM

MgSO₄·7H₂O, 25 mM NaHCO₃, 2.52 mM CaCl₂·2H₂O, 24 mM

HEPES, 0.2% FFA-free BSA) containing 1 mM glucose. After the

preincubation, the buﬀer solution was discarded and KRBH solution

containing various concentration of glucose as indicated in Figure 3,

with or without compound 31 dissolved in dimethylsulfoxide, was

added. After 2 h incubation, the supernatant in each well was collected.

Insulin concentration of each supernatant shown in Figure 3a was

measured by EIA kit (Morinaga, Japan), according to the

manufacturer’s instruction. Insulin concentration shown in Figure 3b

was measured by homemade competitive EIA using human insulin

speciﬁc antibody and biotinated-human insulin.

Oral Glucose Tolerance Test in Female Wistar Rats. Female

Wistar fatty rats were obtained from Takeda RABICS (Osaka, Japan).

Rats were fed regular 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

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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). Thirteen-week-old female

Wistar fatty rats were fasted overnight, and blood samples were

collected from tail vein. Plasma glucose (PG), triglyceride (TG), and

nonesteriﬁed free fatty acids (NEFA) levels of each sample were

measured using the automatic analyzer Hitachi 7080 (Hitachi, Japan).

Rats were divided into several groups based on body weight, PG, TG,

and NEFA (n = 6). Compound 31 (0.1−1 mg/kg) or 0.5%

methylcellulose (vehicle) was orally administered 30 min before oral

glucose load (1 g/kg). Blood samples were collected from tail vein 0

(just before glucose load), 10, 30, 60, and 120 min after the glucose

load. After collecting the plasma, PG levels were measured using

Hitachi 7080. Plasma insulin levels were measured using rat insulin

RIA kit (Linco). Statistical diﬀerences versus control were analyzed by

one-tailed Williams’ test or Shirley−Williams’ test.

Eﬀects on Normal Fasting Plasma Glucose and Insulin

Levels in Normal Rats. Eight-week-old male SD rats were fasted

overnight and divided into 3 groups based on body weight (n = 6).

Compound 35 (30 mg/kg), glibenclamide (10 mg/kg), or vehicle

(0.5% methylcellulose) was orally administered, and blood was

collected from tail vein before 0.5, 1, 2, and 3 h after the

administrations. Plasma glucose and levels were measured as described

above. Plasma insulin levels were measured by rat insulin ELISA kit

(Morinaga, Japan). Statistical diﬀerences versus control were analyzed

by Student’s t test or Aspin−Welch test.

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AUTHOR INFORMATION

■

Corresponding Author

(14) Roduit, R.; Nolan, C.; Alarcon, C.; Moore, P.; Barbeau, A.;

*Phone: +81-466-32-1217. Fax: +81-466-29-4458. E-mail:

Mikami_Satoshi@takeda.co.jp.

́

Delghingaro-Augusto, V.; Przybykowski, E.; Morin, J.; Masse, F.;

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for the malonyl-CoA/long-chain acyl-CoA pathway of lipid signaling in

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Notes

The authors declare no competing ﬁnancial interest.

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ACKNOWLEDGMENTS

■

We thank Dr. Hideo Fukui for toxicological study and Dr. Jyun-

ichi Sakamoto, Dr. Akiyoshi Tani, and Dr. Nobuyuki Amano

for their helpful suggestions and comments.

ABBREVIATIONS USED

■

GPCR, G protein-coupled receptor; GPR40, G protein-coupled

receptor 40; T2DM, type 2 diabetes mellitus; FFA, free fatty

acids; GSIS, glucose-stimulated insulin secretion; PLC,

phospholipase C; IP₃, inositol 1,4,5-triphosphate; DAG,

diacylglycerol; PKC, protein kinase C; FLIPR, ﬂuorometric

imaging plate reader; TBAF, tetrabutylammonium ﬂuoride;

CHO, Chinese hamster ovary; EIA, enzyme immunoassay;

KRBH, Krebs−Ringer bicarbonate Hepes; PG, plasma glucose;

TG, triglyceride; NEFA, nonesteriﬁed free fatty acids

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Article Doi

Discovery of phenylpropanoic acid derivatives containing polar functionalities as potent and orally bioavailable G protein-coupled receptor 40 agonists for the treatment of type 2 diabetes

DOI: 10.1021/jm2016123

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