Design and Identification of a GPR40 Full Agonist (SCO-267) Possessing a 2-Carbamoylphenyl Piperidine Moiety

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Design and Identiﬁcation of a GPR40 Full Agonist (SCO-267)

Possessing a 2‑Carbamoylphenyl Piperidine Moiety

Hideki Furukawa,* Yasufumi Miyamoto, Yasuhiro Hirata, Koji Watanabe, Yuko Hitomi,

Yayoi Yoshitomi, Jumpei Aida, Naoyoshi Noguchi, Nobuyuki Takakura, Kazuaki Takami,

Seiji Miwatashi, Yoshihiko Hirozane, Teruki Hamada, Ryo Ito, Mitsugi Ookawara, Yusuke Moritoh,

Masanori Watanabe, and Tsuyoshi Maekawa *

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ABSTRACT: GPR40/FFAR1 is a G-protein-coupled receptor expressed in pancreatic β-cells and enteroendocrine cells. GPR40

activation stimulates secretions of insulin and incretin, both of which are the pivotal regulators of glycemic control. Therefore, a

GPR40 agonist is an attractive target for the treatment of type 2 diabetes mellitus. Using the reported biaryl derivative 1, we shifted

the hydrophobic moiety to the terminal aryl ring and replaced the central aryl ring with piperidine, generating 2-(4,4-

dimethylpentyl)phenyl piperidine 4a, which had improved potency for GPR40 and high lipophilicity. We replaced the hydrophobic

moiety with N-alkyl-N-aryl benzamides to lower the lipophilicity and restrict the N-alkyl moieties to the presumed lipophilic pocket

using the intramolecular π−π stacking of cis-preferential N-alkyl-N-aryl benzamide. Among these, orally available (3S)-3-

cyclopropyl-3-(2-((1-(2-((2,2-dimethylpropyl)(6-methylpyridin-2-yl)carbamoyl)-5-methoxyphenyl)piperidin-4-yl)methoxy)pyridin-

4-yl)propanoic acid (SCO-267) eﬀectively stimulated insulin secretion and GLP-1 release and ameliorated glucose tolerance in

diabetic rats via GPR40 full agonism.

INTRODUCTION

■

GPR40/FFAR1 is a class A G-protein-coupled receptor, which

is highly expressed in pancreatic β-cells. The membrane

protein GPR40 is activated by medium- and long-chain fatty

acids, the endogenous ligands that stimulate insulin secretion

by elevating cytosolic Ca²⁺levels.¹⁻³A variety of synthetic

molecules have exhibited hypoglycemic properties in rodents

Figure 1. GPR40 partial and full agonists in Takeda.

as insulin secretagogues, thus oﬀering an attractive target for

the treatment of type 2 diabetes mellitus (T2DM).⁴The

GPR40 partial agonist fasiglifam (TAK-875) signiﬁcantly

lowered glucose levels in T2DM patients, with a minimal

risk of hypoglycemia, via glucose-stimulated insulin secretion

(GSIS) mediated by GPR40 signaling.⁵⁻⁸Unlike the partial

agonist TAK-875 (Figure 1), other types of synthetic GPR40

agonists, which are capable of acting as full agonists, have been

reported.⁹⁻¹⁶A prior study using a Ca²⁺mobilization assay in

Chinese hamster ovary (CHO) cells, with relatively low

expression levels of GPR40 (clone #2), revealed that partial

and full agonists can be distinguished by measuring maximal

Ca²⁺response to test compounds. AM-1638, a GPR40 full

agonist, had an EC₅₀value of 150 nM and an E_maxvalue of

Received: May 18, 2020

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A

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Figure 2. (a) Repositioning the hydrophobic group from the central aryl ring to the terminal aryl ring. (b) Structure of compound 2 possessing a

hydrophobic group on the terminal aryl ring. (c) Flexible alignment of compounds 1 (gray) and 2 (green) using Maestro.

Figure 3. Replacement of the phenyl ring with saturated linkers. ^aData are presented as mean values with standard deviation from more than two

b

c

independent experiments in duplicate. The value is from a single experiment in duplicate. % Compared to the reference full agonist.

182% (% of γ-linolenic acid, an endogenous ligand). TAK-875

(a partial agonist) showed EC₅₀values of >1000 nM and E_max

values of 22% (% of γ-linolenic acid).¹⁷Full agonists activate

GPR40 expressed in the gastrointestinal tract and pancreatic β-

cells to stimulate glucagon-like peptide-1 (GLP-1) secretion

and insulin secretion simultaneously, providing a GLP-1-

dependent anorectic eﬀect and an additional mechanism to

control GSIS.^18,19Therefore, GPR40 full agonists may be an

eﬀective treatment for diabetes and obesity.

In an eﬀort to discover full agonists of GPR40 as novel

antidiabetic agents, we identiﬁed a β-cyclopropyl propionic

acid derivative 1.²⁰Compound 1 exhibited moderate agonistic

activity in CHO cells with a relatively low expression of GPR40

(clone #2) with an EC₅₀value of 150 nM and an E_maxvalue of

88% (% of reference full agonist). As a result, we rearranged

the hydrophobic moiety from the central ring to the terminal

ring to access greater chemical space. We assumed that the

ﬂuorine-substituted position of compound I could easily access

the presumed pocket that the neopentyl alkyl chain of

compound 1 occupies (Figure 2a). Therefore, we incorporated

an alkyl moiety to the ortho-position relative to the pyridine

ring of compound II, which led to the identiﬁcation of

compound 2, with an EC₅₀value of 410 nM and an E_maxvalue

of 105% (Figure 2b). The ﬂexible alignment of the biaryl

fragments of compounds 1 and 2 using Maestro²¹shown in

Figure 2c may support this assumption.

We next attempted to replace the central benzene ring of 2

with a series of saturated linkers with the aim of breaking the

aromaticity of compound 2, which may allow us to identify

compounds that better ﬁt the spatial subtleties of the target

protein and improve its overall physicochemical properties.²²

We designed 4-piperidine ether 3, 4-methyl piperidine ether

4a, and 3-methyl azetidine ether 5, to keep the distance

between ring A and C (Figure 3). The agonistic activity for

each compound is depicted in Figure 3. 4-Piperidine ether 3

had poor agonistic activity as its conformation diﬀered from

that of biphenyl compound 2, whereas 4-methyl piperidine

ether 4a and 3-methyl azetidine ether 5 showed potent

agonistic properties, which implies that the saturated

piperidine of compound 4a may more eﬀectively ﬁt the spatial

subtleties of GPR40 relative to the biaryl derivative 2. To get a

better understanding of compound 4a binding, we performed a

liquid chromatography−mass spectrometry (LC−MS)-based

saturation binding assay. Saturation binding curves of allosteric

partial agonists TAK-875 and AM-1638 were generated using

an LC−MS-based saturation binding assay with the WT

B

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hGPR40-expressed VLP protein. The apparent K_dvalue of

TAK-875 was 11.9 nM and AM-1638 was 5.0 nM.^23,24

Competitiveness of 4a between TAK-875 and AM-1638 was

also determined using the LC−MS-based competitive binding

assay. As a result, AM-1638 competed with 4a for binding to

GPR40, whereas TAK-875 did not compete with 4a (Figures 4

to aﬀord 11a as a methyl ester. The pyrolysis of 10 was also

performed in ethanol to provide ethyl ester 11b. 3-Alkoxy-

substituted phenyl propionate 14 was furnished from the

formation of β-hydroxyl esters 13 with lithium diisopropyla-

mide and ethyl acetate. This was followed by silver carbonate-

promoted alkylation and the removal of benzyl ether to aﬀord

two key building blocks 14a and 14b. Compound 18 was

obtained from the coupling of the Grignard reagent with 15.

The resulting ketone 16 was converted to propionate 17 by the

Horner−Wadsworth−Emmons (HWE) reaction, followed by

reduction of the α,β-unsaturated ester. Deprotection of phenol

of 17 aﬀorded intermediate 18. The Wittig reaction with

oxetane 19, followed by Rh-catalyzed 1,4-addition of 3-

(benzyloxy)phenylboronic acid and the removal of benzyl

ether, aﬀorded 3,3-disubstituted derivative 22.

The synthetic route for the modiﬁcation of linker moieties is

illustrated in Scheme 2. 2-Bromo-4-methoxybenzaldehyde 23

was subjected to Suzuki coupling conditions to introduce the

aromatic fragment. The resulting ester 24 was converted to 25

by the Wittig reaction and reduction of oleﬁn, followed by the

reduction with LiAlH₄to produce the key intermediate alcohol

26. The alcohol 26 was condensed with phenol 11a via the

Mitsunobu reaction, followed by ester hydrolysis of the

coupling product to produce the carboxylic acid 2. N-

substituted phenyl derivatives were prepared from 2-ﬂuoro

benzaldehyde by S_NAr, followed by the Wittig reaction,

reduction, and condensation by the Mitsunobu reaction for 3

and 4a or by alkylation with the corresponding mesylate for 5.

A similar reaction sequence to Scheme 2 delivered 4b−d using

Pd-catalyzed amination instead of S_NAr and 4e (Scheme 3).

Several phenylpiperidine derivatives with diversiﬁed sub-

stituents were synthesized from a common precursor 30.

Protection of alcohol with TBSCl, followed by the addition of

the Grignard reagent produced 43. Chlorination of the alcohol

of 43 with thionyl chloride and catalytic hydrogenation with

palladium on carbon followed by deprotection of alcohol

converted it to key intermediate alcohol 44. Further

conversion was performed on 44 through the sequential

Mitsunobu reaction and ester hydrolysis to deliver the

carboxylic acid 4f. Compound 30 was also converted to

intermediate 45 through the corresponding mesylate of 30 and

the condensation with 11a. The resulting aldehyde 45 was

treated with N-methylisobutylamine and NaBH(OAc)₃,

followed by ester hydrolysis to produce the carboxylic acid

4h (Scheme 4). The synthetic route toward the derivative with

a hydroxyl group on the terminal of the alkyl chain is depicted

in Scheme 5. The Wittig reaction of 30, followed by copper-

catalyzed 1,4-reduction produced the ethyl ester 46. The

resulting ester 46 was converted to 47 by the addition of

methyl magnesium chloride; the sequential protection of the

two alcohols proceeded smoothly and regioselectivity through

the initial treatment with TBSCl and imidazole, followed by

Ac₂O and tetraethylammonium (TEA) to aﬀord 49. After the

deprotection of 49 with tetrabutylammonium ﬂuoride

(TBAF), a condensation reaction with 11b and hydrolysis

yielded the carboxylic acid 4g.

Figure 4. LC−MS-based competitive binding assay result of 4a and

two types of GPR40 agonists, TAK-875 and AM-1638. Two

independent experiments in duplicate were performed. Data are

shown separately in this ﬁgure and Figure S1.

and S1, Supporting Information). Furthermore, TAK-875 and

4a showed potential to form a ternary complex with GPR40.

Therefore, 4a does not bind near the TAK-875 binding site.

Based on the results obtained by in vitro characterization of the

compounds, we embarked on a medicinal chemistry campaign

starting with 4a and a phenylpiperidine ring to discover potent

derivatives to reveal the structure−activity relationship (SAR)

of the substituents on ring A of 4a. Herein, we report the

design of a novel phenylpiperidine-bearing amide moiety on

the benzene ring and the subsequent optimization studies that

led to the identiﬁcation of SCO-267, with high potency and

signiﬁcant glucose-lowering properties mediated by the full

agonistic eﬀects of GPR40 (Figure 5).

Figure 5. Structural exploration of GPR40 full agonists.

CHEMISTRY

■

The amide analogues (6a−6h) were generated with

benzylation of benzoic acid 51 under basic conditions,

followed by the addition of 4-piperidinemethanol to aﬀord

53. The resulting 53 was converted to key intermediate benzyl

carboxylic acid 55 by the Mitsunobu reaction and the removal

of the benzyl protection of the ester (Scheme 6). Target

compounds 6a−6h were obtained by a condensation reaction

The derivatization on the β-position of phenyl propionates is

depicted in Scheme 1 (11a−b, 14a−b, 18, and 22). Aldehyde

8 was condensed with Meldrum’s acid, followed by 1,4-

addition with the cyclopropyl Grignard reagent to aﬀord 10.

Conversion of alkyl Meldrum’s acid derivative 10 was

performed in dimethylformamide (DMF) and water, followed

by esteriﬁcation of the carboxylic acid under acidic conditions

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a

Scheme 1. Synthesis of 3-Substituted-3-(3-hydroxyphenyl)propionate

a

Reagents and conditions: (a) Meldrum’s acid, water, r.t., 97%; (b) cyclopropylmagnesium bromide, THF, −10 °C to r.t., 93%; (c) (i) DMF,

water, 90 °C, 92%; (ii) H₂SO₄(cat), MeOH, 70 °C, 82% (for 11a); (d) DMF, EtOH, 100 °C, 54% (for 11b); (e) n-BuLi, diisopropylamine, THF,

ethyl acetate, −78 °C; (f) iodomethane, silver carbonate, toluene, 100 °C, 69% (for 14a); (g) iodoethane, silver carbonate, toluene, 100 °C, 44%

(for 14b); (h) H₂(1 atm), 10% Pd on carbon, EtOH, r.t., 69−77%; (i) 2-(2-tetrahydro-2H-pyranoxy)phenylmagnesium bromide, THF, −78 °C;

(j) 2-(diethoxyphosphoryl) acetate, NaH, THF, 0−50 °C; (k) zinc (powder), AcOH, 50 °C, 5% over 3 steps; (l) H₂(1 atm), 10% Pd on carbon,

EtOH, r.t., 97%; (m) (carbethoxymethylene)triphenylphosphorane, THF, 0 °C to r.t., 72%; (n) 3-(benzyloxy)phenylboronic acid, Rh₂Cl₂(cod)₂,

KOH, dioxane, 100 °C, quant; (o) H₂(1 atm), 10% Pd on carbon, EtOH, r.t., 87%.

a

Scheme 2. Synthesis of Derivatives with a Variety of Linkers

a

Reagents and conditions: (a) 4-(methoxycarbonyl)benzeneboronic acid, Pd(PPh₃)₄, 2 M Na₂CO₃aq, DME, 130 °C, microwave, 99%; (b) (3,3-

dimethylbutyl)triphenylphosphonium tosylate (for 25, 29, 33), t-BuOK, THF, 50 °C; (c) 10% Pd on carbon, MeOH, H₂, r.t., 15−36% over 2

steps; (d) LiAlH₄, THF, 0 °C, 35−56%; (e) 11a, DEAD, PPh₃, THF, r.t., 74%; (f) 1 M NaOH aq, MeOH, THF, r.t., 95% (g) 4-hydroxy piperidine

(for 28), 4-piperidinemethanol (for 30), Cs₂CO₃, TBAI, DMF, 100−110 °C, 42−78%; (h) (3,3-dimethylbutyl)triphenylphosphonium bromide

(for 31), n-BuLi, THF, −78 °C, 53%, (i) 10% Pd on carbon, EtOH, H₂, r.t., 98% (j) 11b, cyanomethylenetributylphosphorane, toluene, 100 °C;

(k) 2 M NaOH, EtOH, THF, 80 °C, 82% over 2 steps; (l) (i) methyl azetidine-3-carboxylate hydrochloride, Cs₂CO₃, TBAI, DMSO, 120 °C; (ii)

MeI, K₂CO₃, DMF, r.t., 41% over 2 steps; (m) mesyl chloride, TEA, THF, r.t.; (n) 11a, K₂CO₃, DMF, 100 °C, 30%.

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a

Scheme 3. Synthesis of Phenylpiperidine Derivatives with Small Substituents

a

Reagents and conditions: (a) ethyl isonipecotate, Pd(OAc)₂, BINAP, Cs₂CO₃, toluene, 100 °C, 67% (for 35b); (b) ethyl isonipecotate,

Pd₂(dba)₃, BINAP, NaO^tBu, toluene, 100 °C, 79% (for 35c); (c) LiAlH₄, THF, 0 °C; (d) 4-piperidinemethanol, Pd₂(dba)₃, BINAP, NaO^tBu,

toluene, 100 °C, 33% (for 36d); (e) 11b, DIAD, PPh₃, THF, r.t., 18% over 2 steps (for 4b); (f) 11b, ADDP, PBu₃, THF, 0 °C to r.t., 34% over 2

steps (for 4c); (g) 11b, cyanomethylenetributylphosphorane, toluene, 100 °C, quant (for 4d); (h) 1 M NaOH aq, THF, MeOH or EtOH, r.t., 85−

i

89%; (i) 4-piperidinemethanol, K₂CO₃, DMSO, 130 °C, 84%; (j) MOMCl, Pr₂EtN, THF, r.t., quant; (k) t-BuOK, methyltriphenylphosphonium

bromide, THF, 50 °C, 79%; (l) H₂(1 atm), 10% Pd on carbon, EtOAc, r.t., 93%; (m) (i) HCl, MeOH, 60 °C; (ii) 11b,

cyanomethylenetributylphosphorane, toluene, 100 °C, 80% over 2 steps; (n) 1 M NaOH aq, THF, EtOH, 50 °C, 38%.

a

Scheme 4. Synthesis of Phenylpiperidine Derivatives with Bulky Substituents

a

Reagents and conditions: (a) TBSCl, imidazole, DMF, 0 °C, 81%; (b) neopentylmagnesium chloride, THF, r.t., 73%; (c) SOCl₂, toluene, r.t.,

95%; (d) H₂(1 atm), 10% Pd on carbon, MeOH, r.t., 88%; (e) 11a, ADDP, PBu₃, THF, 0 °C to r.t., 16%; (f) 1 M NaOH aq, THF, MeOH, r.t.,

74−82%; (g) methane sulfonyl chloride, TEA, THF, r.t.; (h) 11a, NaH, DMF, 100 °C, 64% over 2 steps; (i) N-methylisobutylamine,

NaBH(OAc)₃, THF, r.t., 59%.

through 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo-

[4,5-b]pyridinium 3-oxide hexaﬂuorophosphate (HATU)

activation or acyl chlorination using chloro-N,N,2-trimethyl-

1-propen-1-amine.

The convergent synthesis routes of 7a−d with derivatization

on the β-position to the propionic acid and 7e−f with the

replacement of the C-ring (Figure 3) with a pyridine ring are

depicted in Schemes 7 and 8. 56 was obtained via the

generation of acyl chloride, followed by S_NAr using 4-

piperidinemethanol to aﬀord 57. The resulting 57 was

converted to target 7a−d by the Mitsunobu reaction and

hydrolysis of the ester. For replacement of the C-ring, the

reaction of cyclopropylmagnesium bromide with aldehydes

58a and 58b yielded alcohols 59a and 59b. The obtained

alcohols were subjected to oxidation, followed by the HWE

reaction and reduction of oleﬁn using zinc to furnish 62a and

62b, which were treated with pyridinium hydrochloride to

deliver the key intermediate 63a and 63b. The Mitsunobu

reaction of phenol 63a and 63b with alcohol 57, followed by

ester hydrolysis yielded 7a−7f. The chiral (S)-7f and (R)-7f

were prepared by optical resolution using the chiral column.

The conﬁguration is discussed in Scheme 9 (vide infra).

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a

Scheme 5. Synthesis of a Phenylpiperidine Derivative with a Bulky Substituent

a

Reagents and conditions: (a) (carbethoxymethylene)triphenylphosphorane, toluene, r.t.; (b) CuCl, NaO^tBu, BINAP, t-BuOH, PMHS, r.t., 24%

over 2 steps; (c) methyl magnesium chloride, THF, 0 °C to r.t., 56%; (d) TBSCl, imidazole, DMF, 0 °C, 81%; (e) Ac₂O, TEA, DMAP, THF, 60

°C; (f) TBAF, THF, 60 °C, 76% over 2 steps; (g) 11b, cyanomethylenetributylphosphorane, toluene, 100 °C, 76%; (h) 1 M NaOH aq, THF,

MeOH, r.t., 85%.

a

Scheme 6. Synthesis of Phenylpiperidine Derivatives with Amide Substituents

a

Reagents and conditions: (a) benzyl bromide, K₂CO₃, DMF, r.t., 95%; (b) 4-piperidinemethanol, Cs₂CO₃, TBAI, DMF, 100 °C, 59%; (c) 11b,

cyanomethylenetributylphosphorane, toluene, 100 °C, 90%; (d) H₂, 10% Pd on carbon, EtOH, THF using H-Cube, r.t., 93%; (e) R¹R²NH, HATU,

DIPEA, DMF, r.t., quant (for 6a, 6c) (f) R¹R²NH, 1-chloro-N,N,2-trimethyl-1-propen-1-amine, Et₃N, toluene, 100 °C (for 6b, 6d−6h); (g) 1 M

NaOH aq, THF, MeOH or EtOH, r.t., 53%-quant.

a

Scheme 7. Synthesis of 2-Carbamoylphenyl Piperidine with 3-Substituted-3-(pyridin-4-yl)propionic Acid

a

Reagents and conditions: (a) 6-methyl-N-neopentylpyridine-2-amine, oxalyl chloride, DMF, Et₃N, THF, r.t., 91%; (b) 4-piperidinemethanol,

Cs₂CO₃, TBAI, DMF, 120 °C, 40%; (c) 11a or 11b, cyanomethylenetributylphosphorane, toluene, 100 °C; (d) 1 M NaOH aq, THF, MeOH, r.t.,

55−83% over 2 steps.

an index. Compound 4a exhibited potent agonistic activity in

CHO cells with a relatively low expression of GPR40 (clone

#2) and an EC₅₀value of 11 nM. However, compound 4a

exhibited cytotoxicity potential as evidenced by both decreased

RESULTS AND DISCUSSION

■

The newly synthesized compounds were initially evaluated for

human GPR40 agonist activity with an increase in intracellular

Ca²⁺concentration in CHO cells expressing human GPR40 as

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a

Scheme 8. Synthesis of 2-Carbamoylphenyl Piperidine with 3-Cyclopropyl-3-(pyridin-4-yl) Propionic Acid

a

Reagents and conditions: (a) cyclopropylmagnesium bromide, THF, 0 °C; (b) sulfur trioxide pyridine complex, TEA, DMSO, r.t.; (c) ethyl

diethylphosphonoacetate, NaH, THF, 0 °C to r.t.; (d) zinc (powder), AcOH, r.t., 74% over 4 steps (for 62a), 53% (for 62b); (e) pyridinium

hydrochloride, DMF, 120−130 °C, 71−92%; (f) 11a or 11b, cyanomethylenetributylphosphorane, toluene, 100 °C, 59−84%; (g) 1 M NaOH aq,

THF, MeOH, r.t., 79−84%; (h) CHIRALPAK AD-H SFC separation, CO₂/2-propanol (3/2, v/v), 41%, >99.9% ee as the faster eluting compound

(AD-H), 39%, 97% ee as the slower eluting compound (AD-H).

a

Scheme 9. Chiral Synthesis of Compound (S)-7f (SCO-267) via Diastereomeric Salt Formation

a

Reagents and conditions: (a) NaH, 1-Boc-4-piperidinemethanol, DMF, 0 °C, 74%; (b) cyclopropyl magnesium bromide, THF, 0 °C; (c) ethyl

diethylphosphonoacetate, NaH, THF, 0 °C; (d) zinc (powder), AcOH, 90 °C; (e) 1 M NaOH aq, THF, EtOH, 0 °C; (f) (S)-4-methyl-

methylbenzylamine, EtOH and then recrystallization twice; (g) 1 M HCl in EtOAc, 0 °C; (h) EtOH, H₂SO₄, 80 °C, (i) 56, Cs₂CO₃, neat, 130 °C;

(j) 1 M NaOH aq, THF, EtOH, 60 °C.

intracellular ATP content and Caspase induction activity, as

shown in Table 1, because of its highly lipophilic properties,

which may be reduced by a modiﬁcation of the lipophilic

group.²⁵In an initial attempt to decrease the lipophilicity and

improve the physicochemical properties of 4a, we veriﬁed the

tolerability of the substituent at the ortho-position relative to

the piperidine nitrogen attachment. As shown in Table 1, the

replacement of the long alkyl chain with a hydrogen resulted in

a >250-fold decrease in GPR40 agonistic activity, despite the

reduction in cytotoxicity potential (4b). Meanwhile, the

introduction of a ﬂuorine atom, methyl group, or iso-propyl

group into 4b resulted in tolerable potency (4c, 4d, and 4e vs

4b). This result suggests that the lipophilic moiety in this

position is essential for potent GPR40 agonistic activity.

Further elongation of the alkyl group produced an analogue 4f

with a 3,3-dimethyl butyl moiety; there was a great impact on

the GPR40 agonistic activity, similar to that seen in compound

4a, suggesting that the lipophilic pocket may be located two or

three carbons away from the A ring. Although the more

lipophilic compounds increased the agonistic properties of

GRP40, they were accompanied by an increase in cytotoxic

potential. To reduce lipophilicity of compound 4f, we replaced

the terminal methyl moiety of 4f with a hydroxyl group,

leading to the mitigation of the cytotoxic potential and >10-

fold reduction in agonistic activity (4g). In our search for

acceptable polar functions, we brieﬂy investigated the eﬀects of

introducing a heteroatom near the benzene ring on agonistic

activity. We introduced a cationic amine moiety and a

hydrophilic amide moiety. The amine derivative 4h with the

iso-butyl group exhibited a robust loss in agonistic activity

compared to 4f. In contrast, incorporation of an amide moiety

resulted in only a threefold decline in the agonistic activity of

4f with a robust reduction in cytotoxic potential (6a). Through

this investigation, we found that the binding pocket

encompassing the alkyl chain of 6a favors hydrophobicity

and the hydrophilic amide moiety is allowed near ring A of 6a.

As a result of the retained agonistic activity of compound 6a

with less cytotoxic potential compared to that of alkyl

derivatives (4a and 4f), we obtained a new lead compound

for further optimization. We replaced the tert-butyl group of

compound 6a with a small lipophilic and expandable benzene

ring, which led to a similar agonistic activity. Consequently, the

pyridine ring, characterized as a more polar substituent,

retained the agonistic activity despite the reduction in

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Table 1. Eﬀect of Substituents on the Phenyl Group on GPR40 Agonistic Activity

a

b

Data are presented as means values with the standard deviation from more than two independent experiments in duplicate. Values are from a

c

d

e

single experiment in duplicate. % Compared to the reference full agonist. The clog P values were calculated using Daylight. Intracellular ATP

content (%) at 30 μM relative to the untreated control. Lowered ATP content relative to the untreated control (100%) indicates a decrease in cell

f

viability. Data were obtained from a single experiment in triplicate. Caspase-3/7 activity was measured using a Caspase-GloTM 3/7 assay kit

(Promega) according to the manufacturer’s instruction to conﬁrm the scientiﬁc rigor of cytotoxicity in an orthogonal assay. Caspase-3/7 activity

(%) at 30 μM relative to the response to the reference compound (staurosporine) was set to 100%. Data were obtained from a single experiment in

triplicate.

Figure 6. (A) Conformational preference of N-methylbenzanilide. (B) Elongation of the alkyl chain of N-methylbenzanilide toward the presumed

lipophilic pocket that the t-butyl group of compound 4a occupied.

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Table 2. Eﬀect of Substituents on the Amide Group on GPR40 Agonistic Activity

a

b

Data are presented as mean values with the standard deviation from more than two independent experiments in duplicate. Values are from a

c

d

single experiment in duplicate. % Compared to the reference full agonist. The clog P values were calculated using Daylight.

lipophilicity (clog P values of 6b and 6c are 5.5 and 4.0,

respectively). Several studies have reported that the structures

of the compounds containing the N-methyl-N-(2-phenyl) or

N-methyl-N-(2-pyridyl) benzamide moiety place the aromatic

rings in cis conformation to each other, and the methyl

substituents on nitrogen were cis to the carbonyl group.²⁶⁻²⁹

This implies that the methyl groups of compounds 6b and 6c

are directed to and partially occupy the presumed lipophilic

pocket because of the structural conformation of cis-

preferential aromatic amides. Therefore, we hypothesized

that incorporation of longer and bulky alkyl chains on the

nitrogen group of the amide moiety instead of the methyl

group could enhance the agonistic activity by eﬀectively

occupying the presumed lipophilic pocket (Figure 6).

As expected, the incorporation of iso-butyl dramatically

impacted agonistic activity (6c vs 6d in Table 2); moreover,

further incorporation of the methyl group on the branched

position of the iso-butyl group resulted in a slight enhancement

of potency with good reproducibility (compound 6e).

However, the incorporation of polar substituents such as the

cyano group and methoxy group negatively aﬀected GPR40

potency (6f and 6g). We veriﬁed that these SAR data are

comparable with the results discussed in Table 1.

With (2,2-dimethylpropyl)(6-methylpyridin-2-yl)-

carbamoylphenyl-based 6h, we optimized the β-position of

the propionic acid moiety and the C ring (Table 3).

Replacement of the cyclopropyl group with a methoxy group

reduced the lipophilicity of the molecule with a modest decline

in agonistic activity. As with 7a, other compounds such as

ethoxy 7b, methoxy methyl 7c, and 3-oxetanyl 7d derivatives

were not in the desired potency range. This implies that

cyclopropyl is an appropriate substituent in terms of potency.

As for optimization of the C ring, we selected the 2-alkoxy

substitution pattern on the pyridine ring because, as observed

in our previous study, basic property is not tolerable around

the C ring. As a result, in both 2-alkoxy patterns, 2,6-

disubstituted pyridine derivative 7e exhibited decreased

agonistic activity with a lower clog P value compared to 6h.

Conversely, the 2,4-disubstituted pyridine derivative 7f

retained agonistic activity along with decreased lipophilicity.

To obtain a better understanding of the diﬀerence, we

calculated energy barriers for 2-alkoxy pyridine using Maestro,

as illustrated in Figure 7. As for the 2,6-disubstituted pyridine

derivative, 7e-anti was found to be more stable than 7e-syn.

However, the syn-form 7f-syn is more stable than the 7f-anti.³⁰

We assumed that the repulsion of lone pairs on the oxygen

atom and the pyridine nitrogen could put the diﬀerent spatial

arrangement of whole structure conformation of 7f-syn, which

could be closer to the active conformer of 6h, and this

substitution pattern was deemed to be optimal in this series.

From an optimization study to enhance the potency of the

lead compound 6a discussed in Table 1, we successfully

discovered 7f as a racemate. To determine a eutomer

conformation, we synthesized an enantiomer of 7f via

diastereomeric salt formation shown in Scheme 9. The

synthesis was initiated from the alkoxylation of the 2-position

Based on the abovementioned ﬁndings, we have explored

the optimal substituents on the pyridine ring. Introduction of a

methyl group into the 6-position on the pyridine ring led to a

discovery of 6h with a good balance between agonistic activity

and lipophilicity. From the lead identiﬁcation study described

thus far, (2,2-dimethylpropyl)(6-methylpyridin-2-yl)-

carbamoylphenyl 6h was identiﬁed as a potent GPR40 agonist

possessing an amide moiety. We selected 6h for further

optimization to reduce lipophilicity (clog P: 6.4).

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Table 3. Eﬀect of Variation of β-Position and Polar Aromatic Ring of the Phenyl Propionic Acidic Moiety

a

b

Data are presented as mean values with the standard deviation from more than two independent experiments in duplicate. Values are from a

c

d

single experiment in duplicate. % Compared to the reference full agonist. The clog P values were calculated using Daylight.

of 2-chloro-4-cyanopyridine (65) with sodium hydride,

followed by the nucleophilic addition reaction with cyclo-

propylmagnesium bromide to produce heteroaryl alkyl ketone

66. To prepare 3-cyclopropyl-propanoic acid 67, the conven-

tional HWE reaction of heteroaryl alkyl ketone 66 with ethyl

diethylphosphonoacetate in the presence of sodium hydride

gave the corresponding α,β-unsaturated ester, followed by

reduction of oleﬁn with the zinc metal under acidic conditions

and hydrolysis of the ethyl ester. Diastereomeric salt formation

of the corresponding carboxylic acid derivative with (S)-4-

methyl-methylbenzylamine and recrystallization twice pro-

duced optically active 68 (>99.9% ee). The absolute

conﬁguration of 68 was determined by X-ray crystallographic

analysis of the reference to the known conﬁguration of (1S)-1-

(4-methylphenyl)ethan-1-aminium. The chiral 68 was con-

verted to compound 69 by a two-step sequence consisting of

desalination and subsequent esteriﬁcation. Compound 69 was

converted to the key intermediate 70 through S_NAr

substitution with 56, which was then subjected to the ﬁnal

chiral product 7f by a hydrolysis reaction. X-ray crystallo-

graphic analysis of the ﬁnal chiral product 7f provided further

conﬁrmation of the structure (Figure 8). We concluded (S)-7f

(SCO-267) as a eutomer based on its agonistic activity with an

EC₅₀value of 12 nM. The N-(2,2-dimethylpropyl)-N-(2-

pyridyl) amide moiety was in the geometrically cis

conformation in the literature,²⁶⁻²⁹and the absolute

conﬁguration of compound 7f was retained.

Based on these results, SCO-267 was selected for further

evaluation. SCO-267 showed a good cytotoxicity proﬁle (ATP:

92.1% at 30 μM) and did not display phospholipidosis

potential in an in vitro assay and hERG inhibition potential in

a patch clamp test (IC₅₀> 10 μM). The oral bioavailability (F)

of SCO-267 in rats and mice was 16 and 26%, respectively

(Table 4).

To evaluate their pharmacological eﬀect on hormone

secretion and glucose tolerance, SCO-267 (0.1, 0.3, and 1

mg/kg) and sitagliptin (10 mg/kg) were orally dosed to male

N-STZ-1.5 rats, a diabetic model displaying impairments in

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Figure 7. Conformational preference of the alkoxy moiety of 2,6-pyridine and 2,4-pyridine derivatives.

in insulin secretion in a 2-week-dosing study of N-STZ-1.5 rats

with diabetes (C_maxand AUC_0−24hare as follows: 139 ng/mL

vs 39.8 μg/mL and 626 ng·h/mL vs 255 μg·h/mL,

respectively).³²SCO-267 was also eﬃcacious in reducing

body weight in a 2-week repeated-dosing study of diet-induced

obese rats. These studies demonstrated the eﬃcacy of SCO-

267 in ameliorating glycemic control and reducing body

weight as a novel therapeutic drug for treating diabetes and

obesity. Considering the eﬀective exposure of 50 mg TAK-875

in patients (C_max= 5.3 μg/mL; AUC_0−24h= 100.3 μg·h/mL),

the lower plasma exposure of SCO-267 may be a safety

advantage compared with exposure of TAK-875, which was

terminated in phase 3 because of the unfavorable eﬀects on the

liver.³¹A phase I study of SCO-267, a ﬁrst-in-class oral GPR40

full agonist, has been initiated in healthy adults and people

with impaired glucose tolerance.

Figure 8. ORTEP diagram of (A) compound 68, thermal ellipsoids

are drawn at 30% probability. The minor disorder component is

omitted for clarity, (B) SCO-267, thermal ellipsoids are drawn at 50%

probability.

insulin secretion and action. A single dose of SCO-267

stimulated insulin secretion and GLP-1 release and ameliorated

glucose tolerance in male N-STZ-1.5 rats, whereas the GPR40

partial agonist TAK-875 provided almost no eﬀect on GLP-1

secretion.³¹Sitagliptin elevated insulin secretion and amelio-

rated glucose tolerance. The eﬃcacy of SCO-267 on insulin

secretion, GLP-1 release, and glucose tolerance was much

better than that of sitagliptin. In addition, SCO-267-treated

male N-STZ-1.5 rats showed better glucose tolerance than

normal rats (Figure 9). These ﬁndings conﬁrmed that our

optimization strategy successfully produced a GPR40 full

agonist that results in insulin and GLP-1 secretions.

Consequently, these in vivo results suggest that our lead

optimization strategy is an eﬀective approach to discover novel

GPR40 full agonists with enhanced glucose-lowering proper-

ties by incorporating an amide group as a linker in the left-

hand side. We recently reported that 1 mg/kg SCO-267 was

more eﬃcacious in ameliorating glucose tolerance than 10 mg/

kg TAK-875 at the lower plasma exposure without a decrease

CONCLUSIONS

■

We identiﬁed a novel series of carbamoylphenyl piperidine

derivatives as potent and orally bioavailable full agonists of

GPR40. Starting with alkyl derivatives 4, we explored an

optimal linker of the hydrophobic group as a key

pharmacophoric motif that showed a suﬃcient balance

between potency and lipophilicity. Incorporation of a polar

amide linker reduced the lipophilicity without a loss in

agonistic activity. Consequently, N-methyl-N-(2-pyridyl)

amide derivative 6c was identiﬁed as an attractive lead

compound with potent GPR40 agonistic activity and a

chemically expandable motif. Subsequent optimization of

substituents on the amide moiety was conducted to enhance

agonistic activity by extension of the alkyl chain on the amide

a

Table 4. Pharmacokinetic Parameters of SCO-267 in Rats and Mice

intravenous (0.1 mg/kg)

oral (1 mg/kg)

b

c

d

e

f

g

compound

species

F

(%)

16

26

CL_total(mL/h/kg)

V_ss(mL/kg)

C_max(ng/mL)

AUC_0−8h(ng·h/mL)

MRT (h)

SCO-267

rats

mice

1478

2584

3094

1349

19.9

33.2

126.6

98.7

4.1

2.3

a

The pharmacokinetic parameters of SCO-267 in 8 week-old male SD rats and 8 week-old male ICR mice after intravenous and oral

b

c

d

e

administrations are summarized. Bioavailability. Total body clearance. Volume of distribution at steady state. Maximal plasma concentration.

f

g

Area under the plasma concentration−time curve (0−8 h). Mean residence time. The parameters represent mean (n = 3 for each group).

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Figure 9. Eﬀect of single administration of SCO-267 and sitagliptin on hormone secretion and glucose tolerance in 26 week-old male diabetic N-

STZ-1.5 rats. Plasma insulin (A), total GLP-1 (B), and glucose levels (C) in male N-STZ-1.5 rats. SCO-267 elevated insulin levels, stimulated

GLP-1 secretion, and ameliorated glucose tolerance. # and $ P < 0.025 vs vehicle-treated male N-STZ-1.5 rats by one-tailed Williams’ test and

Shirley−Williams test, respectively. *P < 0.05 vs vehicle-treated male N-STZ-1.5 rats by Student’s t-test. Values are means S.D. (n = 6 for each

group).

standard in a deuterated solvent and coupling constants (J) are in

hertz (Hz). Data are reported as follows: chemical shift, integration,

multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, quint =

quintet, m = multiplet, dd = doublet of doublets, td = triplet of

doublets, and bs = broad signal), and coupling constants. All solvents

and reagents were obtained from commercial suppliers and used

without further puriﬁcation. Reaction progress was determined by

thin layer chromatography (TLC) analysis on Merck silica gel plates

60F₂₅₄. Silica gel column chromatography was performed on Purif-

Pack (SI or NH, Shoko Scientiﬁc). Low-resolution mass spectra (MS)

were acquired using an Agilent LC−MS system (Agilent1200SL/

Agilent6130MS) or Shimadzu UFLC/MS (Prominence UFLC high

pressure gradient system/LCMS-2020) operating in an electrospray

ionization mode (ESI⁺). The column used was an L-column 2 ODS

(3.0 mm × 50 mm ID, 3 μm, CERI) with a temperature of 40 °C and

a ﬂow rate of 1.2 or 1.5 mL/min. Condition 1: mobile phases A and B

under acidic conditions were 0.05% triﬂuoroacetic acid (TFA) in

water and 0.05% TFA in MeCN, respectively. The ratio of mobile

phase B was increased linearly from 5 to 90% over 0.9 min, 90% over

moiety to the presumed lipophilic pocket, resulting in the

identiﬁcation of (3S)-3-cyclopropyl-3-(2-((1-(2-((2,2-

dimethylpropyl)(6-methylpyridin-2-yl)carbamoyl)-5-

methoxyphenyl)piperidin-4-yl)methoxy)pyridin-4-yl)-

propanoic acid (SCO-267). SCO-267 eﬀectively stimulated

insulin secretion and GLP-1 release and ameliorated glucose

tolerance in male N-STZ-1.5 rats. These results indicate that

our lead optimization strategy successfully produced potent,

orally available, full agonists, and this series of GPR40 agonists

may have potential in the treatment of diabetes and obesity.

EXPERIMENTAL SECTION

Chemistry General Method. Melting points were determined in

■

open capillary tubes on a Bu

uncorrected. H NMR spectra were recorded on a Bruker AVANCE

III (300 MHz) or Bruker ADVANCE III plus (400 MHz)

̈

chi melting point apparatus B545 and are

1

spectrometer. Chemical shifts for H NMR are given in parts per

million (ppm) downﬁeld from tetramethylsilane (δ) as the internal

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the next 1.1 min. Condition 2: mobile phases A and B under neutral

conditions were a mixture of 5 mM AcONH₄and MeCN (9/1, v/v)

and a mixture of 5 mM AcONH₄and MeCN (1/9, v/v), respectively.

The ratio of mobile phase B was increased linearly from 5 to 90% over

0.9 min, 90% over the next 1.1 min. Chemical intermediates were

characterized by ¹H NMR or mass spectral data or both. Unless

otherwise stated, the purities of the synthesized compounds for

biological testing were >95% determined by elemental analyses within

0.4% of the calculated values or analytical high-performance liquid

chromatography (HPLC) or both (The purities of 7c and 7d were

92.6 and 92.7%, respectively). Analytical HPLC data were collected

by HPLC with NQAD or Corona charged aerosol detector (CAD) or

photodiode array detector. The column was a Capcell Pak C18AQ

(50 mm × 3.0 mm ID, Shiseido, Japan) or L-column 2 ODS (30 mm

× 2.0 mm ID, CERI, Japan) with a temperature of 50 °C and a ﬂow

rate of 0.5 mL/min. Mobile phases A and B were a mixture of 50 mM

AcONH₄, water, and MeCN (1:8:1, v/v/v) and a mixture of 50 mM

AcONH₄and acetonitrile (1:9, v/v), respectively. The ratio of mobile

phase B was increased linearly from 5 to 95% over 3 min, 95% over

the next 1 min. Mobile phases A and B under acidic conditions were a

mixture of 0.2% formic acid in 10 mM ammonium formate and 0.2%

formic acid in MeCN, respectively. The ratio of mobile phase B was

increased linearly from 14 to 86% over 3 min, 86% over the next 1

min. Preparative HPLC was performed on a Gilson Preparative

HPLC system using a YMC-Actus Triart C18 column (150 mm × 20

mm ID, 5 μm, YMC). Condition 1: mobile phases A and B under

acidic conditions were 0.1% TFA in water and 0.1% TFA in MeCN,

respectively. Condition 2: mobile phases A and B under neutral

conditions were 10 mM ammonium bicarbonate and MeCN,

respectively. The ratio of mobile phase B was increased linearly in

5−10 min. All commercially available solvents and reagents were used

without further puriﬁcation. Yields were not optimized.

3-Cyclopropyl-3-(3-((2′-(4,4-dimethylpentyl)-5′-methoxy-[1,1′-

biphenyl]-4-yl)methoxy)phenyl)propanoic Acid (2). To a solution of

(2′-(4,4-dimethylpentyl)-5′-methoxy-[1,1′-biphenyl]-4-yl)methanol

26 (200 mg, 0.64 mmol), methyl 3-cyclopropyl-3-(3-hydroxyphenyl)-

propanoate 11a (155 mg, 0.70 mmol), and PPh₃(504 mg, 1.92

mmol) in THF (dry) (5 mL) was added DEAD (0.873 mL, 1.92

mmol) under Ar and stirred for 30 min. The reaction mixture was

concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, eluted with 0−25% EtOAc in hexane)

to give methyl 3-cyclopropyl-3-(3-((2′-(4,4-dimethylpentyl)-5′-me-

thoxy-[1,1′-biphenyl]-4-yl)methoxy)phenyl)propanoate (244 mg,

0.474 mmol, 74.1%) as a colorless oil. This product was subjected

to a further step. A mixture of the obtained methyl ester (244 mg)

and 1 M NaOH aq (2 mL) in MeOH and THF (1/1) was stirred at

room temperature for 1 h. The mixture was neutralized with 1 N HCl

and extracted with EtOAc three times. The organic layer was

separated, washed with water and brine, dried over MgSO₄, and

concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, eluted with 0−50% EtOAc in hexane)

to give the title compound (226 mg, 96%) as a colorless oil. ¹H NMR

(400 MHz, DMSO-d₆): δ ppm −0.18 to −0.07 (1H, m), −0.05−0.13

(2H, m), 0.19−0.32 (1H, m), 0.51 (9H, s), 0.74−0.82 (3H, m),

1.03−1.13 (2H, m), 1.99−2.08 (1H, m), 2.21 (2H, br t, J = 7.7 Hz),

2.34−2.47 (2H, m), 3.50 (3H, s), 4.88 (2H, s), 6.46 (1H, d, J = 2.5

Hz), 6.58−6.67 (3H, m), 6.70 (1H, s), 6.93−7.01 (2H, m), 7.08 (2H,

d, J = 7.9 Hz), 7.27 (2H, d, J = 7.8 Hz), 11.77 (1H, br s). ¹³C NMR

(75 MHz, CDCl₃): δ ppm 4.1, 5.3, 17.2, 26.6, 29.3, 30.2, 33.1, 41.3,

43.9, 46.8, 55.3, 69.9, 112.5, 113.2, 114.3, 115.2, 120.0, 127.3, 129.3,

129.4, 130.1, 132.6, 135.6, 141.8, 142.4, 145.6, 157.3, 158.9, 177.5.

MS (ESI/APCI): m/z 499.2 [M − 1] C₃₃H₄₀O₄: 1.3 H₂O/C, 75.63;

H, 8.19; N, 0.00. Found: C, 75.43; H, 8.40; N, 0.00. HPLC purity

98.8%.

EtOAc. The organic layer was separated, washed with brine, dried

over MgSO₄, and concentrated in vacuo to give a pale-yellow oil. The

residue was mixed with sodium phenoxide, which was prepared by the

addition of NaH (60% oil dispersion, 29.5 mg, 0.74 mmol) to the

phenol 11a (108 mg, 0.49 mmol) in DMF (20 mL). The mixture was

stirred at 100 °C for 15 h. The mixture was poured into water and

extracted with EtOAc. The organic layer was separated, washed with

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

puriﬁed by column chromatography (silica gel, eluted with 0−10%

EtOAc in hexane) to give methyl 3-cyclopropyl-3-(3-((1-(2-(4,4-

dimethylpentyl)-5-methoxyphenyl)piperidin-4-yl)oxy)phenyl)-

1

propanoate as a pale-yellow oil (75 mg, 0.18 mmol, 30%). H NMR

(300 MHz, CDCl₃): δ ppm 0.17 (1H, dt, J = 9.3, 4.6 Hz), 0.26 (1H,

dq, J = 9.2, 4.7 Hz), 0.36−0.50 (1H, m), 0.51−0.65 (1H, m), 0.88

(9H, s), 0.93−1.11 (1H, m), 1.20−1.31 (4H, m), 1.55−1.65 (2H, m),

1.84−2.02 (2H, m), 2.06−2.18 (2H, m), 2.29−2.41 (1H, m), 2.49−

2.59 (2H, m), 2.66−2.85 (2H, m), 3.03−3.18 (2H, m), 3.61 (3H, s),

3.79 (3H, s), 4.42 (1H, dt, J = 7.8, 3.9 Hz), 6.59 (1H, dd, J = 8.4, 2.6

Hz), 6.68 (1H, d, J = 2.6 Hz), 6.75−6.86 (3H, m), 7.11 (1H, d, J =

8.4 Hz), 7.17−7.25 (1H, m). MS (ESI/APCI): m/z 508.2 [M + H]⁺.

Compound 3 was prepared from the obtained methyl ester in a

manner similar to that described for compound 2 [puriﬁcation:

column chromatography (silica gel, eluted with 0−50% EtOAc in

1

hexane)]. Colorless oil. (54.0 mg, 0.109 mmol, 74%). H NMR (400

MHz, DMSO-d₆): δ ppm 0.09−0.18 (1H, m), 0.20−0.27 (1H, m),

0.28−0.36 (1H, m), 0.40−0.54 (1H, m), 0.86 (9H, s), 1.18−1.25

(2H, m), 1.44−1.60 (2H, m), 1.76 (2H, d, J = 8.8 Hz), 2.05 (2H, br

s), 2.18−2.33 (1H, m), 2.56−2.70 (2H, m), 2.79 (2H, t, J = 9.3 Hz),

2.99 (2H, d, J = 11.4 Hz), 3.71 (3H, s), 4.51 (1H, br s), 6.59 (1H, dd,

J = 8.3, 2.4 Hz), 6.64 (1H, d, J = 2.3 Hz), 6.76−6.83 (2H, m), 6.87

(1H, s), 7.08 (1H, d, J = 8.4 Hz), 7.18 (1H, t, J = 7.8 Hz), 12.02 (1H,

br s). ¹³C NMR (75 MHz, CDCl₃): δ ppm 4.1, 5.3, 17.2, 26.0, 29.4,

30.4, 31.2, 31.8, 41.4, 44.5, 46.8, 50.3, 55.2, 72.7, 106.9, 108.1, 113.7,

115.5, 119.8, 129.4, 130.0, 130.4, 145.7, 152.9, 157.5, 158.2, 178.0.

MS (ESI/APCI): m/z 494.2 [M + H]⁺. C₃₁H₄₃NO₄: 0.1 H₂O: C,

75.15; H, 8.79; N, 2.83. Found: C, 75.23; H, 8.73; N, 2.72. HPLC

purity 99.5%.

3-Cyclopropyl-3-(3-((1-(2-(4,4-dimethylpentyl)-5-

methoxyphenyl)piperidin-4-yl)methoxy)phenyl)propanoic Acid

(4a). Cyanomethylenetri-n-butylphosphorane (9.85 mL, 37.56

mmol) was added to a solution of (1-(2-(4,4-dimethylpentyl)-5-

methoxyphenyl)piperidin-4-yl)methanol 31 (6 g, 18.78 mmol) and

ethyl 3-cyclopropyl-3-(3-hydroxyphenyl)propanoate 11b (4.40 g,

18.78 mmol) in toluene (50 mL) at room temperature. The mixture

was stirred at 100 °C under N₂for 30 min. The reaction mixture was

concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, eluted with 0−15% EtOAc in hexane)

to give a pale-brown oil. The obtained oil was dissolved in EtOH (50

mL) and NaOH was added (50 mL, 50.00 mmol). The mixture was

stirred at 50 °C for 2 h. The mixture was neutralized with 1 N HCl

and extracted with EtOAc. The organic layer was separated, washed

with water and brine, dried over MgSO₄, and concentrated in vacuo.

The residue was puriﬁed by column chromatography (silica gel, eluted

with 0−25% EtOAc in hexane) to give the title compound (7.80 g,

1

15.36 mmol, 82%) as a white amorphous solid. H NMR (400 MHz,

DMSO-d₆): δ ppm 0.12 (1H, dd, J = 9.0, 4.0 Hz), 0.19−0.27 (1H,

m), 0.28−0.39 (1H, m), 0.41−0.56 (1H, m), 0.86 (9H, s), 0.93−1.07

(1H, m), 1.13−1.27 (2H, m), 1.36−1.61 (4H, m), 1.87 (3H, d, J =

10.2 Hz), 2.17−2.30 (1H, m), 2.47−2.53 (2H, m), 2.55−2.72 (4H,

m), 3.00 (2H, d, J = 11.3 Hz), 3.70 (3H, s), 3.87 (2H, d, J = 5.4 Hz),

6.57 (1H, dd, J = 8.3, 2.4 Hz), 6.62 (1H, d, J = 2.4 Hz), 6.72−6.87

(3H, m), 7.07 (1H, d, J = 8.4 Hz), 7.18 (1H, t, J = 7.9 Hz), 11.98

(1H, br s). ¹³C NMR (75 MHz, CDCl₃): δ ppm 4.1, 5.3, 17.2, 26.0,

29.5, 29.9, 30.4, 31.2, 36.0, 41.1, 44.5, 46.9, 53.0, 55.2, 72.6, 106.9,

107.7, 112.1, 113.8, 119.6, 129.4, 129.9, 130.4, 145.5, 153.4, 158.2,

159.2, 176.7. MS (ESI/APCI): m/z 508.4 [M + H]⁺. C₃₂H₄₅NO₄: 0.1

H₂O: C, 75.43; H, 8.94; N, 2.75. Found: C, 75.68; H, 8.78; N, 2.56.

HPLC purity 98.6%.

3-Cyclopropyl-3-(3-((1-(2-(4,4-dimethylpentyl)-5-

methoxyphenyl)piperidin-4-yl)oxy)phenyl)propanoic Acid (3). To a

solution of 1-(2-(4,4-dimethylpentyl)-5-methoxyphenyl)piperidin-4-

ol 29 (150 mg, 0.49 mmol) and TEA (0.103 mL, 0.74 mmol) in THF

(dry) (20 mL) was added MsCl (0.046 mL, 0.59 mmol) and stirred at

r.t. for 30 min. The mixture was poured into water and extracted with

3-Cyclopropyl-3-(3-((1-(2-(4,4-dimethylpentyl)-5-

methoxyphenyl)azetidin-3-yl)methoxy)phenyl)propanoic Acid (5).

M

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To a solution of (1-(2-(4,4-dimethylpentyl)-5-methoxyphenyl)-

azetidin-3-yl)methanol 33 (100 mg, 0.34 mmol) and TEA (0.143

mL, 1.03 mmol) in THF (dry) (5 mL) was added MsCl (0.064 mL,

0.82 mmol) and stirred at r.t. for 30 min. The mixture was poured

into water and extracted with EtOAc. The organic layer was separated,

washed with brine, dried over MgSO₄, and concentrated in vacuo to

give a pale-yellow oil. To a solution of methyl 3-cyclopropyl-3-(3-

hydroxyphenyl)propanoate 11a (151 mg, 0.69 mmol) in DMF (dry)

(5 mL) were added K₂CO₃(142 mg, 1.03 mmol) and the obtained

mesylate. The mixture was stirred at 100 °C for 15 h. The mixture was

poured into water and extracted with EtOAc. The organic layer was

separated, washed with brine, dried over MgSO₄, and concentrated in

vacuo. The residue was puriﬁed by column chromatography (silica

gel, eluted with 0−10% EtOAc in hexane) to give a mixture of methyl

3-cyclopropyl-3-(3-((1-(2-(4,4-dimethylpentyl)-5-methoxyphenyl)-

azetidin-3-yl)methoxy)phenyl)propanoate (140 mg, 0.284 mmol,

C, 72.68; H, 7.66; N, 3.39. Found: C, 72.68; H, 7.39; N, 3.24. HPLC

purity 100%.

3-Cyclopropyl-3-(3-((1-(2-ﬂuoro-5-methoxyphenyl)piperidin-4-

yl)methoxy)phenyl)propanoic Acid (4c). To a solution of (1-(2-

ﬂuoro-5-methoxyphenyl)piperidin-4-yl)methanol 36c (353 mg, 1.48

mmol), methyl 3-cyclopropyl-3-(3-hydroxyphenyl)propanoate (357

mg, 1.62 mmol), and ADDP (558 mg, 2.21 mmol) in THF (dry) (20

mL) was added Bu₃P (0.587 mL, 2.21 mmol) at 0 °C. The mixture

was stirred at room temperature for 6 h. The mixture was diluted with

EtOAc and quenched with water at room temperature. The organic

layer was separated, washed with water and brine, dried over Na₂SO₄,

and concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, eluted with 1−15% EtOAc in hexane) to

give methyl 3-cyclopropyl-3-(3-((1-(2-ﬂuoro-5-methoxyphenyl)-

piperidin-4-yl)methoxy)phenyl)propanoate (219 mg, 0.496 mmol,

33.6%) as a colorless oil. ¹H NMR (300 MHz, CDCl₃): δ ppm 0.09−

0.21 (1H, m), 0.21−0.32 (1H, m), 0.36−0.50 (1H, m), 0.50−0.64

(1H, m), 0.93−1.11 (1H, m), 1.45−1.73 (2H, m), 1.96 (3H, d, J =

11.7 Hz), 2.34 (1H, dt, J = 9.8, 7.6 Hz), 2.61−2.83 (4H, m), 3.51

(2H, d, J = 12.1 Hz), 3.62 (3H, s), 3.77 (3H, s), 3.85 (2H, d, J = 5.7

Hz), 6.41 (1H, dt, J = 8.8, 3.2 Hz), 6.53 (1H, dd, J = 7.2, 3.0 Hz),

6.71−6.86 (3H, m), 6.93 (1H, dd, J = 12.3, 8.9 Hz), 7.21 (1H, t, J =

7.8 Hz). Compound 4c was prepared from the obtained methyl ester

in a manner similar to that described for compound 2 (puriﬁcation:

column chromatography (silica gel, eluted with 20−80% EtOAc in

1

83%) as a colorless oil. H NMR (400 MHz, CDCl₃): δ ppm 0.15

(1H, dq, J = 9.6, 4.9 Hz), 0.26 (1H, dq, J = 9.4, 4.8 Hz), 0.36−0.50

(1H, m), 0.51−0.63 (1H, m), 0.87 (9H, s), 0.91−1.09 (1H, m), 1.25

(2H, q, J = 7.3 Hz), 1.45−1.61 (2H, m), 2.26−2.38 (1H, m), 2.38−

2.50 (2H, m), 2.60−2.84 (2H, m), 2.95−3.17 (1H, m), 3.61 (3H, s),

3.72−3.81 (5H, m), 4.06 (2H, t, J = 7.3 Hz), 4.19 (2H, d, J = 6.9 Hz),

6.07 (1H, d, J = 2.3 Hz), 6.33 (1H, dd, J = 8.3, 2.3 Hz), 6.71−6.86

(3H, m), 6.96 (1H, d, J = 8.3 Hz), 7.22 (1H, t, J = 7.9 Hz). MS (ESI/

APCI): m/z 494.16. [M + H]⁺. HPLC 96.6% (LC−MS). Compound

5 was prepared from the obtained methyl ester in a manner similar to

that described for compound 2 as a colorless oil (107 mg, 0.223

mmol, 79%). ¹H NMR (400 MHz, CDCl₃): δ ppm 0.15 (1H, dq, J =

9.6, 4.9 Hz), 0.26 (1H, dq, J = 9.4, 4.8 Hz), 0.36−0.50 (1H, m),

0.51−0.63 (1H, m), 0.87 (9H, s), 0.91−1.09 (1H, m), 1.25 (2H, q, J

= 7.3 Hz), 1.45−1.61 (2H, m), 2.26−2.38 (1H, m), 2.38−2.50 (2H,

m), 2.60−2.84 (2H, m), 2.95−3.17 (1H, m), 3.61 (3H, s), 3.72−3.81

(5H, m), 4.06 (2H, t, J = 7.3 Hz), 4.19 (2H, d, J = 6.9 Hz), 6.07 (1H,

d, J = 2.3 Hz), 6.33 (1H, dd, J = 8.3, 2.3 Hz), 6.71−6.86 (3H, m),

6.96 (1H, d, J = 8.3 Hz), 7.22 (1H, t, J = 7.9 Hz). MS (ESI/APCI):

m/z 480.3 C₃₀H₄₁NO₄: 0.1 H₂O: C, 74.84; H, 8.63; N, 2.91. Found:

C, 74.94; H,8.59; N,2.85. HPLC purity 99.6%.

1

hexane)). White solid (166 mg, 0.389 mmol, 85%). H NMR (300

MHz, CDCl₃): δ ppm 0.11−0.24 (1H, m), 0.24−0.36 (1H, m), 0.38−

0.51 (1H, m), 0.53−0.67 (1H, m), 1.04 (1H, ddt, J = 17.9, 8.0, 5.0,

5.0 Hz), 1.57−1.70 (2H, m), 1.84−2.03 (3H, m), 2.35 (1H, dt, J =

9.5, 7.8 Hz), 2.62−2.75 (2H, m), 2.79 (2H, dd, J = 7.4, 3.2 Hz), 3.51

(2H, d, J = 11.7 Hz), 3.77 (3H, s), 3.85 (2H, d, J = 6.1 Hz), 6.41 (1H,

dt, J = 8.9, 3.1 Hz), 6.53 (1H, dd, J = 7.4, 2.8 Hz), 6.72−6.87 (3H,

m), 6.88−6.98 (1H, m), 7.21 (1H, d, J = 8.3 Hz), 1 H was not found.

¹³C NMR (75 MHz, CDCl₃): δ ppm 4.1, 5.3, 17.2, 29.3, 35.9, 41.2,

46.9, 50.9, 50.9, 55.7, 72.4, 105.1, 106.2, 112.2, 113.8, 116.0, 119.6,

129.4, 141.6, 145.5, 150.5, 156.0, 159.1, 177.3. MS (ESI/APCI): m/z

428.3. mp 150−152 °C. C₂₅H₃₀FNO₄: 0.1 H₂O: C, 69.94; H, 7.09; N,

3.26. Found: C, 70.09; H, 6.88; N, 3.21. HPLC purity 99.5%.

3-Cyclopropyl-3-(3-((1-(5-methoxy-2-methylphenyl)piperidin-4-

yl)methoxy)phenyl)propanoic Acid (4d). Compound 4d was

prepared from (1-(5-methoxy-2-methylphenyl)piperidin-4-yl)-

methanol 36d in a manner similar to that described for compound

4a (puriﬁcation: column chromatography (silica gel, eluted with 15−

3-Cyclopropyl-3-(3-((1-(3-methoxyphenyl)piperidin-4-yl)-

methoxy)phenyl)propanoic Acid (4b). To a solution of (1-(3-

methoxyphenyl)piperidin-4-yl)methanol 36b (100 mg, 0.45 mmol),

methyl 3-cyclopropyl-3-(3-hydroxyphenyl)propanoate (119 mg, 0.54

mmol), and PPh₃(237 mg, 0.90 mmol) in THF (dry) (5.0 mL) was

added diisopropyl azodicarboxylate (DIAD) (0.476 mL, 0.90 mmol)

at room temperature. The mixture was stirred at room temperature

for 20 h and evaporated. The residue was puriﬁed by column

chromatography (silica gel, eluted with 5−15% EtOAc in hexane) to

give methyl 3-cyclopropyl-3-(3-((1-(3-methoxyphenyl)piperidin-4-

yl)methoxy)phenyl)propanoate (34.6 mg, 0.082 mmol, 18.1%) as a

1

40% EtOAc in hexane)). White solid (1.100 g, 2.60 mmol, 85%). H

NMR (300 MHz, DMSO-d₆): δ 0.06−0.38 (3H, m), 0.43−0.56 (1H,

m), 0.93−1.08 (1H, m), 1.36−1.57 (2H, m), 1.88 (3H, d, J = 9.7 Hz),

2.15 (3H, s), 2.19−2.32 (1H, m), 2.54−2.69 (4H, m), 3.09 (2H, d, J

= 11.5 Hz), 3.70 (3H, s), 3.88 (2H, d, J = 5.9 Hz), 6.48−6.59 (2H,

m), 6.72−6.88 (3H, m), 7.04 (1H, d, J = 8.3 Hz), 7.18 (1H, t, J = 7.9

Hz), 11.97 (1H, br s). ¹³C NMR (75 MHz, CDCl₃): δ ppm 4.1, 5.3,

17.1, 17.2, 29.8, 36.0, 41.2, 46.9, 52.1, 55.3, 72.6, 106.1, 106.9, 112.2,

113.9, 119.6, 124.6, 129.4, 131.3, 145.5, 153.4, 158.4, 159.2, 176.9.

MS (ESI/APCI): m/z 424.2 [M + H]⁺. C₂₆H₃₃NO₄: 0.1 H₂O: C,

73.42; H, 7.87; N, 3.29. Found: C, 73.70; H, 7.63; N, 3.22. The

corresponding intermediate ethyl 3-cyclopropyl-3-(3-((1-(5-methoxy-

2-methylphenyl)piperidin-4-yl)methoxy)phenyl)propanoate (1380

1

colorless oil. H NMR (300 MHz, CDCl₃): δ ppm 0.09−0.20 (1H,

m), 0.21−0.31 (1H, m), 0.37−0.49 (1H, m), 0.51−0.64 (1H, m),

0.94−1.10 (1H, m), 1.42−1.62 (2H, m), 1.87−2.03 (3H, m), 2.34

(1H, dt, J = 9.4, 7.6 Hz), 2.68−2.83 (4H, m), 3.61 (3H, s), 3.73 (2H,

d, J = 12.5 Hz), 3.80 (3H, s), 3.83 (2H, d, J = 6.1 Hz), 6.40 (1H, dd, J

= 8.0, 2.3 Hz), 6.50 (1H, t, J = 2.3 Hz), 6.58 (1H, dd, J = 8.1, 2.1 Hz),

6.71−6.86 (3H, m), 7.19 (2H, dt, J = 13.1, 8.0 Hz). MS (ESI/APCI):

m/z 424.4 [M + H]⁺. Compound 4b was prepared from the obtained

methyl ester in a manner similar to that described for compound 2

(puriﬁcation: column chromatography (silica gel, eluted with 10−40%

1

mg, 3.06 mmol, 95%) was obtained as a pale-yellow oil. H NMR

(300 MHz, CDCl₃): δ 0.10−0.21 (1H, m), 0.27 (1H, dq, J = 9.3, 4.8

Hz), 0.37−0.48 (1H, m), 0.52−0.63 (1H, m), 0.93−1.09 (1H, m),

1.18 (3H, t, J = 7.1 Hz), 1.47−1.63 (2H, m), 1.87−2.00 (3H, m),

2.23 (3H, s), 2.35 (1H, dt, J = 9.7, 7.6 Hz), 2.59−2.76 (4H, m), 3.18

(2H, d, J = 11.9 Hz), 3.78 (3H, s), 3.86 (2H, d, J = 5.9 Hz), 4.02−

4.11 (2H, m), 6.52 (1H, dd, J = 8.3, 2.6 Hz), 6.61 (1H, d, J = 2.5 Hz),

6.73−6.86 (3H, m), 7.07 (1H, d, J = 8.3 Hz), 7.18−7.24 (1H, m). MS

(ESI/APCI): m/z 452.2 [M + H]⁺. HPLC purity 99.7%.

3-Cyclopropyl-3-(3-((1-(2-isopropyl-5-methoxyphenyl)piperidin-

4-yl)methoxy)phenyl)propanoic Acid (4e). A mixture of ethyl 3-

cyclopropyl-3-(3-((1-(2-isopropyl-5-methoxyphenyl)piperidin-4-yl)-

methoxy)phenyl)propanoate 41 (323.6 mg, 0.67 mmol) and 1 N

NaOH (2.70 mL, 2.70 mmol) in THF (2.7 mL) and EtOH (2.7 mL)

1

EtOAc in hexane)). Colorless oil. (29.9 mg, 0.073 mmol, 89%). H

NMR (300 MHz, CDCl₃): δ ppm 0.11−0.23 (1H, m), 0.30 (1H, dq, J

= 9.3, 4.7 Hz), 0.37−0.49 (1H, m), 0.52−0.65 (1H, m), 0.95−1.11

(1H, m), 1.52 (2H, qd, J = 12.5, 3.4 Hz), 1.86−2.01 (3H, m), 2.34

(1H, dt, J = 9.6, 7.5 Hz), 2.68−2.82 (4H, m), 3.72 (2H, d, J = 12.5

Hz), 3.79 (3H, s), 3.83 (2H, d, J = 6.1 Hz), 6.41 (1H, dd, J = 7.8, 2.1

Hz), 6.51 (1H, t, J = 2.3 Hz), 6.58 (1H, dd, J = 8.1, 2.1 Hz), 6.72−

6.87 (3H, m), 7.12−7.25 (2H, m). ¹³C NMR (75 MHz, CDCl₃): δ

ppm 4.1, 5.3, 17.2, 28.9, 36.1, 41.2, 46.9, 49.7, 55.2, 72.4, 103.1, 104.4,

109.5, 112.2, 113.9, 119.7, 129.4, 129.7, 145.6, 153.1, 159.1, 160.6,

177.1. MS (ESI/APCI): m/z 410.2 [M + H]⁺. C₂₅H₃₁NO₄: 0.2 H₂O:

N

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was stirred at 50 °C for 3 h. The mixture was neutralized with 1 N

HCl and extracted with EtOAc twice. The combined organic layers

were washed with water and brine, dried over MgSO₄and

concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, eluted with 20−25% EtOAc in hexane)

to give compound 4e (114.5 mg, 0.254 mmol, 38%) as a white solid.

¹H NMR (300 MHz, DMSO-d₆): δ 0.06−0.18 (1H, m), 0.18−0.39

¹H NMR (400 MHz, DMSO-d₆): δ 0.07−0.17 (1H, m), 0.18−0.27

(1H, m), 0.27−0.37 (1H, m), 0.44−0.57 (1H, m), 0.97−1.08 (1H,

m), 1.08 (3H, t, J = 7.1 Hz), 1.45 (6H, s), 1.46−1.56 (2H, m), 1.81−

1.91 (3H, m), 1.93 (3H, s), 1.93−2.01 (2H, m), 2.19−2.29 (1H, m),

2.52−2.58 (2H, m), 2.60−2.78 (4H, m), 3.00 (2H, d, J = 11.5 Hz),

3.70 (3H, s), 3.87 (2H, d, J = 4.9 Hz), 3.92−4.00 (2H, m), 6.59 (1H,

d, J = 8.7 Hz), 6.63 (1H, s), 6.77 (1H, d, J = 7.9 Hz), 6.79−6.86 (2H,

m), 7.05 (1H, d, J = 8.3 Hz), 7.18 (1H, t, J = 7.8 Hz).

(2H, m), 0.44−0.55 (1H, m), 0.93−1.06 (1H, m), 1.13 (6H, d, J =

6.9 Hz), 1.36−1.58 (2H, m), 1.87 (3H, d, J = 10.5 Hz), 2.18−2.31

(1H, m), 2.55−2.73 (4H, m), 2.97 (2H, d, J = 11.5 Hz), 3.23−3.38

(1H, m), 3.71 (3H, s), 3.88 (2H, d, J = 5.8 Hz), 6.59−6.67 (2H, m),

6.74−6.87 (3H, m), 7.11−7.23 (2H, m), 11.97 (1H, br s). ¹³C NMR

(101 MHz, DMSO-d₆): δ 3.9, 5.0, 17.4, 24.1, 25.7, 29.4, 35.3, 40.7,

46.4, 53.1, 54.9, 71.9, 106.4, 108.8, 111.8, 113.8, 119.5, 126.7, 129.1,

135.6, 146.1, 152.3, 157.7, 158.6, 173.0. MS (ESI/APCI): m/z 452.3

[M + H]⁺. mp 112−114 °C. C₂₈H₃₇NO₄: 0.1 H₂O: C, 74.17; H, 8.27;

N, 3.09. Found: C, 74.39; H, 8.00; N, 2.82. HPLC purity 99.8%.

3-Cyclopropyl-3-(3-((1-(2-(3, 3-dimethylbutyl)-5-

methoxyphenyl)piperidin-4-yl)methoxy)phenyl)propanoic Acid

(4f). Compound 4f was prepared from (1-(2-(3,3-dimethylbutyl)-5-

methoxyphenyl)piperidin-4-yl)methanol 44 in a manner similar to

that described for compound 4b (puriﬁcation: column chromatog-

raphy (silica gel, eluted with 0−25% EtOAc in hexane)). White

3-Cyclopropyl-3-(3-((1-(2-((isobutyl(methyl)amino)methyl)-5-

methoxyphenyl)piperidin-4-yl)methoxy)phenyl)propanoic Acid

(4h). To a solution of methyl 3-cyclopropyl-3-(3-((1-(2-formyl-5-

methoxyphenyl)piperidin-4-yl)methoxy)phenyl)propanoate 45 (200

mg, 0.44 mmol) and N,2-dimethylpropan-1-amine (77 mg, 0.89

mmol) in THF (dry) (5 mL) was added NaBH(OAc)₃(188 mg, 0.89

mmol) and stirred at r.t. for 1 h. The mixture was poured into water

and extracted with EtOAc. The organic layer was separated, washed

with brine, dried over MgSO₄, and concentrated in vacuo. The residue

was puriﬁed by column chromatography (NH silica gel, eluted with

0−20% EtOAc in hexane) to give methyl 3-cyclopropyl-3-(3-((1-(2-

((isobutyl(methyl)amino)methyl)-5-methoxyphenyl)piperidin-4-yl)-

methoxy)phenyl)propanoate (136 mg, 0.260 mmol, 59%) as a

1

colorless oil that included Et ester. H NMR (300 MHz, CDCl₃): δ

ppm 0.07−0.21 (1H, m), 0.26 (1H, dq, J = 9.2, 4.8 Hz), 0.35−0.50

(1H, m), 0.50−0.64 (1H, m), 0.88 (6H, d, J = 6.6 Hz), 0.94−1.13

(1H, m), 1.41−1.63 (2H, m), 1.72−1.86 (1H, m), 1.86−1.98 (3H,

m), 2.07−2.20 (5H, m), 2.35 (1H, dt, J = 9.7, 7.6 Hz), 2.58−2.83

(4H, m), 3.28 (2H, d, J = 11.6 Hz), 3.42 (2H, s), 3.62 (3H, s), 3.80

(3H, s), 3.86 (2H, d, J = 6.0 Hz), 6.53−6.66 (2H, m), 6.72−6.85

(3H, m), 7.14−7.25 (1H, m), 7.36 (1H, d, J = 8.3 Hz) MS (ESI/

APCI): m/z 523.6. Compound 4h was prepared from the obtained

methyl ester in a manner similar to that described for compound 4a

(puriﬁcation: column chromatography (silica gel, eluted with 0−25%

MeOH in EtOAc)). White amorphous solid (98 mg, 0.193 mmol,

1

amorphous solid (62.0 mg, 0.126 mmol, 82%). H NMR (400 MHz,

DMSO-d₆): δ ppm 0.12 (1H, d, J = 4.9 Hz), 0.20−0.37 (2H, m),

0.42−0.55 (1H, m), 0.95 (9H, s), 0.99 (1H, br s), 1.33−1.58 (4H,

m), 1.87 (3H, d, J = 9.8 Hz), 2.17−2.30 (1H, m), 2.45−2.50 (2H, m),

2.56−2.73 (4H, m), 3.01 (2H, d, J = 12.0 Hz), 3.70 (3H, s), 3.87

(2H, d, J = 5.4 Hz), 6.50−6.65 (2H, m), 6.72−6.87 (3H, m), 7.05

(1H, d, J = 8.2 Hz), 7.18 (1H, t, J = 7.8 Hz), 11.97 (1H, br s). ¹³C

NMR (75 MHz, CDCl₃): δ ppm 4.1, 5.3, 17.2, 25.3, 29.4, 29.9, 30.6,

36.0, 41.1, 46.0, 46.9, 53.2, 55.2, 72.6, 106.9, 108.0, 112.2, 113.8,

119.6, 129.4, 130.2, 130.9, 145.5, 153.3, 158.2, 159.2, 176.7. MS

(ESI/APCI): m/z 494.3. C₃₁H₄₃NO₄: 0.2 H₂O: C, 74.87; H, 8.80; N,

2.82. Found: C, 74.82; H, 8.80; N, 2.62. The corresponding

intermediate methyl 3-cyclopropyl-3-(3-((1-(2-(3,3-dimethylbutyl)-

5-methoxyphenyl)piperidin-4-yl)methoxy)phenyl)propanoate (78

1

74%). H NMR (300 MHz, DMSO-d₆): δ ppm 0.05−0.18 (1H, m),

0.18−0.37 (2H, m), 0.41−0.56 (1H, m), 0.84 (6H, d, J = 6.6 Hz),

0.92−1.12 (1H, m), 1.35−1.58 (2H, m), 1.68−1.91 (4H, m), 1.99

(3H, s), 2.07 (2H, s), 2.16−2.30 (1H, m), 2.55−2.78 (4H, m), 3.23

(2H, d, J = 11.2 Hz), 3.37 (2H, s), 3.72 (3H, s), 3.88 (2H, d, J = 5.8

Hz), 6.59 (2H, dq, J = 4.5, 2.4 Hz), 6.73−6.87 (3H, m), 7.18 (1H, t, J

= 7.8 Hz), 7.23−7.30 (1H, m), 11.99 (1H, br s). ¹³C NMR (75 MHz,

CDCl₃): δ ppm 4.1, 5.2, 17.6, 20.9, 25.2, 29.8, 36.0, 40.2, 43.2, 47.4,

53.3, 53.5, 55.0, 55.2, 63.6, 72.3, 107.0, 108.2, 111.9, 114.1, 120.0,

121.5, 129.0, 132.9, 147.2, 155.0, 159.0, 160.1, 177.2. MS (ESI/

APCI): m/z 509.4 [M + H]⁺. C₃₁H₄₄N₂O₄: 0.5 H₂O: C, 71.92; H,

8.76; N, 5.51. Found: C, 71.67; H, 8.92; N, 5.46. HPLC purity 99.8%.

3-Cyclopropyl-3-(3-((1-(5-methoxy-2-(methyl(neopentyl)-

carbamoyl)phenyl)piperidin-4-yl)methoxy)phenyl)propanoic Acid

(6a). To a solution of 2-(4-((3-(1-cyclopropyl-3-methoxy-3-

oxopropyl)phenoxy)methyl)piperidin-1-yl)-4-methoxybenzoic acid

55 (414.4 mg, 0.89 mmol) in DMF (4.43 mL) were added HATU

(506 mg, 1.33 mmol) and N,N-diisopropylethylamine (DIPEA)

(0.232 mL, 1.33 mmol) at room temperature. The mixture was stirred

at the same temperature for 1.5 h. The mixture was poured into water

and extracted with EtOAc. The organic layer was separated, washed

with brine, dried over Na₂SO₄, and concentrated in vacuo. The

residue was puriﬁed by column chromatography (silica gel, eluted

with 0−30% EtOAc in hexane) to give methyl 3-cyclopropyl-3-(3-((1-

(5-methoxy-2-(methyl(neopentyl)carbamoyl)phenyl)piperidin-4-yl)-

methoxy)phenyl)propanoate (495 mg) as a yellow oil. ¹H NMR (300

MHz, DMSO-d₆): δ 0.05−0.27 (2H, m), 0.27−0.39 (1H, m), 0.43−

0.56 (1H, m), 0.69 (3H, s), 0.91−1.11 (7H, m), 1.22−1.51 (2H, m),

1.66−1.97 (3H, m), 2.18−2.33 (1H, m), 2.65−2.95 (6H, m), 3.02

(1H, s), 3.10−3.28 (2H, m), 3.32−3.46 (1H, m), 3.51 (3H, s), 3.72−

3.79 (3H, m), 3.80−3.92 (2H, m), 6.51−6.67 (2H, m), 6.72−6.88

(3H, m), 6.96−7.12 (1H, m), 7.18 (1H, t, J = 7.9 Hz). The 1H peak

was overlapped with the water peak. MS (ESI/APCI): m/z 551.5 [M

+ H]⁺. Compound 6a was prepared from the obtained methyl ester in

a manner similar to that described for compound 4a. White

amorphous solid (471 mg, 0.878 mmol, 99%). ¹H NMR (300

MHz, DMSO-d₆): δ 0.07−0.17 (1H, m), 0.19−0.37 (2H, m), 0.43−

1

mg, 0.154 mmol, 16.02%) was obtained as a colorless oil. H NMR

(400 MHz, CDCl₃): δ ppm 0.16 (1H, dq, J = 9.5, 5.0 Hz), 0.21−0.33

(1H, m), 0.36−0.49 (1H, m), 0.51−0.64 (1H, m), 0.97 (9H, s),

0.99−1.08 (1H, m), 1.46 (2H, dt, J = 8.4, 4.5 Hz), 1.50−1.62 (2H,

m), 1.93 (3H, d, J = 10.2 Hz), 2.27−2.40 (1H, m), 2.49−2.60 (2H,

m), 2.62−2.84 (4H, m), 3.12 (2H, d, J = 11.5 Hz), 3.62 (3H, s), 3.78

(3H, s), 3.85 (2H, d, J = 5.8 Hz), 6.58 (1H, dd, J = 8.3, 2.4 Hz), 6.66

(1H, d, J = 2.3 Hz), 6.72−6.85 (3H, m), 7.09 (1H, d, J = 8.3 Hz),

7.21 (1H, t, J = 7.8 Hz). MS (ESI/APCI): m/z 508.2 [M + H]⁺.

HPLC purity 100% (LC/MS).

3-(3-((1-(2-(3-Acetoxy-3-methylbutyl)-5-methoxyphenyl)-

piperidin-4-yl)methoxy)phenyl)-3-cyclopropylpropanoicacid (4g).

Compound 4g was prepared from 4-(2-(4-(hydroxymethyl)-

piperidin-1-yl)-4-methoxyphenyl)-2-methylbutan-2-yl acetate 50 in a

manner similar to that described for compound 4a (puriﬁcation:

column chromatography (silica gel, eluted with 0−50% EtOAc in

1

hexane)). White amorphous solid (47.3 mg, 0.095 mmol, 85%). H

NMR (400 MHz, DMSO-d₆): δ 0.04−0.18 (1H, m), 0.19−0.27 (1H,

m), 0.28−0.38 (1H, m), 0.44−0.57 (1H, m), 0.95−1.06 (1H, m),

1.15 (6H, s), 1.48 (2H, d, J = 11.3 Hz), 1.56−1.66 (2H, m), 1.80−

1.90 (2H, m), 2.18−2.30 (1H, m), 2.50−2.59 (3H, m), 2.59−2.73

(4H, m), 3.02 (2H, d, J = 10.5 Hz), 3.70 (3H, s), 3.87 (2H, d, J = 5.1

Hz), 4.16 (1H, br s), 6.51−6.63 (2H, m), 6.77 (1H, d, J = 7.7 Hz),

6.79−6.87 (2H, m), 7.05 (1H, d, J = 8.3 Hz), 7.14−7.23 (1H, m),

11.96 (1H, br s). ¹³C NMR (75 MHz, CDCl₃): δ ppm 4.2, 5.3, 17.1,

25.1, 29.6, 29.6, 29.7, 36.0, 41.5, 45.1, 46.9, 53.5, 53.6, 55.3, 70.9,

72.3, 107.0, 108.8, 112.6, 113.7, 119.6, 129.4, 130.2, 130.7, 145.6,

152.7, 158.6, 159.2, 176.8. MS (ESI/APCI): m/z 496.3 [M + H]⁺.

C₃₀H₄₁NO₅: C, 72.70; H, 8.34; N, 2.83. Found: C, 72.75; H, 8.29; N,

2.75. HPLC purity 98.8%. The corresponding intermediate: ethyl 3-

(3-((1-(2-(3-acetoxy-3-methylbutyl)-5-methoxyphenyl)piperidin-4-

yl)methoxy)phenyl)-3-cyclopropylpropanoate (silica gel, eluted with

5−10% EtOAc in hexane). Colorless oil (289 mg, 0.511 mmol, 76%).

O

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J. Med. Chem. XXXX, XXX, XXX−XXX

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0.55 (1H, m), 0.69 (3H, s), 0.99 (7H, s), 1.25 (1H, br s), 1.29−1.51

(2H, m), 1.69−2.00 (3H, m), 2.17−2.31 (1H, m), 2.57−2.76 (2H,

m), 2.77−2.97 (3H, m), 3.02 (1H, s), 3.10−3.25 (2H, m), 3.35−3.47

(1H, m), 3.73−3.79 (3H, m), 3.81−3.89 (2H, m), 6.47 (1H, d, J =

2.5 Hz), 6.51−6.66 (2H, m), 6.72−6.79 (1H, m), 6.79−6.86 (2H, m),

6.97−7.12 (1H, m), 7.18 (1H, t, J = 8.1 Hz), 11.97 (1H, s). ¹³C NMR

(101 MHz, DMSO-d₆): δ 3.9, 4.9, 17.4, 28.5, 29.0, 34.0, 35.2, 36.8,

40.7, 46.3, 50.0, 53.4, 55.0, 59.5, 71.7, 105.3, 107.3, 111.8, 113.6,

119.5, 125.6, 128.6, 129.1, 146.1, 150.8, 158.6, 160.1, 171.4, 173.0.

Tautomer mixture. MS (ESI/APCI): m/z 537.3 [M + H]⁺.

C₃₂H₄₄N₂O₅: 0.1 H₂O: C, 71.37; H, 8.27; N, 5.20. Found: C,

71.16; H, 8.01; N, 5.15. HPLC purity 100%.

m), 6.30 (1H, s), 6.61 (1H, dd, J = 8.5, 2.3 Hz), 6.72−6.95 (4H, m),

7.05 (1H, dd, J = 6.9, 5.1 Hz), 7.18 (1H, t, J = 8.0 Hz), 7.30 (1H, d, J

= 8.4 Hz), 7.45−7.55 (1H, m), 8.38 (1H, d, J = 3.9 Hz). 3H were

overlapped with the peak of DMSO. MS (ESI/APCI): m/z 558.5 [M

+ H]⁺.

3-Cyclopropyl-3-(3-((1-(2-(isobutyl(pyridin-2-yl)carbamoyl)-5-

methoxyphenyl)piperidin-4-yl)methoxy)phenyl)propanoic Acid

(6d). Compound 6d was prepared from compound 55 (100 mg,

0.21 mmol) in a manner similar to that described for compound 6b.

White amorphous solid (30.3 mg, 0.052 mmol, 60%). ¹H NMR (300

MHz, DMSO-d₆): δ 0.06−0.39 (3H, m), 0.43−0.55 (1H, m), 0.81

(6H, br d, J = 6.6 Hz), 0.92−1.11 (1H, m), 1.11−1.59 (3H, m),

1.60−1.83 (4H, m), 2.20−2.29 (1H, m), 2.52−2.75 (4H, m), 3.11−

3.30 (1H, m), 3.70 (3H, s), 3.83 (2H, br d, J = 5.9 Hz), 3.89−4.06

(2H, m), 6.17−6.29 (1H, m), 6.54 (1H, dd, J = 8.5, 2.3 Hz), 6.67−

6.86 (4H, m), 7.02 (1H, dd, J = 6.9, 5.1 Hz), 7.14−7.24 (2H, m),

7.39−7.50 (1H, m), 8.36 (1H, dd, J = 4.8, 1.2 Hz), 12.00 (1H, br s).

¹³C NMR (101 MHz, DMSO-d₆): δ 3.9, 4.9, 17.4, 20.0, 27.2, 28.2,

3-Cyclopropyl-3-{3-[(1-{5-methoxy-2-[methyl(phenyl)-

carbamoyl]phenyl}piperidin-4-yl)methoxy]phenyl}propanoic Acid

(6b). To a solution of compound 55 (100 mg, 0.21 mmol) in THF

(2 mL) was added 1-chloro-N,N,2-trimethylprop-1-en-1-amine (0.041

mL, 0.31 mmol) at 0 °C. The mixture was stirred at room

temperature under N₂for 30 min. Then, N-methylaniline (44.5 mg,

0.42 mmol) and triethylamine (0.058 mL, 0.42 mmol) were added to

the mixture at 0 °C. The mixture was stirred at room temperature

under N₂overnight. The mixture was quenched with water and

extracted with EtOAc. The organic layer was separated, washed with

brine, dried over Na₂SO₄, and concentrated in vacuo. The residue was

puriﬁed by column chromatography (silica gel, eluted with 0−30%

EtOAc in hexane) to give ethyl 3-cyclopropyl-3-(3-((1-(5-methoxy-2-

(methyl(phenyl)carbamoyl)phenyl)piperidin-4-yl)methoxy)phenyl)-

propanoate (72.3 mg, 0.127 mmol, 61.0%) as (a) colorless oil. MS

(ESI/APCI): m/z 571.4 [M + H]⁺. Compound 6b was prepared from

the corresponding ethyl ester (72.3 mg, 0.127 mmol) in a manner

similar to that described for compound 4a. White amorphous solid

(35 mg, 0.064 mmol, 51%). ¹H NMR (400 MHz, DMSO-d₆): δ

0.07−0.17 (1H, m), 0.18−0.37 (2H, m), 0.43−0.54 (1H, m), 0.94−

1.10 (1H, m), 1.45 (2H, br d, J = 10.8 Hz), 1.79 (3H, br d, J = 6.0

Hz), 2.17−2.31 (1H, m), 2.51−2.74 (4H, m), 3.69 (3H, br s), 3.89

(2H, br d, J = 5.4 Hz), 6.03−6.37 (1H, m), 6.55 (1H, br s), 6.74−

6.87 (3H, m), 6.96−7.31 (7H, m), 11.98 (1H, br s). ¹³C NMR (101

MHz, DMSO-d₆): δ 3.9, 4.9, 17.4, 28.7, 35.2, 40.7, 46.4, 55.0, 71.7,

104.4, 106.5, 111.9, 113.6, 119.5, 124.3, 125.5, 126.0, 127.8, 129.1,

130.2, 143.7, 146.1, 151.0, 158.6, 160.4, 170.1, 173.0. Two C peaks

next to the nitrogen of the piperidine ring and a C peak of methyl on

the amide were not detected. MS (ESI/APCI): m/z 543.4 [M + H]⁺.

C₃₃H₃₈N₂O₅: 0.4 H₂O: C, 72.08; H, 7.11; N, 5.09. Found: C, 71.92;

H, 6.82; N, 5.20. HPLC purity 100%.

35.2, 40.7, 46.4, 52.7, 55.0, 71.8, 104.2, 106.4, 111.8, 113.6, 119.5,

120.1, 120.3, 123.5, 129.1, 131.1, 136.5, 146.1, 147.6, 151.0, 155.1,

158.6, 160.8, 170.2, 173.0. Two C peaks next to the nitrogen of the

piperidine ring were not detected. MS (ESI/APCI): m/z 586.4 (M +

H)⁺. C₃₅H₄₃N₃O₅: 0.1 H₂O: C, 71.55; H, 7.41; N, 7.15. Found: C,

71.33; H, 7.21; N, 7.14. HPLC purity 98.4%. The corresponding

intermediate: methyl 3-cyclopropyl-3-(3-((1-(2-(isobutyl(pyridin-2-

yl)carbamoyl)-5-methoxyphenyl)piperidin-4-yl)methoxy)phenyl)-

propanoic acid propanoate (puriﬁcation: NH silica gel, eluted with 0−

30% EtOAc in hexane). Colorless oil (75.1 mg, 0.122 mmol, 59%).

3-Cyclopropyl-3-(3-((1-(5-methoxy-2-(neopentyl(pyridin-2-yl)-

carbamoyl)phenyl)piperidin-4-yl)methoxy)phenyl)propanoic Acid

(6e). Compound 6e was prepared from compound 55 (114 mg,

0.24 mmol) in a manner similar to that described for compound 6b.

1

White amorphous solid (129 mg, 0.215 mmol, 94%). H NMR (300

MHz, DMSO-d₆): δ ppm 0.05−0.18 (1H, m), 0.18−0.39 (2H, m),

0.43−0.59 (1H, m), 0.77 (9H, s), 0.93−1.10 (1H, m), 1.20−1.39

(1H, m), 1.51 (1H, br s), 1.71 (3H, d, J = 11.6 Hz), 2.19−2.31 (1H,

m), 2.31−2.47 (2H, m), 2.53−2.75 (4H, m), 3.69 (3H, s), 3.84 (2H,

d, J = 5.9 Hz), 3.96−4.26 (2H, m), 6.22 (1H, d, J = 2.2 Hz), 6.52

(1H, dd, J = 8.4, 2.3 Hz), 6.70 (1H, d, J = 7.6 Hz), 6.75−6.89 (3H,

m), 7.00 (1H, dd, J = 6.7, 4.9 Hz), 7.10−7.25 (2H, m), 7.36−7.46

(1H, m), 8.33 (1H, dd, J = 4.8, 1.2 Hz), 11.68−12.42 (1H, m). ¹³C

NMR (75 MHz, CDCl₃): δ 4.1, 5.3, 17.2, 28.4, 28.7, 34.2, 35.9, 41.2,

46.9, 49.8, 54.0, 55.2, 55.9, 72.5, 104.6, 106.1, 112.4, 113.8, 119.7,

120.0, 121.4, 124.2, 129.4, 131.6, 135.8, 145.6, 147.2, 151.3, 156.5,

159.2, 161.4, 171.7, 176.7. MS (ESI/APCI): m/z 600.3 [M + H]⁺.

C₃₆H₄₅N₃O₅: 0.1 H₂O: C, 71.88; H, 7.57; N, 6.99. Found: C, 72.12;

H, 7.53; N, 6.74. HPLC purity 95.3%. The corresponding intermediate:

ethyl 3-cyclopropyl-3-(3-((1-(5-methoxy-2-(neopentyl(pyridin-2-yl)-

carbamoyl) phenyl)piperidin-4-yl)methoxy)phenyl)propanoate (puri-

ﬁcation: NH silica gel, eluted with 0−30% EtOAc in hexane).

Colorless gum (143 mg, 0.228 mmol, 96%).

3-{3-[(1-{2-[(2-Cyano-2-methylpropyl)(pyridin-2-yl)carbamoyl]-

5-methoxyphenyl}piperidin-4-yl)methoxy]phenyl}-3-cyclopropyl-

propanoic Acid (6f). Compound 6f was prepared from compound 55

(100 mg, 0.21 mmol) in a manner similar to that described for

compound 6b. White amorphous solid (98 mg, 0.160 mmol, quant).

¹H NMR (400 MHz, DMSO-d₆): δ ppm 0.09−0.18 (1H, m), 0.20−

0.28 (1H, m), 0.29−0.37 (1H, m), 0.45−0.57 (1H, m), 0.92−1.06

(1H, m), 1.16−1.63 (4H, m), 1.29 (6H, br s), 1.64−1.87 (3H, m),

2.18−2.30 (1H, m), 2.51−2.74 (4H, m), 3.71 (3H, s), 3.84 (2H, d, J

= 6.0 Hz), 4.18−4.63 (2H, m), 6.24 (1H, d, J = 1.9 Hz), 6.57 (1H,

dd, J = 8.4, 2.3 Hz), 6.70 (1H, d, J = 6.0 Hz), 6.75−6.80 (1H, m),

6.81−6.86 (2H, m), 7.06 (1H, dd, J = 7.0, 5.2 Hz), 7.14−7.25 (2H,

m), 7.44 (1H, t, J = 7.7 Hz), 8.37 (1H, d, J = 4.8 Hz), 11.99 (1H, br

s). ¹³C NMR (101 MHz, DMSO-d₆): δ 3.9, 5.0, 17.4, 24.7, 28.1, 33.2,

35.1, 40.7, 46.4, 51.5, 55.1, 71.8, 104.2, 106.6, 111.8, 113.6, 119.5,

120.8, 121.0, 122.8, 123.5, 129.1, 131.2, 136.5, 146.2, 147.4, 151.1,

154.2, 158.6, 161.1, 170.9, 173.1. Two C peaks next to the nitrogen of

the piperidine ring were not detected. MS (ESI/APCI): m/z 611.2

[M + H]⁺. C₃₆H₄₂N₄O₅: 0.4 H₂O: C, 69.97; H, 6.98; N, 9.07. Found:

3-Cyclopropyl-3-(3-((1-(5-methoxy-2-(methyl(pyridin-2-yl)-

carbamoyl)phenyl)piperidin-4-yl)methoxy)phenyl)propanoic Acid

(6c). Compound 6c was prepared from compound 55 (402.6 mg,

0.86 mmol) in a manner similar to that described for compound 6a.

1

White amorphous solid (330 mg, 0.607 mmol, 73%). H NMR (300

MHz, DMSO-d₆): δ 0.07−0.18 (1H, m), 0.18−0.37 (2H, m), 0.44−

0.56 (1H, m), 0.91−1.09 (1H, m), 1.10−1.60 (2H, m), 1.70 (3H, d, J

= 9.6 Hz), 2.18−2.32 (1H, m), 2.53−2.75 (4H, m), 3.42 (3H, s), 3.74

(3H, s), 3.80 (2H, d, J = 5.9 Hz), 6.30 (1H, d, J = 2.1 Hz), 6.61 (1H,

dd, J = 8.5, 2.3 Hz), 6.75 (1H, dd, J = 7.8, 2.0 Hz), 6.79−6.94 (3H,

m), 7.05 (1H, dd, J = 6.5, 4.9 Hz), 7.17 (1H, t, J = 8.0 Hz), 7.30 (1H,

d, J = 8.4 Hz), 7.44−7.56 (1H, m), 8.38 (1H, dd, J = 4.8, 1.2 Hz),

12.00 (1H, br s). 2H were overlapped with the water peak. ¹³C NMR

(75 MHz, CDCl₃): δ 4.2, 5.3, 17.2, 28.8, 34.9, 35.8, 41.3, 46.9, 55.3,

72.4, 104.7, 106.3, 112.5, 113.7, 119.0, 119.6, 119.8, 123.1, 129.4,

131.6, 136.3, 145.6, 147.5, 151.6, 156.1, 159.1, 161.8, 171.6, 176.4.

Two C peaks next to the nitrogen of the piperidine ring were not

detected. MS (ESI/APCI): m/z 544.2 [M + H]⁺. C₃₂H₃₇N₃O₅: 0.6

H₂O: C, 69.32; H, 6.94; N, 7.58. Found: C, 69.55; H, 6.69; N, 7.29.

HPLC purity 99.3%. The corresponding intermediate: methyl 3-

cyclopropyl-3-(3-((1-(5-methoxy-2-(methyl(pyridin-2-yl)carbamoyl)-

phenyl)piperidin-4-yl)methoxy)phenyl)propanoate (puriﬁcation: NH

silica gel, eluted with 0−30% EtOAc in hexane). Colorless oil (311

1

mg, 0.558 mmol, 65%). H NMR (300 MHz, DMSO-d₆): δ ppm

0.06−0.26 (2H, m), 0.26−0.38 (1H, m), 0.43−0.58 (1H, m), 0.93−

1.11 (1H, m), 1.61−1.81 (3H, m), 2.18−2.30 (1H, m), 2.58 (2H, br

s), 2.65−2.82 (3H, m), 3.42 (3H, s), 3.51 (3H, s), 3.69−3.87 (5H,

P

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C, 69.78; H, 6.88; N, 9.02. HPLC purity 95.9%. The corresponding

intermediate: ethyl 3-(3-((1-(2-((2-cyano-2-methylpropyl)(pyridin-2-

yl)carbamoyl)-5-methoxyphenyl)piperidin-4-yl)methoxy)phenyl)-3-

cyclopropylpropanoate (NH silica gel, eluted with 15−25% EtOAc in

mmol) at room temperature. The mixture was stirred at 100 °C under

N₂for 1 h. After cooling to room temperature, the reaction mixture

was concentrated in vacuo. The residue was puriﬁed by column

chromatography (NH silica gel, eluted with 0−40% EtOAc in hexane)

to give ethyl 3-methoxy-3-(3-((1-(5-methoxy-2-((6-methylpyridin-2-

yl)(neopentyl)carbamoyl)phenyl)piperidin-4-yl)methoxy)phenyl)-

propanoate as a pale-yellow gum. Compound 7a was prepared from

the obtained ethyl ester in a manner similar to that described for

compound 4a (puriﬁcation: HPLC). White amorphous solid (98 mg,

1

hexane). White amorphous solid (108.3 mg, 0.170 mmol, 82%). H

NMR (400 MHz, CDCl₃): δ ppm 0.12−0.21 (1H, m), 0.23−0.32

(1H, m), 0.38−0.49 (1H, m), 0.53−0.64 (1H, m), 0.96−1.10 (1H,

m), 1.19 (3H, t, J = 7.2 Hz), 1.39 (6H, br s), 1.38−1.68 (2H, m),

1.69−1.92 (3H, m), 2.30−2.39 (1H, m), 2.35−2.71 (3H, m), 2.67−

2.80 (2H, m), 3.14−3.42 (1H, m), 3.78 (3H, s), 3.82 (2H, d, J = 6.3

Hz), 4.00−4.12 (2H, m), 4.33−4.86 (2H, m), 6.20 (1H, d, J = 2.1

Hz), 6.54 (2H, dd, J = 8.4, 2.3 Hz), 6.70−6.88 (3H, m), 6.90−7.01

(1H, m), 7.18−7.25 (2H, m), 7.37 (1H, d, J = 8.4 Hz), 8.40 (1H, d, J

= 3.6 Hz).

1

0.193 mmol, 74%, 2 steps). H NMR (300 MHz, DMSO-d₆): δ ppm

0.77 (9H, s), 1.57 (1H, br s), 1.65−1.84 (3H, m), 2.35 (3H, s), 2.53−

2.69 (3H, m), 3.11 (3H, s), 3.24−3.29 (3H, m), 3.69 (3H, s), 3.86

(2H, d, J = 4.3 Hz), 3.96−4.26 (2H, m), 4.53 (1H, dd, J = 8.7, 4.9

Hz), 6.22 (1H, d, J = 2.2 Hz), 6.43−6.56 (2H, m), 6.82−6.94 (4H,

m), 7.14 (1H, d, J = 8.4 Hz), 7.28 (2H, td, J = 7.8, 2.9 Hz), 12.20

(1H, s). ¹³C NMR (75 MHz, CDCl₃): δ 24.3, 28.4, 28.8, 34.2, 35.9,

43.2, 50.0, 54.2, 55.2, 55.8, 56.9, 72.7, 79.8, 104.7, 106.1, 112.3, 114.3,

118.1, 118.8, 119.2, 124.6, 129.7, 131.4, 136.1, 141.8, 151.3, 155.7,

156.0, 159.5, 161.2, 171.7, 175.2. MS (ESI/APCI): m/z 604.4 [M +

H]⁺. C₃₅H₄₅N₃O₆: 0.4 H₂O: C, 68.81; H, 7.56; N, 6.88. Found: C,

68.65; H, 7.35; N, 6.88. HPLC purity 99.2%.

3-Cyclopropyl-3-{3-[(1-{5-methoxy-2-[(2-methoxy-2-

methylpropyl)(pyridin-2-yl)carbamoyl]phenyl}piperidin-4-yl)-

methoxy]phenyl}propanoic Acid (6g). Compound 6g was prepared

from compound 55 (100 mg, 0.21 mmol) in a manner similar to that

described for compound 6b. White amorphous solid (77.9 mg, 0.127

1

mmol, 96%). H NMR (400 MHz, DMSO-d₆): δ ppm 0.08−0.16

(1H, m), 0.20−0.28 (1H, m), 0.29−0.39 (1H, m), 0.44−0.55 (1H,

m), 0.93−1.19 (7H, m), 1.20−1.63 (2H, m), 1.66−1.87 (3H, m),

2.18−2.30 (1H, m), 2.40−2.72 (6H, m), 2.75 (3H, s), 3.68 (3H, s),

3.84 (2H, d, J = 5.9 Hz), 4.03−4.43 (2H, m), 6.24 (1H, s), 6.49 (1H,

dd, J = 8.5, 2.2 Hz), 6.67 (1H, br s), 6.74−6.86 (3H, m), 6.95−7.03

(1H, m), 7.08 (1H, d, J = 8.5 Hz), 7.15−7.23 (1H, m), 7.31−7.45

(1H, m), 8.32 (1H, d, J = 4.8 Hz), 11.97 (1H, br s). ¹³C NMR (101

MHz, DMSO-d₆): δ 3.9, 5.0, 17.4, 23.5, 28.3, 35.2, 40.7, 46.4, 48.5,

51.0, 55.0, 71.8, 75.9, 104.3, 106.4, 111.8, 113.7, 119.5, 120.3, 121.3,

123.9, 129.1, 130.7, 136.1, 146.1, 147.1, 151.1, 155.5, 158.6, 160.6,

170.5, 173.0. Two C peaks next to the nitrogen of the piperidine ring

were not detected. MS (ESI/APCI): m/z 616.3 [M + H]⁺.

C₃₆H₄₅N₃O₆: 0.5 H₂O: C, 69.21; H, 7.42; N, 6.73. Found: C,

69.31; H, 7.33; N, 6.43. HPLC purity 97.0%. The corresponding

intermediate: ethyl 3-cyclopropyl-3-(3-((1-(5-methoxy-2-((2-me-

thoxy-2-methylpropyl)(pyridin-2-yl)carbamoyl)phenyl)piperidin-4-

yl)methoxy)phenyl)propanoate (NH silica gel, eluted with 15−25%

EtOAc in hexane). Colorless oil (85 mg, 0.132 mmol, 64%) ¹H NMR

(400 MHz, CDCl₃): δ ppm 0.11−0.21 (1H, m), 0.24−0.35 (1H, m),

0.38−0.49 (1H, m), 0.52−0.64 (1H, m), 0.97−1.08 (1H, m), 1.12−

1.23 (9H, m), 1.71−1.91 (3H, m), 2.27−2.79 (4H, m), 2.29−2.39

(1H, m), 2.73 (3H, tt, J = 14.6, 7.4 Hz), 2.83 (3H, s), 3.40 (1H, br s),

3.75 (3H, s), 3.83 (2H, d, J = 6.0 Hz), 3.98−4.12 (2H, m), 4.22−4.63

(2H, m), 6.20 (1H, s), 6.47 (1H, dd, J = 8.4, 2.3 Hz), 6.57 (1H, br s),

6.73−6.92 (4H, m), 7.21 (2H, t, J = 7.8 Hz), 7.24−7.32 (1H, m),

8.35 (1H, dd, J = 4.8, 1.2 Hz).

3-Ethoxy-3-(3-((1-(5-methoxy-2-((6-methylpyridin-2-yl)-

(neopentyl)carbamoyl)phenyl)piperidin-4-yl)methoxy)phenyl)-

propanoic Acid (7b). Compound 7b was prepared from compound

57 (103.6 mg, 0.24 mmol) in a manner similar to that described for

compound 7a. White amorphous solid (100 mg, 0.162 mmol, 77%).

¹H NMR (300 MHz, DMSO-d₆): δ ppm 0.77 (9H, s), 1.07 (3H, t, J =

7.0 Hz), 1.24 (1H, s), 1.42−1.83 (4H, m), 2.36 (4H, s), 2.53−2.68

(3H, m), 3.22−3.38 (4H, m), 3.69 (3H, s), 3.79−3.93 (2H, m),

3.93−4.25 (2H, m), 4.64 (1H, dd, J = 8.6, 5.1 Hz), 6.22 (1H, d, J =

2.2 Hz), 6.42−6.56 (2H, m), 6.80−6.94 (4H, m), 7.14 (1H, d, J = 8.4

Hz), 7.22−7.32 (2H, m), 12.20 (1H, br s). ¹³C NMR (101 MHz,

DMSO-d₆): δ 15.1, 23.8, 28.2, 33.8, 35.2, 43.1, 49.3, 53.4, 55.0, 55.1,

63.5, 72.0, 77.6, 104.1, 106.3, 112.3, 113.6, 117.6, 118.5, 119.3, 124.2,

129.4, 130.9, 136.5, 143.2, 150.8, 155.1, 155.5, 158.8, 160.6, 170.7,

171.7. MS (ESI/APCI): m/z 618.3 [M + H]⁺. C₃₆H₄₇N₃O₆: 0.4 H₂O:

C, 69.18; H, 7.71; N, 6.72. Found: C, 69.42; H, 7.62; N, 6.44. HPLC

purity 98.3%. The corresponding intermediate: ethyl 3-ethoxy-3-(3-((1-

(5-methoxy-2-((6-methylpyridin-2-yl)(neopentyl)carbamoyl)-

phenyl)piperidin-4-yl)methoxy)phenyl)propanoate (NH silica gel,

eluted with 0−40% EtOAc in hexane). Pale-yellow gum (135.5 mg,

0.21 mmol). ¹H NMR (300 MHz, CDCl₃): δ ppm 0.82 (9H, s), 1.16

(3H, t, J = 7.0 Hz), 1.21−1.31 (4H, m), 1.75 (3H, d, J = 11.2 Hz),

2.00 (1H, s), 2.35−2.71 (7H, m), 2.78 (1H, dd, J = 15.2, 9.3 Hz),

3.29−3.52 (3H, m), 3.76 (3H, s), 3.84 (2H, d, J = 6.1 Hz), 4.07−4.40

(4H, m), 4.72 (1H, dd, J = 9.2, 4.6 Hz), 6.20 (1H, d, J = 2.3 Hz), 6.34

(1H, br s), 6.49 (1H, dd, J = 8.5, 2.4 Hz), 6.70 (1H, d, J = 7.4 Hz),

6.80−6.89 (1H, m), 6.89−6.97 (2H, m), 7.09 (1H, t, J = 7.6 Hz),

7.21−7.35 (2H, m).

4-Methoxy-3-(3-((1-(5-methoxy-2-((6-methylpyridin-2-yl)-

(neopentyl)carbamoyl)phenyl)piperidin-4-yl)methoxy)phenyl)-

butanoic Acid (7c). Compound 7c was prepared from compound 57

(195 mg, 0.46 mmol) in a manner similar to that described for

compound 7a. White amorphous solid (235 mg, 0.380 mmol, 83%).

¹H NMR (400 MHz, DMSO-d₆): δ ppm 0.77 (9H, s), 0.86 (1H, t, J =

6.5 Hz), 1.12−1.31 (2H, m), 1.70 (2H, d, J = 11.3 Hz), 2.36 (3H, s),

2.58−2.72 (2H, m), 3.21 (3H, s), 3.23−3.30 (3H, m), 3.44 (2H, d, J

= 6.8 Hz), 3.69 (3H, s), 3.83 (2H, d, J = 5.6 Hz), 4.03 (1H, d, J = 7.2

Hz), 4.16 (1H, br s), 6.22 (1H, br s), 6.47 (1H, br s), 6.52 (1H, d, J =

8.5 Hz), 6.72−6.92 (4H, m), 7.14 (1H, d, J = 8.4 Hz), 7.19 (1H, t, J =

7.8 Hz), 7.28 (1H, t, J = 7.8 Hz), 12.04 (1H, br s). ¹³C NMR (101

MHz, DMSO-d₆): δ 13.9, 22.0, 23.9, 28.2, 30.9, 33.8, 35.2, 37.0, 41.5,

55.0, 58.0, 71.9, 75.8, 104.1, 106.3, 112.2, 114.1, 117.6, 119.3, 119.9,

124.2, 129.1, 130.9, 136.5, 143.6, 150.8, 155.1, 155.5, 158.6, 160.6,

170.6, 173.1. MS (ESI/APCI): m/z 618.3 [M + H]⁺. C₃₆H₄₇N₃O₆:

0.4 H₂O: C, 69.18; H, 7.71; N, 6.72. Found: C, 69.30; H, 7.67; N,

6.42. HPLC purity 92.6%.

3-Cyclopropyl-3-(3-((1-(5-methoxy-2-((6-methylpyridin-2-yl)-

(neopentyl)carbamoyl)phenyl)piperidin-4-yl)methoxy)phenyl)-

propanoic Acid (6h). Compound 6h was prepared from compound

55 (112.7 mg, 0.23 mmol) in a manner similar to that described for

1

compound 6b. White solid (27.5 mg, 0.045 mmol, 53%). H NMR

(300 MHz, DMSO-d₆): δ ppm 0.06−0.18 (1H, m), 0.18−0.38 (2H,

m), 0.41−0.56 (1H, m), 0.77 (9H, s), 0.91−1.10 (1H, m), 1.42−1.82

(4H, m), 2.19−2.30 (1H, m), 2.36 (4H, s), 2.56−2.74 (3H, m), 3.69

(3H, s), 3.84 (2H, d, J = 5.9 Hz), 3.91−4.27 (2H, m), 6.22 (1H, s),

6.42−6.58 (2H, m), 6.73−6.89 (4H, m), 7.09−7.24 (2H, m), 7.28

(1H, t, J = 7.7 Hz). ¹³C NMR (75 MHz, CDCl₃): δ 4.1, 5.3, 17.3,

24.3, 28.5, 28.8, 34.2, 36.0, 41.2, 46.9, 55.2, 72.6, 77.2, 104.6, 106.0,

112.2, 113.9, 118.0, 119.2, 119.6, 124.6, 129.4, 131.4, 136.0, 145.6,

151.3, 155.7, 156.0, 159.2, 161.2, 171.7, 176.4. Two C peaks next to

the nitrogen of the piperidine ring were not detected. MS (ESI/

APCI): m/z 614.4 [M + H]⁺. C₃₇H₄₇N₃O₅: 0.1 H₂O: C, 72.19; H,

7.73; N, 6.83. Found: C, 72.03; H, 7.60; N, 6.73. HPLC purity 95.3%.

3-Methoxy-3-(3-((1-(5-methoxy-2-((6-methylpyridin-2-yl)-

(neopentyl)carbamoyl)phenyl)piperidin-4-yl)methoxy)phenyl)-

propanoic Acid (7a). To a solution of 2-(4-(hydroxymethyl)-

piperidin-1-yl)-4-methoxy-N-(6-methylpyridin-2-yl)-N-neopentylben-

zamide 57 (110.1 mg, 0.26 mmol) in toluene (2.59 mL) were added

ethyl 3-(3-hydroxyphenyl)-3-methoxypropanoate (63.8 mg, 0.28

mmol) and cyanomethylenetri-n-butylphosphorane (136 μL, 0.52

2-(3-(3-((1-(5-Methoxy-2-((6-methylpyridin-2-yl)(neopentyl)-

carbamoyl)phenyl)piperidin-4-yl)methoxy)phenyl)oxetan-3-yl)-

acetic Acid (7d). Compound 7d was prepared from compound 57

Q

https://dx.doi.org/10.1021/acs.jmedchem.0c00843

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(144 mg, 0.34 mmol) in a manner similar to that described for

compound 7a. White amorphous solid (114 mg, 0.185 mmol, 55%).

¹H NMR (300 MHz, DMSO-d₆): δ ppm 0.77 (9H, s), 1.70 (3H, d, J

155.8, 156.1, 161.2, 164.4, 171.7, 175.7. Two C peaks next to the

nitrogen of the piperidine ring were not detected. MS (ESI/APCI):

m/z 615.3 [M + H]⁺. Calcd for C₃₆H₄₆N₄O₅: C, 70.33; H, 7.54; N,

9.11. Found: C, 70.24; H, 7.48; N, 9.09. HPLC purity 97.6%.

3-Cyclopropyl-3-{2-[(1-{2-[(2,2-dimethylpropyl)(6-methylpyridin-

2-yl)carbamoyl]-5-methoxyphenyl}piperidin-4-yl)methoxy]pyridin-

4-yl}propanoic Acid ((S)-7f, tR1). The racemic 7f (197.7 mg, 0.31

mmol) was puriﬁed by chiral HPLC. The desired fraction was

concentrated in vacuo to give 3-cyclopropyl-3-(2-((1-(5-methoxy-2-

((6-methylpyridin-2-yl)(neopentyl)carbamoyl)phenyl)piperidin-4-

yl)methoxy)pyridin-4-yl)propanoic acid (78 mg, 0.127 mmol, 41%,

= 11.5 Hz), 2.36 (3H, s), 3.30 (8H, br s), 3.05 (2H, s), 3.69 (3H, s),

3.85 (2H, d, J = 5.9 Hz), 4.68−4.81 (4H, m), 6.22 (1H, d, J = 2.0

Hz), 6.38−6.59 (2H, m), 6.76−6.89 (4H, m), 7.14 (1H, d, J = 8.4

Hz), 7.21−7.33 (2H, m), 12.14 (1H, br s). ¹³C NMR (101 MHz,

DMSO-d₆): δ 23.9, 28.2, 33.8, 35.2, 44.0, 44.7, 49.3, 53.3, 55.0, 55.1,

71.9, 81.0, 104.1, 106.3, 112.1, 112.6, 117.6, 118.4, 119.3, 124.2,

129.2, 130.9, 136.5, 146.0, 150.8, 155.0, 155.5, 158.6, 160.6, 170.6,

172.0. MS (ESI/APCI): m/z 616.3 [M + H]⁺. C₃₆H₄₅N₃O₆: 0.4 H₂O:

C, 69.41; H, 7.41; N, 6.75. Found: C, 69.29; H, 7.29; N, 6.52. HPLC

purity 92.7%.

1

>99.9% ee) as a white powder. H NMR (300 MHz, DMSO-d₆): δ

ppm 0.18 (1H, dd, J = 7.9, 4.6 Hz), 0.22−0.42 (2H, m), 0.44−0.60

(1H, m), 0.77 (9H, s), 0.93−1.06 (1H, m), 1.23 (1H, br s), 1.42−

1.90 (4H, m), 2.17−2.30 (2H, m), 2.36 (5H, s), 2.53−2.71 (3H, m),

3.69 (3H, s), 3.94−4.27 (4H, m), 6.21 (1H, d, J = 2.3 Hz), 6.39−6.57

(2H, m), 6.70 (1H, s), 6.84 (1H, d, J = 7.6 Hz), 6.91 (1H, dd, J = 5.3,

1.3 Hz), 7.14 (1H, d, J = 8.4 Hz), 7.28 (1H, t, J = 7.8 Hz), 8.04 (1H,

d, J = 5.3 Hz), 12.09 (1H, br s). ¹³C NMR (75 MHz, CDCl₃): δ 4.2,

5.3, 16.6, 24.3, 28.4, 28.7, 34.1, 35.6, 40.3, 46.1, 55.2, 55.8, 70.6,

104.6, 106.0, 109.5, 116.1, 118.0, 119.2, 124.6, 131.3, 136.0, 146.7,

151.4, 155.6, 155.8, 156.1, 161.2, 164.3, 171.8, 176.1. Two C peaks

next to the nitrogen of the piperidine ring were not detected. MS

(ESI/APCI): m/z 615.5[M + H]⁺. mp 153−154 °C. Calcd for

C₃₆H₄₆N₄O₅: C, 70.33; H, 7.54; N, 9.11. Found: C, 70.24; H, 7.48; N,

9.09. HPLC purity 100%. [α]²_D⁰+17.49 (c 1.0, MeOH).

3-Cyclopropyl-3-{2-[(1-{2-[(2,2-dimethylpropyl)(6-methylpyridin-

2-yl)carbamoyl]-5-methoxyphenyl}piperidin-4-yl)methoxy]pyridin-

4-yl}propanoic Acid ((R)-7f, tR2). Compound (R)-7f was prepared in

a manner similar to that described for compound (S)-7f. The desired

fraction was concentrated in vacuo to give 3-cyclopropyl-3-(2-((1-(5-

methoxy-2-((6-methylpyridin-2-yl)(neopentyl)carbamoyl)phenyl)-

piperidin-4-yl)methoxy)pyridin-4-yl)propanoic acid (73.6 mg, 0.120

mmol, 38.9%, 97% ee) as a powder. ¹H NMR (300 MHz, DMSO-d₆):

δ ppm 0.10−0.22 (1H, m), 0.22−0.41 (2H, m), 0.45−0.58 (1H, m),

0.77 (9H, s), 0.91−1.09 (1H, m), 1.11−1.32 (1H, m), 1.42−1.86

(4H, m), 2.17−2.30 (2H, m), 2.36 (5H, s), 2.53−2.71 (3H, m), 3.69

(3H, s), 3.92−4.29 (4H, m), 6.21 (1H, d, J = 2.5 Hz), 6.40−6.55

(2H, m), 6.70 (1H, s), 6.84 (1H, d, J = 7.6 Hz), 6.91 (1H, dd, J = 5.3,

1.3 Hz), 7.14 (1H, d, J = 8.4 Hz), 7.28 (1H, t, J = 7.9 Hz), 8.04 (1H,

d, J = 5.4 Hz), 12.09 (1H, s). ¹³C NMR (75 MHz, CDCl₃): δ 4.2, 5.3,

16.6, 24.3, 28.4, 28.8, 34.1, 35.6, 40.2, 46.1, 55.2, 55.7, 70.6, 104.6,

106.0, 109.5, 116.1, 118.0, 119.1, 124.6, 131.3, 136.0, 146.7, 151.4,

155.7, 155.8, 156.1, 161.2, 164.4, 171.7, 175.7. Two C peaks next to

the nitrogen of the piperidine ring were not detected. MS (ESI/

APCI): m/z 615.5 [M + H]⁺. Calcd for C₃₆H₄₆N₄O₅: C, 68.66; H,

7.52; N, 11.12. Found: C, 68.58; H, 7.47; N, 11.05. HPLC purity

99.5%.

3-Cyclopropyl-3-(6-((1-(5-methoxy-2-((6-methylpyridin-2-yl)-

(neopentyl)carbamoyl)phenyl)piperidin-4-yl)methoxy)pyridin-2-

yl)propanoic Acid (7e). Compound 7e was prepared from 2-(4-

(hydroxymethyl)piperidin-1-yl)-4-methoxy-N-(6-methylpyridin-2-yl)-

N-neopentylbenzamide 57 (100 mg, 0.43 mmol) in a manner similar

to that described for compound 7a. White solid (175 mg, 0.284

1

mmol, 79%). H NMR (400 MHz, DMSO-d₆): δ ppm 0.19−0.31

(2H, m), 0.33−0.41 (1H, m), 0.46−0.55 (1H, m), 0.76 (9H, s),

0.94−1.06 (1H, m), 1.08−1.31 (1H, m), 1.41−1.85 (4H, m), 2.27−

2.44 (2H, m), 2.36 (3H, s), 2.44−2.63 (2H, m), 2.64 (1H, dd, J =

15.4, 5.5 Hz), 2.88 (1H, dd, J = 15.2, 9.0 Hz), 3.14−3.34 (1H, m),

3.69 (3H, s), 3.92−4.19 (2H, m), 4.16 (2H, d, J = 6.1 Hz), 6.21 (1H,

br s), 6.44 (1H, br s), 6.52 (1H, d, J = 8.7 Hz), 6.62 (1H, d, J = 8.0

Hz), 6.80−6.93 (2H, m), 7.14 (1H, d, J = 8.4 Hz), 7.28 (1H, t, J = 7.3

Hz), 7.60 (1H, t, J = 7.8 Hz), 11.94 (1H, br s). ¹³C NMR (101 MHz,

DMSO-d₆): δ 4.0, 4.4, 16.6, 23.8, 28.2, 33.7, 35.0, 38.8, 47.2, 49.3,

53.4, 55.0, 69.4, 104.1, 106.3, 107.9, 115.0, 117.5, 119.3, 124.1, 130.9,

136.5, 139.2, 150.8, 155.1, 155.5, 160.6, 160.8, 162.6, 170.6, 173.3.

MS (ESI/APCI): m/z 615.5 [M + H]⁺. mp 139−141 °C.

C₃₆H₄₆N₄O₅: 0.1 H₂O: C, 70.13; H, 7.55; N, 9.09. Found: C,

70.19; H, 7.37; N, 8.98. HPLC purity 99.8%. The corresponding

intermediate: ethyl 3-cyclopropyl-3-(6-((1-(5-methoxy-2-((6-methyl-

pyridin-2-yl)(neopentyl)carbamoyl) phenyl)piperidin-4-yl)methoxy)-

pyridin-2-yl)propanoate (100 mg, 0.358 mmol, 84%). White

amorphous solid (243 mg, 0.358 mmol, 84%). ¹H NMR (400

MHz, CDCl₃): δ 0.20−0.36 (2H, m), 0.38−0.51 (1H, m), 0.53−0.64

(1H, m), 0.82 (9H, s), 1.04−1.15 (1H, m), 1.19 (3H, t, J = 7.2 Hz),

1.22−1.34 (1H, m), 1.58−1.86 (4H, m), 2.04 (1H, s), 2.35−2.49

(2H, m), 2.44 (3H, s), 2.60 (2H, br s), 2.77 (1H, dd, J = 15.1, 6.1

Hz), 3.01 (1H, dd, J = 15.4, 8.3 Hz), 3.39 (1H, br s), 3.75 (3H, s),

3.97−4.41 (6H, m), 6.19 (1H, br s), 6.33 (1H, br s), 6.48 (1H, d, J =

8.8 Hz), 6.56 (1H, d, J = 8.2 Hz), 6.70 (1H, d, J = 7.2 Hz), 6.78 (1H,

d, J = 7.2 Hz), 7.09 (1H, br s), 7.29 (1H, d, J = 8.4 Hz), 7.48 (1H, t, J

= 7.7 Hz).

3-Cyclopropyl-3-{2-[(1-{2-[(2,2-dimethylpropyl)(6-methylpyridin-

2-yl)carbamoyl]-5-methoxyphenyl}piperidin-4-yl)methoxy]pyridin-

4-yl}propanoic Acid (7f). NaOH (20 mL, 20.00 mmol, 1 M) was

added to a solution of ethyl 3-cyclopropyl-3-(2-((1-(5-methoxy-2-((6-

methylpyridin-2-yl)(neopentyl)carbamoyl)phenyl)piperidin-4-yl)-

methoxy)pyridin-4-yl)propanoate 64b (1.95 g, 3.03 mmol) in THF

(20 mL) and MeOH (10.00 mL) at room temperature. The mixture

was stirred at 50 °C for 30 min. The mixture was neutralized with 1 N

HCl at r.t. and extracted with EtOAc. The organic layer was separated,

washed with water and brine, dried over MgSO₄, ﬁltered through a

silica gel pad, and concentrated in vacuo. The residue was crystallized

from EtOAc (8 mL)/hexane (45 mL) to give the target compound as

5-(3-Hydroxybenzylidene)-2,2-dimethyl-1,3-dioxane-4,6-dione

(9). To a solution of 3-hydroxybenzaldehyde 8 (20 g, 163.77 mmol)

in water (600 mL) was added 2,2-dimethyl-1,3-dioxane-4,6-dione

(23.60 g, 163.77 mmol) at room temperature. The mixture was stirred

at room temperature for 24 h. After evaporation and addition of

water, the mixture was extracted with EtOAc. The organic layer was

separated, dried over MgSO₄, and concentrated in vacuo to give the

title compound (39.2 g, 158 mmol, 97%) as a pale-yellow solid. This

1

product was used without further puriﬁcation. H NMR (300 MHz,

CDCl3): δ 1.81 (6H, s), 5.36 (1H, s), 7.00−7.15 (1H, m), 7.29−7.41

(1H, m), 7.43−7.52 (1H, m), 7.73−7.80 (1H, m), 8.36 (1H, s). MS

(ESI/APCI): m/z not detected. HPLC 97.6% (LC−MS).

1

a white solid (washed with hex/AcOEt = 6:1). H NMR (400 MHz,

DMSO-d₆): δ ppm 0.11−0.21 (1H, m), 0.22−0.40 (2H, m), 0.46−

0.57 (1H, m), 0.77 (9H, s), 0.92−1.07 (1H, m), 1.08−1.32 (1H, m),

1.40−1.84 (4H, m), 2.18−2.28 (1H, m), 2.31−2.66 (3H, m), 2.36

(3H, s), 2.68 (2H, d, J = 7.2 Hz), 3.21−3.32 (1H, m), 3.69 (3H, s),

3.91−4.27 (4H, m), 6.21 (1H, s), 6.45 (1H, br s), 6.52 (1H, d, J = 8.8

Hz), 6.71 (1H, s), 6.85 (1H, d, J = 7.2 Hz), 6.91 (1H, d, J = 5.3 Hz),

7.14 (1H, d, J = 8.4 Hz), 7.28 (1H, t, J = 7.5 Hz), 8.04 (1H, d, J = 5.3

Hz), 12.08 (1H, br s). ¹³C NMR (75 MHz, CDCl₃): δ 4.2, 5.3, 16.6,

24.3, 28.4, 28.8, 34.1, 35.6, 40.2, 46.1, 55.2, 55.8, 70.6, 104.6, 106.0,

109.5, 116.1, 117.9, 119.1, 124.6, 131.3, 136.0, 146.7, 151.4, 155.7,

5-(Cyclopropyl(3-hydroxyphenyl)methyl)-2,2-dimethyl-1,3-diox-

ane-4,6-dione (10). To a solution of compound 9 (26.1 g, 105.1

mmol) in THF (dry) (780 mL) was added cyclopropylmagnesium

bromide (900 mL, 630 mmol) at −10−0 °C. The mixture was stirred

at room temperature for 1.5 h. After addition of 1 N HCl aq (1050

mL), EtOAc (270 mL) was added. The organic layer was separated,

washed with brine, dried over MgSO₄, and concentrated in vacuo.

The residue was puriﬁed by column chromatography (silica gel, eluted

with 0−50% EtOAc in hexane) to give the title compound (28.5 g, 98

mmol, 93%) as a colorless oil. ¹H NMR (300 MHz, CDCl_s): δ 0.16−

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0.28 (1H, m), 0.32−0.46 (1H, m), 0.58−0.79 (2H, m), 1.32 (3H, s),

1.68 (3H, s), 1.85−2.04 (1H, m), 2.80−2.89 (1H, m), 3.86 (1H, d, J

= 3.0 Hz), 5.14 (1H, br s), 6.75 (1H, d, J = 0.8 Hz), 6.84−6.94 (2H,

m), 7.10−7.22 (1H, m). MS (ESI/APCI): m/z 291.1 [M + H]⁺.

HPLC 95.6% (LC−MS).

puriﬁcation. ¹H NMR (300 MHz, DMSO-d₆): δ ppm 1.10−1.17 (3H,

m), 2.53−2.71 (2H, m), 3.09 (3H, s), 3.98−4.09 (2H, m), 4.46 (1H,

dd, J = 8.7, 5.2 Hz), 6.63−6.77 (3H, m), 7.14 (1H, td, J = 7.5, 0.8

Hz), 9.40 (1H, s). MS (ESI/APCI) not detected. HPLC 94.9% (LC−

MS).

Methyl 3-Cyclopropyl-3-(3-hydroxyphenyl)propanoate (11a).

H₂SO₄(2.56 mL, 48.02 mmol) was added to a solution of 3-

cyclopropyl-3-(3-hydroxyphenyl)propanoic acid (99 g, 480.03 mmol)

in MeOH (990 mL) at room temperature. The mixture was stirred at

64 °C for 2 h. The mixture was quenched with water and NaCl at

room temperature and extracted with EtOAc. The organic layer was

separated, washed with sat. NaHCO₃aq and brine, dried over

Na₂SO₄, and concentrated in vacuo. The residue was puriﬁed by

column chromatography (silica gel, eluted with 25% EtOAc in

hexane) to give methyl 3-cyclopropyl-3-(3-hydroxyphenyl)propanoate

Ethyl 3-Ethoxy-3-(3-hydroxyphenyl)propanoate (14b). Com-

pound 14b was prepared from compound 13 (2.5 g, 8.32 mmol)

and iodoethane (5.19 g, 33.29 mmol) in a manner similar to that

described for compound 14a. Colorless oil (0.660 g, 2.77 mmol,

1

77%). H NMR (300 MHz, DMSO-d₆): δ ppm 1.05 (3H, t, J = 7.0

Hz), 1.10−1.19 (3H, m), 2.53−2.68 (2H, m), 3.19−3.34 (2H, m),

3.95−4.13 (2H, m), 4.57 (1H, dd, J = 8.5, 5.5 Hz), 6.62−6.76 (3H,

m), 7.08−7.17 (1H, m), 9.38 (1H, s). MS (ESI/APCI) not detected.

HPLC 95.5% (LC−MS).

2-Methoxy-1-(3-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)-

ethanone (16). To a solution of methyl 2-methoxyacetate 15 (3.79 g,

36.36 mmol) in THF (dry) (50 mL) was added (3-((tetrahydro-2H-

pyran-2-yl)oxy)phenyl)magnesium bromide (80 mL, 40.00 mmol)

dropwise at −78 °C and stirred for 1 h. To the mixture was added

methyl 2-methoxyacetate (3.79 g, 36.36 mmol) and stirred at r.t. for 3

h. The mixture was poured into water and insoluble materials were

removed by ﬁltration and extracted with EtOAc. The organic layer

was separated, washed with brine, dried over MgSO₄, and

concentrated in vacuo to give the title compound (9.00 g, 36.0

mmol, 99%) as a brown oil. This product was used without further

puriﬁcation.

(Z)-Ethyl 3-(3-Hydroxyphenyl)-4-methoxybut-2-enoate (17). To

a suspension of NaH (2.88 g, 71.92 mmol) in THF (100 mL) was

added ethyl 2-(diethoxyphosphoryl)acetate (16.12 g, 71.92 mmol) at

0 °C and stirred for 30 min. To the mixture was added compound 16

(9 g, 35.96 mmol) and stirred at 50 °C for 15 h. The reaction mixture

was quenched with sat. NH₄Cl aq and extracted with EtOAc. The

organic layer was separated, washed with brine, dried over MgSO₄,

and concentrated in vacuo to give (Z)-ethyl 4-methoxy-3-(3-

((tetrahydro-2H-pyran-2-yl)oxy)phenyl)but-2-enoate (11.00 g, 34.3

mmol) as a brown oil. The obtained product was dissolved in AcOH

(50 mL). Zinc (22.45 g, 343.34 mmol) was added portionwise thereto

at room temperature. The mixture was stirred at 50 °C for 3 h. The

reaction mixture was ﬁltered through Celite pad, and the ﬁltrate was

concentrated in vacuo. The residue was dissolved in 1 N HCl and

washed with EtOAc. The aqueous layer was basiﬁed with NaHCO₃

and extracted with AcOEt, washed with sat. NaHCO₃aq and brine,

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

puriﬁed by column chromatography (silica gel, eluted with 0−25%

EtOAc in hexane) to give the title compound (0.390 g, 1.651 mmol,

4.8%, over 3 steps) as a pale-yellow oil. ¹H NMR (400 MHz, CDCl₃):

δ ppm 1.20 (3H, t, J = 7.2 Hz), 3.49 (2H, s), 3.72 (3H, s), 4.12 (2H,

q, J = 7.1 Hz), 5.02 (1H, br s), 6.52 (1H, s), 6.66 (1H, dd, J = 8.0, 2.0

Hz), 6.75 (1H, t, J = 1.9 Hz), 6.84 (1H, d, J = 7.7 Hz), 7.14 (1H, t, J =

7.9 Hz). MS (ESI/APCI): m/z not detected. HPLC 93.1% (LC−

MS).

1

(84 g, 381 mmol, 79%) as a pale-yellow oil. H NMR (400 MHz,

DMSO-d₆): δ 0.04−0.14 (1H, m), 0.14−0.23 (1H, m), 0.27−0.37

(1H, m), 0.44−0.53 (1H, m), 0.92−1.04 (1H, m), 2.13−2.23 (1H,

m), 2.60−2.76 (2H, m), 3.51 (3H, s), 6.58 (1H, d, J = 8.0 Hz), 6.63

(1H, s), 6.66 (1H, d, J = 7.7 Hz), 7.06 (1H, t, J = 7.7 Hz), 9.24 (1H,

s).

Ethyl 3-Cyclopropyl-3-(3-hydroxyphenyl)propanoate (11b). A

solution of compound 10 (62.34 g, 214.74 mmol) in EtOH (63

mL) and DMF (126 mL) was stirred at 100 °C for 7 h. The resulting

mixture was concentrated in vacuo and diluted with ethyl acetate. The

mixture was washed with brine, dried over Na₂SO₄, and concentrated

in vacuo to yield a brown oil. The oil was puriﬁed by column

chromatography (silica gel, eluted with 10−25% EtOAc in hexane) to

give the title compound (27.0 g, 115 mmol, 54%) as a pale-yellow oil.

¹H NMR (400 MHz, CDCl₃): δ 0.14 (1H, dq, J = 9.5, 4.9 Hz), 0.26

(1H, dq, J = 9.4, 4.8 Hz), 0.37−0.47 (1H, m), 0.52−0.61 (1H, m),

0.94−1.05 (1H, m), 1.17 (3H, t, J = 7.2 Hz), 1.67 (1H, s), 2.26−2.36

(1H, m), 2.59−2.79 (2H, m), 3.99−4.10 (2H, m), 5.22 (1H, s), 6.68

(1H, dd, J = 8.0, 1.6 Hz), 6.71 (1H, s), 6.79 (1H, d, J = 7.5 Hz), 7.16

(1H, t, J = 7.8 Hz).

Ethyl 3-(3-(Benzyloxy)phenyl)-3-hydroxypropanoate (13). To a

solution of diisopropylamine (4.32 g, 42.7 mmol) in THF (dry) (100

mL) was added butyllithium (12.07 mL, 19.32 mmol) dropwise at

−78 °C and stirred for 15 min. To the mixture was added ethyl

acetate (3.44 g, 39.1 mmol) and stirred for 1 h and then 3-

(benzyloxy)benzaldehyde 12 (4.1 g, 19.32 mmol) was added. The

mixture was stirred for 1h and quenched with sat. NH₄Cl aq and

extracted with EtOAc. The organic layer was separated, washed with

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

puriﬁed by column chromatography (silica gel, eluted with 0−25%

EtOAc in hexane) to give the title compound (5.05 g, 16.81 mmol,

87%) as a yellow oil. ¹H NMR (400 MHz, CDCl₃): δ ppm 1.27 (3H,

t, J = 7.2 Hz), 2.63−2.81 (2H, m), 3.24 (1H, d, J = 3.4 Hz), 4.19 (2H,

q, J = 7.2 Hz), 5.07 (2H, s), 5.11 (1H, dt, J = 8.0, 3.8 Hz), 6.90 (1H,

dd, J = 8.2, 1.8 Hz), 6.96 (1H, d, J = 7.5 Hz), 7.05 (1H, s), 7.22−7.49

(6H, m). MS (ESI/APCI): m/z 299.1 [M − H]⁻. HPLC 95.9%

(LC−MS).

Ethyl 3-(3-Hydroxyphenyl)-4-methoxybutanoate (18). A mixture

of compound 17 (390 mg, 1.65 mmol) and 10% Pd−C (50 mg, 0.47

mmol) in EtOH (5 mL) was hydrogenated under balloon pressure at

room temperature overnight. The catalyst was removed by ﬁltration,

and the ﬁltrate was concentrated in vacuo to give the title compound

(380 mg, 1.595 mmol, 97%) as a colorless oil. This product was used

Ethyl 3-(3-Hydroxyphenyl)-3-methoxypropanoate (14a). A mix-

ture of compound 13 (2.5 g, 8.32 mmol), iodomethane (4.73 g, 33.29

mmol), and silver(I) oxide (3.86 g, 16.65 mmol) in toluene (50 mL)

was stirred at 100 °C under N₂for 15 h. The insoluble material was

removed by ﬁltration, and the ﬁltrate was concentrated in vacuo. The

residue was puriﬁed by column chromatography (silica gel, eluted

with 0−10% EtOAc in hexane) to give the corresponding methyl

1

without further puriﬁcation. H NMR (300 MHz, CDCl₃): δ ppm

1.17 (3H, t, J = 7.1 Hz), 2.51−2.63 (1H, m), 2.71−2.86 (1H, m),

3.32 (3H, s), 3.34−3.59 (3H, m), 4.00−4.11 (2H, m), 4.93 (1H, br

s), 6.62−6.73 (2H, m), 6.75−6.83 (1H, m), 7.10−7.23 (1H, m). MS

(ESI/APCI): m/z 237.1 [M-H]^‑

Ethyl 2-(Oxetan-3-ylidene)acetate (20). (Carbethoxymethylene)-

triphenylphosphorane (3.83 g, 11.00 mmol) was added to a solution

of oxetan-3-one 19 (0.721 g, 10 mmol) in THF (dry) (20 mL) at 0

°C. The mixture was stirred at room temperature under N₂overnight.

To the reaction mixture were added silica gel and hexane, and the

precipitate was removed by ﬁltration. The ﬁltrate was concentrated in

vacuo to give the title compound (1.09 g, 7.21 mmol, 72%) as a

colorless oil. This product was subjected to the next reaction without

1

ether (1.810 g, 5.76 mmol, 69%) as a colorless oil. H NMR (300

MHz, CDCl₃): δ ppm 1.18−1.27 (3H, m), 2.56 (1H, dd, J = 15.3, 4.6

Hz), 2.77 (1H, dd, J = 15.3, 9.2 Hz), 3.22 (3H, s), 4.09−4.25 (2H,

m), 4.61 (1H, dd, J = 9.2, 4.6 Hz), 5.07 (2H, s), 6.86−6.95 (2H, m),

6.95−7.01 (1H, m), 7.18−7.49 (6H, m). MS (ESI/APCI): m/z not

detected. A mixture of the obtained compound and 10% Pd−C (150

mg, 1.41 mmol) in EtOH (10 mL) and THF (10 mL) was

hydrogenated under balloon pressure at room temperature overnight.

The catalyst was removed by ﬁltration, and the ﬁltrate was

concentrated in vacuo to give the title compound (0.900 g, 4.01

mmol, 69%) as a colorless oil. This product was used without further

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further puriﬁcation. ¹H NMR (300 MHz, DMSO-d₆): δ 1.19 (3H, t, J

= 7.0 Hz), 4.08 (2H, q, J = 7.2 Hz), 5.20−5.28 (2H, m), 5.33−5.41

(2H, m), 5.75 (1H, quin, J = 2.4 Hz).

hexane) to give the title compound (1.13 g, 3.62 mmol, 56%) as a

colorless oil. ¹H NMR (400 MHz, DMSO-d₆): δ 0.75 (9H, s), 0.93−

1.03 (2H, m), 1.25−1.36 (2H, m), 2.43 (2H, t, J = 7.7 Hz), 3.74 (3H,

s), 4.54 (2H, d, J = 5.6 Hz), 5.19 (1H, t, J = 5.7 Hz), 6.67 (1H, d, J =

2.6 Hz), 6.86 (1H, dd, J = 8.5, 2.6 Hz), 7.17−7.28 (3H, m), 7.36 (2H,

d, J = 7.8 Hz). MS (ESI/APCI): m/z 295.2. HPLC 99.2% (LC−MS).

2-(4-Hydroxypiperidin-1-yl)-4-methoxybenzaldehyde (28). A

mixture of 2-ﬂuoro-4-methoxybenzaldehyde 27 (9.83 g, 63.8

mmol), piperidin-4-ol (4.3 g, 42.5 mmol), Cs₂CO₃(27.7 g, 85.0

mmol), and tetrabutylammonium iodide (TBAI) (1.57 g, 4.25 mmol)

in DMF (dry) (100 mL) was stirred at 110 °C for 15 h. The mixture

was poured into water and extracted with EtOAc. The organic layer

was separated, washed with brine, dried over MgSO₄, and

concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, eluted with 0−50% EtOAc in hexane)

to give the title compound Cs₂CO₃(4.24 g, 18.02 mmol, 42%) as a

yellow oil. ¹H NMR (400 MHz, CDCl₃): δ 1.55 (1H, d, J = 4.1 Hz),

1.69−1.86 (2H, m), 2.01−2.11 (2H, m), 2.85−3.01 (2H, m), 3.21−

3.41 (2H, m), 3.86 (3H, s), 3.91 (1H, td, J = 8.1, 4.2 Hz), 6.56 (1H,

d, J = 2.3 Hz), 6.62 (1H, dd, J = 8.6, 2.1 Hz), 7.79 (1H, d, J = 8.7 Hz),

10.13 (1H, s).

Ethyl 2-(3-(3-(Benzyloxy)phenyl)oxetan-3-yl)acetate (21). To a

solution of chloro(1,5-cyclooctadiene)rhodium(I), dimer (73.5 mg,

0.15 mmol) in dioxane (5 mL) was added KOH (3.73 mL, 5.59

mmol), and the yellow solution was stirred for 15 min. Then, a

mixture of (3-(benzyloxy)phenyl)boronic acid (1701 mg, 7.46 mmol)

and compound 20 (530 mg, 3.73 mmol) in dioxane (5 mL) was

slowly added, and the color of the solution turned to orange. After the

mixture was stirred for 30 min at room temperature, the reaction was

quenched by the addition of brine (40 mL). The aqueous phase was

extracted two times with AcOEt. The combined organic phases were

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

was puriﬁed by ﬂash chromatography (silica gel, eluted with 0−25%

EtOAc in hexane) to give the title compound (1.3 g) as a slightly

1

yellow oil. This product was used without further puriﬁcation. H

NMR (400 MHz, CDCl₃): δ ppm 1.13 (3H, t, J = 7.2 Hz), 3.09 (2H,

s), 3.97−4.05 (2H, m), 4.81−4.87 (2H, m), 4.99 (2H, d, J = 6.3 Hz),

5.05 (2H, s), 6.72−6.77 (1H, m), 6.78−6.81 (1H, m), 6.86 (1H, ddd,

J = 8.3, 2.5, 0.7 Hz), 7.22−7.28 (1H, m), 7.29−7.47 (5H, m).

Ethyl 2-(3-(3-Hydroxyphenyl)oxetan-3-yl)acetate (22). Com-

pound 22 was prepared from compound 21 (1.3 g, 3.98 mmol) in

a manner similar to that described for compound 14a. Colorless oil

1-(2-(4,4-Dimethylpentyl)-5-methoxyphenyl)piperidin-4-ol (29).

Compound 29 was prepared from compound 28 in a manner similar

to that described for compound 25. Colorless oil (220 mg, 0.720

1

(0.818 g, 3.46 mmol, 87%). H NMR (300 MHz, CDCl₃): δ ppm

mmol, 73%). H NMR (400 MHz, CDCl₃): δ ppm 0.87 (9H, s),

1.13 (3H, t, J = 7.1 Hz), 3.10 (2H, s), 4.02 (2H, q, J = 7.2 Hz), 4.86

(2H, d, J = 6.3 Hz), 5.00 (2H, d, J = 6.2 Hz), 5.42 (1H, s), 6.63−6.70

(2H, m), 6.72 (1H, d, J = 2.1 Hz), 7.20 (1H, t, J = 7.9 Hz).

1.19−1.31 (2H, m), 1.47−1.62 (2H, m), 1.65−1.81 (2H, m), 2.00

(2H, d, J = 9.7 Hz), 2.53 (2H, t, J = 7.9 Hz), 2.64−2.77 (2H, m),

2.99−3.14 (2H, m), 3.78 (3H, s), 3.79−3.88 (1H, m), 6.58 (1H, dd, J

= 8.3, 2.4 Hz), 6.64 (1H, d, J = 2.5 Hz), 7.10 (1H, d, J = 8.3 Hz).

2-(4-(Hydroxymethyl)piperidin-1-yl)-4-methoxybenzaldehyde

(30). Compound 30 was prepared from 27 in a manner similar to that

described for compound 28. Light-yellow oil (12.6 g, 50.4 mmol,

78%). ¹H NMR (300 MHz, CDCl₃): δ 1.40 (1H, t, J = 5.5 Hz), 1.42−

1.56 (2H, m), 1.58−1.75 (1H, m), 1.87 (2H, dd, J = 12.5, 1.8 Hz),

2.79−2.90 (3H, m), 3.36 (2H, d, J = 12.4 Hz), 3.59 (2H, t, J = 5.8

Hz), 3.86 (3H, s), 6.50−6.64 (2H, m), 7.79 (1H, d, J = 8.6 Hz), 10.12

(1H, d, J = 0.6 Hz). MS (ESI/APCI): m/z 250.1 [M + H]⁺. HPLC

95.3% (LC−MS).

Methyl 2′-Formyl-5′-methoxy-[1,1′-biphenyl]-4-carboxylate

(24). A mixture of 2-bromo-4-methoxybenzaldehyde 23 (1 g, 4.65

mmol), (4-(methoxycarbonyl)phenyl)boronic acid (1.26 g, 6.98

mmol), Pd(Ph₃P)₄(0.269 g, 0.23 mmol), and 2 M Na₂CO₃(4.65

mL, 9.30 mmol) in dimethyl ether (DME) (13 mL) was heated at

130 °C for 1 h under microwave irradiation under N₂. The mixture

was poured into water, and the aqueous phase was extracted with

EtOAc twice. The combined organic layers were washed with water

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

was puriﬁed by column chromatography (silica gel, eluted with 3−

20% EtOAc in hexane) to give the title compound(1.25 g, 4.62 mmol,

(1-(2-(4,4-Dimethylpentyl)-5-methoxyphenyl)piperidin-4-yl)-

methanol (31). A mixture of diisopropylamine (74.08 g, 173.49

mmol) in dried THF (500 mL) was cooled to −78 °C. Then, to the

mixture was dropwise added n-BuLi (130.1 mL, 2.5 N, 325.30 mmol)

at −78 °C. The mixture was stirred at −78 °C for 30 min. Then, to

the mixture was dropwise added a solution of 30 (27 g, 108.43 mmol)

in THF (100 mL) at −78 °C and the mixture was stirred at −78 °C

for 1 h. The reaction mixture was slowly poured into ice water (500

mL) and extracted with ethyl acetate (1000 mL). The organic phase

was washed with brine, dried over Na₂SO₄, and concentrated in

vacuo, and the residue was puriﬁed by silica gel column

chromatography (petroleum ether/ethyl acetate, from 20/1 to 5/1)

to give the oleﬁn product (18.5 g, yield: 53%) as an oﬀ-white solid. ¹H

NMR (CDCl₃, 500 MHz): δ 0.94−0.96 (9H, m), 1.42−1.48 (2H, m),

1.62−1.69 (1H, m), 1.84 (2H, d, J = 10.5 Hz), 2.11−2.26 (2H, m),

2.61−2.65 (2H, m), 3.32−3.38 (2H, m), 3.59−3.62 (2H, m), 3.79

(3H, s), 5.68−6.12 (1H, m), 6.49−6.58 (3H, m), 7.18−7.38 (1H, m).

An OH peak was not observed. MS (ESI/APCI): m/z 318.3 [M +

H]⁺. The solution of the obtained product (22.2 g, 69.9 mmol) and

Pd−C (1 g, 0.94 mmol) in EtOH (200 mL) was stirred at r.t. for 21.5

h under a H₂atmosphere. The reaction mixture was ﬁltered with

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

chromatography (silica gel, eluted with 0−70% EtOAc in hexane) to

give the title compound (21.90 g, 68.5 mmol, 98%) as a pale-yellow

1

99%) as a white solid. H NMR (300 MHz, CDCl₃): δ 3.92 (3H, s),

3.97 (3H, s), 6.87 (1H, d, J = 2.5 Hz), 7.04 (1H, ddd, J = 8.8, 2.5, 0.8

Hz), 7.43−7.51 (2H, m), 8.04 (1H, d, J = 8.8 Hz), 8.10−8.17 (2H,

m), 9.81 (1H, d, J = 0.8 Hz). MS (ESI/APCI): m/z 271.1 [M + H]⁺.

HPLC 95.8% (LC−MS).

Methyl 2′-(4,4-Dimethylpentyl)-5′-methoxy-[1,1′-biphenyl]-4-

carboxylate (25). To a solution of (3,3-dimethylbutyl)-

triphenylphosphate(III), MsOH (16.41 g, 37.00 mmol) in THF

(dry) (100 mL) was added potassium 2-methyl-propan-2-olate (8.30

g, 74.00 mmol) and stirred for 30 min. To the reaction mixture was

added compound 24 (2 g, 7.40 mmol) and stirred at 50 °C for 10

min. The mixture was poured into water and extracted with EtOAc.

The organic layer was separated, washed with brine, dried over

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

column chromatography (silica gel, eluted with 0−10% EtOAc in

hexane) to give the corresponding oleﬁn (5.5 g) as a colorless oil. MS

(ESI/APCI): m/z 339.2 [M + H]⁺. The title compound was reduced

in a manner similar to that described for compound 18. Colorless oil

(2.200 g, 6.46 mmol, 40%). ¹H NMR (300 MHz, DMSO-d₆): δ

0.67−0.76 (9H, m), 0.91−1.02 (2H, m), 1.20−1.30 (2H, m), 1.57

(3H, s), 2.43 (2H, t, J = 7.7 Hz), 3.75 (3H, s), 6.66−6.74 (1H, m),

6.91 (1H, dd, J = 8.5, 2.8 Hz), 7.24 (1H, d, J = 8.5 Hz), 7.41−7.50

(2H, m), 7.90−8.03 (2H, m).

(2′-(4,4-Dimethylpentyl)-5′-methoxy-[1,1′-biphenyl]-4-yl)-

methanol (26). To a solution of compound 25 (2.2 g, 6.46 mmol) in

THF (20 mL) was added LiAlH₄(0.270 g, 7.11 mmol) portionwise at

0 °C and stirred for 30 min. To the reaction mixture were added H₂O

(0.33 mL), 15% NaOH (0.33 mL), and H₂O (0.99 mL) and stirred

for 1 h. The insoluble material was removed by ﬁltration, and the

ﬁltrate was concentrated in vacuo. The residue was puriﬁed by

column chromatography (silica gel, eluted with 0−15% EtOAc in

1

oil. H NMR (300 MHz, DMSO-d₆): δ 0.76−0.91 (9H, m), 1.10−

1.61 (7H, m), 1.74 (2H, d, J = 10.1 Hz), 2.38−2.68 (4H, m), 2.95

(2H, d, J = 11.8 Hz), 3.23−3.39 (2H, m), 3.70 (3H, s), 4.45 (1H, t, J

= 5.3 Hz), 6.48−6.66 (2H, m), 7.05 (1H, d, J = 8.3 Hz). MS (ESI/

APCI): m/z 320.2 [M + H]⁺. HPLC 94.1% (LC−MS).

Methyl 1-(2-Formyl-5-methoxyphenyl)azetidine-3-carboxylate

(32). The title compound was prepared from compound 27 (1.65

g, 10.7 mmol) and methyl azetidine-3-carboxylate hydrochloride

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(1.48 g, 9.76 mmol) in DMSO (30 mL) in a manner similar to that

described for compound 28. Yellow oil (1.00 g, 4.02 mmol, 41%). ¹H

NMR (300 MHz, CDCl₃): δ 3.53−3.64 (1H, m), 3.75 (3H, s), 3.85

(3H, s), 4.13−4.19 (2H, m), 4.28−4.36 (2H, m), 5.94 (1H, d, J = 2.3

Hz), 6.44 (1H, dd, J = 8.8, 2.3 Hz), 7.62 (1H, d, J = 8.8 Hz), 9.80

(1H, s). MS (ESI/APCI): m/z 250.1 [M + H]⁺. HPLC 100% (LC−

MS).

(1-(2-(4,4-Dimethylpentyl)-5-methoxyphenyl)azetidin-3-yl)-

methanol (33). Compound 33 was prepared from compound 32

(300 mg, 1.20 mmol) in a manner similar to that described for

compound 24 and 25 (puriﬁcation: silica gel, eluted with 0−30%

EtOAc in hexane). Colorless oil (18.3 mg, 0.063 mmol, 5% over 2

steps). ¹H NMR (300 MHz, CDCl₃): δ 0.88 (9H, s), 1.20−1.27 (2H,

m), 1.46−1.59 (2H, m), 1.63 (1H, t, J = 4.9 Hz), 2.39−2.47 (2H, m),

2.70−2.84 (1H, m), 3.68 (2H, dd, J = 7.2, 4.8 Hz), 3.77 (3H, s),

3.86−3.92 (2H, m), 3.98 (2H, t, J = 7.5 Hz), 6.06 (1H, d, J = 2.5 Hz),

6.33 (1H, dd, J = 8.3, 2.5 Hz), 6.96 (1H, d, J = 8.3 Hz). MS (ESI/

APCI): m/z 292.2 [M + H]⁺. HPLC 100% (LC−MS).

J = 12.3, 2.0 Hz), 2.21 (3H, s), 2.61 (2H, td, J = 11.7, 2.3 Hz), 3.11−

3.22 (2H, m), 3.57 (2H, d, J = 1.9 Hz), 3.78 (3H, s), 6.51 (1H, dd, J =

8.2, 2.6 Hz), 6.59 (1H, d, J = 2.5 Hz), 7.06 (1H, dd, J = 8.3, 0.4 Hz).

1-(2-(4-(Hydroxymethyl)piperidin-1-yl)-4-methoxyphenyl)-

ethanone (38). Compound 38 was prepared from compound 37

(5.01 g, 29.77 mmol) in a manner similar to that described for

compound 28. Yellow solid (6.59 g, 25.0 mmol, 84%). ¹H NMR (300

MHz, CDCl₃): δ 1.33−1.54 (3H, m), 1.57−1.71 (1H, m), 1.79−1.89

(2H, m), 2.62 (3H, s), 2.73 (2H, ddd, J = 12.0, 2.3 Hz), 3.22−3.31

(2H, m), 3.58 (2H, dd, J = 5.7 Hz), 3.83 (3H, s), 6.50−6.58 (2H, m),

7.48−7.53 (1H, m). MS (ESI/APCI): m/z 264.1 [M + H]⁺. HPLC

100% (LC−MS).

1-(4-Methoxy-2-(4-((methoxymethoxy)methyl)piperidin-1-yl)-

phenyl)ethanone (39). To a solution of compound 38 (6.59 g, 25.0

mmol) and N,N′-diisopropylethylamine (17.4 mL, 100.11 mmol) in

THF (60 mL) was added chloromethyl methyl ether (5.70 mL, 75.1

mmol) at room temperature. The mixture was stirred at the same

temperature overnight. The mixture was poured into water and

extracted with EtOAc. The organic layer was separated, washed with

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

puriﬁed by column chromatography (silica gel, eluted with 5−30%

EtOAc in hexane) to give the title compound (8.08 g, 26.3 mmol,

Ethyl 1-(3-Methoxyphenyl)piperidine-4-carboxylate (35b). To a

mixture of ethyl isonipecotate (0.978 mL, 6.36 mmol), m-

bromoanisole (34b, 0.797 mL, 6.36 mmol), BINAP (0.436 g, 0.70

mmol), and Cs₂CO₃(6.22 g, 19.08 mmol) was added Pd(OAc)₂

(0.143 g, 0.64 mmol). The mixture was stirred at 100 °C for 24 h.

The mixture was quenched with water and extracted with EtOAc. The

organic layer was separated, washed with water and brine successively,

dried over Na₂SO₄, and concentrated in vacuo. The residue was

puriﬁed by column chromatography (silica gel, eluted with 0−30%

EtOAc in hexane) to give the title compound (1.33 g, 5.05 mmol,

79%) as a pale-yellow oil. ¹H NMR (300 MHz, CDCl₃): δ 1.27 (3H,

t, J = 7.1 Hz), 1.75−1.95 (2H, m), 1.95−2.07 (2H, m), 2.34−2.49

(1H, m), 2.72−2.86 (2H, m), 3.64 (2H, dt, J = 12.7, 3.3 Hz), 3.79

(3H, s), 4.15 (2H, q, J = 7.2 Hz), 6.35−6.45 (1H, m), 6.47 (1H, t, J =

2.3 Hz), 6.55 (1H, dd, J = 8.0, 2.0 Hz), 7.16 (1H, t, J = 8.2 Hz). MS

(ESI/APCI): m/z 264.2 [M + H]⁺. HPLC 95.3% (LC−MS).

Ethyl 1-(2-Fluoro-5-methoxyphenyl)piperidine-4-carboxylate

(35c). Compound 35c was prepared from compound 34c (1.0 g,

4.88 mmol) and ethyl piperidine-4-carboxylate (1.15 g, 7.32 mmol) in

a manner similar to that described for compound 35c. Pale-yellow oil

1

quant.) as a yellow oil. H NMR (300 MHz, CDCl₃): δ 1.40−1.54

(2H, m), 1.64−1.90 (3H, m), 2.63 (3H, s), 2.73 (2H, ddd, J = 12.1,

2.4 Hz), 3.21−3.30 (2H, m), 3.37 (3H, s), 3.44 (2H, d, J = 6.3 Hz),

3.83 (3H, s), 4.63 (2H, s), 6.50−6.58 (2H, m), 7.50 (1H, d, J = 8.6

Hz). MS (ESI/APCI): m/z 308.1 [M + H]⁺. HPLC 100% (LC−MS).

1-(2-Isopropyl-5-methoxyphenyl)-4-((methoxymethoxy)methyl)-

piperidine (40). Compound 40 was prepared from compound 39

(353 mg, 1.15 mmol) in a manner similar to that described for

compound 25. Colorless oil (259 mg, 0.841 mmol, 93%). This

product was used without further puriﬁcation. MS (ESI/APCI): m/z

308.2 [M + H]⁺. HPLC 92.2% (LC−MS).

Ethyl 3-Cyclopropyl-3-(3-((1-(2-isopropyl-5-methoxyphenyl)-

piperidin-4-yl)methoxy)phenyl)propanoate (41). HCl (0.701 mL,

8.41 mmol) was added to a solution of compound 40 (259 mg, 0.84

mmol) in MeOH (3 mL) at room temperature. The mixture was

stirred at 60 °C for 1 h. The mixture was neutralized with sat.

NaHCO₃aq at r.t. and extracted with EtOAc. The organic layer was

separated, washed with water and brine, dried over MgSO₄, and

concentrated in vacuo. The residue and compound 11b (256 mg, 1.09

mmol) were dissolved in toluene (3 mL). To the solution was added

2-(tributylphosphoranylidene)acetonitrile (0.441 mL, 1.68 mmol) at

r.t.. The mixture was stirred at 100 °C under N₂for 2 h. The reaction

mixture was concentrated in vacuo. The residue was puriﬁed by

column chromatography (silica gel, eluted with 2−5% EtOAc in

hexane) to give the title compound (324 mg, 0.675 mmol, 80%) as a

1

(916 mg, 3.26 mmol, 67%). H NMR (300 MHz, CDCl₃): δ 1.27

(3H, t, J = 7.2 Hz), 1.86−2.08 (4H, m), 2.35−2.49 (1H, m), 2.72

(2H, td, J = 11.4, 3.0 Hz), 3.43 (2H, dt, J = 11.9, 3.1 Hz), 3.76 (3H,

s), 4.16 (2H, q, J = 7.1 Hz), 6.40 (1H, dt, J = 8.7, 3.2 Hz), 6.49 (1H,

dd, J = 7.2, 3.0 Hz), 6.92 (1H, dd, J = 12.1, 8.7 Hz). HPLC 96.6%

(LC−MS).

(1-(3-Methoxyphenyl)piperidin-4-yl)methanol (36b). Compound

36b was prepared from compound 35b (325 mg, 1.23 mmol) in a

manner similar to that described for compound 26. Colorless oil (306

mg, 1.383 mmol, quant). ¹H NMR (300 MHz, CDCl₃): δ 0.88 (1H, t,

J = 1.0 Hz), 1.23−1.47 (2H, m), 1.61−1.74 (1H, m), 1.85 (2H, dt, J =

6.9, 3.2 Hz), 2.72 (2H, td, J = 12.3, 2.7 Hz), 3.55 (2H, t, J = 6.1 Hz),

3.66−3.76 (2H, m), 3.79 (3H, s), 6.39 (1H, dd, J = 7.6, 2.3 Hz), 6.49

(1H, t, J = 2.5 Hz), 6.56 (1H, dd, J = 8.0, 1.9 Hz), 7.16 (1H, t, J = 8.1

Hz).

(1-(2-Fluoro-5-methoxyphenyl)piperidin-4-yl)methanol (36c).

Compound 36c was prepared from compound 35c (400 mg) in a

manner similar to that described for compound 26 (puriﬁcation:

column chromatography (silica gel, eluted with 20−60% EtOAc in

hexane)). Colorless oil (354 mg, 1.479 mmol, quant). This product

was used without further puriﬁcation. ¹H NMR (300 MHz, CDCl₃): δ

1.33 (1H, t, J = 5.5 Hz), 1.39−1.54 (2H, m), 1.58−1.75 (1H, m),

1.85 (2H, dd, J = 12.7, 1.7 Hz), 2.65 (2H, td, J = 11.8, 2.1 Hz), 3.48

(2H, d, J = 12.1 Hz), 3.56 (2H, t, J = 5.9 Hz), 3.77 (3H, s), 6.40 (1H,

dt, J = 8.8, 3.2 Hz), 6.51 (1H, dd, J = 7.2, 3.0 Hz), 6.92 (1H, dd, J =

12.3, 8.9 Hz).

1

colorless oil. H NMR (300 MHz, CDCl₃): δ 0.10−0.21 (1H, m),

0.28 (1H, dq, J = 9.3, 4.7 Hz), 0.37−0.49 (1H, m), 0.52−0.63 (1H,

m), 0.95−1.10 (1H, m), 1.14−1.21 (9H, m), 1.46−1.65 (2H, m),

1.85−1.99 (3H, m), 2.35 (1H, dt, J = 9.6, 7.6 Hz), 2.63−2.81 (4H,

m), 3.07 (2H, d, J = 11.6 Hz), 3.29−3.46 (1H, m), 3.78 (3H, s), 3.86

(2H, d, J = 5.9 Hz), 4.00−4.11 (2H, m), 6.59−6.70 (2H, m), 6.74−

6.85 (3H, m), 7.13−7.25 (2H, m). MS (ESI/APCI): m/z 480.3 [M +

H]⁺. HPLC 90.4% (LC−MS).

2-(4-(((tert-Butyldimethylsilyl)oxy)methyl)piperidin-1-yl)-4-me-

thoxybenzaldehyde (42). To a solution of compound 30 (11.1 g,

44.52 mmol) in DMF (25 mL) were added 1H-imidazole (6.06 g,

89.05 mmol) and tert-butylchlorodimethylsilane (7.38 g, 48.98

mmol). The reaction mixture was stirred at r.t for 2 h, cooled to

r.t., quenched with sat. aqueous NH₄Cl (20 mL), and extracted with

EtOAc (50 mL). The combined organic layers were washed with

brine (3 × 20 mL), dried with MgSO₄, and concentrated in vacuo.

The residue was puriﬁed by column chromatography (silica gel, eluted

with 90−10% EtOAc in hexane) to give the title compound (12.5 g,

34.4 mmol, 77%) as a yellow oil. ¹H NMR (300 MHz, CDCl₃): δ 0.00

(5H, s), 0.03 (1H, s), 0.84−0.86 (9H, m), 1.31−1.47 (2H, m), 1.52−

1.65 (1H, m), 1.72−1.82 (2H, m), 2.77 (2H, td, J = 11.9, 2.2 Hz),

3.27 (2H, br d, J = 12.3 Hz), 3.46 (2H, d, J = 6.2 Hz), 3.79 (3H, s),

(1-(5-Methoxy-2-methylphenyl)piperidin-4-yl)methanol (36d).

Compound 36d was prepared from compound 34d (2 g, 9.95

mmol) and piperidin-4-ylmethanol (3.44 g, 29.8 mmol) in a manner

similar to that described for compound 34c. Pale-brown oil (0.760 g,

1

3.23 mmol, 33%). H NMR (300 MHz, CDCl₃): δ 1.29−1.39 (1H,

m), 1.45 (2H, td, J = 12.1, 3.8 Hz), 1.57−1.73 (1H, m), 1.82 (2H, dd,

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6.48 (1H, d, J = 2.3 Hz), 6.53 (1H, dd, J = 8.6, 2.4 Hz), 7.72 (1H, d, J

= 8.7 Hz), 9.99−10.09 (1H, m).

br s), 1.68−1.83 (2H, m), 2.59−2.70 (2H, m), 3.08 (2H, d, J = 11.8

Hz), 3.32−3.37 (2H, m), 3.78 (3H, s), 4.16 (2H, q, J = 7.2 Hz), 4.51

(1H, t, J = 5.3 Hz), 6.40 (1H, d, J = 16.1 Hz), 6.58−6.67 (2H, m),

7.49−7.69 (1H, m), 7.83 (1H, d, J = 16.2 Hz). A mixture of copper(I)

chloride (0.426 g, 4.30 mmol), sodium 2-methylpropan-2-olate (0.413

g, 4.30 mmol), and 2,2′-bis(diphenylphosphino)-1,1′-binaphthalene

(2.68 g, 4.30 mmol) in toluene (100 mL) was stirred at r.t. for 5 min.

To the reaction mixture were added sequentially PMHS (5.20 mL,

86.00 mmol), the obtained compound (13.73 g, 43 mmol), and 2-

methylpropan-2-ol (20.27 mL, 215.00 mmol) at r.t.. The mixture was

stirred at room temperature under Ar for 30 min. The mixture was

quenched with EtOH and sat. NH₄Cl aq at room temperature and

extracted with EtOAc. The organic layer was separated, washed with

sat. NH₄Cl aq and brine, dried over MgSO₄, and concentrated in

vacuo. The residue was puriﬁed by column chromatography (NH

silica gel, eluted with 35−45% EtOAc in hexane and then silica gel,

eluted with 25−35% EtOAc in hexane) to give the title compound

1-(2-(4-(((tert-Butyldimethylsilyl)oxy)methyl)piperidin-1-yl)-4-

methoxyphenyl)-3,3-dimethylbutan-1-ol (43). To a solution of

compound 42 (1.55 g, 4.26 mmol) in THF (dry) (10 mL) was

added neopentylmagnesium chloride (5.12 mL, 5.12 mmol) and

stirred at r.t. for 5 min. The mixture was poured into sat. NH₄Cl aq

and extracted with EtOAc. The organic layer was separated, washed

with brine, dried over MgSO₄, and concentrated in vacuo. The residue

was puriﬁed by column chromatography (silica gel, eluted with 0−

10% EtOAc in hexane) to give the title compound (1.360 g, 3.12

1

mmol, 73.2%) as a white powder. H NMR (400 MHz, CDCl₃): δ

0.00 (6H, s), 0.85 (9H, s), 0.93 (9H, br s), 1.26−1.42 (2H, m), 1.49

(1H, dd, J = 14.2, 2.8 Hz), 1.66 (1H, dd, J = 14.2, 9.3 Hz), 1.77 (2H,

d, J = 12.4 Hz), 2.54−2.78 (2H, m), 2.98 (1H, d, J = 11.2 Hz), 3.08

(1H, d, J = 11.2 Hz), 3.45 (2H, d, J = 6.3 Hz), 3.72 (3H, s), 4.91 (1H,

d, J = 3.8 Hz), 5.31 (1H, br s), 6.61 (1H, dd, J = 8.4, 2.5 Hz), 6.70

(1H, d, J = 2.5 Hz), 7.03 (1H, d, J = 8.4 Hz). MS (ESI/APCI): m/z

436.2. HPLC 93.5% (LC−MS).

1

(3.31 g, 10.29 mmol, over 24%) as a colorless oil. H NMR (400

MHz, DMSO-d₆): δ 1.12−1.18 (3H, m), 1.21−1.35 (2H, m), 1.47

(1H, br s), 1.74 (2H, d, J = 11.4 Hz), 2.53−2.64 (4H, m), 2.73−2.83

(2H, m), 2.96 (2H, d, J = 11.5 Hz), 3.25−3.36 (2H, m), 3.70 (3H, s),

3.98−4.08 (2H, m), 4.47 (1H, t, J = 5.2 Hz), 6.57 (1H, dd, J = 8.3, 2.2

Hz), 6.62 (1H, d, J = 2.1 Hz), 7.06 (1H, d, J = 8.4 Hz).

(1-(2-(3,3-Dimethylbutyl)-5-methoxyphenyl)piperidin-4-yl)-

methanol (44). A mixture of compound 43 (500 mg, 1.15 mmol) and

SOCl₂(0.168 mL, 2.30 mmol) in toluene (5 mL) was stirred at r.t. for

2 h. The mixture was neutralized with sat. NaHCO₃aq at 0 °C and

extracted with EtOAc. The organic layer was separated, washed with

water and brine, dried over MgSO₄, and concentrated in vacuo. A

mixture of the residue (330 mg) and 10% Pd−C (50 mg, 0.05 mmol)

in MeOH (10 mL) was hydrogenated under balloon pressure at room

temperature for 15 h. The catalyst was removed by ﬁltration, and the

ﬁltrate was concentrated in vacuo. The residue was puriﬁed by

column chromatography (silica gel, eluted with 0−25% EtOAc in

hexane) to give (293 mg, 0.959 mmol, 88%) a colorless oil. ¹H NMR

(400 MHz, CDCl3): δ 0.96 (9H, s), 1.33−1.49 (4H, m), 1.59−1.69

(1H, m), 1.82 (2H, d, J = 11.3 Hz), 2.49−2.57 (2H, m), 2.60−2.68

(2H, m), 3.09 (2H, d, J = 11.7 Hz), 3.57 (2H, br s), 3.77 (3H, s), 6.57

(1H, dd, J = 8.3, 2.6 Hz), 6.64 (1H, d, J = 2.5 Hz), 7.08 (1H, d, J =

8.3 Hz). MS (ESI/APCI): m/z 306.4. HPLC 98.7% (LC−MS).

Methyl 3-Cyclopropyl-3-(3-((1-(2-formyl-5-methoxyphenyl)-

piperidin-4-yl)methoxy)phenyl)propanoate (45). To a solution of

compound 30 (5.7 g, 22.86 mmol) and TEA (4.78 mL, 34.30 mmol)

in THF (dry) (50 mL) was added MsCl (2.124 mL, 27.44 mmol) and

stirred at r.t. for 30 min. The mixture was poured into water and

extracted with EtOAc. The organic layer was separated, washed with

brine, dried over MgSO₄, and concentrated in vacuo to give a pale-

yellow oil. To a solution of the obtained 11a (5.04 g, 22.86 mmol) in

DMF (dry) (50 mL) were added NaH (0.914 g, 22.86 mmol) and the

obtained methanesulfonate. The mixture was stirred at 100 °C for 1 h.

The mixture was poured into water and extracted with EtOAc. The

organic layer was separated, washed with brine, dried over MgSO₄,

and concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, eluted with 0−20% EtOAc in hexane) to

give the title compound (6.60 g, 14.62 mmol, 64%) as a pale-yellow

4-(2-(4-(Hydroxymethyl)piperidin-1-yl)-4-methoxyphenyl)-2-

methylbutan-2-ol (47). Methylmagnesium chloride in THF (5.54

mL, 16.61 mmol) was added to a solution of compound 46 (890 mg,

2.77 mmol) in THF (dry) (20 mL) at 0 °C. The mixture was stirred

at room temperature under N₂for 20 min. The mixture was quenched

with sat. NH₄Cl aq at room temperature and extracted with EtOAc.

The organic layer was separated, washed with water and brine, dried

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

column chromatography (silica gel, eluted with 50−60% EtOAc in

hexane) to give the title compound (476 mg, 1.547 mmol, 56%) as a

colorless oil. ¹H NMR (400 MHz, DMSO-d₆): δ 1.14 (6H, s), 1.21−

1.35 (2H, m), 1.47 (1H, br s), 1.54−1.62 (2H, m), 1.74 (2H, d, J =

11.9 Hz), 2.50−2.61 (4H, m), 2.97 (2H, d, J = 11.3 Hz), 3.27−3.31

(2H, m), 3.69 (3H, s), 4.17 (1H, s), 4.46 (1H, t, J = 5.2 Hz), 6.51−

6.61 (2H, m), 7.04 (1H, d, J = 8.2 Hz).

4-(2-(4-(((tert-Butyldimethylsilyl)oxy)methyl)piperidin-1-yl)-4-

methoxyphenyl)-2-methylbutan-2-ol (48). The title compound 48

was obtained from compound 47 (336 mg, 1.09 mmol) in a manner

similar to that described for compound 42. Colorless oil (372 mg,

0.883 mmol, 81%). ¹H NMR (400 MHz, DMSO-d₆): δ 0.04 (6H, s),

0.88 (9H, s), 1.13 (6H, s), 1.28−1.43 (2H, m), 1.46−1.63 (3H, m),

1.71 (2H, d, J = 11.4 Hz), 2.49−2.63 (4H, m), 2.97 (2H, d, J = 11.3

Hz), 3.50 (2H, d, J = 5.9 Hz), 3.69 (3H, s), 4.14 (1H, s), 6.50−6.63

(2H, m), 7.04 (1H, d, J = 8.0 Hz).

4-(2-(4-(Hydroxymethyl)piperidin-1-yl)-4-methoxyphenyl)-2-

methylbutan-2-yl Acetate (50). Ac₂O (0.167 mL, 1.77 mmol) was

added to a solution of compound 48 (372 mg, 0.88 mmol), DMAP

(216 mg, 1.77 mmol), and TEA (0.369 mL, 2.65 mmol) in THF

(dry) (3 mL) at room temperature. The mixture was stirred at 60 °C

for 3 h. The mixture was quenched with water at room temperature

and extracted with EtOAc. The organic layer was separated, washed

with 1 N HCl aq, sat. NaHCO₃aq, and brine, dried over MgSO₄, and

concentrated in vacuo. The residue was dissolved in THF (3 mL). To

the mixture was added TBAF (2.65 mL, 2.65 mmol). The mixture was

stirred at 60 °C for 10 min. The mixture was quenched with water at

room temperature and extracted with EtOAc. The organic layer was

separated, washed with water and brine, dried over MgSO₄, and

concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, eluted with 30−40% EtOAc in hexane)

to give the title compound (236 mg, 0.674 mmol, 76%) as (a)

colorless oil. ¹H NMR (400 MHz, DMSO-d₆): δ 1.22−1.35 (2H, m),

1.37−1.55 (1H, m), 1.44 (6H, s), 1.75 (2H, d, J = 11.5 Hz), 1.89−

1.97 (2H, m), 1.93 (3H, s), 2.46−2.64 (4H, m), 2.95 (2H, d, J = 11.0

Hz), 3.22−3.35 (2H, m), 3.70 (3H, s), 4.45 (1H, t, J = 4.6 Hz), 6.45−

6.68 (2H, m), 7.04 (1H, d, J = 8.2 Hz).

1

oil. H NMR (400 MHz, CDCl₃): δ 0.09−0.20 (1H, m), 0.20−0.31

(1H, m), 0.37−0.49 (1H, m), 0.52−0.63 (1H, m), 0.94−1.08 (1H,

m), 1.60−1.73 (1H, m), 1.98 (2H, br d, J = 10.5 Hz), 2.26−2.40 (1H,

m), 2.65−2.82 (2H, m), 2.89 (1H, br t, J = 11.4 Hz), 3.38 (1H, br d, J

= 11.7 Hz), 3.56−3.65 (3H, m), 3.83−3.88 (3H, m), 6.55−6.63 (1H,

m), 6.66−6.88 (3H, m), 7.13−7.26 (2H, m), 7.80 (1H, d, J = 8.7 Hz),

10.14 (1H, s). MS (ESI/APCI): m/z 452.5 [M + H]⁺.

Ethyl 3-(2-(4-(Hydroxymethyl)piperidin-1-yl)-4-methoxyphenyl)-

propanoate (46). (Carbethoxymethylene)triphenyl phosphorane

(22.68 g, 65.12 mmol) was added to a solution of compound 30

(10.82 g, 43.41 mmol) in toluene (80 mL) at room temperature. The

mixture was reﬂuxed under N₂for 2 h. The reaction mixture was

concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, eluted with 35−45% EtOAc in hexane)

to give (E)-ethyl 3-(2-(4-(hydroxymethyl)piperidin-1-yl)-4-

methoxyphenyl)acrylate (13.73 g, 43.0 mmol, 99%) as a pale-yellow

oil containing triphenylphosphine oxide. This product was subjected

to the next reaction without further puriﬁcation. ¹H NMR (400 MHz,

DMSO-d₆): δ 1.25 (3H, t, J = 7.1 Hz), 1.28−1.41 (2H, m), 1.50 (1H,

Benzyl 2-Fluoro-4-methoxybenzoate (52). To a solution of 2-

ﬂuoro-4-methoxybenzoic acid 51 (15.46 g, 90.87 mmol) in DMF

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(363 mL) were added K₂CO₃(18.84 g, 136.30 mmol) and benzyl

bromide (11.9 mL, 100.0 mmol) at room temperature. The mixture

was stirred at the same temperature under N₂for 8 h. The mixture

was quenched with iced water at 0 °C and extracted with EtOAc. The

organic layer was separated, washed with brine, dried over Na₂SO₄,

and concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, eluted with 0−20% EtOAc in hexane) to

give the title compound (22.5 g, 86 mmol, 95%) as a colorless oil. ¹H

NMR (300 MHz, DMSO-d₆): δ 3.85 (3H, s), 5.32 (2H, s), 6.84−7.00

(2H, m), 7.31−7.49 (5H, m), 7.88 (1H, t, J = 8.7 Hz). MS (ESI/

APCI): m/z 261.2.

(NH silica gel, eluted with 0−50% EtOAc in hexane) crystallization:

EtOAc/hexane). White powder (0.773 g, 1.817 mmol, 40%). ¹H

NMR (300 MHz, CDCl₃): δ 0.81 (9H, s), 1.14 (1H, d, J = 4.8 Hz),

1.24−1.35 (1H, m), 1.43−1.53 (2H, m), 1.56−1.70 (2H, m), 2.35−

2.48 (4H, m), 2.48−2.66 (2H, m), 3.38 (1H, br s), 3.47−3.60 (2H,

m), 3.75 (3H, s), 4.09−4.23 (1H, m), 4.27 (1H, br s), 6.18 (1H, d, J

= 2.1 Hz), 6.32 (1H, br s), 6.48 (1H, dd, J = 8.4, 2.4 Hz), 6.70 (1H, d,

J = 7.3 Hz), 7.08 (1H, t, J = 7.5 Hz), 7.29 (1H, d, J = 8.5 Hz).

Cyclopropyl(6-methoxypyridin-2-yl)methanol (59a). The title

compound was obtained from cyclopropyl magnesium bromide

THF solution derived from cyclopropyl bromide (3.50 mL, 43.75

mmol) and 58a (1.75 mL, 14.6 mmol) in a manner similar to that

described for compound 43. Pale-brown oil. This product was

Benzyl 2-(4-(Hydroxymethyl)piperidin-1-yl)-4-methoxybenzoate

(53). Compound 53 was prepared from compound 52 (1.02 g, 3.91

mmol) in a manner similar to that described for compound 28. Pale-

1

subjected to the next reaction without further puriﬁcation. H NMR

1

yellow oil (813 mg, 2.29 mmol, 59%). H NMR (300 MHz, DMSO-

(400 MHz, DMSO-d₆): δ 0.25−0.49 (4H, m), 1.02−1.23 (1H, m),

3.83 (3H, s), 3.99−4.08 (1H, m), 5.19 (1H, d, J = 4.9 Hz), 6.65 (1H,

d, J = 8.2 Hz), 7.04 (1H, d, J = 7.3 Hz), 7.66 (1H, dd, J = 8.1, 7.5 Hz).

Cyclopropyl(2-methoxypyridin-4-yl)methanol (59b). Compound

59b was prepared from compound 58b (4.33 g) in a manner similar

to that described for compound 59a. Brown oil (5.30 g). This

compound was used for the next step without further puriﬁcation. ¹H

NMR (400 MHz, DMSO-d₆): δ 0.34−0.50 (4H, m), 0.90−1.04 (1H,

m), 3.83 (3H, s), 3.95 (1H, dd, J = 7.2, 4.6 Hz), 5.35 (1H, d, J = 4.6

Hz), 6.77 (1H, s), 6.99 (1H, d, J = 5.3 Hz), 8.07 (1H, d, J = 5.3 Hz).

Cyclopropyl(6-methoxypyridin-2-yl)methanone (60a). Py·SO₃

(9.28 g, 58.3 mmol) was added to a solution of compound 59a

(2.29 mL, 14.6 mmol) and TEA (16.3 mL, 116.6 mmol) in DMSO

(80 mL) at room temperature. The mixture was stirred at room

temperature for 5 min. The mixture was quenched with water at room

temperature and extracted with EtOAc. The organic layer was

separated, washed with water and brine, dried over MgSO₄, and

concentrated in vacuo to give a brown oil. This product was subjected

to the next reaction without further puriﬁcation. ¹H NMR (400 MHz,

CDCl₃): δ 1.02−1.12 (2H, m), 1.18−1.30 (2H, m), 3.40−3.55 (1H,

m), 4.04 (3H, s), 6.94 (1H, d, J = 8.0 Hz), 7.61−7.66 (1H, m), 7.67−

7.75 (1H, m).

Cyclopropyl(2-methoxypyridin-4-yl)methanone (60b). Com-

pound 60b was prepared from compound 59b (5.30 g) in a manner

similar to that described for compound 60a. Brown oil. This

compound was used for the next step without further puriﬁcation. ¹H

NMR (400 MHz, DMSO-d₆): δ 1.00−1.19 (4H, m), 2.82−2.95 (1H,

m), 3.92 (3H, s), 7.34 (1H, s), 7.45 (1H, d, J = 5.3 Hz), 8.37 (1H, d, J

= 5.3 Hz).

Ethyl 3-Cyclopropyl-3-(6-methoxypyridin-2-yl)acrylate (61a).

Triethyl phosphonoacetate (7.23 mL, 36.5 mmol) was added to a

suspension of NaH (1.28 g, 32.1 mmol) in THF (80 mL) at 0 °C.

The mixture was stirred at room temperature under N₂for 5 min. To

the solution was added compound 60a (2.27 mL, 14.6 mmol) at r.t..

The mixture was reﬂuxed under N₂overnight. The mixture was

quenched with water at room temperature and extracted with EtOAc.

The organic layer was separated, washed with water and brine, dried

over MgSO₄, and concentrated in vacuo to give the title compound as

brown oil. This product was subjected to the next reaction without

further puriﬁcation. ¹H NMR (400 MHz, CDCl₃): δ 0.65−0.75 (2H,

m), 0.78−0.91 (2H, m), 1.22−1.29 (3H, m), 1.73−1.90 (1H, m),

3.91 (3H, s), 4.08−4.27 (2H, m), 5.89 (1H, s), 6.64−6.69 (1H, m),

6.80 (1H, d, J = 7.0 Hz), 7.50−7.57 (1H, m).

d₆): δ 1.11−1.28 (2H, m), 1.33−1.52 (1H, m), 1.64 (2H, d, J = 10.3

Hz), 2.56−2.70 (2H, m), 3.17−3.28 (4H, m), 3.78 (3H, s), 4.43 (1H,

t, J = 5.3 Hz), 5.25 (2H, s), 6.49−6.58 (2H, m), 7.29−7.48 (5H, m),

7.63−7.70 (1H, m).

Benzyl 2-(4-((3-(1-Cyclopropyl-3-ethoxy-3-oxopropyl)phenoxy)-

methyl)piperidin-1-yl)-4-methoxybenzoate (54). The title com-

pound 54 was obtained from compound 53 (3.01 g, 8.47 mmol)

and compound 11a (1.98 g, 8.47 mmol) in a manner similar to that

described for compound 4a. Colorless gum (4.36 g, 7.63 mmol, 90%).

¹H NMR (300 MHz, DMSO-d₆): δ 0.07−0.18 (1H, m), 0.18−0.28

(1H, m), 0.28−0.40 (1H, m), 0.44−0.56 (1H, m), 0.96−1.12 (4H,

m), 1.27−1.46 (2H, m), 1.67−1.90 (3H, m), 2.19−2.32 (1H, m),

2.61−2.79 (4H, m), 3.20−3.29 (2H, m), 3.74−3.84 (5H, m), 3.89−

4.00 (2H, m), 5.26 (2H, s), 6.52−6.59 (2H, m), 6.75 (1H, dd, J = 8.2,

1.6 Hz), 6.79−6.86 (2H, m), 7.19 (1H, t, J = 8.0 Hz), 7.27−7.42 (3H,

m), 7.42−7.49 (2H, m), 7.69 (1H, d, J = 8.9 Hz).

2-(4-((3-(1-Cyclopropyl-3-ethoxy-3-oxopropyl)phenoxy)methyl)-

piperidin-1-yl)-4-methoxybenzoic Acid (55). A solution of com-

pound 54 (4.36 g, 7.63 mmol) in EtOH (102 mL) and THF (50.8

mL) was deprotected using H-Cube (70 mm 10% Pd/C, r.t., ﬂow: 1.0

mL/min, reaction time: 5 h). The mixture was concentrated in vacuo.

The residue was puriﬁed by column chromatography (silica gel, eluted

with 0−100% EtOAc in hexane) to give (3.41 g, 7.08 mmol, 93%) a

1

white amorphous solid. H NMR (300 MHz, DMSO-d₆): δ 0.07−

0.17 (1H, m), 0.17−0.27 (1H, m), 0.27−0.39 (1H, m), 0.45−0.56

(1H, m), 0.97−1.13 (4H, m), 1.43−1.62 (2H, m), 2.01 (3H, d, J =

14.0 Hz), 2.19−2.31 (1H, m), 2.63−2.80 (2H, m), 3.04−3.18 (4H,

m), 3.85 (3H, s), 3.89−4.03 (4H, m), 6.76−6.89 (3H, m), 6.99 (1H,

dd, J = 8.8, 2.5 Hz), 7.19 (1H, t, J = 7.8 Hz), 7.26 (1H, d, J = 2.4 Hz),

7.98 (1H, d, J = 8.8 Hz). MS (ESI/APCI): m/z 482.4 [M + H]⁺.

HPLC 96.8% (LC−MS).

2-Fluoro-4-methoxy-N-(6-methylpyridin-2-yl)-N-neopentylben-

zamide (56). Oxalyl dichloride (8.10 mL, 92.6 mmol) was added to a

solution of compound 51 (14.3 g, 84.1 mmol) and 5 drops of DMF in

THF (150 mL) at room temperature. The mixture was stirred at

room temperature under N₂for 10 min. The mixture was

concentrated in vacuo. To a THF (150 mL) solution of 6-methyl-

N-neopentylpyridin-2-amine (7.5 g, 42.07 mmol) and triethylamine

(17.59 mL, 126.21 mmol) was added dropwise THF solution (15

mL) of the acid chloride at 0 °C. The mixture was stirred at room

temperature under N₂overnight. The mixture was quenched with

water at room temperature and extracted with EtOAc. The organic

layer was separated, washed with water, 1 N NaOH aq, and brine,

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

ﬁltered through NH silica gel (eluted with 5−15% EtOAc in hexane),

and the residue was solidiﬁed. The solid was washed with cold hexane

to give the title compound (12.7 g, 38.4 mmol, 91%) as a white solid.

¹H NMR (400 MHz, CDCl₃): δ 0.86 (9H, s), 2.47 (3H, s), 3.73 (3H,

s), 4.13 (2H, s), 6.39 (1H, d, J = 11.5 Hz), 6.53 (1H, d, J = 8.4 Hz),

6.60 (1H, d, J = 7.9 Hz), 6.84 (1H, d, J = 7.5 Hz), 7.14 (1H, t, J = 8.3

Hz), 7.24−7.32 (1H, m).

2-(4-(Hydroxymethyl)piperidin-1-yl)-4-methoxy-N-(6-methylpyr-

idin-2-yl)-N-neopentylbenzamide (57). Compound 57 was prepared

from compound 56 (1.5 g, 4.54 mmol) in a manner similar to that

described for compound 28 (puriﬁcation: column chromatography

Ethyl 3-Cyclopropyl-3-(2-methoxypyridin-4-yl)acrylate (61b).

Compound 61b was prepared from compound 60b (4.42 g) in a

manner similar to that described for compound 61a. This compound

was used for the next step without further puriﬁcation.

Ethyl 3-Cyclopropyl-3-(6-methoxypyridin-2-yl)propanoate (62a).

Zinc (9.53 g, 145.80 mmol) was added to a solution of compound

61a (3.61 g, 14.6 mmol) in AcOH (50 mL) at room temperature. The

mixture was stirred at room temperature for 20 min. The reaction

mixture was ﬁltered, and the ﬁltrate was concentrated in vacuo

(azeotropy with PhMe). The residue was puriﬁed by column

chromatography (silica gel, eluted with 0−5% EtOAc in hexane) to

give (2.68 g, 10.75 mmol, 74% over 4 steps) a colorless oil. ¹H NMR

(400 MHz, CDCl₃): δ 0.18−0.34 (2H, m), 0.39−0.50 (1H, m),

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0.52−0.64 (1H, m), 1.01−1.13 (1H, m), 1.18 (3H, t, J = 7.2 Hz),

2.34−2.49 (1H, m), 2.77 (1H, dd, J = 15.3, 6.0 Hz), 3.02 (1H, dd, J =

15.2, 8.5 Hz), 3.90 (3H, s), 3.98−4.17 (2H, m), 6.56 (1H, d, J = 8.3

Hz), 6.79 (1H, d, J = 7.3 Hz), 7.47 (1H, t, J = 7.7 Hz).

at the same temperature for 2 h. The resulting mixture was slowly

quenched with 1 N aq HCl below 10 °C and extracted with EtOAc at

this temperature. The organic layer was washed with water and brine,

dried over Na₂SO₄, and concentrated in vacuo to give the title

compound (2250 g) as a brown oil which was used for the next

reaction without further puriﬁcations.

Ethyl 3-Cyclopropyl-3-(2-methoxypyridin-4-yl)propanoate (62b).

Compound 62b was prepared from compound 61b in a manner

similar to that described for compound 62a. Pale-yellow oil (4.17 g,

tert-Butyl 4-({[4-(1-Cyclopropyl-3-ethoxy-3-oxopropyl)pyridin-2-

yl]oxy}methyl)piperidine-1-carboxylate (67). To a suspension of

NaH (499.4 g, 12.49 mol) in THF (10 L) was added ethyl 2-

(diethoxyphosphoryl)acetate (2785 g, 12.49 mmol) at 0 °C and

stirred for 30 min. To the mixture was added a solution of compound

66 (2250 g, 6.243 mol) in THF (10 L) and stirred at 80 °C for 3 h.

The reaction mixture was poured into sat. NH₄Cl aq (10 L), and the

mixture was extracted with EtOAc. The organic layer was separated,

washed with brine, dried over Na₂SO₄, and concentrated in vacuo to

give tert-butyl 4-[({4-[(1E)-1-cyclopropyl-3-ethoxy-3-oxoprop-1-en-

1-yl]pyridin-2-yl}oxy)methyl]piperidine-1-carboxylate (2450 g) as a

brown oil which was used for the next reaction without further

puriﬁcations. Zinc (2215 g, 34.15 mol) was added portionwise to a

solution of the obtained product (2450 g, 5.691 mol) in AcOH (10

L) at room temperature. The mixture was stirred at room temperature

for 2 h. The reaction mixture was ﬁltered through Celite pad, and the

ﬁltrate was concentrated in vacuo. The residue was basiﬁed with

NaHCO₃and extracted with EtOAc. The organic layer was separated,

washed with sat. NaHCO₃aq and brine, dried over Na₂SO₄, and

concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, eluted with 0−25% EtOAc in hexane)

1

53% over 4 steps). H NMR (400 MHz, DMSO-d₆): δ 0.09−0.20

(1H, m), 0.20−0.29 (1H, m), 0.29−0.38 (1H, m), 0.44−0.57 (1H,

m), 0.94−1.05 (1H, m), 1.08 (3H, t, J = 7.1 Hz), 2.19−2.30 (1H, m),

2.75 (2H, d, J = 7.5 Hz), 3.82 (3H, s), 3.90−402 (2H, m), 6.71 (1H,

s), 6.92 (1H, d, J = 5.1 Hz), 8.05 (1H, d, J = 5.1 Hz).

Ethyl 3-Cyclopropyl-3-(6-hydroxypyridin-2-yl)propanoate (63a).

Pyridine hydrochloride (12.4 g, 107.5 mmol) was added to a solution

of compound 62a (2.68 g, 10.75 mmol) in DMF (5 mL) at room

temperature. The mixture was stirred at 120 °C under N₂for 30 min.

The reaction mixture was diluted with THF at r.t.. The resulting

precipitate was removed by ﬁltration through Celite, and the ﬁltrate

was concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, eluted with 3−8% MeOH in EtOAc) to

give pale-yellow oil. The residue was solidiﬁed and washed with i-

Pr₂O to give the title compound (1.79 g, 7.61 mmol, 71%) as a white

solid. ¹H NMR (400 MHz, DMSO-d₆): δ 0.14−0.23 (1H, m), 0.24−

0.32 (1H, m), 0.32−0.40 (1H, m), 0.45−0.55 (1H, m), 0.97−1.07

(1H, m), 1.09 (3H, t, J = 7.1 Hz), 2.11−2.25 (1H, m), 2.69−2.78

(1H, m), 2.79−2.89 (1H, m), 3.90−4.05 (2H, m), 5.95−6.09 (1H,

m), 6.14 (1H, d, J = 8.4 Hz), 7.33 (1H, t, J = 7.6 Hz), 11.51 (1H, br

s).

1

to give the title compound (1750 g, 71%) as a pale-yellow oil. H

NMR (300 MHz, CDCl₃): δ 0.17 (1H, dt, J = 9.6, 4.8 Hz), 0.28 (1H,

dt, J = 9.6, 4.8 Hz), 0.39−0.52 (1H, m), 0.54−0.66 (1H, m), 0.94−

1.08 (1H, m), 1.15−1.22 (3H, m), 1.27−1.35 (2H, m), 1.46 (9H, s),

1.81 (2H, d, J = 12.0 Hz), 1.88−2.01 (1H, m), 2.30 (1H, dt, J = 9.6,

7.5 Hz), 2.58−2.84 (4H, m), 4.02−4.10 (2H, m), 4.10−4.23 (4H, m),

6.58−6.62 (1H, m), 6.76 (1H, dd, J = 5.4, 1.5 Hz), 7.86−8.16 (1H,

m).

Ethyl 3-Cyclopropyl-3-(2-hydroxypyridin-4-yl)propanoate (63b).

Compound 63b was prepared from compound 62b (4.17 g) in a

manner similar to that described for compound 63a. Yellow oil (3.61

1

g). H NMR (300 MHz, DMSO-d₆): δ 0.10−0.28 (2H, m), 0.29−

0.43 (1H, m), 0.43−0.58 (1H, m), 0.86−1.03 (1H, m), 1.11 (3H, t, J

= 7.1 Hz), 2.07 (1H, dt, J = 9.7, 7.6 Hz), 2.68 (2H, d, J−7.5 Hz),

3.91−4.07 (2H, m), 6.19 (1H, s), 6.20−6.25 (1H, m), 7.30 (1H, d, J

= 6.7 Hz), 11.48 (1H, br s).

(3S)-3-(2-{[1-(tert-Butoxycarbonyl)piperidin-4-yl]methoxy}-

pyridin-4-yl)-3-cyclopropylpropanoic Acid-(1S)-1-(4-

Methylphenyl)ethan-1-amine (1/1) (68). To a solution of compound

67 (1750 g, 4.045 mol) in MeOH (6 L) was added 4 N NaOH (4.045

L, 16.18 mmol) at room temperature. After stirring at ambient

temperature for 2 h, TLC showed that the reaction was completed.

The mixture was concentrated in vacuo to remove MeOH. The

residue was dissolved into H₂O and extracted with Et₂O. The aqueous

phase was acidiﬁed with 1 N HCl to pH = 4−5. The solid was

collected by ﬁltration, washed with PE, and dried in vacuo to give the

Ethyl 3-Cyclopropyl-3-(2-((1-(5-methoxy-2-((6-methylpyridin-2-

yl)(neopentyl)carbamoyl)phenyl)piperidin-4-yl)methoxy)pyridin-4-

yl)propanoate (64b). Compound 64b was prepared from compound

57 (221.5 mg, 0.52 mmol) and 61b (135 mg, 0.57 mmol) in a manner

similar to that described for compound 54 (puriﬁcation: column

chromatography (silica gel, eluted with 0−40% EtOAc in hexane)).

1

Colorless gum (198 mg, 0.308 mmol, 59%). H NMR (300 MHz,

CDCl₃): δ 0.11−0.22 (1H, m), 0.30 (1H, dq, J = 9.5, 4.7 Hz), 0.41−

0.53 (1H, m), 0.54−0.67 (1H, m), 0.82 (9H, s), 0.90−1.07 (1H, m),

1.14−1.43 (4H, m), 1.60−1.89 (4H, m), 2.25−2.37 (1H, m), 2.44

(4H, s), 2.48−2.83 (4H, m), 3.39 (1H, br s), 3.75 (3H, s), 3.99−4.40

(6H, m), 6.19 (1H, d, J = 2.0 Hz), 6.35 (1H, br s), 6.48 (1H, dd, J =

8.5, 2.4 Hz), 6.63 (1H, d, J = 0.7 Hz), 6.69 (1H, d, J = 7.5 Hz), 6.76

(1H, dd, J = 5.3, 1.4 Hz), 7.08 (1H, t, J = 7.6 Hz), 7.29 (1H, d, J = 8.4

Hz), 8.07 (1H, d, J = 5.3 Hz).

tert-Butyl 4-(((4-(Cyclopropanecarbonyl)pyridin-2-yl)oxy)-

methyl)piperidine-1-carboxylate (66). To a solution of compound

65 (2000 g, 9.29 mol) in DMF (6 L) was added NaH (440 g, 11.0

mol) at 0 °C. After being stirred at 0 °C for 40 min, a solution of N-

Boc-4-hydroxymethylpiperidine (1250 g, 9.00 mol) in DMF (3 L)

was added to the reaction mixture. The mixture was stirred at 0 °C for

2 h. The mixture was diluted with EtOAc and water, and the mixture

was extracted with EtOAc. The organic layer was separated, washed

with brine, dried over Na₂SO₄, and concentrated in vacuo to give a

white powder. The powder was collected by ﬁltration, washed with

PE, and dried to give tert-butyl 4-{[(4-cyanopyridin-2-yl)oxy]-

methyl}piperidine-1-carboxylate (2100 g) as a white powder. ¹H

NMR (300 MHz, CDCl₃): δ 1.26 (2H, qd, J = 12.3, 4.5 Hz), 1.46

(9H, s), 1.79 (2H, d, J = 12.6 Hz), 1.85−2.09 (1H, m), 2.74 (2H, t, J

= 12.1 Hz), 4.13 (2H, br s), 4.19 (2H, d, J = 6.3 Hz), 6.98 (1H, t, J =

1.2 Hz), 7.06 (1H, dd, J = 5.1, 1.2 Hz), 8.28 (1H, dd, J = 5.1, 0.8 Hz).

A solution of the obtained product (2100 g, 6.616 mol) in THF (15

L) was added dropwise to cyclopropylmagnesium bromide (1 M in

THF, 13.3 L, 13.3 mol) at room temperature. The mixture was stirred

1

corresponding carboxylic acid (1510 g, 92%) as a white powder. H

NMR (300 MHz, CDCl₃): δ 0.12−0.20 (1H, m), 0.30−0.34 (1H, m),

0.41−0.51 (1H, m), 0.56−0.65 (1H, m), 0.95−1.01 (1H, m), 1.18−

1.32 (2H, m), 1.46 (9H, s), 1.77−1.82 (2H, m), 1.91−1.98 (1H, m),

2.28 (1H, dd, J = 17.1, 7.5 Hz), 2.69−2.84 (4H, m), 4.10−4.13 (4H,

m), 6.60 (1H, 2), 6.75 (1H, dd, J = 5.4, 1.2 Hz), 8.03 (1H, d, J = 5.4

Hz). MS (ESI/APCI): m/z 405 [M + H]⁺. To a solution of 3-(2-((1-

(tert-butoxycarbonyl)piperidin-4-yl)methoxy)pyridin-4-yl)-3-cyclo-

propylpropanoic acid (870 g, 2150.80 mmol) in EtOH (5.4 L) was

added EtOAc (10.80 L) solution of (S)-1-(p-tolyl)ethanamine (291 g,

2150.80 mmol) at room temperature. The mixture was stirred at

room temperature under a dry atmosphere (CaCl₂tube) for 16.5 h.

The resulting white precipitate was collected by ﬁltration and washed

with AcOEt/EtOH (2:1, 2.4 L). The precipitate was dried in vacuo to

give the title compound (477 g, 884 mmol, 41%) as a white crude

solid. The obtained product (237 g, 439.13 mmol) was dissolved in

EtOH (3.1 L) at 70 °C. To the solution was added dropwise heptane

(4.7 L) at 60−50 °C. After addition of a small amount of the seed

crystal, the mixture was stirred at 50 °C for 1.5 h, then r.t. for 12 h,

and then cooled with a water bath for 1 h. The resulting white solid

was collected, washed with EtOH/heptane (1:2), and dried in vacuo

1

to give the crude material (182.3 g, 338 mmol) as a white solid. H

NMR (300 MHz, DMSO-d₆): δ 0.41−0.54 (1H, m), 0.87−1.21 (4H,

m), 1.25 (3H, d, J = 6.7 Hz), 1.39 (9H, s), 1.72 (2H, d, J = 11.2 Hz),

1.81−1.98 (1H, m), 2.15−2.30 (4H, m), 2.58 (2H, d, J = 7.5 Hz),

X

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2.72 (1H, br s), 3.88−4.11 (5H, m), 6.67 (1H, s), 6.88 (1H, dd, J =

5.3, 1.2 Hz), 7.11 (2H, d, J = 7.8 Hz), 7.25 (2H, d, J = 8.1 Hz), 8.00

(1H, d, J = 5.3 Hz). MS (ESI/APCI): m/z 405.3 [M + H]⁺. 99.3% ee

(HPLC on CHIRALPAK IC 4.6 mm ID × 150 mmL), mobile phase:

H₂O/MeCN/TFA = 700/300/1, ﬂow rate = 1 mL/min, 30 °C, UV

detection: 254 nm.

(170 mL, 170 mmol, 1 N) was added to a solution of ethyl 3-

cyclopropyl-3-(2-((1-(5-methoxy-2-((6-methylpyridin-2-yl)-

(neopentyl)carbamoyl)phenyl)piperidin-4-yl)methoxy)pyridin-4-yl)-

propanoate (5, 17 g, 25 mmol) in THF (170 mL) and MeOH (85

mL) at room temperature. The mixture was stirred at 60 °C under N₂

for 30 min. The mixture was quenched with 1 N HCl aq (170 mL) at

room temperature (water bath) and extracted with EtOAc (150 mL ×

2). The organic layer was separated, washed with brine (150 mL),

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

solidiﬁed and dissolved in isopropylacetate (110 mL) at 60 °C. To the

solution was added heptane (120 mL) dropwise at 60 °C and stirred

at 30 min. Additional heptane (65 mL) was added at the same

temperature and then stirred at 60 °C for 30 min and room

temperature for 1 h. The solid was collected and washed with

heptane/isopropylacetate (3:1) to give 3-cyclopropyl-3-(2-((1-(5-

methoxy-2-((6-methylpyridin-2-yl)(neopentyl)carbamoyl)phenyl)-

piperidin-4-yl)methoxy)pyridin-4-yl)propanoic acid (13 g, 22 mmol,

87%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 0.10−0.22

(1H, m), 0.23−0.39 (2H, m), 0.45−0.56 (1H, m), 0.77 (9H, s),

0.93−1.05 (1H, m), 1.10−1.30 (1H, m), 1.45−1.60 (1H, m), 1.61−

1.86 (3H, m), 2.18−2.28 (1H, m), 2.30−2.66 (4H, m), 2.36 (3H, s),

2.68 (2H, d, J = 7.4 Hz), 3.69 (3H, s), 3.93−4.27 (4H, m), 6.21 (1H,

br s), 6.45 (1H, br s), 6.52 (1H, d, J = 8.4 Hz), 6.71 (1H, s), 6.85

(1H, d, J = 7.5 Hz), 6.91 (1H, d, J = 5.1 Hz), 7.14 (1H, d, J = 8.4 Hz),

7.28 (1H, t, J = 7.7 Hz), 8.04 (1H, d, J = 5.3 Hz), 12.09 (1H, br s).

MS m/z 615.5 (M + H)⁺. C₃₆H₄₆N₄O₅: C, 70.33; H, 7.54; N, 9.11.

Found: C, 70.24; H, 7.48; N, 9.09.

Crystallography Methods. All measurements were performed

on a Rigaku R-AXIS RAPID-191R diﬀractometer using multilayer

mirror monochromated Cu Kα radiation. The structure was solved by

direct methods with SHELXS-97³³and was reﬁned using full-matrix

least-squares on F2 with SHELXL-97. All non-H atoms were reﬁned

with anisotropic displacement parameters.

CCDC1978712 for SCO-267 and CCDC 1978713 for compound

68 contain the supplementary crystallographic data for this paper.

These data can be obtained free of charge from The Cambridge

Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/

structures.

Ethyl (3S)-3-Cyclopropyl-3-{2-[(piperidin-4-yl)methoxy]pyridin-

4-yl}propanoate (69). HCl aq (460 mL, 460 mmol, 1 N) was

added to a suspension of (S)-1-(p-tolyl)ethanamine 3-(2-((1-(tert-

butoxycarbonyl)piperidin-4-yl)methoxy)pyridin-4-yl)-3-cyclopropyl-

propanoate 68 (46 g, 86 mmol) in EtOAc (750 mL) at 0 °C. The

mixture was stirred at 0 °C for 5 min. The mixture was extracted with

EtOAc/THF (1:1, 200 mL, three times). The organic layer was

separated, washed with brine (200 mL), dried over MgSO₄, and

concentrated in vacuo to give a white solid. The white solid was

washed with i-Pr₂O to give 3-(2-((1-(tert-butoxycarbonyl)piperidin-4-

yl)methoxy)pyridin-4-yl)-3-cyclopropylpropanoic acid (35 g, 83

mmol, 97%) as a white solid (containing 4 wt % of (S)-1-(p-

1

tolyl)ethanamine). H NMR (400 MHz, DMSO-d₆): δ 0.10−0.20

(1H, m), 0.22−0.39 (2H, m), 0.44−0.58 (1H, m), 0.90−1.05 (1H,

m), 1.06−1.21 (2H, m), 1.40 (9H, s), 1.72 (2H, d, J = 13.8 Hz),

1.82−1.99 (1H, m), 2.15−2.27 (1H, m), 2.60−2.83 (2H, m), 2.68

(2H, d, J = 7.2 Hz), 3.97 (2H, d, J = 11.5 Hz), 4.08 (2H, d, J = 6.5

Hz), 6.69 (1H, s), 6.90 (1H, d, J = 5.4 Hz), 8.02 (1H, d, J = 5.3 Hz),

12.04 (1H, br s). H₂SO₄(4.8 mL, 91 mmol) was added to a solution

of 3-(2-((1-(tert-butoxycarbonyl)piperidin-4-yl)methoxy)pyridin-4-

yl)-3-cyclopropylpropanoic acid (15 g, 36 mmol) in EtOH (360

mL) at room temperature. The mixture was stirred at 80 °C for 1 h.

The reaction mixture was concentrated in vacuo. The residue was

diluted with AcOEt/THF (150/150 mL). To the suspension was

added 2 N NaOH aq (73 mL, 150 mmol) at 0 °C. The mixture was

extracted with EtOAc/THF (50/50 mL) with NaCl. The organic

layer was separated, washed with brine (50 mL), dried over MgSO₄,

and concentrated in vacuo to give the title compound (14 g, 37 mmol,

101%, 86 wt %, mixture with PhMe used for azeotropy) as colorless

oil. This product was subjected to the next reaction without further

1

puriﬁcation. H NMR (400 MHz, CDCl₃): δ 0.07−0.20 (1H, m),

0.24−0.35 (1H, m), 0.38−0.51 (1H, m), 0.55−0.66 (1H, m), 0.86−

1.05 (1H, m), 1.18 (3H, t, J = 7.2 Hz), 1.26−1.45 (2H, m), 1.85 (2H,

d, J = 13.4 Hz), 1.89−2.03 (1H, m), 2.24−2.33 (1H, m), 2.61−2.79

(4H, m), 3.17 (2H, d, J = 12.3 Hz), 3.99−4.13 (2H, m), 4.12 (2H, d,

J = 6.5 Hz), 6.61 (1H, s), 6.75 (1H, d, J = 5.3 Hz), 8.05 (1H, d, J =

5.3 Hz).

Crystal Data for SCO-267. (CCDC 1978712): C₃₆H₄₆N₄O₅, MW

= 614.78; crystal size, 0.10 × 0.07 × 0.04 mm; colorless, needle;

monoclinic, space group P1, a = 10.3497 (2) Å, b = 13.5736 (3) Å, c

= 14.4222 (3) Å, α = 103.627 (2)°, β = 107.2110 (8)°, γ =

109.413(2)°, V = 1695.28(8) Å³, Z = 2, dx = 1.204 g/cm³, T = 100 K,

μ = 0.647 mm⁻¹, λ = 1.54184 Å, R₁= 0.0415, wR₂= 0.1118, S =

1.039, Flack parameter³⁰= 0.04(9).

Ethyl (3S)-3-Cyclopropyl-3-{2-[(1-{2-[(2,2-dimethylpropyl)(6-

methylpyridin-2-yl)carbamoyl]-5-methoxyphenyl}piperidin-4-yl)-

methoxy]pyridin-4-yl}propanoate (70). A mixture of 2-ﬂuoro-4-

methoxy-N-(6-methylpyridin-2-yl)-N-neopentylbenzamide 56 (12 g,

36 mmol), ethyl 3-cyclopropyl-3-(2-(piperidin-4-ylmethoxy)pyridin-

4-yl)propanoate 69 (14 g, 37 mmol, 86 wt %), and cesium carbonate

(18 g, 54 mmol) was stirred at 130 °C under N₂for 19 h (200 mL

ﬂask, neat). After dilution with PhMe, the mixture was ﬁltered

through Celite pad. The ﬁltrate was concentrated in vacuo. The

residue was puriﬁed by column chromatography (silica gel, eluted

with 5−15% EtOAc in hexane twice) to give the desired product as a

mixture with 4. The mixture was dissolved in AcOEt and extracted

with 2 N HCl aq, and the aqueous layer was basiﬁed with 2 N NaOH

aq and sat. NaHCO₃aq. The aqueous layer was extracted with

AcOEt, washed with water and brine, dried over MgSO₄, and

concentrated in vacuo to give the title compound (17 g, 25 mmol,

69%) as a pale-orange amorphous solid. ¹H NMR (400 MHz,

CDCl₃): δ 0.11−0.24 (1H, m), 0.25−0.36 (1H, m), 0.39−0.54 (1H,

m), 0.55−0.66 (1H, m), 0.82 (9H, s), 0.93−1.06 (1H, m), 1.19 (3H,

t, J = 7.2 Hz), 1.52−1.89 (5H, m), 2.25−2.35 (1H, m), 2.40−2.86

(5H, m), 2.44 (3H, s), 3.40 (1H, br s), 3.75 (3H, s), 3.98−4.38 (6H,

m), 6.19 (1H, br s), 6.33 (1H, br s), 6.48 (1H, d, J = 8.3 Hz), 6.63

(1H, s), 6.69 (1H, d, J = 7.5 Hz), 6.76 (1H, d, J = 5.3 Hz), 7.08 (1H,

br s), 7.29 (1H, d, J = 8.4 Hz), 8.07 (1H, d, J = 5.1 Hz).

Crystal Data for Compound 68. (CCDC 1978713):

−

C₂₂H₃₁N₂O₅·C₉H₁₄N⁺, MW = 539.71; crystal size, 0.19 × 0.05 ×

0.05 mm; colorless, needle; monoclinic, space group P2₁, a =

13.1146(3) Å, b = 6.3865(2) Å, c = 17.9946(4) Å, α = γ = 90°, β =

94.942(7)°, V = 1501.56(5) Å³, Z = 2, dx = 1.194 g/cm³, T = 100 K,

μ = 0.648 mm⁻¹, λ = 1.54187 Å, R₁= 0.0924, wR₂= 0.2416, S =

1.064, Flack parameter³⁴= 0.2(5). The absolute conﬁguration was

determined using the reference to the known conﬁguration of the

(1S)-1-(4-methylphenyl)ethan-1-aminium.

Cell Lines. To establish CHO cells (dhfr-) stably expressing

hFFAR1, CHO/dhfr- (ATCC catalog # CRL-9096) cells were

transfected with hFFAR1-pAKKO-111H using Lipofectamine 2000

(Thermo Fisher Scientiﬁc). After transfection, a limiting dilution was

performed, and clone cells were selected with a selective medium,

nucleotide-free α-minimum essential medium (Thermo Fisher

Scientiﬁc) supplemented with 10% dialyzed fetal bovine serum

(Thermo Fisher Scientiﬁc), 100 IU/mL penicillin, and 100 mg/mL

streptomycin (Thermo Fisher Scientiﬁc). The expression level of

human hFFAR1 mRNA was conﬁrmed by TaqMan PCR (Thermo

Fisher Scientiﬁc), the intracellular calcium mobilization activity of γ-

linolenic acid as an hFFAR1 ligand was conﬁrmed using a

ﬂuorometric imaging plate reader (FLIPR) system (Molecular

Devices), and clones of hFFAR1-expressing CHO cells, including

clone #2 herein, were selected.

(3S)-3-Cyclopropyl-3-{2-[(1-{2-[(2,2-dimethylpropyl)(6-methyl-

pyridin-2-yl)carbamoyl]-5-methoxyphenyl}piperidin-4-yl)-

methoxy]pyridin-4-yl}propanoic Acid ((S)-7f, SCO-267). NaOH aq

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In Vitro Ca²⁺Mobilization Assay. CHO cells stably expressing

human GPR40 (clones #2) were cultured with minimum essential

medium-α (Nikken Bio Medical Laboratory) containing 10% dialyzed

fetal bovine serum (Gemini Bio-Products), 10 mmol/L N-(2-

hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES) (Thermo

Fisher Scientiﬁc), and 1× penicillin/streptomycin (Thermo Fisher

Scientiﬁc) in 5% CO₂at 37 °C. Cells were seeded at 1 × 10⁴cells/

well in a 384-well black/clear plate and then incubated overnight.

After removing the medium, cells were incubated with 30 μL of

loading buﬀer Hanks’ balanced salt solution (Thermo Fisher

Scientiﬁc) containing 20 mmol/L HEPES (Thermo Fisher Scientiﬁc),

0.1% fatty acid-free bovine serum albumin (Sigma), 2.5 mmol/L

Probenecid (Dojindo), and 2.5 μL of calcium probe (Dojindo) for 60

min in 5% CO₂at 37 °C. Test compounds at indicated concentrations

(10⁻¹⁰, 10⁻⁹, 10⁻⁸, 10⁻⁷, 10⁻⁶, and 10⁻⁵mol/L) were added to the

cells, and the increase in the intracellular Ca²⁺concentration was

monitored using an FLIPR tetra system (Molecular Devices) for 150

s. Regarding determination of the agonist activity rate, the Ca²⁺inﬂux

of no compound was set as 0% of agonist activity and the Ca²⁺inﬂux

of (S)-3-cyclopropyl-3-(3-((2-((R)-2,2-dimethylcyclopentyl)-2′-ﬂuo-

ro-5′-methoxy-[1,1′-biphenyl]-4-yl)methoxy)-2-ﬂuorophenyl)-

propanoic acid¹⁴was set as 100% of agonist activity. The agonist

activity rate (% of control) of test compounds was calculated as

state distribution volume (V_ss) was determined as the product of the

CL_totalmultiplied by the MRT after intravenous administration. The

maximum plasma concentration (C_max) was recorded from the

experimental data after oral administration. The bioavailability (F)

was determined as the quotient of dose-normalized AUC at oral

administration divided by that at intravenous administration.

LC−MS-Based Binding Assay. To get a better understanding of

compound 4a binding to GPR40, we performed an LC−MS-based

binding assay.²³GPR40 protein samples were diluted to 3 μg/mL in

the binding assay buﬀer [50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 5

mM ethylenediaminetetraacetic acid (EDTA), and 0.005% Tween 20]

and dispensed in a 96-well plate (Corning). Compound 4a was added

at 10⁻⁶to 10⁻¹²M of the ﬁnal concentration. In turn, TAK-875 and

AMG-1638 were added at the ﬁnal concentration of their K_dand 10

times of K_dat room temperature for 2 h. The K_dvalue of TAK-875 to

GPR40 was 11.9 nM and that of AMG-1638 was 5.0 nM to be

evaluated by the LC−MS-based binding assay.^23,24After the

incubation, free compounds which were not bound to GPR40 was

separated by 96-well plate-based size exclusion chromatography.³⁵

Subsequently, 70% acetonitrile and 0.2% formic acid were added to

each well to dissociate the compound bound to GPR40. The samples

were subjected to to LC−MS analysis, and the peak area of TAK-875

and AMG-1638 was quantiﬁed. Sigmoidal dose response curves were

calculated using Prism 5.03 software (GraphPad Software). Two

independent experiments in duplicate were performed. Data are

shown separately in Figures 4 and S1.

N-STZ-1.5 Rat Study. The care of the animals and use of the

experimental protocols in the current studies were approved by the

Institutional Animal Care and Use Committee (IACUC) in a Shonan

Health Innovation Park accredited by the American Association for

Accreditation of Laboratory Animal Care (AAALAC). All rats were

housed in a room with controlled temperature (23 °C), lighting

(lights remained on between 7:00 am and 7:00 pm), and humidity

(55%) and were allowed free access to standard chow diet (CE-2,

CLEA, Japan Inc.) and water. Male N-STZ-1.5 Wistar Kyoto rats (N-

STZ-1.5 rats), which are diabetic, were developed via subcutaneous

administration of 120 mg/kg streptozotocin (STZ) to Wistar Kyoto

rats (Rabics, Ltd. Kanagawa, Japan) at 1.5 days after birth. N-STZ rats

display impairments in insulin secretion and action which resemble

those observed in human patients with diabetes. To evaluate the

eﬃcacy of single dosing, 26 week-old male N-STZ-1.5 rats and normal

male rats (Wistar Kyoto rats) were fasted (12 h) and fasting glucose

levels were determined. Rats were randomized into groups (n = 6 for

each group) based on glycosylated hemoglobin levels, body weight,

and fasting glucose levels. After 15 h fasting, the vehicle (0.5%

methylcellulose solution) and the test materials (SCO-267, 0.3−1

mg/kg; Sitagliptin phosphate 10 mg/kg [Cayman]) were orally

administered. Glucose solution (1.5 g/kg) was orally loaded 60 min

after the vehicle and the test material dosing (total 16 h fasting).

Blood samples were collected before the glucose load (time 0) and 10,

30, 60, and 120 min after the glucose load, and plasma glucose was

determined using an autoanalyzer 7180 (Hitachi). Plasma insulin was

determined using an ELISA kit (M1101, Takara), and plasma total

GLP-1 was determined using an ELISA kit (EZGLP1T-36K, Merck).

Statistical Analysis. Statistical signiﬁcance was ﬁrst analyzed

using Bartlett’s test for homogeneity of variances, followed by the

Williams’ test (P > 0.05) and Shirley−Williams test (P ≤ 0.05) for

dose-dependent studies. Alternatively, statistical signiﬁcance was

analyzed using the F test for homogeneity of variances, followed by

Student’s t-test (P > 0.2) or the Aspin−Welch test (P ≤ 0.2). The

Williams’ and Shirley−Williams tests were conducted using a one-

tailed signiﬁcance level of 2.5% (0.025). Other tests were conducted

using a two-tailed signiﬁcance level of 5% (0.05). All data are

presented as mean standard deviation (S.D.).

(C_sample− C_{0% of agonist activity})/(C_{100% of agonist activity}− C_{0% of agonist activity}

)

× 100 where C indicates Ca²⁺inﬂux values. Data were expressed as %

of control. Data of 2, 4a, 4e, 4g, 5, 6a, 6b, 6c, 6d, 6e, 6h, 7f, (S)-7f,

and (R)-7f were obtained from more than two experiments in

duplicate. Data of 3, 4b, 4c, 4d, 4f, 4h, 6f, 6g, 7a, 7b, 7c, 7d, and 7e

were obtained from a single experiment in duplicate. Furthermore, the

agonist activity of test compounds for hGPR40 was shown as the EC₅₀

value from the dose−response curve. Their EC₅₀values and standard

deviation were calculated using Prism 6 software (GraphPad

Software).

Cytotoxicity Test. HepG2 cells were cultured at 37 °C, 5% CO₂

in Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented with

10% fetal bovine serum, 50 IU/mL penicillin, and 50 μg/mL

streptomycin. Cells were seeded at 2 × 10⁴cells/well in a 96-well

white plate (Corning) and cultured with test compounds or DMSO

control 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 using ATPlite-M (PerkinElmer) according to the

manufacturer’s instruction. ATP content was calculated as follows:

ATP content (% of control) = (RLU of compound/RLU of 1%

DMSO) × 100. Data were obtained from a single experiment in

triplicate. Caspase-3/7 activity was measured using a Caspase-GloTM

3/7 assay kit (Promega) according to the manufacturer’s instruction.

Caspase-3/7 activity was calculated as follows: Caspase-3/7 activity

(%) = (RLU of compound − RLU of 1% DMSO)/(RLU of 30 μM

staurosporine − RLU of 1% DMSO) × 100. Data were obtained from

a single experiment in triplicate.

Pharmacokinetic Analysis in Rat and Mouse Cassette

Dosing Studies. Three male SD rats (8 weeks old) and three

male ICR mice (8 weeks old) were used in the pharmacokinetic study.

Test compounds were administered to nonfasted rats and mice as a

cassette dosing study, which is a technique for evaluation of the

pharmacokinetic proﬁle by simultaneous administration of multiple

compounds to a single animal. After oral and intravenous

administration, blood samples were collected and centrifuged to

obtain the plasma fraction. The plasma samples were deproteinized

with acetonitrile containing an internal standard. After centrifugation,

the supernatant was diluted with 10 mmol/L ammonium acetate and

centrifuged again. The compound concentrations in the supernatant

were measured by high-performance LC−tandem MS. The area under

the plasma concentration−time curve (AUC) and the area under the

ﬁrst moment of plasma concentration−time curve (AUMC) were

calculated using a linear trapezoidal method. The mean residence time

(MRT) was derived from the AUMC divided by the AUC. The total

body clearance (CL_total) was determined as the quotient of the dose

divided by the AUC after intravenous administration, and the steady-

ASSOCIATED CONTENT

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00843.

Z

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Journal of Medicinal Chemistry

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Article

Author Contributions

Analytical RP-HPLC traces and another competitive

The manuscript was written through contributions of all

authors. All authors have given approval to the ﬁnal version of

the manuscript.

binding assay result of 4a (PDF)

Molecular formula strings with human GPR40 EC₅₀data

and E_max(%) (CSV)

Notes

The authors declare no competing ﬁnancial interest.

Accession Codes

Atomic coordinates and structure factors for the crystal

structures of SCO-267 and compounds 68 are deposited in

The Cambridge Crystallographic Data Centre with the

deposition number 1978712 and 1978713, respectively.

Authors will release the atomic coordinates and experimental

data upon article publication.

ACKNOWLEDGMENTS

■

We thank Motoo Iida for conducting X-ray analysis to

determine the structure of the compounds, Tomoyuki Odani

and Takeshi Matsubayashi for in vitro proﬁling activities, and

Shinichi Abe for initial exploration of the OGTT system. We

are grateful to Ikumi Chisaki, Nobuyuki Amano, and Dr.

Junichi Sakamoto for their helpful suggestions and comments

on the manuscript.

AUTHOR INFORMATION

■

Corresponding Authors

ABBREVIATIONS

■

Hideki Furukawa − Research, Takeda Pharmaceutical

Company, Ltd., Fujisawa, Kanagawa 251-8555, Japan;

orcid.org/0000-0002-9894-7264; Phone: +81 466 32

1078; Email: hideki.furukawa@takeda.com; Fax: +81 466

29 4475

Tsuyoshi Maekawa − Research Division, SCOHIA PHARMA

Inc., Fujisawa, Kanagawa 251-8555, Japan; Phone: +81-70-

3875-1081; Email: tsuyoshi.maekawa@scohia.com;

Fax: +81-466-29-4427

GPR40, G protein-coupled receptor 40; T2DM, type 2

diabetes mellitus; FFA, free fatty acid; GSIS, glucose-

stimulated insulin secretion; FLIPR, ﬂuorometric imaging

plate reader; TBAF, tetrabutylammonium ﬂuoride; CHO,

Chinese hamster ovary; PG, plasma glucose; OGTT, oral

glucose tolerance test

REFERENCES

■

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Authors

Yasufumi Miyamoto − Research, Takeda Pharmaceutical

Company, Ltd., Fujisawa, Kanagawa 251-8555, Japan

Yasuhiro Hirata − Research, Takeda Pharmaceutical Company,

Ltd., Fujisawa, Kanagawa 251-8555, Japan

Koji Watanabe − Research Division, SCOHIA PHARMA Inc.,

Fujisawa, Kanagawa 251-8555, Japan

Yuko Hitomi − Research, Takeda Pharmaceutical Company,

Ltd., Fujisawa, Kanagawa 251-8555, Japan

Yayoi Yoshitomi − Research, Takeda Pharmaceutical Company,

Ltd., Fujisawa, Kanagawa 251-8555, Japan

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2003, 93, 249−258.

Jumpei Aida − Research, Takeda Pharmaceutical Company,

Ltd., Fujisawa, Kanagawa 251-8555, Japan; orcid.org/

0000-0002-7546-1915

Naoyoshi Noguchi − Research Division, SCOHIA PHARMA

Inc., Fujisawa, Kanagawa 251-8555, Japan

Nobuyuki Takakura − Research, Takeda Pharmaceutical

Company, Ltd., Fujisawa, Kanagawa 251-8555, Japan

Kazuaki Takami − Research, Takeda Pharmaceutical Company,

Ltd., Fujisawa, Kanagawa 251-8555, Japan

Seiji Miwatashi − Research, Takeda Pharmaceutical Company,

Ltd., Fujisawa, Kanagawa 251-8555, Japan

Yoshihiko Hirozane − Research, Takeda Pharmaceutical

Company, Ltd., Fujisawa, Kanagawa 251-8555, Japan

Teruki Hamada − Research, Takeda Pharmaceutical Company,

Ltd., Fujisawa, Kanagawa 251-8555, Japan

Ryo Ito − Research, Takeda Pharmaceutical Company, Ltd.,

Fujisawa, Kanagawa 251-8555, Japan

Mitsugi Ookawara − Research Division, SCOHIA PHARMA

Inc., Fujisawa, Kanagawa 251-8555, Japan

Yusuke Moritoh − Research Division, SCOHIA PHARMA Inc.,

Fujisawa, Kanagawa 251-8555, Japan

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Masanori Watanabe − Research Division, SCOHIA PHARMA

Inc., Fujisawa, Kanagawa 251-8555, Japan

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c00843

AA

https://dx.doi.org/10.1021/acs.jmedchem.0c00843

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

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J. Med. Chem. XXXX, XXX, XXX−XXX

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Design and Identification of a GPR40 Full Agonist (SCO-267) Possessing a 2-Carbamoylphenyl Piperidine Moiety

DOI: 10.1021/acs.jmedchem.0c00843

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