Discovery of (1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide (E2006): A Potent and Efficacious Oral Orexin Receptor Antagonist

Article

pubs.acs.org/jmc

Discovery of (1R,2S)‑2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-

(3-ﬂuorophenyl)‑N‑(5-ﬂuoropyridin-2-yl)cyclopropanecarboxamide

(E2006): A Potent and Eﬃcacious Oral Orexin Receptor Antagonist

Yu Yoshida,*^,†Yoshimitsu Naoe,^†Taro Terauchi,^†Fumihiro Ozaki,^†Takashi Doko,^†Ayumi Takemura,^†

Toshiaki Tanaka,^†Keiichi Sorimachi,^†Carsten T. Beuckmann,^‡Michiyuki Suzuki,^‡Takashi Ueno,^§

Shunsuke Ozaki,^∥and Masahiro Yonaga^†

^†Medicinal Chemistry, ^‡Biopharmacology, ^§Physical Chemistry, and ^∥Drug Metabolism and Pharmacokinetics, Eisai Product Creation

Systems, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan

S

* Supporting Information

ABSTRACT: The orexin/hypocretin receptors are a family of

G protein-coupled receptors and consist of orexin-1 (OX₁)

and orexin-2 (OX₂) receptor subtypes. Orexin receptors are

expressed throughout the central nervous system and are

involved in the regulation of the sleep/wake cycle. Because

modulation of these receptors constitutes a promising target

for novel treatments of disorders associated with the control of

sleep and wakefulness, such as insomnia, the development of

orexin receptor antagonists has emerged as an important focus

in drug discovery research. Here, we report the design, synthesis, characterization, and structure−activity relationships (SARs) of

novel orexin receptor antagonists. Various modiﬁcations made to the core structure of a previously developed compound (-)-5,

the lead molecule, resulted in compounds with improved chemical and pharmacological proﬁles. The investigation aﬀorded a

potential therapeutic agent, (1R,2S)-2-{[(2,4-dimethylpyrimidin-5-yl)oxy]methyl}-2-(3-ﬂuorophenyl)-N-(5-ﬂuoropyridin-2-yl)-

cyclopropanecarboxamide (E2006), an orally active, potent orexin antagonist. The eﬃcacy was demonstrated in mice in an in

vivo study by using sleep parameter measurements.

safety and eﬃcacy of treatments that target the GABA signaling

pathway.^3,4Notably, two new non-GABA-related sleep drugs

have recently been approved for the treatment of insomnia.

Ramelteon, a melatonin (MT) receptor MT₁/MT₂agonist, was

approved in 2005, and doxepin, a histamine H₁receptor

antagonist, received regulatory approval in 2010. In contrast to

benzodiazepine and nonbenzodiazepine agents, these two

sleep-aid drugs are nonscheduled drugs in the U.S.. However,

questions remain about the eﬀectiveness of ramelteon and

doxepin, owing to the limited number of reports demonstrating

their superiority to other sleep drugs, including those that

positively modulate GABA_A.^5,6The medical needs of patients

with insomnia warrant the development of sleep medications

with novel mechanisms of action.

Toward that end, antagonism of the orexin receptor may

serve as a promising route. Orexin A and orexin B, also known

as hypocretin 1 and hypocretin 2, are endogenous neuropeptide

ligands for orexin/hypocretin receptors (OXRs), which are G

protein-coupled receptors (GPCRs). Orexin-1 receptor

(OX₁R) and orexin-2 receptor (OX₂R) mediate the action of

these ligands by postsynaptic neuronal signal transduction.

OX₁R and OX₂R are expressed throughout the central nervous

INTRODUCTION

■

Insomnia, a disorder in which the patient suﬀers from an

inability to sleep or stay asleep for as long as desired,

constitutes a widespread issue in today’s society, as

approximately 30% of adults in the United States experience

some symptoms of insomnia and approximately 10% describe

their insomnia as chronic and/or severe. Despite the prevalence

of this disorder, research regarding medical treatments for

insomnia is relatively limited.¹

The eﬀects of insomnia are dramatic, as it leads to a

reduction in the quality of life, poor productivity, and a high

risk of traﬃc and work-related safety incidents. Additionally,

insomnia is a costly disorder, as it is estimated to cost over $63

billion per year in the United States.²Commonly prescribed

medications for the treatment of insomnia include agents that

positively modulate GABA_Areceptors, with zolpidem, a

nonbenzodiazepine sleep drug, being the current market leader.

Nonbenzodiazepine sleep aids are structurally distinct from

benzodiazepines, as reﬂected in their naming, and bind to

benzodiazepine sites with high speciﬁcity. Additionally, their

pharmacological proﬁles are believed to be better in

comparison with those of benzodiazepines owing to the less

severe side eﬀects. Despite the availability of various sleep-

modifying drugs, the prevalence of insomnia has not decreased

substantially because of remaining concerns about the overall

Received: February 6, 2015

A

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Figure 1. Orexin receptor dual antagonists that have advanced to clinical trials.

system and are involved in the regulation of the sleep/wake

cycle.⁷⁻⁹To date, the speciﬁc contribution of OXR to the

regulation of the sleep/wake cycle has recently starting

emerged. A number of genetic and pharmacological studies

have suggested that the orexin pathway plays an important role

in regulating the sleep/wake cycle.¹⁰⁻¹⁹Studies performed in

orexin/ataxin-3 transgenic mice and rats, which lack orexin

peptide-producing neurons, and OXR-deﬁcient mice have

shown that both receptors are involved in the regulation of

the sleep/wake cycle because the lack of activation of both

receptors results in a narcolepsy−cataplexy phenotype, while

the lack of activation of either receptor alone elicits an

attenuated sleep phenotype.²⁰⁻²³Furthermore, very low levels

of orexin in cerebrospinal ﬂuid (CSF), indicative of nearly

complete or complete loss of orexinergic neurons, have been

observed in humans suﬀering from narcolepsy−cataplexy

syndrome.²⁴⁻²⁶

has entered another dual OXR antagonist ﬁlorexant (4) into a

phase II clinical trial for multiple indications, including the

treatment of insomnia (Figure 1).³⁵

In this paper, we report the synthesis, structure−activity

relationships (SARs), optimization of drug-likeness parameters,

and in vivo eﬃcacy of a series of cyclopropane compounds

reported previously.³⁶Our eﬀorts in this area led to the

discovery of 34 (E2006) as a promising dual OXR antagonist

that has advanced into clinical trials.

A novel series of compounds containing a cyclopropane core

structure were identiﬁed as promising orally active orexin

receptor antagonists, as exempliﬁed by (−)-5³⁶(Table 1).

(−)-5 exhibits low-nanomolar aﬃnity toward human OX₂R, as

measured by radio ligand-displacement binding assays.

However, this compound exhibits a number of drawbacks in

its drug-likeness parameters that need to be improved, such as

A dual OXR antagonist, almorexant, evaluated by Actelion

Pharmaceuticals/GlaxoSmithKline was found to signiﬁcantly

decrease the behavioral indices of wakefulness in animals and

has shown eﬀectiveness in clinical trials.¹¹Recently, a number

of orally active selective OX₂R antagonists have been evaluated

in animal studies.^27,28However, whether a dual receptor

antagonist²⁹or an OX₂R-selective antagonist³⁰will be better

suited for use as a sleep aid is a topic of ongoing debate. We

think that it is feasible to speculate that a sleep drug which

promotes both non-REM and REM sleep would provide

patients with a more natural sleep architecture, and we are

providing animal data in this manuscript which suggest that a

dual orexin antagonist is a potential candidate to fulﬁll this

requirement.

Table 1. Properties of Compound (−)-5

in vitro

binding

a

aﬃnity

solubility

(μM)

metabolic

e

d

(K_i, nM)

TDI of CYP3A

stability

pH

(% of control at

(residual

ratio %)

b

c

compd OX₂R OX₁R 7.4

1.2

10 μM)

To date, a number of dual OXR antagonists have been

described. Almorexant (1) and was tested in the treatment of

insomnia,³¹but a phase III clinical trial was discontinued. SB-

649868 (2) from GlaxoSmithKline proceeded to a phase II

clinical trial.³²Suvorexant (3), developed by Merck, received

approval from the Pharmaceutical and Medical Devices Agency

in Japan and the US Food and Drug Administration (FDA) in

2014 and is marketed for the treatment of insomnia.^33,34Merck

(−)-5 106 15

5

25

49

80

DMSO solution precipitation method.³⁶Dulbecco’s phosphate-

a

b

c

buﬀered saline (PBS). Japanese Pharmacopoeia 1st ﬂuid (aqueous

d

HCl solution containing 34 mM NaCl). Time-dependent inhibition

evaluated using a cocktail of probe substrates with human liver

e

microsomes. Metabolism after incubation of compounds at 0.3 μM in

0.1% DMSO with human liver microsomes for 15 min.

B

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a

Scheme 1

a

Reagents and conditions: (a) (1) NaHMDS, THF, 0 °C, 3 h, (2) KOH, EtOH, reﬂux, 8 h, (3) HCl, 0 °C−rt, 3 h; (b) NaBH₄, MeOH−THF, 0

°C−rt; (c) TBDPS-Cl, imidazole, DMF, −10 °C−rt, or vinyl acetate, lipase acrylic resin from Candida antarctica, rt; (d) Ar₁-OH, DIAD, PPh₃, 0

°C−rt, overnight or (1) MsCl, TEA, DCM, (2) Ar₁-OH, Cs₂CO₃, MeCN, 70 °C; (e) TBAF, THF, rt, 1 h or NaOH, EtOH−H₂O, rt, 1 h (2 steps);

(f) (COCl)₂, DMSO, TEA, DCM, −78 °C−rt, 1 h; (g) 2-methyl-2-butene, NaClO₂, NaH₂PO₄, acetone−H₂O, rt, 2 h; (h) amine, HATU, DIPEA,

DMF, 60 °C, overnight or (COCl)₂, cat. DMF, DCM, rt, 1 h, then amine, DIPEA, THF, 60 °C.

its time-dependent inhibition (TDI) of cytochrome P450 3A

(CYP3A) and low aqueous solubility under both acidic and

neutral conditions (pH 1.2 and pH 7.4). (−)-5 also

demonstrated moderate reversible inhibition of CYPs (IC₅₀

values of 8 and 8.6 μM for CYP2C8 and CYP2C19,

respectively, and >10 μM for CYP1A2, CYP2C9, and

CYP2D6). In an eﬀort to ﬁnd a clinical candidate within the

chemical series, the A, B, and C rings in the molecule were

optimized through chemical modiﬁcations.

aﬀorded carboxylic acid intermediates 18a−i. Subsequent

amidation of 18a−i with various amines yielded the desired

products 19−38.

Components of the heteroaromatic A ring were synthesized

as outlined in Schemes 2 and 3. Pyrazole derivatives 40a and

a

Scheme 2

RESULTS AND DISCUSSION

■

Chemistry. The general synthetic route used to produce the

cyclopropane compounds is presented in Scheme 1. Chiral

cyclopropane ring formation was carried out by the reaction of

corresponding aryl acetonitriles 6−12 with (R)-epichlorohydrin

to yield the desired lactones 13a−f.³⁷The reported racemic

compounds³⁶can be obtained using racemic epichlorohydrin

instead of (R)-epichlorohydrin by following a procedure similar

to the one described in Scheme 1. Reduction of the lactone

with sodium borohydride aﬀorded diol products 14a−f in

excellent yields. Protection of the hydroxy group of 14a with

TBDPS-Cl gave the desired monoprotected compound 15a. In

the selective monoacylation of diols 14b−f, enzymatic acylation

with lipase acrylic resin from Candida antarctica was

successfully applied in the presence of vinyl acetate to obtain

compounds 15b−f in good yields. The Mitsunobu reaction of

the corresponding alcohols with various phenols led to the

production of 16a−i. Deprotection of 16a and 16g−i using

TBAF, or hydrolysis of the acyl group of 16b−f with sodium

hydroxide, followed by Swern oxidation and Pinnick oxidation,

a

Reagents and conditions: (i) m-CPBA, CHCl₃, rt, overnight.

40b were synthesized from commercially available aldehydes

39a and 39b via Baeyer−Villiger oxidation (Scheme 2). The

Pd-catalyzed coupling of commercially available 2,4-dichloro-5-

methoxy pyrimidine 41 with trimethylaluminum aﬀorded 42 in

a good yield. An analogous transformation yielded 45 from

chloro intermediate 44, which was accessed by iron-catalyzed

ethylation of 41 with ethyl magnesium chloride (Scheme 3).

Deprotection of the methoxy group of 42 and 45 in the

presence of BBr₃produced hydroxypyrimidines 43 and 46.

Compound 49 was synthesized from 43. Protection of the

hydroxyl group with benzyl bromide followed by selective

bromination of the methyl group in the 4 position yielded the

crude bromomethyl-containing compound. Subsequent sub-

stitution of the alkyl bromide in the presence of sodium

C

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a

Scheme 3

a

Reagents and conditions: (a) Me₃Al, Pd(PPh₃)₄, THF, 75 °C, overnight; (b) BBr₃, DCM, rt, 4 d; (c) EtMgCl, Fe(acac)₃, THF, rt, overnight; (d)

Me₃Al, Pd(PPh₃)₄, THF, 70 °C, 2 d; (e) BBr₃, DCM, rt, 4 d; (f) BnBr, NaH, THF, 0 °C to rt, overnight; (g) Br₂, CHCl₃, 0 °C to rt, overnight, then

NaOMe, MeOH, 90 °C, 12 h; (h) Pd-C, H₂gas, EtOAc, rt, 1 h.

methoxide aﬀorded methoxymethyl product 48. Deprotection

of the benzyl group gave desired product 49.

Pharmacology. Our eﬀorts were primarily aimed at

reducing the TDI eﬀect on CYP3A and improving the aqueous

solubility. The TDI eﬀect was believed to be related to the

demethylation of the 4-OCH₃or 3-OCH₃groups on the A-

ring, where a second oxidative metabolism step may produce

quinone intermediates that can react with nucleophiles.³⁸

Therefore, to resolve the TDI and solubility issues, we tried to

introduce a substituted hetero Ar into the A-ring position

instead of 3,4-di-OMe-Ph. According to our previous work, the

methoxy groups are critical pharmacophores; the orientations

of the small lipophilic group and lone pair were shown to

strongly inﬂuence in vitro binding aﬃnity.³⁶On the basis of the

information regarding 3,4-di-OMe-Ph in the Cambridge

Structural Database (CSD), the Me groups face opposite

directions (Figure 2). Therefore, two types of dimethylpyr-

idines (50 and 51) were designed with the hope of imitating

the pharmacophores of 3,4-di-OMe-Ph, the lone pair and small

lipophilic group (Table 2).

Experimentally, dramatically diﬀerent in vitro binding

aﬃnities were observed between 50 and 51, as 3-pyridine-

containing 50 exhibited a moderate aﬃnity, whereas

Figure 2. Overlay of (−)-5 and 50 and 51.

introduction of the 4-pyridine-containing 51 resulted in

reduced in vitro binding aﬃnity toward both OX₁R and

OX₂R. From these results, computational simulations of

superposition using Molecular Operating Environment

(MOE, Ryoka System, Inc., Tokyo, Japan) were conducted

with (−)-5 and 50 or 51, with a particular focus on the position

of the three aromatic rings and the di-OMe substituents (Figure

2). Accordingly, we considered the superposition of the

aromatic rings of 50 and 51 with (−)-5, together with the

overall molecular shape including directionality of the ether

linker, the angle of the upper-left aromatic ring (C-ring), and

the direction of the NH of amide bond. On the basis of these

observations, it is reasonable to exhibit better in vitro binding

aﬃnity for 50 than 51. We successfully converted 3,4-di-OMe-

Ph to a hetero Ar group with reasonable activity, and 50

showed improved solubility. This ﬁnding encouraged us to

further investigate hetero Ar rings other than dimethylpyridine

to improve the parameters, although the compound did not

show an improvement in the TDI.

Dimethyl pyrazole 52, which was expected to meet the

structural requirements, was synthesized and it showed an

aﬃnity similar to that shown by 50 as well as a signiﬁcant

improvement in the TDI. To further increase the aﬃnity,

various substituents were installed at the 1-position of pyrazole

52, owing to the results of the computational simulations.

Ethyl-substituted 53 was thought to have more favorable

interactions with the small lipophilic site, and it exhibited an in

vitro binding aﬃnity comparable to that of (−)-5. This racemic

mixture could be resolved by chiral high-performance liquid

chromatography (HPLC) to provide (+)-53 and (−)-53. The

D

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Table 2. A-Ring Modiﬁcations: Compounds 50−53 and 19−22

a

b

c

K_ivalues are calculated from single experiments run in triplicate. Measured in Dulbecco’s PBS by DMSO solution precipitation method.³⁶Time-

dependent inhibition using a cocktail of probe substrates with human liver microsomes. P-Glycoprotein (P-gp) transport assay.³⁶Derived from

rac-epichlorohydrin. Derived from (R)-epichlorohydrin.

d

e

f

two stereoisomers exhibited dramatically diﬀerent receptor

aﬃnities; the (−)-53 isomer exhibited potent aﬃnity toward

both OX₂R and OX₁R, while the (+)-53 isomer did not.

Notably, it was found that (−)-53 could be synthesized

stereoselectively from (R)-epichlorohydrin using the procedure

shown in Scheme 1,³⁶suggesting that (−)-53 had the chiral

conﬁguration of (1R,2S), which is the preferred conﬁguration

for OXR antagonist activity.

Another approach for enhancing the in vitro receptor binding

aﬃnity was attempted in diazine derivatives, as exempliﬁed by

pyrimidine- or pyrazine-containing compounds 19 and 21.

Speciﬁcally, this approach was carried out to modulate the pK_a

of the ring because the di-OMe phenyl moiety of (−)-5 is

nonbasic. As a result, a 5-fold increase in the in vitro binding

aﬃnity was achieved with compound 19. Compound 20, which

contained an Et group instead of the Me group in 19, was

synthesized with the same expectations as we had for Et

substituted pyrazole (−)-53; it exhibited increased in vitro

binding aﬃnity compared to that shown by 19 and exhibited an

aﬃnity comparable to that of (−)-5. Moreover, the pyrazole

and pyrimidine derivatives, especially 19, showed some

improved properties compared to those of (−)-5, namely a

reduced TDI (96% for 19 vs 49% for (−)-5) and aqueous

solubility: 80 μM for 19 vs 15 μM for (−)-5. Pyrazine 21

exhibited a similar aﬃnity to those of the pyrimidine derivatives

but exhibited a concomitant deterioration in certain aspects of

its physicochemical and drug likeness such as its aqueous

solubility and reversible inhibition of CYPs (data not shown).

Further reduction of the lipophilicity by the introduction of an

OMe group in the 4-position of the dimethyl pyrimidine, as

exempliﬁed in 22, resulted in a stronger binding aﬃnity than

that of 19. However, 22 served as a substrate for P-glycoprotein

(P-gp) with a corrected ﬂux ratio of 4.0. Evaluation of the SARs

of the A ring revealed that dimethyl pyrimidine-containing 19

possessed the optimal proﬁle in terms of overall balance

because it exhibited a low lipophilicity (ClogP of 3.3 for 19 vs

3.9 for (−)-5) with acceptable aﬃnity toward OX₂R, improved

TDI and solubility, and moderate liability in a P-gp transport

assay. Therefore, compound 19 was selected for optimization of

the B-ring moiety.

First, 2-, 3-, and 4-pyridine were introduced to conﬁrm the

SAR regarding the position of nitrogen, as a comparison with

our previous work (Table 3).³⁶As a consequence, increased P-

gp susceptibility was observed in 3- and 4-pyridine-containing

E

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Table 3. Results of B-Ring Exploration: Compounds 23−32

a

b

K_ivalues are calculated from single experiments run in triplicate. Time-dependent inhibition using a cocktail of probe substrates with human liver

microsomes. P-gp transport assay.³⁶Sleep measurements conducted using an established method.³⁶

c

d

Figure 3. Wakefulness time measured after oral administration of 26, 28, and 31. The vehicle (10% Cremophor EL, 5% DMSO in saline; n = 4 for

26, n = 7 for 28 and 31), 26 (n = 4), 28 (n = 5), and 31 (n = 6) at 30 mg/kg doses were administered immediately prior to the beginning of the dark

cycle; cumulative wakefulness times were measured during the ﬁrst 3 h after administration. Values are mean SEM.* P < 0.05, ** P < 0.01, and

*** P < 0.001 versus vehicle control (unpaired parametric two-tailed t test).

derivatives (24 and 25, respectively), with a close resemblance

to previously identiﬁed SARs involving the derivatization of the

di-OMe phenyl series.³⁶Subsequently, we evaluated the SARs

of 2-pyridine-containing moieties in the B-ring, with the aim of

enhancing the in vitro binding aﬃnity and in vivo eﬃcacy

without adversely aﬀecting other parameters. Introduction of

substituents in the 5-position, such as in compounds 26 and 28,

resulted in a greater in vitro binding aﬃnity than that of 23. In

contrast, the introduction of an Me substituent in the 4- or 5-

position (29, 30) also resulted in increased in vitro binding

aﬃnity relative to that of 23 although the magnitude of the

observed increase was smaller with an Me group than with a

halogen. These results suggested that the basicity of the B-ring

may inﬂuence the in vitro binding aﬃnity. Accordingly,

incorporation of ﬂuorine in the 5-position and an Me

substituent in the 4-position (31) resulted in increased in

vitro binding aﬃnity, which was more potent than that of

compound 30. However, 31 exhibited decreased solubility

compared to that of 26 (31, 51 μM, vs 26, 92 μM, at pH 7.4).

F

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Table 4. Exploration of C-Ring with Diﬀerent Combinations of B and C-Rings in Compounds 33−38

a

b

K_ivalues are calculated from single experiments run in triplicate. Time-dependent inhibition using a cocktail of probe substrates with human liver

microsomes. P-gp transport assay.³⁶Exploratory screening of inhibition of the human ether-a-go-go related gene (hERG) potassium channel.³⁹

c

d

Introduction of an OMe group in the 4-position (32) also

elicited a strong increase in the in vitro binding aﬃnity.

With highly potent compounds 26, 28, and 31 in hand, in

vivo characterizations were performed. The eﬀects of these

three compounds on wakefulness were evaluated in sleep

experiments with mice via electroencephalogram/electromyo-

gram (EEG/EMG) recordings during the ﬁrst 3 h of the dark

phase, as shown in Figure 3.³⁶All compounds elicited a

signiﬁcant reduction in the time of wakefulness following an

oral dose of 30 mg/kg (Figure 3). Because of its well-balanced

in vitro proﬁles taking into account the in vitro binding aﬃnity,

solubility, and other parameters, compound 26 was selected for

optimization of the C ring.

Previous synthetic eﬀorts regarding the cyclopropane series

were focused on modifying the A- and B-rings, while

modiﬁcations of the C-ring were not explored. To investigate

the SARs owing to changes in this component, a ﬂuorine scan

was conducted to explore favorable positions for substitutions

(Table 4). Substitution at the 3-position (34) slightly increased

the in vitro binding aﬃnity relative to that of 26 without

altering other parameters, while 2- or 4-F derivatives (33, 35)

exhibited 2−3-fold decreases in the in vitro binding aﬃnity or

increased hERG inhibition. 3,5-Diﬂuoro-containing derivative

36 and 3,4-diﬂuoro-containing derivative 37 exhibited strong in

vitro binding aﬃnities, but 37 also exhibited increased hERG

inhibitory activity. Compound 38 was derived from 31, which

exhibited strong in vivo eﬃcacy and relatively weak hERG

inhibitory activity (31: hERG IC₅₀> 10 μM). Compound 38

exhibited an enhanced in vitro binding aﬃnity compared to that

of 26, with reduced hERG inhibition, as expected. However, 38

exhibited decreased solubility (34, 74 μM, vs 38, 46 μM at pH

7.4).

for further proﬁling (Table 5). Compound 34 exhibited very

weak reversible inhibition of CYP with IC₅₀values of >20 μM

Table 5. In Vitro Properties of Selected Compound 34

assay

34

hOX2R RBA K_i(nM)

3

a

b

hOX2R cell-based functional assay K_i(nM)

hOXIR RBA K_i(nM)

0.44

6

a

b

hOXIR cell-based functional assay K_i(nM)

5.7

1.6

76

c

corrected P-gp FR (MDR1 ﬂux ratio/PK1 ﬂux ratio)

d

TDI (CYP3A) (%)

e

solubility (pM)

f

pH 1.2

>100

74

g

pH 7.4

h

metabolic stability (residual ratio %)

human

90

a

b

See cell-based functional assay section in Experimental section. K_i

c

values are calculated from single experiments run; P-gp transport

assay;³⁶Time-dependent inhibition using cocktail of probe substrates

d

e

with human liver microsomes; DMSO solution precipitation

method;³⁶Japanese Pharmacopoeia 1st ﬂuid; Dulbecco’s PBS;

f

g

h

Metabolism after incubation at 0.1 μM in 0.1% DMSO with

human liver microsomes for 15 min.

for CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and

CYP3A. The aqueous solubility of 34 at room temperature was

74 μM at pH 7.4 and >100 μM at pH 1.2. Compound 34

showed weak TDI eﬀects on CYP3A, measured as 76% of the

control at 10 μM. Further evaluations of the possible risk of

drug−drug interactions caused by CYP3A TDI are warranted,

but the potential risk is expected to be marginal when the in

vitro binding aﬃnity and TDI screening data are considered.

The pharmacokinetic properties of 34 were evaluated in male

mice after intravenous and oral administration at 3 mg/kg iv

On the basis of the SAR studies, compound 34 showed the

most promising balance of in vitro properties and was selected

G

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Table 6. Pharmacokinetic Parameters for 34 (iv and po) in Mice

iv (1 mg/kg)

po (10 mg/kg)

po (30 mg/kg)

a

PK parameter

mean

1060

SD

375

mean

SD

mean

SD

CL (mL/h/kg)

V_ss(mL/kg)

AUC_(0−inf)(ng·h/mL)

t_1/2(h)

2710

1030

1380

368

1910

2.20

0.25

488

18.5

403

1.00

4130

1.94

1.00

961

13.4

555

0.585

2.16

0.289

t_max(h)

(0.25−0.50)

25.2

(0.50−1.00)

C_max(ng/mL)

127

b

F (%)

a

b

See the Experimental section F (%) = ((mean AUC_(0−inf), po/dose_po)/(mean AUC_(0−inf), iv/dose_iv)) × 100.

and 10 mg/kg po doses (Table 6). The results at 10 mg/kg po

dose exhibited a plasma clearance of 1060 mL/h/kg, a half-life

of 2.16 h for iv and 2.20 h for po administration, a T_maxof 0.25

h, and an oral bioavailability of 18.5%. A C_maxexhibited 488 ng/

mL for 10 mg/kg and 961 ng/mL for 30 mg/kg po

administration. In addition, brain penetration of 34 was

evaluated in mice after 1 and 3 h following a single oral

administration of 10 mg/kg. After 1 h postdose, the

concentration of 34 reached 32.8 nmol/L in CSF and 633.3

nmol/L in plasma and reached 7.6 nmol/L in CSF and 157.6

nmol/L in plasma at 3 h, with 85.2% of the compound bound

to plasma protein, indicating that 34 had suﬃcient brain

penetrability and exposure was clearly above K_ion the target

(Tables 5 and 7). Therefore, 34 was subjected to eﬃcacy

evaluations through measurements of its eﬀects on sleep in

mice.

compounds probably can be explained by the diﬀerence of

OXRs selectivity proﬁles, (−)-5 is more OX₂R selective

antagonist than 34, which is nearly dual antagonist (about 3-

fold selective for OX₂R). Our results strongly indicate that

REM sleep increase is caused via OX₁R antagonism. This result

is in alignment with orexin receptor KO mouse studies,^40,41

which demonstrate that both receptors are involved in sleep/

wake regulation, with OX₂R more regulating non-REM sleep

and OX₁R more regulating REM sleep. In addition, another

sleep study, where orexin receptors were functionally inabled by

pharmacological intervention by applying OX₂R selective or

dual orexin receptor antagonists, has indicated similar results

regarding the roles of OX₁R and OX₂R for sleep architecture.⁴²

Thus, in our humble opinion, the selectivity ratio for OX₁R and

OX₂R can clearly explain the sleep architecture induced by both

compounds. When clinical data using a SORA will become

available in the future, it will be important for deepening our

understanding of the predictability of rodent’s sleep architec-

ture data for humans. Nonetheless, we believe that both

receptors should be targeted to promote physiological sleep by

increasing both non-REM and REM sleep. Therefore,

compound 34 is thought to induce a more favorable sleep

architecture than (−)-5.

Table 7. Mouse Brain Penetration Data of 34

34 (10 mg/kg)

a

CSF/plasma (nmol/L)

1 h

3 h

plasma

CSF

633.3 67.8

32.8 22.1

157.6 22.3

7.6 2.3

85.2

plasma

CSF

b

PPB (%)

C57BL/6N mice

CONCLUSION

■

a

b

See the Experimental section. See the Experimental section.

In conclusion, we optimized the structure of cyclopropane

compounds based on (−)-5 as a lead OXR antagonist

compound. Modiﬁcations of the A, B, and C-ring, and

appropriate combinations of these alterations, aﬀorded several

potentially valuable compounds. Compared to (−)-5, signiﬁ-

cant improvements in multiple aspects were achieved by our

eﬀorts. Speciﬁcally, 34 exhibited an overall improvement in

other properties such as the TDI of CYP3A and aqueous

solubility, which were previously recognized as the major

drawbacks of (−)-5. Additionally, 34 exhibited high OXRs

antagonist activity and suﬃcient brain penetration. Further-

more, 34 demonstrated high eﬃcacy in preclinical sleep

experiments. On the basis of these ﬁndings, it has been

selected as a candidate compound, E2006, for further clinical

evaluations toward the treatment of sleep disorders. Additional

preclinical and clinical studies regarding this series will be

reported in due course.

Compound 34 was administered orally at 10 and 30 mg/kg

in mice, and the eﬀect on sleep was evaluated (Figure 4).³⁶

Accordingly, 34 resulted in a signiﬁcant decrease in time of

wakefulness during the ﬁrst 3 h following administration of 10

and 30 mg/kg doses (77.1 and 75.3 min, respectively, for 34 vs

116.7 min for vehicle, P < 0.001, Figure 4D). Both non-REM

and REM sleep were increased following oral administration of

10 and 30 mg/kg doses (99.3 min at 10 mg/kg and 95.5 min at

30 mg/kg for 34 vs 62.6 min for vehicle, P < 0.01, Figure 4E;

3.6 min at 10 mg/kg and 9.3 min at 30 mg/kg for 34 vs 0.6 min

for vehicle, not signiﬁcant and P < 0.001, respectively, Figure

4F). Non-REM sleep-promoting eﬀect in this sleep experiment

was thought to be saturated at 10 and 30 mg/kg dose by taking

of the PK results into consideration (Table 6). These data

suggested that the compound 34 possessed robust sleep-

promoting eﬀects³⁶and potential as a drug to treat sleep

disorders such as insomnia.

EXPERIMENTAL SECTION

Chemistry. H NMR spectra were recorded on a Bruker Avance

spectrometer (operating at 600 MHz) or Varian Mercury 400

spectrometer (operating at 400 MHz). ¹³C NMR spectra were

recorded on a Bruker Avance spectrometer (operating at 150 MHz).

Chemical shifts were calculated in ppm (δ) from the residual CHCl₃

signal at (δH) 7.26 ppm or DMSO signal at (δH) 2.50 and (δC) 77.0

■

Interestingly, the eﬀects on sleep architecture were diﬀerent

between lead compound (−)-5³⁶and lead optimized

compound 34 (Figure 4). Especially, 34 caused a dose-

dependent increase of REM sleep time, while (−)-5 did not

provoke REM-sleep even up to highest dose (100 mg/kg po).³⁶

The diﬀerence of the eﬀects on REM sleep by these

1

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Figure 4. Wake and sleep time measurements after administration of 34. (A−C) Vehicle (10% Cremophor EL, 5% DMSO in saline; n = 7) or 34

(10 or 30 mg/kg; n = 6) were orally administered immediately prior to the dark cycle (lights oﬀ), and (A) wakefulness, (B) non-REM sleep, and (C)

REM sleep were measured hourly. (D−F) Cumulative amounts of (D) wakefulness, (E) non-REM sleep, and (F) REM sleep times measured during

the ﬁrst 3 h following administration of vehicle or 34 (derived from the time courses shown in A−C). Values are mean SEM * P < 0.05, ** P <

0.01, *** P < 0.001 versus vehicle control (one-way ANOVA followed by Dunnett’s multiple comparison test).

1

ppm in CDCl₃. Then 600 MHz H or ¹³C NMR data were processed

using ACD Spectrus processor from ACD Laboratories. High-

resolution mass spectra (HRMS) were recorded on a ThermoFisher-

Scientiﬁc LTQ-Orbitrap XL spectrometer (using electrospray

ionization).

added to the residue. The obtained mixture was heated to reﬂux for 8

h. The reaction solution was allowed to cool to room temperature, and

concentrated hydrochloric acid was added to adjust the pH to <2. This

mixture was stirred at 0 °C for 2 h then was concentrated under

reduced pressure. Ethyl acetate and water were added to the

concentrated solution to carry out liquid separation. The organic

layer was washed successively with a saturated sodium bicarbonate

aqueous solution and a saturated sodium chloride aqueous solution.

The organic layer was dried over magnesium sulfate, the drying agent

was removed by ﬁltration, and the ﬁltrate was concentrated under

reduced pressure. The residue was puriﬁed by silica gel column

The purity of the tested biological compounds was determined by

an analytical LC−MS or UPLC method and was found to be greater

than or equal to 95%. LC−MS analyses were performed using a

Shimadzu LCMS-2010 EV and UPLC analyses were performed using

a ACQUITY UPLC. The tested biological compounds were not

PAINS compound. Optical rotation( ) was measured by Shimadzu

HPLC SCL-10A system with a corresponding chiral column and

JASCO OR-2090 optical rotation detector. Column chromatography

was carried out using a Hi-Flash column (40 μm, silica gel and NH-

silica gel, Yamazen Corporation). Chemicals and solvents were

purchased from commercial sources.

1

chromatography, giving the title compound (9.0 g). H NMR (400

MHz, CDCl₃) δ (ppm): 1.37 (t, J = 4.8 Hz, 1H), 1.65 (dd, J = 7.8, 4.4

Hz, 1H), 2.54−2.58 (m, 1H), 4.30 (d, J = 9.2 Hz, 1H), 4.47 (dd, J =

9.4, 4.4 Hz, 1H), 7.25−7.45 (m, 5H).

(1S,5R)-1-(2-Fluorophenyl)-3-oxabicyclo[3.1.0]hexan-2-one

(13b). The title compound was synthesized adapting the procedure

described for compound 13a (6.25 g, 67.5% yield) as a white solid. ¹H

NMR (600 MHz, CDCl₃) δ (ppm): 1.38 (t, J = 4.9 Hz, 1H), 1.73 (dd,

J = 7.9, 4.9 Hz, 1H), 2.43−2.52 (m, 1H), 4.34 (d, J = 9.4 Hz, 1H), 4.57

(dd, J = 9.1, 4.5 Hz, 1H), 7.09 (t, J = 9.1 Hz, 1H), 7.14−7.18 (m, 1H),

7.31−7.42 (m, 2H). ¹³C NMR (150 MHz, CDCl₃) δ (ppm): 17.2,

24.6, 27.9, 68.4, 115.6, 121.6, 124.3, 130.2, 131.6, 162.2, 175.5. HRMS

(ESI(+)) calcd for C₁₁H₁₀FO₂[M + H]⁺, 193.0659; found, 193.0660.

(1S,5R)-1-Phenyl-3-oxabicyclo[3.1.0]hexan-2-one (13a).

NaHMDS (85 mL, 2.0 M) was added dropwise to a solution of 2-

phenylacetonitrile (7.96g) in THF (300 mL) under cooling in an ice−

salt bath. The mixture was stirred for 2 h, then R-(−)-epichlorohydrin

(7.548 g) was added dropwise (3 h, 0 °C). Stirring was continued for 2

h at 0 °C then overnight at room temperature. The reaction solution

was then cooled on ice, and a small amount of water was added. The

resulting mixture was concentrated under reduced pressure, then

ethanol (100 mL) and 1.5 N/KOH aqueous solution (100 mL) were

I

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(1S,5R)-1-(3-Fluorophenyl)-3-oxabicyclo[3.1.0]hexan-2-one

(13c). The title compound was synthesized adapting the procedure

described for compound 13a (9.96 g, 49.8% yield) as a white solid. ¹H

NMR (400 MHz, CDCl₃) δ (ppm): 1.41 (t, J = 5.2 Hz, 1H), 1.64 (dd,

J = 8.0, 5.2 Hz, 1H), 2.56−2.63 (m, 1H), 4.30 (d, J = 9.2 Hz, 1H), 4.47

(dd, J = 9.2, 4.8 Hz, 1H), 6.96−7.02 (m, 1H), 7.16−7.21 (m, 2H),

7.28−7.35 (m, 1H). ¹³C NMR (150 MHz, CDCl₃) δ (ppm): 20.7,

25.4, 31.2, 68.0, 114.7, 115.3, 123.6, 130.2, 136.6, 162.8, 175.3. HRMS

(ESI(+)) calcd for C₁₁H₁₀FO₂[M + H]⁺, 193.0659; found, 193.0659.

(1S,5R)-1-(4-Fluorophenyl)-3-oxabicyclo[3.1.0]hexan-2-one

(13d). The title compound was synthesized adapting the procedure

described for compound 13a (8.73g, 43.7% yield) as a colorless oil. ¹H

NMR (600 MHz, CDCl₃) δ (ppm): 1.39 (t, J = 4.7 Hz, 1H), 1.62 (dd,

J = 7.9, 4.9 Hz, 1H), 2.54−2.58 (m, 1H), 4.31 (d, J = 9.1 Hz, 1H), 4.49

(dd, J = 9.4, 4.5 Hz, 1H), 7.06 (m, 2H), 7.39−7.43 (m, 2H). ¹³C NMR

(150 MHz, CDCl₃) δ (ppm): 20.2, 25.0, 31.2, 68.1, 115.6, 129.9,

130.2, 162.3, 175.8. HRMS (ESI(+)) calcd for C₁₁H₁₀FO₂[M + H]⁺,

193.0659; found, 193.0658.

Hz, 1H), 1.63−1.69 (m, 1H), 2.50 (br s, 1H), 2.86 (d, J = 6.0 Hz, 1H),

3.43 (t, J = 11.3 Hz, 1H), 3.59 (dd, J = 12.1, 2.3 Hz, 1H), 4.13 (dd, J =

11.9, 4.7 Hz, 1H), 4.18−4.25 (m, 1H), 6.99−7.05 (m, 2H), 7.35−7.43

(m, 2H). ¹³C NMR (150 MHz, CDCl₃) δ (ppm): 16.7, 25.9, 31.8,

63.6, 67.7, 115.2, 131.0, 139.6, 161.7. HRMS (ESI(+)) calcd for

C₁₁H₁₄FO₂[M + H]⁺, 197.0972; found, 197.0970.

(1S,2R)-1-(3,4-Diﬂuorophenyl)cyclopropan-1,2-dimethanol (14f).

The title compound was synthesized as a colorless oil (7.1 g, 98%

yield) from (1S,5R)-1-(3,4-diﬂuorophenyl)-3-oxabicyclo[3.1.0]hexan-

2-one 13f by adapting a previously described procedure.³⁶¹H NMR

(600 MHz, CDCl₃) δ (ppm): 0.81 (t, J = 5.5 Hz, 1H), 1.10 (dd, J =

8.7, 5.3 Hz, 1H), 1.60−1.68 (m, 1H), 2.59−2.67 (m, 1H), 2.77 (d, J =

5.3 Hz, 1H), 3.43 (t, J = 11.3 Hz, 1H), 3.59 (dd, J = 11.9, 2.8 Hz, 1H),

4.13 (dd, J = 12.1, 6.4 Hz, 1H), 4.19−4.27 (m, 1H), 7.06−7.16 (m,

2H), 7.24 (ddd, J = 11.4, 7.8, 1.9 Hz, 1H). ¹³C NMR (150 MHz,

CDCl₃) δ (ppm): 16.9, 26.3, 31.7, 63.4, 67.4, 117.0, 118.2, 125.0,

141.0, 149.3, 150.0. HRMS (ESI(+)) calcd for C₁₁H₁₃F₂O₂[M + H]⁺,

215.0878; found, 215.0875.

(1S,2R)-1-(3,5-Diﬂuorophenyl)cyclopropan-1,2-dimethanol (14e).

The title compound was synthesized as a colorless oil (3.2 g, 72.9%

yield) from (1S,5R)-1-(3,5-diﬂuorophenyl)-3-oxabicyclo[3.1.0]hexan-

2-one 13e by adapting a previously described procedure.³⁶¹H NMR

(600 MHz, CDCl₃) δ (ppm): 0.84 (t, J = 5.5 Hz, 1H), 1.14 (dd, J =

8.7, 5.3 Hz, 1H), 1.61−1.72 (m, 1H), 2.70 (br s, 1H), 2.74 (br s, 1H),

3.44 (t, J = 11.5 Hz, 1H), 3.61 (d, J = 12.1 Hz, 1H), 4.16−4.31 (m,

2H), 6.65−6.73 (tt, J = 8.9, 2.3 Hz, 1H), 6.92−6.97 (m, 2H). ¹³C

NMR (150 MHz, CDCl₃) δ (ppm): 17.3, 26.7, 31.9, 63.4, 67.0, 102.2,

111.7, 148.0, 162.9. HRMS (ESI(+)) calcd for C₁₁H₁₃F₂O₂[M + H]⁺,

215.0878; found, 215.0875.

(1S,2R)-2-(tert-Butyldiphenylsilyloxymethyl)-1-phenylcyclopro-

pylmethanol (15a). The title compound was synthesized as a

colorless oil (340 mg, 67.7% yield)from (1S,2R)-1-phenylcyclopro-

pan-1,2-dimethanol 14a by adapting a previously described proce-

dure.³⁶¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.71 (t, J = 5.6 Hz,

1H), 1.04 (dd, J = 9.6, 5.2 Hz, 1H), 1.10 (s, 9H), 1.50−1.58 (m, 1H),

3.50 (dd, J = 12.4, 1.6 Hz, 1H), 3.53 (dd, J = 11.6, 1.6 Hz, 1H), 3.71

(dd, J = 12.4, 1.6 Hz, 1H), 4.10 (t, J = 12.0 Hz, 1H), 4.20 (dd, J = 12.0,

5.6 Hz, 1H), 7.21−7.46 (m, 10H). 7.7−7.76 (m, 5H).

[(1R,2S)-2-(2-Fluorophenyl)-2-(hydroxymethyl)cyclopropyl]-

methyl Acetate (15b). Lipase acrylic resin from Candida antarctica

was added to a THF (50 mL)−vinyl acetate (1.51 g, 17.6 mmol)

solution of (1S,2R)-1-(2-ﬂuorophenyl)cyclopropan-1,2-dimethanol

(2.28 g, 11.7 mmol) under cooling on ice. This mixture was stirred

at room temperature for 17 h, then was ﬁltered, and ﬁltrate was

concentrated, so as to give the title compound (2.1 g, 82.6% yield). ¹H

NMR (600 MHz, CDCl₃) δ (ppm): 0.87 (t, J = 5.7 Hz, 1H), 1.15 (dd,

J = 8.7, 5.3 Hz, 1H), 1.64 (ddd, J = 9.4, 5.7, 3.4 Hz, 1H), 2.08 (dd, J =

8.5, 3.6 Hz, 1H), 2.17 (s, 3H), 3.70 (dd, J = 12.1, 3.4 Hz, 1H), 3.96

(dd, J = 12.1, 8.7 Hz, 1H), 4.06 (dd, J = 11.9, 10.0 Hz, 1H), 4.65 (dd, J

= 12.1, 5.7 Hz, 1H), 6.99−7.06 (m, 1H), 7.11 (td, J = 7.5, 0.9 Hz, 1H),

7.22−7.27 (m, 1H), 7.30 (td, J = 7.5, 1.7 Hz, 1H). ¹³C NMR (150

MHz, CDCl₃) δ (ppm): 15.1, 21.1, 22.1, 28.6, 64.5, 66.2, 115.6, 124.0,

128.9, 130.0, 132.1, 162.2, 171.1. HRMS (ESI(+)) calcd for

C₁₃H₁₆FO₃[M + H]⁺, 239.1078; found, 239.1075.

[(1R,2S)-2-(3-Fluorophenyl)-2-(hydroxymethyl)cyclopropyl]-

methyl Acetate (15c). The title compound was synthesized as a light-

yellow oil (1.21 g, quant) from (1S,2R)-1-(3-ﬂuorophenyl)-

cyclopropan-1,2-dimethanol 14c by adapting the procedure described

for compound 15b. HRMS (ESI(+)) calcd for C₁₃H₁₆FO₃[M + H]⁺,

239.1078; found, 239.1074.

[(1R,2S)-2-(4-Fluorophenyl)-2-(hydroxymethyl)cyclopropyl]-

methyl Acetate (15d). The title compound was synthesized as a

colorless oil (2.8 g, 92.3% yield) from (1S,2R)-1-(4-ﬂuorophenyl)-

cyclopropan-1,2-dimethanol by adapting the procedure described for

compound 15b. HRMS (ESI(+)) calcd for C₁₃H₁₆FO₃[M + H]⁺,

239.1078; found, 239.1072.

(1S,5R)-1-(3,5-Diﬂuorophenyl)-3-oxabicyclo[3.1.0]hexan-2-one

(13e). The title compound was synthesized adapting the procedure

1

described for compound 13a (6.9 g, 50.3% yield). H NMR (600

MHz, CDCl₃) δ (ppm): 1.46 (t, J = 4.9 Hz, 1H), 1.64 (dd, J = 7.7, 5.1

Hz, 1H), 2.62 (dt, J = 8.1, 4.6 Hz, 1H), 4.32 (d, J = 9.4 Hz, 1H), 4.47

(dd, J = 9.4, 4.5 Hz, 1H), 6.76 (tt, J = 8.9, 2.3 Hz, 1H), 7.01 (m, 2H).

¹³C NMR (150 MHz, CDCl₃) δ (ppm): 21.3, 25.7, 31.0, 67.8, 103.2,

110.9, 138.0, 162.2, 163.9, 174.6. HRMS (ESI(+)) calcd for

C₁₁H₉F₂O₂[M + H]⁺, 211.0565; found, 211.0561.

(1S,5R)-1-(3,4-Diﬂuorophenyl)-3-oxabicyclo[3.1.0]hexan-2-one

(13f). The title compound was synthesized adapting the procedure

1

described for compound 13a (7.1 g, 51.7% yield). H NMR (600

MHz, CDCl₃) δ (ppm): 1.42 (t, J = 4.9 Hz, 1H), 1.61 (dd, J = 7.7, 5.1

Hz, 1H), 2.57−2.61 (m, 1H), 4.31 (d, J = 9.4 Hz, 1H), 4.48 (dd, J =

9.3, 4.7 Hz, 1H), 7.12−7.19 (m, 2H), 7.32 (m, 1H). ¹³C NMR (150

MHz, CDCl₃) δ (ppm): 20.7, 25.2, 30.9, 68.0, 117.5, 117.6, 124.2,

131.1, 149.2, 150.9, 175.2. HRMS (ESI(+)) calcd for C₁₁H₉F₂O₂[M +

H]⁺, 211.0565; found, 211.0562.

(1S,2R)-1-Phenylcyclopropan-1,2-dimethanol (14a). The title

compound was synthesized as a colorless oil (340 mg, 67.7%) from

(1S,5R)-1-phenyl-3-oxabicyclo[3.1.0]hexan-2-one by adapting a pre-

viously described procedure.³⁶¹H NMR (400 MHz, CDCl₃) δ (ppm):

0.78 (t, J = 5.2 Hz, 1H), 1.87 (dd, J = 8.6, 5.2, 1H), 1.60−1.76 (m,

1H), 3.42 (t, J = 11.6, 1H), 3.57 (dd, J = 9.4, 4.4 Hz, 1H), 4.14−4.28

(m, 2H) 7.22−7.44 (m, 5H).

(1S,2R)-1-(2-Fluorophenyl)cyclopropan-1,2-dimethanol (14b).

The title compound was synthesized by adapting a previously

described procedure³⁶to give 14b (745 mg, 33.3% yield) as a

1

colorless oil. H NMR (600 MHz, CDCl₃) δ (ppm): 0.88 (t, J = 5.5

Hz, 1H), 1.08 (dd, J = 8.7, 5.3 Hz, 1H), 1.67−1.75 (m, 1H), 2.50 (br s,

1H), 2.90 (br s, 1H), 3.46 (t, J = 11.3 Hz, 1H), 3.58 (d, J = 12.1 Hz,

1H), 4.15 (d, J = 12.1 Hz, 1H), 4.23 (dd, J = 11.3, 4.5 Hz, 1H), 7.04 (t,

J = 9.3 Hz, 1H), 7.12 (t, J = 7.5 Hz, 1H), 7.22−7.27 (m, 1H), 7.42 (td,

J = 7.6, 1.9 Hz, 1H). ¹³C NMR (150 MHz, CDCl₃) δ (ppm): 16.3,

24.8, 28.0, 63.4, 67.0, 115.6, 124.0, 128.8, 130.5, 132.7, 162.1. HRMS

(ESI(+)) calcd for C₁₁H₁₄FO₂[M + H]⁺, 197.0972; found, 197.0972.

(1S,2R)-1-(3-Fluorophenyl)cyclopropan-1,2-dimethanol (14c).

The title compound was synthesized as a colorless oil (10.3 g,

quant) from (1S,5R)-1-(3-ﬂuorophenyl)-3-oxabicyclo[3.1.0]hexan-2-

one by adapting a previously described procedure.³⁶¹H NMR (400

MHz, CDCl₃) δ (ppm): 0.80 (t, J = 5.0 Hz, 1H), 1.10 (dd, J = 8.6, 5.0

Hz, 1H), 1.62−1.71 (m, 1H), 3.41 (t, J =11.4 Hz, 1H), 3.58 (d, J =

12.0 Hz, 1H), 4.12−4.25 (m, 2H), 6.90−6.96 (m, 1H), 7.08−7.14 (m,

1H), 7.16−7.21 (m, 1H) 7.24−7.32 (m, 1H). ¹³C NMR (150 MHz,

CDCl₃) δ (ppm): 17.1, 26.3, 32.1, 63.5, 67.3, 113.7, 116.0, 124.6,

129.8, 146.5, 162.8. HRMS (ESI(+)) calcd for C₁₁H₁₄FO₂[M + H]⁺,

197.0972; found, 197.0970.

(1S,2R)-1-(4-Fluorophenyl)cyclopropan-1,2-dimethanol (14d).

The title compound was synthesized as a colorless oil (8.56g, 96.1%

yield) from (1S,5R)-1-(4-ﬂuorophenyl)-3-oxabicyclo[3.1.0]hexan-2-

one by adapting a previously described procedure.³⁶¹H NMR (600

MHz, CDCl₃) δ (ppm): 0.79 (t, J = 5.3 Hz, 1H), 1.08 (dd, J =8.7, 5.3

[(1R,2S)-2-(3,5-Diﬂuorophenyl)-2-(hydroxymethyl)cyclopropyl]-

methyl Acetate (15e). The title compound was synthesized as a

reddish oil (41.2 g, 99.2% yield) from (1S,2R)-1-(3,5-diﬂuorophenyl)-

cyclopropan-1,2-dimethanol by adapting the procedure described for

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compound 15b. HRMS (ESI(+)) calcd for C₁₃H₁₅F₂O₃[M + H]⁺,

257.0984; found, 257.0979.

Hz, 1H), 1.82−1.88 (m, 1H), 2.21 (dd, J = 9.3, 2.8 Hz, 1H), 2.35 (s,

3H), 2.60 (s, 3H), 3.60 (ddd, J = 12.2, 9.9, 2.8 Hz, 1H), 4.09−4.16 (m,

2H), 4.42 (d, J = 9.8 Hz, 1H), 7.05 (t, J =9.3 Hz, 1H), 7.13 (td, J = 7.6,

1.1 Hz, 1H), 7.25−7.29 (m, 1H), 7.46 (td, J = 7.6, 1.5 Hz, 1H), 7.98

(s, 1H). ¹³C NMR (150 MHz, CDCl₃) δ (ppm): 15.9, 18.8, 25.0, 25.3,

25.9, 62.9, 73.3, 115.7, 124.1, 129.3, 129.7, 132.4, 139.0, 149.1, 157.2,

160.2, 162.1. HRMS (ESI(+)) calcd for C₁₇H₂₀FN₂O₂[M + H]⁺,

303.1503; found, 303.1500.

[(1R,2S)-2-(3,4-Diﬂuorophenyl)-2-(hydroxymethyl)cyclopropyl]-

methyl Acetate (15f). The title compound was synthesized as a

colorless oil (2.9 g, 81% yield) from (1S,2R)-1-(3,4-diﬂuorophenyl)-

cyclopropan-1,2-dimethanolby adapting the procedure described for

compound 15b. HRMS (ESI(+)) calcd for C₁₃H₁₅F₂O₃[M + H]⁺,

257.0984; found, 257.0979.

5-(((1S,2R)-2-(((tert-Butyldiphenylsilyl)oxy)methyl)-1-

phenylcyclopropyl)methoxy)-2,4-dimethylpyrimidine (16a). The

title compound was synthesized as a colorless oil (340 mg, 67.7%

yield) from ((1S,2R)-2-(((tert-butyldiphenylsilyl)oxy)methyl)-1-

phenylcyclopropyl)methanol (15a) and 2,4-dimethyl-5-hydroxypyr-

imidine by adapting a previously described procedure.³⁶¹H NMR

(600 MHz, CDCl₃) δ (ppm): 0.95 (t, J = 5.7 Hz, 1H), 1.09 (s, 9H),

1.24 (dd, J = 8.7, 5.3 Hz, 1H), 1.64−1.70 (m, 1H), 2.30 (s, 3H), 2.61

(s, 3H), 3.80 (dd, J = 11.3, 8.3 Hz, 1H), 4.06 (dd, J = 11.3, 5.7 Hz,

1H), 4.18 (d, J = 9.8 Hz, 1H), 4.24 (d, J = 9.8 Hz, 1H), 7.24−7.28 (m,

1H), 7.31−7.39 (m, 6H), 7.41−7.45 (m, 4H), 7.66−7.70 (m, 4H),

7.89 (s, 1H). HRMS (ESI(+)) calcd for C₃₃H₃₉N₂O₂Si [M + H]⁺,

523.2775; found, 523.2783.

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-(2-

ﬂuorophenylcyclopropyl)methyl Acetate (16b). Diisopropyl azodi-

carboxylate (310 μL, 1.57 mmol) was added dropwise to a THF

solution (10 mL) of the compound 15b (300 mg, 1.26 mmol),

triphenylphosphine (413 mg, 1.57 mmol), and 2,4-dimethyl-

pyrimidin-5-ol 43 (195 mg, 1.57 mmol) at 0 °C. The reaction

mixture was stirred at this temperature for 30 min, then allowed to

warm to ambient temperatureand stirred overnight. The reaction

mixture was concentrated under reduced pressure, and the residue was

puriﬁed by silica gel column chromatography using n-heptane/ethyl

acetate (4/1 to 0/1, v/v) to give 16b (154 mg, 35.5% yield) as a

colorless oil. HRMS (ESI(+)) calcd for C₁₉H₂₂FN₂O₃[M + H]⁺,

345.1609; found, 345.1607.

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-(4-

ﬂuorophenylcyclopropyl)methyl Acetate (16d). The title compound

was synthesized as a colorless oil (276 mg, 63.6% yield) from

[(1R,2S)-2-(4-ﬂuorophenyl)-2-(hydroxymethyl)cyclopropyl]methyl

acetate 15d by adapting the procedure described for compound 16b.

HRMS (ESI(+)) calcd for C₁₉H₂₂FN₂O₃[M + H]⁺, 345.1609; found,

345.1606.

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-(3,4-

diﬂuorophenylcyclopropyl)methyl Acetate (16f). The title com-

pound was synthesized as a colorless oil (87 mg, 20.5% yield) from

[(1R,2S)-2-(3,4-diﬂuorophenyl)-2-(hydroxymethyl)cyclopropyl]-

methyl acetate 15f by adapting the procedure described for compound

16b. HRMS (ESI(+)) calcd for C₁₉H₂₁F₂N₂O₃[M + H]⁺, 363.1515;

found, 363.1511.

((1R,2S)-2-(((2,4-Dimethylpyrimidin-5-yl)oxy)methyl)-2-

phenylcyclopropyl)methanol (17a). The title compound was

synthesized as a white solid (155 mg, 83.9% yield) from 5-

(((1S,2R)-2-(((tert-butyldiphenylsilyl)oxy)methyl)-1-

phenylcyclopropyl)methoxy)-2,4-dimethylpyrimidine by adapting a

previously described procedure.³⁶¹H NMR (600 MHz, CDCl₃) δ

(ppm): 0.98 (t, J = 5.7 Hz, 1H), 1.29 (dd, J = 8.7, 5.3 Hz, 1H), 1.85

(ddd, J = 9.4, 5.6, 3.6 Hz, 1H), 2.25 (br s, 1H), 2.39 (s, 3H), 2.61 (s,

3H), 3.60 (t, J = 11.0 Hz, 1H), 4.06−4.15 (m, 2H), 4.46 (d, J = 10.2

Hz, 1H), 7.25−7.27 (m, 1H), 7.34 (m, 2H), 7.45 (m, 2H), 7.99 (s,

1H). ¹³C NMR (150 MHz, CDCl₃) δ (ppm): 16.4, 19.0, 25.0, 26.9,

30.0, 63.2, 74.4, 127.1, 128.5, 129.4, 139.1, 143.0, 149.1, 157.1, 160.3.

HRMS (ESI(+)) calcd for C₁₇H₂₁N₂O₂[M + H]⁺, 285.1598; found,

285.1599.

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-(2-

ﬂuorophenylcyclopropyl)methanol (17b). The acetate 16b was

dissolved in EtOH−1N sodium hydroxide aqueous solution (5−0.34

mL). The reaction mixture was stirred for 1 h then was concentrated

under reduced pressure. The residue was puriﬁed by NH−silica gel

column chromatography using n-heptane/ethyl acetate (9/1 to 0/1, v/

v) to give 17b (84.5 mg, 62.5% yield) as a white solid. ¹H NMR (600

MHz, CDCl₃) δ (ppm): 1.05 (t, J = 5.9 Hz, 1H), 1.26 (dd, J = 8.9, 5.5

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-(3-

ﬂuorophenylcyclopropyl)methanol (17c). Diisopropyl azodicarbox-

ylate (45.8 mL, 217 mmol) was added dropwise to a THF solution

(400 mL) of the compound 15c (43.1 g, 181 mmol), triphenylphos-

phine (57 g, 217 mmol), and 2,4-dimethyl-pyrimidin-5-ol 43 (24.7 g,

199 mmol) at 0 °C. The reaction mixture was allowed to warm to

ambient temperature and then was stirred overnight. The reaction

mixture was partitioned between sat. NaHCO₃aq and EtOAc. The

organic layer was washed with brine and dried over MgSO₄, ﬁltered to

remove the drying agent, then concentrated under reduced pressure.

The residue was dissolved in EtOH−1N NaOH (200−200 mL), and

this solution was stirred for 1 h at room temperature. Additional 5 N

NaOH (100 mL) was added to the reaction mixture, which was then

concentrated under reduced pressure. The residue was extracted with

EtOAc. The organic layer was washed with brine and dried over with

MgSO₄, ﬁltered to remove the drying agent, and then concentrated

under reduced pressure. The residue was puriﬁed ﬁrst by silica gel

column chromatography using n-heptane/ethyl acetate/methanol (1/

4/0 to 0/1/1, v/v) and thereafter by using a NH−silica gel pad with

EtOAc as the eluent to give 17c (39.3 g, 71.8% yield) as a light-red oil.

¹H NMR (400 MHz, CDCl₃) δ (ppm): 1.00 (t, J = 5.2 Hz, 1H), 1.24−

1.30 (m, 1H), 1.79−1.85 (m, 1H), 2.39 (s, 3H), 2.60 (s, 3H), 3.55−

3.61 (m, 1H), 4.03−4.13 (m, 1H), 4.12 (d, J = 9.6 Hz, 1H), 4.43 (d, J

= 9.6 Hz, 1H), 6.92−6.98 (m, 1H), 7.11−7.15 (m, 1H), 7.19−7.22 (m,

1H), 7.25−7.31 (m, 1H), 8.00 (s, 1H). ¹³C NMR (150 MHz, CDCl₃)

δ (ppm): 16.6, 19.0, 25.0, 27.2, 29.7, 63.0, 73.9, 114.1, 116.3, 124.8,

129.9, 139.1, 145.6, 149.0, 157.1, 160.4, 162.7. HRMS (ESI(+)) calcd

for C₁₇H₂₀FN₂O₂[M + H]⁺, 303.1503; found, 303.1502.

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-(4-

ﬂuorophenylcyclopropyl)methanol (17d). The title compound was

synthesized as a colorless oil (222 mg, 93% yield) from (1R,2S)-2-

{ [ ( 2 , 4 - d i m e t h y l p y r i m i d i n - 5 - y l ) o x y ] m e t h y l } - 2 - ( 4 -

ﬂuorophenylcyclopropyl)methyl acetate by adapting the procedure

described for compound 17b. ¹H NMR (600 MHz, CDCl₃) δ (ppm):

0.98 (t, J = 5.5 Hz, 1H), 1.25 (dd, J = 8.7, 5.3 Hz, 1H), 1.76−1.83 (m,

1H), 2.22 (dd, J = 9.3, 2.8 Hz, 1H), 2.38 (s, 3H), 2.61 (s, 3H), 3.56−

3.62 (m, 1H), 4.05−4.13 (m, 1H), 4.10 (d, 9.8 Hz, 1H), 4.39 (d, J =

9.8 Hz, 1H), 7.02 (m, 2H), 7.39−7.44 (m, 2H), 7.99 (s, 1H). ¹³C

NMR (150 MHz, CDCl₃) δ (ppm): 16.3, 19.0, 25.0, 26.9, 29.5, 63.0,

74.3, 115.3, 131.1, 138.8, 139.1, 149.0, 157.1, 160.4, 161.8. HRMS

(ESI(+)) calcd for C₁₇H₂₀FN₂O₂[M + H]⁺, 303.1503; found,

303.1503.

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-(3,5-

diﬂuorophenylcyclopropyl)methanol (17e). To a solution of acetate

15e (597 mg, 2.33 mmol) was added triethyl amine (357 μL, 2.56

mmol) and methanesulfonyl chloride (180 μL, 2.33 mmol) at 0 °C.

The reaction mixture was stirred for 1 h at this temperature, then

water was added and the mixture was extracted with dichloromethane.

The organic layer was dried over MgSO₄, ﬁltered to remove the drying

agent, and concentrated under reduced pressure. The residue was

dissolved in acetonitrile (20 mL), and to the solution were added 2,4-

dimethylpyrimidin-5-ol 43 (347 mg, 2.8 mmol) and cesium carbonate

(1.52 g, 4.66 mmol). The reaction mixture was stirred for 4 h at 70 °C,

then was ﬁltered through a glass ﬁlter. The ﬁltrate was concentrated

under reduced pressure. The residue was dissolved in EtOH−1N

NaOH (2.45−2.45 mL), after which it was stirred for 1 h at 70 °C.

After cooling the reaction mixture, it was extracted with EtOAc,

washed with brine, and concentrated undwer reduced pressure. The

residue was puriﬁed by silica gel column chromatography using n-

heptane/AcOEt/MeOH (4/1/0 to 0/1/0 to 0/1/1, v/v) to give 17e

(520 mg, 69.7% yield) as a reddish oil. ¹H NMR (600 MHz, CDCl₃) δ

(ppm): 1.03 (t, J = 5.67 Hz, 1H), 1.31 (dd, J = 8.9, 5.5 Hz, 1H), 1.77−

K

DOI: 10.1021/acs.jmedchem.5b00217

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1.84 (m, 1H), 2.12 (dd, J = 8.9, 2.8 Hz, 1H), 2.41 (s, 3H), 2.63 (s,

3H), 3.57−3.63 (m, 1H), 4.04−4.10 (m, 1H), 4.13 (d, J = 10.2 Hz,

1H), 4.42 (d, J = 10.2 Hz, 1H), 6.72 (tt, J = 8.9, 2.3 Hz, 1H), 6.93−

6.99 (m, 2H), 8.03 (s, 1H). ¹³C NMR (150 MHz, CDCl₃) δ (ppm):

16.7, 19.0, 25.0, 27.4, 29.7, 62.7, 73.4, 102.7, 112.0, 139.0, 146.9, 148.9,

157.1, 160.5, 162.9. HRMS (ESI(+)) calcd for C₁₇H₁₉F₂N₂O₂[M +

H]⁺, 321.1409; found, 321.1407.

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-(3,4-

diﬂuorophenylcyclopropyl)methanol (17f). The title compound was

synthesized as a colorless oil (70 mg, 97% yield) from [(1R,2S)-2-(3,4-

ﬂuorophenyl)-2-(hydroxymethyl)cyclopropyl]methyl acetate by adapt-

ing the procedure described for compound 17b. ¹H NMR (600 MHz,

CDCl₃) δ (ppm): 1.00 (t, J = 5.7 Hz, 1H), 1.27 (dd, J = 8.9, 5.5 Hz,

1H), 1.76−1.82 (m, 1H), 2.13 (dd, J = 9.1, 3.0 Hz, 1H), 2.40 (s, 3H),

2.62 (s, 3H), 3.59 (ddd, J = 12.3, 9.6, 3.0 Hz, 1H), 4.05−4.10 (m, 1H),

4.11(d, J = 9.8 Hz, 1H), 4.38 (d, J = 9.8 Hz, 1H), 7.09−7.19 (m, 2H),

7.24−7.28 (m, 1H), 8.01 (s, 1H). ¹³C NMR (150 MHz, CDCl₃) δ

(ppm): 16.4, 19.0, 25.0, 27.1, 29.4, 62.8, 73.8, 117.2, 118.5, 125.3,

139.0, 140.1, 148.9, 149.4, 150.0, 157.1, 160.5. HRMS (ESI(+)) calcd

for C₁₇H₁₉F₂N₂O₂[M + H]⁺. 321.1409; found, 321.1408.

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-phenylcy-

clopropanecarboxylic Acid (18a). The title compound was

synthesized as a white solid (569 mg, 60.4% yield) from ((1R,2S)-2-

(((2,4-dimethylpyrimidin-5-yl)oxy)methyl)-2-phenylcyclopropyl)-

methanol (17a) by adapting a previously described procedure.³⁶¹H

NMR (600 MHz, CDCl₃) δ (ppm): 1.57 (dd, J = 8.3, 4.9 Hz, 1H),

1.77 (t, J = 5.5 Hz, 1H), 2.28 (dd, J = 8.3, 6.0 Hz, 1H), 2.34 (s, 3H),

2.58 (s, 3H), 4.50 (q, J = 9.4 Hz, 2H), 7.28−7.33 (m, 1H), 7.37 (m,

2H), 7.51 (m, 2H), 8.20 (s, 1H). ¹³C NMR (150 MHz, CDCl₃) δ

(ppm): 18.8, 19.4, 24.0, 25.5, 35.0, 71.9, 127.5, 128.5, 129.3, 138.4,

141.8, 149.6, 158.7, 158.7, 174.1. HRMS (ESI(+)) calcd for

C₁₇H₁₉N₂O₃[M + H]⁺, 299.1390; found, 299.1388.

pound was synthesized as a white solid (886 mg, 83.9% yield) from

(1R,2S)-2-{[(2,4-dimethylpyrimidin-5-yl)oxy]methyl}-2-(3,5-

diﬂuorophenylcyclopropyl)methanol by adapting a previously de-

scribed procedure.³⁶¹H NMR (600 MHz, CDCl₃) δ (ppm): 1.57 (dd,

J = 8.3, 5.3 Hz, 1H), 1.79 (t, J = 5.7 Hz, 1H), 2.26 (dd, J = 8.3, 6.0 Hz,

1H), 2.37 (s, 3H), 2.58 (s, 3H), 4.50 (s, 2H), 6.77 (tt, J = 8.8, 2.1 Hz,

1H), 7.00−7.09 (m, 2H), 8.26 (s, 1H). ¹³C NMR (150 MHz, CDCl₃)

δ (ppm): 18.9, 19.4, 23.9, 25.8, 34.2, 71.3, 103.1, 112.1, 138.6, 145.5,

149.5, 159.0, 159.0, 162.9, 173.4. HRMS (ESI(+)) calcd for

C₁₇H₁₇F₂N₂O₃[M + H]⁺, 335.1202; found, 335.1198.

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-(3,4-

diﬂuorophenyl)cyclopropanecarboxylic Acid (18f). The title com-

pound was synthesized as a white amorphous solid (662 mg, 63.1%

yield) from (1R,2S)-2-{[(2,4-dimethylpyrimidin-5-yl)oxy]methyl}-2-

(3,4-diﬂuorophenylcyclopropyl)methanol by adapting a previously

described procedure.³⁶¹H NMR (600 MHz, CDCl₃) δ (ppm): 1.40

(br s, 1H), 1.65 (br s, 1H), 2.13 (br s, 1H), 2.28 (br s, 3H), 2.53 (s,

3H), 4.35−4.48 (m, 2H), 7.05−7.19 (m, 2H), 7.23−7.33 (m, 1H),

8.17 (br s, 1H). ¹³C NMR (150 MHz, CDCl₃) δ (ppm): 18.8, 19.2,

24.1, 26.6, 33.7, 71.5, 117.2, 118.4, 125.2, 138.7, 138.9, 149.4, 149.6,

150.0, 158.5, 159.1, 175.3. HRMS (ESI(+)) calcd for C₁₇H₁₇F₂N₂O₃

[M + H]⁺, 335.1202; found, 335.1198.

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-N-(pyridin-2-

yl)-2-phenylcyclopropanecarboxamide (23). To a solution of

carboxylic acid 18a (40 mg, 0.13 mmol) in DMF (1 mL) were

added 2-aminopyridine (37.8 mg, 0.40 mmol), DIPEA (35.4 μL, 0.27

mmol), and HATU (56 mg, 0.15 mmol). The reaction mixture was

stirred at room temperature for 5 h, then was warmed up to 50 °C and

stirred overnight. The reaction mixture was concentrated under

reduced pressure. The residue was puriﬁed ﬁrst by silica gel column

chromatography using chloroform/methanol (1/0 to 19/1, v/v) and

subsequently by using Prep-TLC (SiO₂, chloroform/methanol = 10/1

1

v/v) to give amide 23 (27.3 mg, 54.4% yield) as a white solid. H

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-(2-

ﬂuorophenyl)cyclopropanecarboxylic Acid (18b). The title com-

pound was synthesized as a white solid by adapting the procedure

NMR (600 MHz, CDCl₃) δ (ppm): 1.63 (dd, J = 8.1, 5.1 Hz, 1H),

1.93 (t, J = 5.3 Hz, 1H), 2.13−2.19 (m, 1H), 2.22 (s, 3H), 2.56 (s,

3H), 4.43 (d, J = 9.4 Hz, 1H), 4.53 (d, J = 9.4 Hz, 1H), 7.02−7.06 (m,

1H), 7.30−7.34 (m, 1H), 7.38 (t, J = 7.4 Hz, 2H), 7.46−7.51 (m, 2H),

7.65−7.70 (m, 1H), 7.98 (s, 1H), 8.07 (d, J = 7.9 Hz, 1H), 8.27−8.30

(m, 1H), 8.37 (br s, 1H). ¹³C NMR (150 MHz, CDCl₃) δ (ppm):

15.7, 15.9, 22.2, 26.2, 32.6, 68.3, 111.0, 117.1, 124.8, 125.9, 126.1,

135.7, 136.1, 139.0, 145.1, 146.4, 148.4, 154.3, 156.8, 165.7. HRMS

(ESI(+)) calcd for C₂₂H₂₃N₄O₂[M + H]⁺, 375.1816; found, 375.1810.

Purity: >95%.

previously described³⁶for compound 18a (47 mg, 73.8% yield). H

1

NMR (600 MHz, CDCl₃) δ (ppm): 1.49−1.55 (m, 1H), 1.74−1.81

(m, 1H), 2.21−2.30 (m, 4H), 2.53−2.60 (m, 3H), 4.47 (d, J = 9.4 Hz,

1H), 4.54 (d, J = 9.4 Hz, 1H), 7.02−7.18 (m, 2H), 7.28−7.33 (m,

1H), 7.45−7.54 (m, 1H), 8.20 (s, 1H). ¹³C NMR (150 MHz, CDCl₃)

δ (ppm): 18.6, 19.0, 24.0, 24.9, 30.0, 70.9, 115.7, 124.0, 128.6, 129.4,

132.3, 138.1, 149.6, 158.6, 158.7, 161.8, 174.3. HRMS (ESI(+)) calcd

for C₁₇H₁₈FN₂O₃[M + H]⁺, 317.1296; found, 317.1293.

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-N-(pyridin-3-

yl)-2-phenylcyclopropanecarboxamide (24). The title compound

was synthesized as a white amorphous solid (28.6 mg, 57% yield) from

(1R,2S)-2-(((2,4-dimethylpyrimidin-5-yl)oxy)methyl)-2-phenylcyclo-

propanecarboxylic acid 18a by adapting the procedure described for

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-(3-

ﬂuorophenyl)cyclopropanecarboxylic Acid (18c). The title com-

pound was synthesized as a white solid (203 mg, 81.7% yield) from

(1R,2S)-2-{[(2,4-dimethylpyrimidin-5-yl)oxy]methyl}-2-(3-

ﬂuorophenylcyclopropyl)methanol by adapting a previously described

procedure.³⁶¹H NMR (600 MHz, CDCl₃) δ (ppm): 1.58 (dd, J = 8.1,

5.1 Hz, 1H), 1.78 (t, J = 5.5 Hz, 1H), 2.28 (dd, J = 7.9, 6.0 Hz, 1H),

2.37 (s, 3H), 2.59 (s, 3H), 4.48−4.54 (m, 2H), 7.01 (td, J = 8.3, 2.3

Hz, 1H), 7.20−7.25 (m, 1H), 7.26−7.30 (m, 1H), 7.30−7.37 (m, 1H),

8.25 (s, 1H). ¹³C NMR (150 MHz, CDCl₃) δ (ppm): 18.9, 19.4, 23.8,

25.7, 34.5, 71.7, 114.5, 116.4, 124.8, 130.0, 138.2, 144.2, 149.6, 158.7,

159.1, 162.7, 173.8. HRMS (ESI(+)) calcd for C₁₇H₁₈FN₂O₃[M +

H]⁺, 317.1296; found, 317.1293.

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-(4-

ﬂuorophenyl)cyclopropanecarboxylic Acid (18d). The title com-

pound was synthesized as an oil (223 mg, 92.5% yield) from (1R,2S)-

2- { [( 2 , 4- d i m e t h y l p y r i m i d i n- 5 - y l ) o x y ] m e t h y l } - 2 -( 4 -

ﬂuorophenylcyclopropyl)methanol by adapting a previously described

procedure.³⁶¹H NMR (600 MHz, CDCl₃) δ (ppm): 1.54 (dd, J = 8.3,

4.9 Hz, 1H), 1.76 (t, J = 5.5 Hz, 1H), 2.24 (dd, J = 8.3, 6.0 Hz, 1H),

2.34 (s, 3H), 2.58 (s, 3H), 4.44−4.50 (m, 2H), 7.06 (m, 2H), 7.46−

7.51 (m, 2H), 8.22 (s, 1H). ¹³C NMR (150 MHz, CDCl₃) δ (ppm):

18.8, 19.4, 24.0, 25.5, 34.3, 71.9, 115.3, 131.1, 137.6, 138.6, 149.6,

158.8, 158.9, 162.1, 173.9. HRMS (ESI(+)) calcd for C₁₇H₁₈FN₂O₃

[M + H]⁺, 317.1296; found, 317.1292.

1

compound 23. H NMR (600 MHz, CDCl₃) δ (ppm): 1.66 (dd, J =

7.7, 5.1 Hz, 1H), 1.93 (t, J = 5.5 Hz, 1H), 2.13 (dd, J = 7.9, 5.7 Hz,

1H), 2.24 (s, 3H), 2.57 (s, 3H), 4.47−4.51 (m, 1H), 4.53−4.57 (m,

1H), 7.25−7.27 (m, 1H), 7.29−7.34 (m, 1H), 7.38 (m, 2H), 7.47 (m,

2H), 7.69 (br s, 1H), 8.01 (s, 1H), 8.11 (d, J = 7.9 Hz, 1H), 8.36 (d, J

= 4.2 Hz, 1H), 8.55 (br s, 1H). ¹³C NMR (150 MHz, CDCl₃) δ

(ppm): 18.7, 18.9, 25.0, 29.0, 35.2, 71.0, 123.8, 127.1, 127.6, 128.6,

128.7, 134.6, 139.1, 140.8, 141.7, 145.5, 149.2, 156.9, 159.7, 168.7.

HRMS (ESI(+)) calcd for C₂₂H₂₃N₄O₂[M + H]⁺, 375.1816; found,

375.1809. Purity: >95%.

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-N-(pyridin-4-

yl)-2-phenylcyclopropanecarboxamide (25). The title compound

was synthesized as a white amorphous solid (37.4 mg, 74.5% yield)

from (1R,2S)-2-(((2,4-dimethylpyrimidin-5-yl)oxy)methyl)-2-phenyl-

cyclopropanecarboxylic acid 18a by adapting the procedure described

for compound 23. ¹H NMR (600 MHz, CDCl₃) δ (ppm): 1.68 (dd, J

= 8.1, 5.1 Hz, 1H), 1.94 (t, J = 5.5 Hz, 1H), 2.11 (dd, J = 7.9, 6.0 Hz,

1H), 2.22 (s, 3H), 2.57 (s, 3H), 4.46 (d, J = 9.8 Hz, 1H), 4.55 (d, J =

9.4 Hz, 1H), 7.29−7.34 (m, 1H), 7.38 (m, 2H), 7.42−7.47 (m, 4H),

7.78 (br s, 1H), 8.01 (s, 1H), 8.50 (m, 2H). ¹³C NMR (150 MHz,

CDCl₃) δ (ppm): 18.7, 19.1, 25.0, 29.2, 35.5, 70.9, 113.36, 127.7,

128.5, 128.7, 139.2, 141.5, 144.7, 149.2, 150.9, 156.9, 159.8, 168.9.

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-(3,5-

diﬂuorophenyl)cyclopropanecarboxylic Acid (18e). The title com-

L

DOI: 10.1021/acs.jmedchem.5b00217

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

HRMS (ESI(+)) calcd for C₂₂H₂₃N₄O₂[M + H]⁺, 375.1816; found,

375.1809. Purity: >95%.

7.37 (m, 2H), 7.45−7.51 (m, 2H), 7.90 (br s, 1H), 7.99 (s, 1H), 8.11

(d, J = 5.3 Hz, 1H), 8.44 (br s, 1H). ¹³C NMR (150 MHz, CDCl₃) δ

(ppm): 18.5, 18.7, 21.4, 24.9, 29.0, 35.3, 71.0, 114.3, 121.1, 127.6,

128.6, 128.8, 138.8, 141.8, 147.4, 149.2, 150.0, 151.2, 157.1, 159.5,

168.5. HRMS (ESI(+)) calcd for C₂₃H₂₅N₄O₂[M + H]⁺, 389.1972;

found, 389.1966. Purity: >95%.

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-N-(5-ﬂuoro-

pyridin-2-yl)-2-phenylcyclopropanecarboxamide (26). The title

compound was synthesized as a white amorphous solid (30.5 mg,

46.3% yield) from (1R,2S)-2-(((2,4-dimethylpyrimidin-5-yl)oxy)-

methyl)-2-phenylcyclopropanecarboxylic acid 18a by adapting the

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-N-(5-ﬂuoro-

4-methylpyridin-2-yl)-2-phenylcyclopropanecarboxamide (31). The

title compound was synthesized as a white amorphous solid (2.4 g,

61.7% yield) from (1R,2S)-2-(((2,4-dimethylpyrimidin-5-yl)oxy)-

methyl)-2-phenylcyclopropanecarboxylic acid by adapting the proce-

1

procedure described for compound 23. H NMR (600 MHz, CDCl₃)

δ (ppm): 1.64 (dd, J = 8.1, 5.1 Hz, 1H), 1.92 (t, J = 5.3 Hz, 1H),

2.10−2.16 (m, 1H), 2.22 (s, 3H), 2.56 (s, 3H), 4.42 (d, J = 9.4 Hz,

1H), 4.52 (d, J = 9.4 Hz, 1H), 7.29−7.34 (m, 1H), 7.35−7.43 (m,

3H), 7.46−7.50 (m, 2H), 7.98 (s, 1H), 8.10 (dd, J = 9.1, 3.8 Hz, 1H),

8.14 (d, J = 3.0 Hz, 1H), 8.29 (br s, 1H). ¹³C NMR (150 MHz,

CDCl₃) δ (ppm): 18.6, 18.7, 25.0, 28.9, 35.4, 71.1, 114.5, 125.3, 127.7,

128.7, 128.8, 135.4, 138.8, 141.7, 147.4, 149.2, 156.4, 157.0, 159.6,

168.3. HRMS (ESI(+)) calcd for C₂₂H₂₂FN₄O₂[M + H]⁺, 393.1721;

found, 393.1716. Purity: >95%.

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-N-(6-ﬂuoro-

pyridin-2-yl)-2-phenylcyclopropanecarboxamide (27). The title

compound was synthesized as a white solid (8.8 mg, 16.7% yield)

from (1R,2S)-2-(((2,4-dimethylpyrimidin-5-yl)oxy)methyl)-2-phenyl-

cyclopropanecarboxylic acid 18a by adapting the procedure described

for compound 23. ¹H NMR (600 MHz, CDCl₃) δ (ppm): 1.64 (dd, J

= 7.9, 5.5 Hz, 1H), 1.92 (t, J = 5.5 Hz, 1H), 2.09−2.16 (m, 1H), 2.22

(s, 3H), 2.56 (s, 3H), 4.41 (d, J = 9.4 Hz, 1H), 4.50 (d, J = 9.4 Hz,

1H), 6.66 (dd, J = 7.9, 2.3 Hz, 1H), 7.29−7.34 (m, 1H), 7.38 (m, 2H),

7.47 (m, 2H), 7.75 (q, J = 8.2 Hz, 1H), 7.94 (br d, J = 7.6 Hz, 1H),

7.98 (s, 1H), 8.23 (s, 1H). ¹³C NMR (150 MHz, CDCl₃) δ (ppm):

18.7, 24.9, 28.8, 35.7, 71.1, 104.3, 104.5, 110.1, 127.7, 128.7, 128.9,

138.8, 141.6, 143.4, 149.2, 149.3, 157.0, 159.6, 161.9, 168.6. HRMS

(ESI(+)) calcd for C₂₂H₂₂FN₄O₂[M + H]⁺, 393.1721; found,

393.1717. Purity: >95%.

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-N-(5-chloro-

pyridin-2-yl)-2-phenylcyclopropanecarboxamide (28). The title

compound was synthesized as a light-yellow solid (105.7 mg, 30.8%

yield) from (1R,2S)-2-(((2,4-dimethylpyrimidin-5-yl)oxy)methyl)-2-

phenylcyclopropanecarboxylic acid by adapting the procedure

described for compound 23. ¹H NMR (600 MHz, CDCl₃) δ

(ppm): 1.64 (dd, J = 7.9, 4.9 Hz, 1H), 1.92 (t, J = 5.5 Hz, 1H),

2.11−2.16 (m, 1H), 2.22 (s, 3H), 2.56 (s, 3H), 4.41 (d, J = 9.4 Hz,

1H), 4.52 (d, J = 9.4 Hz, 1H), 7.29−7.34 (m, 1H), 7.38 (t, J = 7.4 Hz,

2H), 7.45−7.50 (m, 2H), 7.63 (dd, J = 9.1, 2.6 Hz, 1H), 7.98 (s, 1H),

8.06 (d, J = 8.7 Hz, 1H), 8.24 (d, J = 2.6 Hz, 1H), 8.29 g(s, 1H). ¹³C

NMR (150 MHz, CDCl₃) δ (ppm): 18.7, 18.7, 25.0, 28.9, 35.6, 71.0,

114.4, 126.9, 127.7, 128.7, 128.8, 138.1, 138.9, 141.6, 146.5, 149.2,

149.4, 157.0, 159.6, 168.5. HRMS (ESI(+)) calcd for C₂₂H₂₂ClN₄O₂

[M + H]⁺, 409.1426; found, 409.1422. Purity: >95%.

1

dure described for compound 23. H NMR (600 MHz, CDCl₃) δ

(ppm): 1.63 (dd, J = 8.1, 5.1 Hz, 1H), 1.91 (t, J = 5.5 Hz, 1H), 2.09−

2.14 (m, 1H), 2.23 (s, 3H), 2.29 (s, 3H), 2.56 (s, 3H), 4.42 (d, J = 9.4

Hz, 1H), 4.52 (d, J = 9.4 Hz, 1H), 7.29−7.34 (m, 1H), 7.38 (m, 2H),

7.45−7.50 (m, 2H), 7.96 (d, J = 5.3 Hz, 1H), 7.98 (s, 1H), 8.02 (s,

1H), 8.23 (br s, 1H). ¹³C NMR (150 MHz, CDCl₃) δ (ppm): 14.7,

18.5, 18.7, 25.0, 28.9, 35.4, 71.0, 115.8, 127.6, 128.7, 128.8, 134.5,

136.6, 138.8, 141.7, 147.1, 149.2, 155.8, 157.0, 159.5, 168.2. HRMS

(ESI(+)) calcd for C₂₃H₂₄FN₄O₂[M + H]⁺, 407.1878; found,

407.1871. Purity: >95%.

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-N-(5-ﬂuoro-

4-methoxypyridin-2-yl)-2-phenylcyclopropanecarboxamide (32).

To a solution of carboxylic acid 18a (1 g, 3.35 mmol) was added

oxalyl chloride (575 μL, 6.7 mmol). The reaction mixture was stirred

for 2 h, then was concentrated under reduced pressure. The residue

was dissolved in THF (15 mL), and 28% aqueous ammonia (4.08 mL,

67 mmol) was added at 0 °C. This mixture was stirred for 30 min, after

which it was concentrated under reduced pressure to aﬀord crude

carboxamide. A mixture of this carboxamide (300 mg, 1.01 mmol), 2-

chloro-5-ﬂuoro-4-methoxypyridine (235 mg, 1.52 mmol), 4,5-bis-

(diphenylphosphino)-9,9-dimethylxanthene (351 mg, 0.606 mmol),

potassium triphosphate (429 mg, 2.02 mmol), and tris(dibenzylidene)-

dipalladium(0) (185 mg, 0.202 mmol) in 1,4-dioxane (20 mL) was

heated to 95 °C. and stirred for 15 h. The reaction solution was cooled

to room temperature and ﬁltered using Celite. The ﬁltrate was

concentrated under reduced pressure, and the residue was partitioned

between EtOAc and H₂O. The organic layer was washed with brine

and dried with MgSO₄, ﬁltered to remove the drying agent, and

concentrated under reduced pressure. The residue was puriﬁed by

silica gel chromatography using n-heptane/ethyl acetate (7/3 to ethyl

acetate, v/v) then by NH−silica gel chromatography using n-heptane/

ethyl acetate (4:1 to 2:3, v/v). The crude product obtained was

dissolved in chloroform, and n-hexane was added. The precipitated

solid was colected by ﬁltration and dried to give compound (304 mg,

71.2% yield). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 1.63 (dd, J = 8.0,

5.6 Hz, 1H), 1.89 (t, J = 5.6 Hz, 1H), 2.11 (dd, J = 8.0, 5.6 Hz, 1H),

2.23 (s, 3H), 2.55 (s, 3H), 3.88 (s, 3H), 4.41 (d, J = 9.6 Hz, 1H), 4.51

(d, J = 9.6 Hz, 1H), 7.28−7.39 (m, 3H), 7.45−7.48 (m, 2H), 7.82 (d, J

= 6.4 Hz, 1H), 7.97 (d, J = 2.8 Hz, 1H), 7.98 (s, 1H), 8.30 (br s, 1H).

¹³C NMR (150 MHz, CDCl₃) δ (ppm): 18.5, 18.7, 24.9, 29.0, 35.5,

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-N-(5-methyl-

pyridin-2-yl)-2-phenylcyclopropanecarboxamide (29). The title

compound was synthesized as a white amorphous solid (5.5 mg,

44.9% yield) from (1R,2S)-2-(((2,4-dimethylpyrimidin-5-yl)oxy)-

methyl)-2-phenylcyclopropanecarboxylic acid 18a by adapting the

1

56.1, 71.0, 98.4, 127.7, 128.7, 128.8, 134.7, 138.8, 141.6, 147.1, 148.6,

149.2, 155.2, 157.0, 159.6, 168.5. HRMS (ESI(+)) calcd for

C₂₃H₂₄FN₄O₃[M + H]⁺, 423.1827; found, 423.1820. Purity: >95%.

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-(2-ﬂuoro-

phenyl)-N-(5-ﬂuoropyridin-2-yl)cyclopropanecarboxamide (33).

The title compound was synthesized as a white solid (1.7 mg, 8.74%

yield) from (1R,2S)-2-{[(2,4-dimethylpyrimidin-5-yl)oxy]methyl}-2-

(2-ﬂuorophenyl)cyclopropanecarboxylic acid by adapting the proce-

procedure described for compound 23. H NMR (400 MHz, CDCl₃)

δ (ppm): 1.60 (dd, J = 8.0, 5.2 Hz, 1H), 1.90 (t, J = 5.2 Hz, 1H), 2.11

(br, 1H), 2.20 (s, 3H), 2.28 (s, 3H), 2.54 (s, 3H), 4.40 (d, J = 9.4 Hz,

1H), 4.51 (d, J = 9.4 Hz,1H), 7.27−7.38 (m, 3H), 7.45−7.48 (m, 3H),

7.94 (br, 1H), 7.96(s,1H), 8.08 (q, J = 0.8 Hz, 1H), 8.27 (br s, 1H).

¹³C NMR (150 MHz, CDCl₃) δ (ppm): 17.8, 18.4, 18.7, 25.0, 28.9,

35.2, 71.1, 113.3, 127.6, 128.6, 128.9, 129.3, 138.8, 139.0, 141.9, 147.7,

149.0, 149.2, 157.1, 159.5, 168.3. HRMS (ESI(+)) calcd for

C₂₃H₂₅N₄O₂[M + H]⁺, 389.1972; found, 389.1965. Purity: >95%.

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-N-(4-methyl-

pyridin-2-yl)-2-phenylcyclopropanecarboxamide (30). The title

compound was synthesized as a white amorphous solid (6.5 mg,

53.0% yield) from (1R,2S)-2-(((2,4-dimethylpyrimidin-5-yl)oxy)-

methyl)-2-phenylcyclopropanecarboxylic acid 18a by adapting the

1

dure described for compound 23. H NMR (600 MHz, CDCl₃) δ

(ppm): 1.53 (dd, J = 8.3, 5.3 Hz, 1H), 1.94 (t, J = 5.5 Hz, 1H), 2.15−

2.27 (m, 4H), 2.55 (s, 3H), 4.34 (d, J = 9.8 Hz, 1H), 4.43 (d, J = 9.4

Hz, 1H), 7.08−7.14 (m, 1H), 7.17 (t, J = 7.4 Hz, 1H), 7.29−7.35 (m,

1H), 7.39−7.49 (m, 2H), 7.93 (s, 1H), 8.10−8.19 (m, 2H), 8.35 (br s,

1H). ¹³C NMR (150 MHz, CDCl₃) δ (ppm): 17.6, 18.6, 24.9, 28.0,

30.3, 70.6, 114.5, 115.9, 124.4, 125.3, 128.5, 129.7, 131.9, 135.4, 138.5,

147.4, 149.2, 156.4, 157.1, 159.6, 161.9, 168.0. HRMS (ESI(+)) calcd

for C₂₂H₂₁F₂N₄O₂[M + H]⁺, 411.1627; found, 411.1621. Purity:

>95%.

1

procedure described for compound 23. H NMR (600 MHz, CDCl₃)

δ (ppm): 1.58−1.62 (m, 1H), 1.91 (t, J = 5.5 Hz, 1H), 2.11−2.16 (m,

1H), 2.22 (s, 3H), 2.32 (s, 3H), 2.56 (s, 3H), 4.43 (d, J = 9.4 Hz, 1H),

4.53 (d, J = 9.4 Hz, 1H), 6.85 (d, J = 5.3 Hz, 1H), 7.29−7.33 (m, 1H),

M

DOI: 10.1021/acs.jmedchem.5b00217

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-(3-ﬂuoro-

phenyl)-N-(5-ﬂuoropyridin-2-yl)cyclopropanecarboxamide (34).

The title compound was synthesized as a white solid (3.66 g, 56.4%

yield) from (1R,2S)-2-{[(2,4-dimethylpyrimidin-5-yl)oxy]methyl}-2-

(3-ﬂuorophenyl)cyclopropanecarboxylic acid 18c by adapting the

149.1, 155.8, 157.0, 159.7, 162.8, 167.8. HRMS (ESI(+)) calcd for

C₂₃H₂₃F₂N₄O₂[M + H]⁺, 425.1784; found, 425.1779. Purity: >95%.

Animals. All animal experiments were approved by the Animal

Care and Use Committee of Eisai Co., Ltd. Male C57BL/6NCrlCrlj

mice (Charles River, Yokohama, Japan) were maintained under a 12 h

light−dark cycle with food and water available ad libitum.

1

procedure described for compound 23. H NMR (400 MHz, DMSO-

d) δ (ppm): 1.46−1.50 (m, 1H), 1.68 (t, J = 6.0 Hz, 1H), 2.01 (s, 3H),

2.36 (s, 3H), 2.59−2.63 (m, 1H), 4.27 (d, J = 10.4 Hz, 1H), 4.66 (d, J

= 10.4 Hz, 1H), 7.06−7.11 (m, 1H), 7.37−7.44 (m, 3H), 7.60−7.65

(m, 1H), 7.85−7.89 (m, 1H), 8.11 (s, 1H), 8.30 (d, J = 3.2 Hz, 1H),

11.20 (br s, 1H). ¹³C NMR (150 MHz, CDCl₃) δ (ppm): 18.7, 18.7,

25.0, 29.0, 34.9, 70.7, 114.5, 114.7, 115.9, 124.2, 125.4, 130.2, 135.5,

138.9, 144.1, 147.3, 149.1, 156.4, 157.0, 159.8, 162.8, 167.9. HRMS

(ESI(+)) calcd for C₂₂H₂₁F₂N₄O₂[M + H]⁺, 411.1627; found,

411.1622. Purity: >95%.

In Vivo Sleep Experiments. Recordings of Wake/Sleep

Behavior. The sleep assay was quantitated using polysomnography

in mice implanted with electrodes for recording EEG and EMG

activity as previously described.³⁶

In male mice (C57BL/6NCrlCrlj; Charles River Laboratories,

Japan), compounds 26, 28, 31, and 34 were formulated in 10%

Cremophor EL, 5% DMSO in saline. The compounds or vehicle were

administered prior to switching the lights oﬀ at ZT (zeitgeber time)

12:00, and EEG/EMG data were recorded from ZT 12:00 until ZT

20:00.

After administration of compounds or vehicle, EEG/EMG activity

recording results were analyzed by software (SleepSign 3.0, Kissei

Comtech, Japan), followed up by visual conﬁrmation by an

experienced sleep researcher blinded as to treatment, to determine

wakefulness, non-REM sleep, or REM sleep. The data were expressed

as vigilance state time every hour (for time course) or for 3 h after

lights out (for cumulative analysis).³⁶

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-(4-ﬂuoro-

phenyl)-N-(5-ﬂuoropyridin-2-yl)cyclopropanecarboxamide (35).

The title compound was synthesized as a white solid (102 mg,

39.3% yield) from (1R,2S)-2-{[(2,4-dimethylpyrimidin-5-yl)oxy]-

methyl}-2-(4-ﬂuorophenyl)cyclopropanecarboxylic acid by adapting

the procedure described for compound 23. ¹H NMR (400 MHz,

DMSO-d) δ (ppm): 1.43−1.45 (m, 1H), 1.66 (t, J = 4.4 Hz, 1H), 2.02

(s, 3H), 2.36 (s, 3H), 2.55−2.58 (m, 1H), 4.26 (d, J = 10.4 Hz, 1H),

4.59 (d, J = 10.4 Hz, 1H), 7.15−7.20 (m, 2H), 7.57−7.65 (m, 3H),

7.86−7.89 (m, 1H), 8.09 (s, 1H), 8.30 (d, J = 3.2 Hz, 1H), 11.18 (br s,

1H). ¹³C NMR (150 MHz, CDCl3) δ (ppm): 18.6, 18.7, 24.9, 28.8,

34.8, 71.1, 114.5, 115.6, 125.4, 130.6, 135.4, 137.5, 138.9, 147.3, 149.1,

156.4, 157.0, 159.7, 162.1, 168.1. HRMS (ESI(+)) calcd for

C₂₂H₂₁F₂N₄O₂[M + H]⁺, 411.1627; found, 411.1620. Purity: >95%.

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-(3,4-di-

ﬂuorophenyl)-N-(5-ﬂuoropyridin-2-yl)cyclopropanecarboxamide

(37). The title compound was synthesized as a white amorphous solid

(123 mg, 53.4% yield) from (1R,2S)-2-{[(2,4-dimethylpyrimidin-5-

yl)oxy]methyl}-2-(3,4-diﬂuorophenyl)cyclopropanecarboxylic acid by

Data were expressed as mean SEM. Unpaired parametric two-

tailed t test for 26, 28, and 31 or one-way analysis of variance

(ANOVA) followed by Dunnett’s multiple comparison test for 34

were applied to test the null hypothesis. A value of p < 0.05 was

considered statistically signiﬁcant. Statistical analyses were performed

using GraphPad Prism version 6.02 (GraphPad Software, Inc.).

Cell-Based Calcium Accumulation Functional Assay. HEK-

293 cells expressing human OX₁R or OX₂R were cultured in

Dulbecco’s Minimum Essential Medium (DMEM)/10% fetal bovine

serum and frozen in aliquots until use. On the day before the

experiment, cells were thawed and seeded at 16000 cells/well into

poly-^D-lysine-coated 384-well plates (black walls with transparent

bottom) in a culture medium and incubated overnight at 37 °C. On

the day of the experiment, the cell supernatant medium was replaced

with an assay buﬀer (115 mM NaCl, 5.4 nM KCl, 0.8 mM MgCl₂, 1.8

mM CaCl₂, 13.8 mM D-glucose, 20 mM HEPES, 0.1% bovine serum

albumin), containing Calcium 4 calcium-sensitive dye (FLIPR

Calcium4 assay kit, Molecular Devices; Sunnyvale, CA, USA), and

cells were subsequently incubated at 37 °C for 1 h, followed by 15 min

at room temperature. After transfer to an FDSS6000 device

(Hamamatsu Photonics; Hamamatsu, Japan), test compounds

dissolved in the assay buﬀer were added to the cells and allowed to

equilibrate for 30 min at room temperature. Baseline ﬂuorescence was

measured for approximately 10 s (9 measurements, 1.1 s each) before

buﬀer alone or orexin-A in assay buﬀer were added in the 10th

measurement, followed by 101 additional measurements (1.1 s each).

All measurements were obtained using excitation and detection

wavelengths of 480 and 540 nm, respectively. Calcium accumulation

was expressed as the ratio of peak ﬂuorescence intensity to

ﬂuorescence intensity at the baseline (9th measurement). In every

experiment, the dose−response of orexin-A was measured to conﬁrm

the conditions of the cells and to calculate the half-maximal eﬀective

concentration (EC₅₀). Test compounds were challenged with orexin-A

at a concentration (L) equivalent to EC₈₀on both receptors. The

inhibition potential of the test compounds was expressed as the

concentration necessary to reduce the orexin-A-elicited response by

50% (IC₅₀), which was then converted to the inhibition constant, K_i,

by using the Cheng−Prusoﬀ formula: K_i= IC₅₀/[1 + (L/EC₅₀)].

Evaluation of Pharmacokinetic Proﬁle in Mice. Compound 34

was formulated for intravenous (1 mg/kg) and oral (10 mg/kg)

administrations in 10% DMSO in water and 10% Cremophor EL, 5%

DMSO in saline, respectively, and adeministered to male C57BL/

6NCrlCrlj mice. After administration, blood was collected from the tail

vein at the designated time points and centrifuged to obtain the plasma

sample. Plasma samples were precipitated with 20 volumes of

acetonitrile containing the internal standard (10 ng/mL imipramine).

Following vortex mixing and centrifugation, the supernatant was

1

adapting the procedure described for compound 23. H NMR (400

MHz, CDCl₃) δ (ppm): 1.58−1.61 (m, 1H), 1.91 (t, J = 5.2 Hz, 1H),

2.07 (br t, J = 8.0 Hz, 1H), 2.21 (s, 3H), 2.56 (s, 3H), 4.39 (d, J = 9.6

Hz, 1H), 4.45 (d, J = 9.6 Hz, 1H), 7.12−7.41 (m, 4H), 7.97 (s, 1H),

8.04−8.08 (m, 1H), 8.12 (d, J = 2.4 Hz, 1H), 8.30 (br s, 1H). ¹³C

NMR (150 MHz, CDCl₃) δ (ppm): 18.6, 18.7, 25.0, 28.9, 34.6, 70.7,

114.5, 117.5, 118.1, 124.8, 125.4, 135.5, 138.6, 138.9, 147.2, 149.0,

149.8, 150.1, 156.4, 156.87, 159.9, 167.7. HRMS (ESI(+)) calcd for

C₂₂H₂₀F₃N₄O₂[M + H]⁺, 429.1533; found, 429.1527. Purity: >95%.

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-(3,5-di-

ﬂuorophenyl)-N-(5-ﬂuoropyridin-2-yl)cyclopropanecarboxamide

(36). The title compound was synthesized as a white amorphous solid

(140 mg, 60.7% yield) from (1R,2S)-2-{[(2,4-dimethylpyrimidin-5-

yl)oxy]methyl}-2-(3,5-diﬂuorophenyl)cyclopropanecarboxylic acid by

1

adapting the procedure described for compound 23. H NMR (400

MHz, CDCl₃) δ (ppm): 1.61−1.64 (m, 1H), 1.93 (t, J = 5.2 Hz, 1H),

2.09 (br t, J = 8.0 Hz, 1H), 2.22 (s, 3H), 2.56 (s, 3H), 4.40 (d, J = 9.6

Hz, 1H), 4.49 (d, J = 9.6 Hz, 1H), 6.74−6.79 (m, 1H), 6.98 (d, J = 6.0

Hz, 2H), 7.36−7.41 (m, 1H), 7.99 (s, 1H), 8.05 (dd, J = 3.6, 9.2 Hz,

1H), 8.11 (d, J = 2.8 Hz, 1H), 8.35 (br t, 1H). ¹³C NMR (150 MHz,

CDCl₃) δ (ppm): 18.7, 18.7, 25.0, 29.1, 34.7, 70.3, 103.3, 111.7, 114.5,

125.4, 135.5, 138.9, 145.4, 147.2, 149.0, 156.5, 156.9, 160.0, 163.0,

167.4. HRMS (ESI(+)) calcd for C₂₂H₂₀F₃N₄O₂[M + H]⁺, 429.1533;

found, 429.1526. Purity: >95%.

(1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-N-(5-ﬂuoro-

4-methylpyridin-2-yl)-2-(3-ﬂuorophenyl)cyclopropanecarboxamide

(38). The title compound was synthesized as a white amorphous solid

(289 mg, 55.4% yield) from (1R,2S)-2-{[(2,4-dimethylpyrimidin-5-

yl)oxy]methyl}-2-(3-ﬂuorophenyl)cyclopropanecarboxylic acid by

1

adapting the procedure described for compound 23. H NMR (400

MHz, CDCl₃) d (ppm): 1.55−1.65 (m, 1H), 1.91 (t, J = 5.6 Hz, 1H),

2.05−2.13 (m, 1H), 2.22 (s, 3H), 2.27 (s, 3H), 2.56 (s, 3H), 4.41 (d, J

= 10.0 Hz, 1H), 4.50 (d, J = 9.2 Hz, 1H), 6.97−7.04 (m, 1H), 7.14−

7.20 (m, 1H), 7.22−7.28 (m, 1H), 7.33 (td, J = 8.0, 5.8 Hz, 1H), 7.93

(d, J = 5.2 Hz, 1H), 7.99 (s, 1H), 8.00 (s, 1H), 8.24 (br s, 1H). ¹³C

NMR (150 MHz, CDCl₃) δ (ppm): 14.7, 18.6, 18.7, 25.0, 29.0, 34.8,

70.6, 114.7,115.8, 115.8, 124.2, 130.2, 134.5, 136.7, 138.8, 144.1, 147.0,

N

DOI: 10.1021/acs.jmedchem.5b00217

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

ﬁltered, and the resulting ﬁltrate was injected into the LC−MS/MS

system. Detection was performed by multiple reaction monitoring in

positive ionization mode. The standard curve was obtained from the

ratios of the peak area responses relative to the internal standard using

linear least-squares regression with a 1/x²weighting. On the basis of

the plasma concentrations, pharmacokinetic parameters were calcu-

lated using a software of Phoenix WinNonlin Ver. 6.3 (Certara USA,

Inc., Missouri, US) via noncompartment analysis.

Exploratory Determination of Plasma and CSF Concen-

trations in Mice. Compound 34 was formulated as a suspension in

0.5% aqueous methylcellulose and orally administered to male

C57BL/6NCrlCrlj mice at a dose of 10 mg/kg. Subsequently, 1 or

3 h after administration, the mice were anesthetized with sodium

pentobarbital and CSF was collected from the cisterna magna.

Immediately after CSF sampling, a blood sample was collected from

the abdominal aorta and centrifuged to obtain the plasma sample. The

plasma and CSF samples were stored below −20 °C until analysis by

liquid chromatography−tandem mass spectroscopy (LC−MS/MS).

Plasma samples were precipitated with 25 volumes of acetonitrile

containing the internal standard (1 ng/mL deuterated 34). Following

vortex mixing and centrifugation, the supernatant was ﬁltered and the

resulting ﬁltrate was injected into the LC−MS/MS system. CSF

samples were mixed with 9 volumes of acetonitrile and 50 volumes of

acetonitrile containing the internal standard. After vortex mixing, the

mixture was injected onto the LC−MS/MS system. Plasma and CSF

concentrations were determined by LC−MS/MS, and the values are

reported as means SEM.

residual activity in the presence of NADPH relative to that in the

absence of NADPH was evaluated.⁴³

ASSOCIATED CONTENT

* Supporting Information

■

S

Synthesis and analytical data of all compounds and

intermediates not described in the Experimental Section. The

Supporting Information is available free of charge on the ACS

Publications website at DOI: 10.1021/acs.jmedchem.5b00217.

AUTHOR INFORMATION

Corresponding Author

*Phone: +81 (0)29 847 7395. Fax: +81 (0)29 847 4952. E-

mail: y11-yoshida@hhc.eisai.co.jp.

■

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We express our gratitude to the colleagues who supported the

generation of data reported in this manuscript. Special thanks

to Makoto Nakagawa for in vivo experiment, Eri Ena, Satoko

Naito, Masaki Kato, and Yumi Asai for NMR and HRMS

analyses, and to Misako Watanabe and Kazuya Nagaoka for

chemo-informatic support.

Exploratory Assessment of Plasma Protein Binding in Mice.

The plasma protein binding of compound 34 in C57BL/6N mice was

determined by equilibrium dialysis. Blood was collected from the

abdominal vein under anesthesia, and the plasma was obtained via

centrifugation. Plasma samples spiked with 34 (1000 ng/mL) were

dialyzed against an equal volume of PBS over a cellulose membrane

with a 14000 Da molecular weight cutoﬀ for 24 h at 37 °C. Following

the dialysis, plasma and PBS fractions were collected and mixed with 4

volumes of acetonitrile containing the internal standard (100 ng/mL

propranolol). After vortex mixing, the mixture was injected into the

LC−MS/MS system. Plasma protein binding was calculated based on

the concentrations measured in the plasma and PBS fractions using the

following equation:

ABBREVIATIONS USED

■

OX₁R, orexin 1 receptor; OX₂R, orexin 2 receptor; NaHMDS,

sodium bis(trimethylsilyl)amide; TBDPS, tert-butyldiphenylsil-

yl; DIAD, diisopropyl azodicarboxylate; TEA, triethylamine;

HATU, O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluro-

niumhexaﬂuorophosphate; DIPEA, N,N-diisopropylethylamine;

m-CPBA, m-chloroperoxybenzoic acid; acac, acetylacetone; Bn,

benzyl; EMG, electromyogram; ZT, zeitgeber time

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⎛

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Metabolic Stability Screening in Liver Microsomes. Test

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J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Discovery of (1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide (E2006): A Potent and Efficacious Oral Orexin Receptor Antagonist

DOI: 10.1021/acs.jmedchem.5b00217

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Article abstract of DOI:10.1021/acs.jmedchem.5b00217

Full text of DOI:10.1021/acs.jmedchem.5b00217

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