Identification of a novel series of orexin receptor antagonists with a distinct effect on sleep architecture for the treatment of insomnia

Article

pubs.acs.org/jmc

Identiﬁcation of a Novel Series of Orexin Receptor Antagonists with a

Distinct Eﬀect on Sleep Architecture for the Treatment of Insomnia

Claudia Betschart,*^,†,○Samuel Hintermann,*^,†,○Dirk Behnke,^†Simona Cotesta,^†Markus Fendt,^‡,∞

Christine E. Gee,^‡,#Laura H. Jacobson,^‡,●Grit Laue,^§Silvio Ofner,^†Vinod Chaudhari,^∥,×

Sangamesh Badiger,^∥Chetan Pandit,^∥Juergen Wagner,^†and Daniel Hoyer^‡,⊥

‡

§

^†Global Discovery Chemistry, Neuroscience, and Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research,

CH-4002 Basel, Switzerland

^∥Medicinal Chemistry, Aurigene Discovery Technologies, 39-40, Electronic City Phase 2, Bangalore 560 100, India

ABSTRACT: Dual orexin receptor (OXR) antagonists

(DORAs) such as almorexant, 1 (SB-649868), or suvorexant

have shown promise for the treatment of insomnias and sleep

disorders in several recent clinical trials in volunteers and

primary insomnia patients. The relative contribution of

antagonism of OX₁R and OX₂R for sleep induction is still a

matter of debate. We therefore initiated a drug discovery

project with the aim of creating both OX₂R selective

antagonists and DORAs. Here we report that the OX₂R

selective antagonist 26 induced sleep in mice primarily by

increasing NREM sleep, whereas the DORA suvorexant

induced sleep largely by increasing REM sleep. Thus, OX₂R selective antagonists may also be beneﬁcial for the treatment of

insomnia.

with some fragmentation of sleep, whereas mice lacking OX₂R

show a more pronounced sleep phenotype albeit milder than

that seen with a complete loss of orexin signaling.²⁸In mice, the

sleep inducing properties of the DORA almorexant depend

primarily on the presence of OX₂R,²⁹suggesting that

antagonism of OX₂R alone may be suﬃcient for inducing

sleep. Conclusions from experiments using receptor selective

antagonists in rats are partially contradictory. Many reports

suggest that OX₂R selective antagonists induce sleep, whereas

OX₁R selective antagonists are usually reported to lack this

eﬀect.³⁰⁻³³Only one group reported on an increase in total

sleep time after administration of an OX₁R selective

antagonist.³⁴More controversial is the eﬀect of dual antagonists

or coadministration of OX₁R and OX₂R antagonists. While

combined antagonism of both receptors was reported to

promote less sleep than OX₂R inhibition alone,³¹it has also

been reported that dual antagonism is more eﬀective than

antagonism of either receptor alone.³⁴It is unclear whether

variations in time and route of application may be responsible

for some of the discrepancy (with the exception of the OX₁R

selective antagonist (R)-2-((S)-1-(3,4-dimethoxybenzyl)-6,7-

dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-N-isopropyl-2-

phenylacetamide (ACT335827),³³application of compounds in

these studies was ip, sc, or icv, as properties of the tool

compounds did not allow for oral application). Overall, there is

strong evidence that at least antagonism of OX₂R is required

INTRODUCTION

■

The orexin system is composed of two widely expressed G-

protein-coupled receptors orexin 1 receptor (OX₁R) and orexin

2 receptor (OX₂R) and two peptide agonists orexin A and

orexin B (also known as hypocretin 1 and hypocretin 2) that

are selectively expressed in a small cluster of neurons in the

lateral hypothalamus.^1,2The orexin system is involved in a

number of systems including feeding, addiction, depression,

anxiety, panic, and lung/respiratory function as suggested from

animal studies.³⁻⁶A key role for the orexin system in the

regulation of sleep/wake was indicated by the observations that

mice lacking orexin peptides or both orexin receptors display a

strong narcoleptic-like phenotype with cataplexy⁷⁻¹⁰and that

familial canine narcolepsy is caused by defective OX₂R

signaling.¹¹Shortly after this discovery, nearly undetectable

CSF levels of orexin and the loss or near complete loss of the

orexinergic neurons were reported in humans suﬀering from

narcolepsy with cataplexy.^12,13Conversely, orexin increases

arousal¹⁴and orexin neurons ﬁre during wake and excite

neurons in brain regions important for maintaining wakeful-

ness.¹⁵⁻¹⁷Novel treatments for sleep disorders have therefore

targeted the orexin system, and the discovery of dual OX₁R/

OX₂R as well as subtype selective orexin receptor antagonists

has been reported.^6,18−22Positive clinical proof of concept

studies have demonstrated sleep induction by the three DORAs

shown in Figure 1.²³⁻²⁷However, it remains unclear whether

targeting one or both receptors is required for sleep induction

or conversely if antagonism at both receptors may be

detrimental. In mice, loss of OX₁R results in a mild phenotype

Received: May 22, 2013

A

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Figure 1. Orexin receptor antagonists in clinical trials.

Scheme 1. Scaﬀold Morphing of Z- and E-Amide Conformations of a Literature Compound

a

Scheme 2. Synthesis of Compounds 3 and 6

a

Reaction and conditions: (a) TFA, CH₂Cl₂, rt, quant; (b) 2-chloroquinoxaline, K₂CO₃, DMF, 80 °C, 71%; (c) for 3, benzyl bromide, NaH, THF,

60 °C, 19%; for 6, 2,5-dimethylbenzyl chloride, NaH, THF, 60 °C, 22%.

for sleep induction.²⁹⁻³⁷In our drug discovery program, we

sought to generate OX₂R selective antagonists as well as

DORAs. Here we present the design of a novel series of orally

active orexin receptor antagonists and report on the distinctive

eﬀect of an OX₂R selective compound on sleep EEG in mouse,

in comparison with the DORA suvorexant.

Therefore, we decided to include this acceptor function in our

pharmacophore model. Using this pharmacophore to search

our compound library, we identiﬁed several novel core

structures. Among them were a number of compounds with a

carbonyl group as acceptor directly incorporated into the

aliphatic group of the spacer. While we decided not to follow

up these speciﬁc hits, this observation inspired us to apply the

same design principle to a spiro scaﬀold of published orexin

antagonists (Scheme 1).^39,40

RESULTS AND DISCUSSION

■

Ligand Design. Analysis of published structures of orexin

receptor antagonists revealed in several cases a general motif

comprising two aromatic moieties linked by a spacer. In most

cases this spacer comprises an aliphatic group and at least one

acceptor such as an amide, a urea, or a sulfonamide. For

example, in 1 (SB-649868)³⁸the aromatic elements are the

benzofuran and the methylthiazole, and the piperidine is the

aliphatic part of the spacer. In suvorexant, the aromatic features

are the chlorobenzoxazole and the tolyl, and the diazepane is

part of the spacer. To the best of our knowledge, the exact role

of the acceptor group has never been clariﬁed. It could be

involved in polar interactions with the receptors or may simply

be important for constraining a speciﬁc ligand conformation.

On the basis of our own and published SAR information, we

decided to use compound 2³⁹as the starting point. Morphing

of 2 led to two novel core scaﬀolds each mimicking either the

E- or Z-conﬁguration of the amide. This is illustrated by the

alignments of low energy conformers in Scheme 1. We were

expecting to gain some information on the bioactive

conformation of 2, anticipating that only one of the mimics

would show potency on the receptors. In order to test our

1

hypothesis, we synthesized compounds 2−4. The H NMR

spectrum of 2 at room temperature showed only one set of

resonances, suggesting a fast equilibrium between E- and Z-

amides. Calculations support this interpretation, as the

B

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a

Scheme 3. Synthesis of Compound 4

a

Reagents and conditions: (a) (1) LDA, THF, −78 °C; (2) 3-bromopropionitrile, THF, −78 °C to rt, 40%; (b) Raney Ni, H₂, NH₃(5% in EtOH),

rt, 41%; (c) NaH, BnBr, TBAI, THF/DMF, 0−60 °C, 67%; (d) 4 M HCl in dioxane, rt, 83%; (e) 2-chloroquinoxaline, NEt₃, EtOH, microwave, 160

°C, 66%.

a

Scheme 4. Synthesis of Compounds 14−17

a

Reagents and conditions: (a) (1-tosyl-1H-indol-4-yl)methanamine, 2-allyl-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, toluene, 4 Å molecular sieves,

reﬂux, 70%; (b) 1-tosyl-1H-indole-3-carbaldehyde, 1,2-dichloroethane, AcOH, 60 °C, then NaBH(OAc)₃, 60 °C, 60%; (c) acryloyl chloride, DIPEA,

CH₂Cl₂, 0 °C to rt, 67−71%; (d) Grubbs second generation catalyst, CH₂Cl₂, rt, 84−90%; (e) H₂, 10% Pd/C, MeOH, rt, 96−99%; (f) TFA,

CH₂Cl₂, 0 °C to rt, 95% to quant; (g) R¹Cl, DBU, NMP, 110 °C, microwave; (h) Cs₂CO₃, MeOH, reﬂux, 20−94% (two steps). Residue R²for

intermediates 10−13 is deﬁned as follows: (a) (1-tosyl-1H-indol-4-yl)methyl; (b) (1-tosyl-1H-indol-3-yl)methyl.

rotational barrier for the amide is predicted to be around 19

kcal/mol.⁴²Similar observations with respect to E- and Z-amide

rotamers were also reported for other orexin receptor

antagonists.^38,43−47

Synthesis. Compounds 3 and 4 were synthesized as

described in Schemes 2 and 3. For the E-amide mimic 3,

synthesis started from commercial 9-boc-1,9-diazaspiro[5.5]-

undecan-2-one which was deprotected and then reacted with 2-

chloroquinaxoline under basic conditions to give the inter-

mediate 5 which was subsequently alkylated with benzyl

bromides using sodium hydride as base. By use of 2,5-

dimethylbenzyl chloride as alkylating agent, analogue 6 was

obtained under the same reaction conditions.

Synthesis of the Z-amide mimic 4 ﬁrst required a reliable

access to the spiro-lactam 8 (Scheme 3) that was also necessary

for later SAR exploration. Deprotonation of 1-tert-butyl 4-ethyl

piperidine-1,4-dicarboxylate with LDA at low temperature

followed by reaction with 3-bromopropionitrile yielded

intermediate 7 reproducibly and in moderate yield. Reduction

of the nitrile using Raney nickel as a catalyst under hydrogen

atmosphere in ethanolic ammonia yielded the corresponding

primary amine as intermediate, which cyclized spontaneously to

yield the desired lactam 8. Finally, benzylation of the lactam

using sodium hydride as a base followed by boc deprotection

and reaction with 2-chloroquinoxaline gave the desired

compound 4 in good yields.

For our initial SAR work around the benzyl group of core 1,

we intended to rely on the alkylation strategy that was used for

the synthesis of 3. However, it became rapidly evident that

alkylation on the rather hindered lactam worked well only for

simple and highly reactive alkylating agents such as benzyl

bromide. In the case of less reactive reagents, such as

indolylmethyl halogenides, decomposition of the agents was

faster than the desired alkylation and yields were extremely low.

In these cases, incorporation of the side chain before closure of

the lactam proved to be eﬀective (Scheme 4).

Secondary amines 10 could be synthesized eﬃciently by two

diﬀerent routes as exempliﬁed in Scheme 4: either by an α-

aminoallylation reaction of N-boc-piperidine-4-one in a one-pot

addition of the allyl group to the in situ generated ketimine

using pinacol allylboronate⁴⁸or by reductive amination of N-

boc-4-allylpiperidin-4-amine with the corresponding indolylcar-

C

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a

Scheme 5. Synthesis of Compound 19

a

Reagents and conditions: (a) HCl 4 M in dioxane, CH₂Cl₂, rt, 82%; (b) 2-bromoquinoxaline, K₂CO₃, DMF, 60 °C, 76%; (c) NaH, 4-

(bromomethyl)-1-tosyl-1H-indole, THF, 50 °C, 55%.

a

Scheme 6. Synthesis of Compounds 22−28

a

Reagents and conditions: (a) LDA, 3-(bromomethyl)-1-tosyl-1H-indole, THF, 0 °C to rt, quant; (b) TFA, CH₂Cl₂, rt, quant.; (c) Cs₂CO₃, MeOH,

reﬂux, 99%; (d) R¹-Cl, DIPEA, DBU, MeCN, 120 °C, microwave, 41−74%; (e) NaH, MeI, THF, 0 °C to rt, 25%.

a

Scheme 7. Synthesis of Compounds 30−32

a

Reagents and conditions: (a) TFA, CH₂Cl₂, 0 °C to rt, quant; (b) 2-chloro-4,6-dimethylpyrimidine, EtOH, DIPEA, DMAP, 160 °C, microwave,

75%; (c) R²-Br, NaH, TBAI, THF, 0 °C to rt; (d) Cs₂CO₃, MeOH, reﬂux, 67−88% (two steps).

a

Scheme 8. Synthesis of Compounds 34−37

a

Reagents and conditions: (a) NaH, TBAI, THF, 0 °C to rt, (b) 6 M aq HCl, EtOH, 90 °C, 32−91% (two steps); (c) NaH, MeI, THF, 0 °C to rt,

30−74%.

D

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Figure 2. Orexin receptor antagonistic activity of original (2) and designed analogous scaﬀolds (3 and 4).

baldehyde. Both routes yielded the desired intermediates 10 in

good yields.

potencies on both OX₁R and OX₂R (Figure 2). In addition, the

potency was in the same range as for 2, the analogous example

of the original scaﬀold, thereby validating our design principle.

The similar activity observed for the E- and Z-amide

mimetics 3 and 4 can be interpreted in two ways. Either the

carbonyl is not involved in polar interactions with the receptor

and its main role is to ﬁx the conformation of the ligand or the

two molecules bind to the receptor by diﬀerent binding modes.

In the latter case the carbonyl could be involved in forming

polar interactions with diﬀerent residues.

Acylation with acryloyl chloride followed by ring-closing

metathesis and hydrogenation yielded the substituted lactams

13 in excellent yields. Finally, boc deprotection followed by

arylation with the appropriate aryl chlorides under microwave

irradiation using cesium carbonate as a base and removal of the

tosyl-protecting group led to the ﬁnal products 14−17.

For SAR exploration around core 2, we were delighted to

ﬁnd that the alkylation route worked well for a range of

electrophiles. It was found that even the order of the alkylation

and the arylation steps was interchangeable, thus allowing

incorporation of diversity for both the benzyl and the

quinoxalyl portions of the molecule late in the synthesis.

Scheme 5 exempliﬁes the synthesis of the 4-indolyl regioisomer

19 using the arylation/alkylation strategy.

Intermediate 8 was boc-deprotected under acidic conditions

and then reacted with 2-bromoquinoxaline in good yield to

obtain the desired lactam 18. Deprotonation of the lactam with

sodium hydride and reaction with tosyl-protected 4-

(bromomethyl)indole at slightly elevated temperature led to

the alkylation product, which under the reaction conditions was

also slowly tosyl-deprotected to yield 19.

Incorporation of a diverse set of quinoxaline replacements,

this time using the alkylation/arylation strategy, is exempliﬁed

in Scheme 6. The previously described intermediate 8 was

alkylated with tosyl protected 3-(bromomethyl)indole using

LDA as base. Removal of both protecting groups followed by

arylation under microwave irradiation gave direct access to

compounds 22−27 in good overall yields. Indole N-alkylation

of 27 with iodomethane using sodium hydride as base gave N-

methyl analogue 28.

Synthetic access to substituted indole and indole isosteres is

described in Scheme 7. Starting from intermediate 8, the boc

group was removed and the resulting amine reacted with 2-

chloro-4,6-dimethylpyrimidine to give the intermediate lactam

29. Subsequent alkylation using sodium hydride, catalytic

amounts of TBAI, and the appropriate alkyl bromides followed

by tosyl deprotection using cesium carbonate yielded the

respective analogues 30−32.

Initially, we focused our derivatization eﬀorts on 3 (core 1)

mainly because of its more attractive lipophilicity compared to

4 and therefore the resulting favorable lipophilic eﬃciency

(LipE)⁴⁹(Table 1).

First we investigated the eﬀects of small substituents on the

benzyl moiety. A gain in potency by 1 log unit could easily be

achieved as exempliﬁed with 6, however, only at the expense of

increased lipophilicity (Table 1). In parallel, we explored

heteroaryl replacements of the phenyl group to keep the

lipophilicity in a more favorable range. Whereas monoaryl

derivatives such as pyridines lost potency (data not shown),

some fused biaryls looked promising. For example, the indole

moiety reduced lipophilicity in parallel with increased potency

(14 and 15), resulting in considerably improved ligand

eﬃciency (LE)⁵⁰and LipE. In particular, the 4-indolyl

derivative 15 showed excellent activity, reaching subnanomolar

K_ion OX₂R. Next we investigated modiﬁcations of the

quinoxaline. Interestingly, when the 4-indolyl moiety was

kept in place, high potency on OX₂R was retained for a range of

substitutions (e.g., 15-17). On the other hand, activity on

OX₁R was more sensitive to structural changes, resulting in

some cases in high selectivity for OX₂R (16). As we were

interested in both OX₂R selective and dual orexin receptor

antagonists, the on-target potency optimization was primarily

directed toward OX₂R. Nonetheless, the compounds were also

routinely tested for antagonism of OX₁R.

With highly potent compounds in hand, in vivo character-

ization was initiated. In mouse PK studies, by use of a cassette

dosing approach, very high blood clearances and low absolute

oral bioavailabilities were found for all derivatives of core 1, as

exempliﬁed for 15 in Table 2. The investigation of metabolism

in liver microsomes or in vivo in mice revealed multiple

metabolic weak spots. This liability could not be overcome

despite major synthetic eﬀorts. Therefore, the focus of

optimization was shifted toward derivatives of core 2, despite

their intrinsic higher lipophilicity and lower LipE.

Finally, the alkylation strategy was also successfully applied

for the introduction of substituted pyrazoles and triazoles on

core 2, as illustrated in Scheme 8. Lactam 29 was reacted with

SEM-protected bromides 33 under the same conditions as

described in Scheme 7. Then the intermediates were

deprotected under acidic conditions to yield the desired

compounds 34 and 35. Methylation under standard conditions

gave the corresponding analogues 36 and 37.

In Vitro SAR. Assuming that only one of the rotamers of 2

would represent the bioactive conformation, we expected a

clear diﬀerence in potency for the E- and Z-amide mimics 3 and

4. To our surprise, the two antagonists showed comparable

While 3 (E-amide mimic) and 4 (Z-amide mimic) showed

comparable potencies, other modiﬁcations revealed clear

diﬀerences in the SAR for the two cores, supporting the

hypothesis that the two scaﬀolds could adopt diﬀerent binding

modes. It is for instance interesting to note that for core 1,

going from the 3-indolyl derivative 14 to the corresponding 4-

E

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Table 1. In Vitro SAR Data

F

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Table 1. continued

a

b

Average values from at least three independent measurement. In the cases of n = 1 or n = 2, individal measurements are reported. Single

c

d

measurements (SD of the assay was determined to be 0.34). Calculated using HTlogP (LipE = pK_i− HTlogP). log P.

a

Table 2. Mouse PK Data of Selected Compounds

b

c

intravenous dose, 1 mg/kg

oral dose, 3 mg/kg

d

e

f

compd

Cl_bl(mL min⁻¹kg⁻¹

)

V_ss(L/kg)

C_max,bl(nM)

AUC_bl(nM·h)

F

(%)

[br]/[bl] at 15 min pd SD

g

15

26

188

28

3.1

1.6

18

31

5

0.2

864

2086

47

1.1 0.1

a

The absorption and disposition parameters were estimated by a noncompartmental analysis of the mean blood concentration (n = 3) versus time

b

c

proﬁle after oral and intravenous administration. Dosed as a solution in NMP/blank plasma (10/90, v/v). Dosed as a suspension in

carboxymethylcellulose 0.5% w/v in water/Tween 80 (99.5%/0.5%, v/v). AUC was extrapolated to inﬁnity. Absolute oral bioavailability was

calculated by dividing the dose-normalized AUC after oral and intravenous dosing assuming linear pharmacokinetics between these two doses. Mean

brain/blood concentration ratio from n = 3. Only 2 out of 3 animals showed blood or brain levels above the quantitation limit.

d

e

f

g

indolyl derivative 15 resulted in a potency gain of almost 2

orders of magnitude. This is in contrast to core 2 where no

large diﬀerence for the corresponding regioisomers 19 and 22

was found. Their potencies were both in the range of the

weaker isomer 14 of core 1. Overall, single digit nanomolar

potency for derivatives of core 2 was more diﬃcult to reach

than for core 1. Together with the higher lipophilicity, this

generally resulted in lower LipE in this series.

Therefore, in an attempt to reduce the lipophilicity, the next

round of optimization focused on the quinoxaline part of 22.

G

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a

Table 3. Mouse Speciﬁc Data for 26 and Suvorexant

FLIPR, pK_iSEM

b

compd

26

suvorexant

mOX₁R

mOX₂R

[brain] 60 min pd SD (pmol/g)

[blood] 60 min pd SD (nM)

brain f_uSD (%)

6.34 0.06

8.77 0.14

7.23 0.04

8.06 0.09

8778 1205

10329 3667

6912 698

0.61 0.06

0.65 0.05

10461 3584

a

All compounds were dosed per os at 50 mg/kg as a suspension in methylcellulose 0.5% w/v in water. Shown are the mean values (n = 3) SD.

b

The free fraction in mouse brain homogenate was assessed using a slightly adapted literature method.⁵²

Pyrazine as a monocyclic replacement (23) led to the expected

reduction of log P by 1 unit, albeit at the expense of a lower

potency. In the case of the pyrimidine (24), lipophilicity

reduction was more signiﬁcant, leading to an increase of LipE

by half a unit. In addition, potency could be recovered by

appropriate substitution of the pyrimidine. For instance,

incorporation of a methyl (25) or a methoxy group (26) in

position 4 or 4,6-dimethyl substitution (27) led to increased

potencies and a net increase in LipE. A further jump in potency

and LipE could be achieved by substitution of the indole 5-

position with small lipophilic substituents, as exempliﬁed by

methoxy analogue 30, leading to highly potent OX₂R-preferring

antagonists.

A locomotor activity assay in mouse was used as a ﬁlter

before performing more resource demanding sleep measure-

ments. In addition, brain and blood levels were determined 1 h

postdose in satellite groups of animals (Table 3). In the

locomotor paradigm, reduction of motility (i.e., increase in

inactivity) was measured as a surrogate for sleep.⁵³Animals

were dosed orally immediately before lights oﬀ (start of the

active phase). Inactivity was deﬁned as at least 1 min with no

infrared beam breaks. The increase in inactivity during the ﬁrst

4 h postdose was calculated relative to the same 4 h on the

previous day during the habituation phase.²⁹Suvorexant was

used as positive control. Dosed at 50 mg/kg, 26 and suvorexant

had a signiﬁcant eﬀect in this model (Figure 3). Estimated free

N-Methylation of the indole (28) led to only a marginal drop

in potency, suggesting that the NH is not involved in a direct

interaction with the receptors. The corresponding indazole

(31) and aza-indole (32) derivatives were found to be

equipotent OX₂R antagonists. As expected, the measured

log P was lower for the two analogues when compared to 27.

In a next round of optimization, the indazole was morphed

into a 4-phenylpyrazole (this can formally be seen as a ring-

opening of the annulated system) leading to analogue 34 which

turned out to be a very potent OX₂R antagonist. Interestingly,

in contrast to the indole, N-methylation of the phenylpyrazole

(35) led to a loss of 1 order of magnitude in potency. Finally,

when going from the pyrazole to the triazole, a very potent

antagonist (36) with increased LipE (5.2) was identiﬁed. As for

the indoles, N-methylation (37) had only a small impact on

potency but still led to a decrease of LipE. Interestingly,

compounds 34−37 contain a biaryl moiety, a common motif in

known orexin antagonists (see, for example, 1 and suvorexant),

indicating that the core 2 compounds could interact with the

receptors through similar interactions.

In Vivo Characterization. Compounds with promising in

vitro properties were tested for their blood pharmacokinetic

and brain penetration properties in mice. A cassette dosing

setup was applied; i.e., ﬁve experimental compounds and a

reference compound were administered together intravenously

at 1 mg/kg and per os at 3 mg/kg. The exposure in blood after

oral administration mainly appeared to be driven by blood

clearance. In particular, 26 that had low blood clearance (see

Table 2) showed high maximal blood exposure and AUC after

oral dosing. It exhibited an acceptable absolute oral

bioavailability and a brain/blood concentration ratio that

indicated favorable brain penetration. On the basis of the

overall properties, 26 was selected for testing its pharmaco-

logical activity in mice.

Figure 3. Eﬀect of 26 and suvorexant in the locomotor paradigm

(increase of inactivity [min] in the 4 h following application). Both

compounds were administered at a dose of 50 mg/kg (n = 6): (∗) p <

0.05, (∗∗) p < 0.01 one-way ANOVA with post hoc Dunnett’s tests

(comparison with vehicle).

levels of compound in the brain were calculated by multiplying

the fraction unbound as measured in a separate mouse brain

homogenate assay with the brain levels of compound achieved

in a satellite group. The data suggested that both compounds

were present at similar levels to interact with orexin receptors in

the brain (Table 3). These levels of both compounds were high

enough to reach the maximal achievable increase in inactivity

during the ﬁrst 4 h. Because of its signiﬁcant activity in the

locomotor paradigm, 26 was selected for studying the eﬀects on

mouse sleep.

The eﬀect of orexin receptor antagonists on sleep in mice

was determined by EEG (electrocorticogram/electroencephalo-

gram) and EMG (electromyogram) recordings. Freely moving

C57Bl/6 mice with chronically implanted electrodes were well

habituated to the experiment boxes and had access to food and

water ad libitum. The test compounds or vehicle were

administered per os as a suspension in 0.5% methylcellulose

immediately prior to lights oﬀ (start of the active phase) and

start of recording. Movement was recorded using infrared

sensors in the roof of the box. EEG/EMG signals and motility

data were used to score 10 s epochs into wake, NREM sleep,

and REM sleep. Each animal served as its own control by

application and recording of vehicle the day before compound

dosing.

For a comprehensive interpretation of the in vivo data, the

potencies of 26 and suvorexant were determined at the mouse

orexin receptors (mOX₁R, mOX₂R) in a functional assay

(Table 3).⁵¹Suvorexant was found to be slightly selective for

mOX₁R over mOX₂R, whereas 26 showed a 10-fold preference

for mOX₂R (Table 3).

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Figure 4. Sleep induction by 26 and suvorexant. (a, b) Vehicle or compounds were dosed just before lights oﬀ (hour 0) in a crossover design, and

total sleep time (TST) increased during the ﬁrst 4 h with both compounds (26, n = 11; suvorexant, n = 10). (c, d) Amount of NREM and REM

sleep during the ﬁrst hours after lights oﬀ and quantiﬁed during the ﬁrst four hours. (e, f) REM proportion of total sleep time. Dotted line indicates

the mean proportion on the vehicle day during the inactive (lights on) phase. Shading indicates lights oﬀ. Time course graphs: (∗) p < 0.05; (∗∗) p <

0.01; (∗∗∗) p < 0.001; Fisher’s LSD. Bar graphs: (∗) p < 0.05; (∗∗) p < 0.01; (∗∗∗) p < 0.001; paired t test.

The eﬀect of 26 and suvorexant both at 50 mg/kg po were

compared. Both compounds showed a fast onset of action, with

a clear increase in total sleep time during the ﬁrst hour after

dosing (Figure 4a,b). The eﬀect lasted 4−5 h, after which time

the total sleep time per hour was the same as on vehicle day.

The increase in total sleep time during the ﬁrst four hours was

in a similar range for both compounds, despite the fact that the

apparent free brain levels in relation to the potency on the

orexin receptors were much higher for suvorexant than for 26

(Table 3). Analysis of the sleep architecture revealed a

surprising diﬀerence between the two compounds. Suvorexant

induced a very strong increase in REM sleep, which accounted

for much of the increase in total sleep time (Figure 4d).

Suvorexant also increased NREM sleep during the ﬁrst four

hours but to a lesser extent. This was in sharp contrast to 26,

which primarily increased sleep by a strong eﬀect on NREM

sleep, accompanied by a smaller increase in REM sleep that was

not signiﬁcant following the ﬁrst hour (Figure 4c). When the

same data were used to examine the percentage of total sleep

time spent in REM, treatment with 26 did not change the

balance between REM and NREM sleep (Figure 4e), whereas

treatment with suvorexant dramatically increased the propor-

tion of REM sleep to levels considerably higher than the mean

level during normal sleep in the inactive period (Figure 4f).

These ﬁndings are particularly interesting in connection with

recently published data from clinical trials with suvorexant and

other DORAs in both healthy subjects and in patients suﬀering

from primary insomnia.^27,54Despite the fact that mice show a

very distinct sleep behavior compared to humans (nocturnal

activity, more fragmented sleep pattern), the observations in

this species appear to be quite predictive of the eﬀects seen in

humans. As described above, in mice suvorexant induced a

strong increase in REM sleep, accounting for around half of the

increase in total sleep time. In patients suﬀering from insomnia,

about half of the increase in total sleep time due to suvorexant

was attributable to an increase in time spent in REM and when

the percentage of total sleep time spent in a particular sleep

stage was analyzed, only REM sleep showed a nominally

signiﬁcant increase versus placebo for all tested doses (10−80

mg).⁵⁴Similarly, in healthy subjects suvorexant increased sleep

with about half the increase attributable to an increase in time

spent in REM sleep.²⁷An earlier report on a clinical study with

1 revealed similar eﬀects of this DORA, with a very strong

increase in REM sleep and at the highest dose the appearance

of sleep onset REM periods.⁵⁵Whether the diﬀerence in

selectivity for OX₁R and OX₂R is the key explanation for the

distinctive architecture of the sleep induced by 26 can currently

only be speculated. Interestingly, mouse sleep induced by

almorexant also has a very diﬀerent architecture than sleep

induced by suvorexant and is not accompanied by an

overproportional increase in REM.²⁹However, while almorex-

ant is generally considered a DORA, it has the property that the

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acetate; B, acetonitrile + 0.04% formic acid; 2−98% B in 1.4 min, 98%

B 0.45 min, ﬂow 1.2 mL/min; column temperature 50 °C.

relative aﬃnity for OX₂R increases with time because of its very

slow dissociation from OX₂R,^29,56,57suggesting it may behave

in vivo more like an OX₂R preferring antagonist.

Method B: Agilent 1100 series; LC−MS; column Zorbax SB-C18 1.8

μm, 3.0 mm × 30 mm; A, water + 0.05% triﬂuoroacetic acid; B,

acetonitrile + 0.05% triﬂuoroacetic acid; 30−100% B in 3.25 min,

100% B 0.75 min; ﬂow 0.7 mL/min; column temperature 35 °C.

Method C: Agilent 1100 series; Agilent MSD vsl single quad mass

spectrometer; column Mercury MS Synergi 2 μm, 20 mm × 4.0 mm;

A, water + 0.1% formic acid; B, acetonitrile; 30−95% B in 1.5 min,

95% B 1 min; ﬂow 2.0 mL/min; column temperature 30 °C.

Method D: Agilent 1100 series; LC−MS; column Zorbax SB-C18

1.8 μm, 3.0 mm × 30 mm; A, water + 0.05% triﬂuoroacetic acid; B,

acetonitrile + 0.05% triﬂuoroacetic acid in; 10−100% B in 3.25 min,

100% B in 0.75 min; ﬂow 0.7 mL/min; column temperature 35 °C.

The accurate mass analyses were performed by using electrospray

ionization in positive mode after separation by liquid chromatography.

The elemental composition was derived from the averaged mass

spectra acquired at a high resolution of about 30 000 on an LTQ

Orbitrap XL mass spectrometer (Thermo Scientiﬁc). The high mass

accuracy below 1 ppm was obtained by using a lock mass.

1-Benzyl-9-(quinoxalin-2-yl)-1,9-diazaspiro[5.5]undecan-2-

one (3). NaH (34.8 mg, 0.871 mmol, 60% in mineral oil) was added

to an ice-cold solution of 9-(quinoxalin-2-yl)-1,9-diazaspiro[5.5]-

undecan-2-one (5) (86 mg, 0.29 mmol) in THF (5 mL), and the

suspension was stirred for 1 h at 0 °C. Then benzyl bromide (64.5 mg,

0.377 mmol) was added at 0 °C, and the reaction mixture was allowed

to warm to room temperature and stirred for 24 h. The mixture was

poured into water, and the solution was extracted twice with ethyl

acetate. The combined organic layers were washed with water and

brine, ﬁltered, and dried over anhydrous sodium sulfate. The organic

layer was concentrated under reduced pressure. The crude product

was puriﬁed by ﬂash column chromatography (dichloromethane/

methanol 97:3) to yield the title compound 3 (24 mg, 19%). ¹H NMR

(400 MHz, DMSO-d₆) δ ppm 8.78 (s, 1H), 7.79 (d, J = 8.07 Hz, 1H),

7.55 (d, J = 4.16 Hz, 2H), 7.36 (ddd, J = 3.55, 4.83, 8.25 Hz, 1H),

7.18−7.27 (m, 2H), 7.04−7.17 (m, 3H), 4.39−4.62 (m, 4H), 3.09 (t, J

= 12.35 Hz, 2H), 2.41 (t, J = 6.60 Hz, 2H), 2.03−2.16 (m, 2H), 1.96

(dt, J = 4.52, 12.90 Hz, 2H), 1.72−1.86 (m, 2H), 1.52−1.67 (m, 2H).

HRMS (ESI⁺) calcd for C₂₄H₂₇N₄O (MH⁺) 387.217 60, found

387.217 94. UPLC−MS t_R,A= 1.07 min, [M + H]⁺= 387.4.

2-Benzyl-9-(quinoxalin-2-yl)-2,9-diazaspiro[5.5]undecan-1-

one (4). To a solution of tert-butyl 2-benzyl-1-oxo-2,9-diazaspiro-

[5.5]undecane-9-carboxylate (9) (440 mg, 1.20 mmol) in dichloro-

methane (5 mL) was added 4 M HCl in dioxane (10 mL). The

solution was stirred for 18 h at room temperature. After completion of

the reaction the mixture was evaporated to dryness. The residue was

crystallized from THF/heptane 3:1 to yield 2-benzyl-2,9-

diazaspiro[5.5]undecan-1-one hydrochloride as white crystals (293

mg, 83%).

To the solution of 2-benzyl-2,9-diazaspiro[5.5]undecan-1-one

hydrochloride (100 mg, 0.34 mmol) in ethanol (1 mL) in a microwave

tube were added 2-chloroquinoxaline (95 mg, 0.58 mmol) and

triethylamine (0.14 mL, 1.02 mmol). The tube was sealed, and the

reaction mixture was heated at 160 °C for 15 min under microwave

conditions. The solvent was removed under reduced pressure. The

resulting crude product was puriﬁed by ﬂash column chromatography

(hexane/ethyl acetate 2:1) to yield the title compound 4 (114 mg,

66%). ¹H NMR (600 MHz, DMSO-d₆) δ ppm 8.84 (s, 1H), 7.81 (d, J

= 8.07 Hz, 1H), 7.53−7.63 (m, 2H), 7.36−7.41 (m, 1H), 7.34 (t, J =

7.57 Hz, 2H), 7.22−7.29 (m, 1H), 7.19 (d, J = 7.27 Hz, 2H), 4.50 (s,

2H), 4.24−4.34 (m, 2H), 3.39−3.49 (m, 2H), 3.22 (t, J = 6.26 Hz,

2H), 2.01−2.15 (m, 2H), 1.86−1.93 (m, 2H), 1.73−1.84 (m, 2H),

1.55−1.66 (m, 2H). HRMS (ESI⁺) calcd for C₂₄H₂₇N₄O (MH⁺)

387.217 94, found 387.218 16. LC−MS t_R,D= 3.29 min, [M + H]⁺=

387.2.

Animal experiments applying selective or dual orexin

receptor antagonists have produced somewhat contradictory

conclusions regarding the importance of antagonizing OX₁R

and OX₂R for sleep induction and architecture.^{29−32,34,35}

Intracerbroventricular injection of the OX₂R antagonist (S)-1-

(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-3,3-dimeth-

yl-2-((pyridin-4-ylmethyl)amino)butan-1-one hydrochloride

(TCS-OX2-29) increased REM, at odds with our results.³²

On the other hand, addition of an OX₁R antagonist to an OX₂R

antagonist further decreased latency to REM, while suppressing

both the decrease in latency and increase in NREM produced

by OX₂R antagonism is in line with our results³¹as are the

eﬀects of OX₁R knockdown in locus coeruleus³⁷and ip

application of 1-(2-methylbenzoxazol-6-yl)-3-[1,5]-

naphthyridin-4-ylurea hydrochloride (SB-334867-A).³⁵Diﬀer-

ences in experimental design, properties of the compounds, and

the species used make it diﬃcult to determine the extent to

which receptor selectivity, rather than other factors, accounts

for the observed diﬀerences in sleep architecture. What is clear

is that antagonism of OX₂R is suﬃcient to promote sleep in

rodents. At present, no clinical data using a selective OX₂R

antagonist have been published. When such data become

available, they will be important for further understanding how

the sleep proﬁle found in rodents translates to humans and

whether or not OX₂R is a superior target for the treatment of

insomnia in man.

In conclusion, two novel series of orexin receptor antagonists

were designed based on a pharmacophore analysis of published

orexin receptor antagonists. They mimic two potential low

energy conformations of known spiro-type orexin receptor

antagonists. Representative compounds of core 2 showed a

good overall proﬁle supporting in vivo testing. 26, an OX₂R

selective antagonist, was chosen for comparison with the dual

antagonist suvorexant in mouse sleep studies. Suvorexant and

26 both induced sleep in mice. While suvorexant mainly

increased REM sleep in this model, the balance between

NREM and REM sleep was preserved under 26. Our studies

demonstrate that selective OX₂R antagonists may be a viable

alternative to DORAs for the treatment of insomnia, with the

potential to induce sleep without strongly increasing the REM

component. Whether these distinct eﬀects are linked to

diﬀerences in the selectivity proﬁle for the two orexin receptors

requires further study. The translatability of rodent sleep

architecture to human sleep will be better understood after

OX₂R selective compounds are tested in the clinic.

EXPERIMENTAL SECTION

■

A. Chemistry. All reagents and solvents were purchased from

commercial suppliers and used without further puriﬁcation or were

prepared according to published procedures. All reactions were

1

performed under inert conditions (argon) unless otherwise stated. H

NMR spectra were recorded on a Bruker 400 MHz or a Bruker 600

MHz NMR spectrometer. Chemical shifts are reported in parts per

million (ppm) relative to an internal solvent reference. Signiﬁcant

peaks are tabulated in the order multiplicity (s, singlet; d, doublet; t,

triplet; q, quartet; quintet; m, multiplet; br, broad), coupling constants,

and number of protons. Final compounds were puriﬁed to ≥95%

purity as assessed by analytical liquid chromatography with one of the

following methods.

9-(Quinoxalin-2-yl)-1,9-diazaspiro[5.5]undecan-2-one (5).

To a solution of tert-butyl 2-oxo-1,9-diazaspiro[5.5]undecane-9-

carboxylate [1031927-12-4] (920 mg, 3.26 mmol) in dichloromethane

(10 mL) was added TFA (2.53 mL, 32.6 mmol). The solution was

stirred for 40 min at room temperature. The mixture was evaporated

Method A: Waters Acquity UPLC−MS; column HSS T3 1.8 μm, 2.1

mm × 50 mm; A, water + 0.05% formic acid + 3.75 mM ammonium

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under reduced pressure and dried under high vacuum to yield 1,9-

diazaspiro[5.5]undecan-2-one triﬂuoroacetate (1.90 g, 100%). ¹H

NMR (400 MHz, DMSO-d₆) δ ppm 8.59−8.35 (m, 2H), 7.83 (s, 1

H), 3.27−3.14 (m, 2H), 3.12−2.98 (m, 2H), 2.17−2.04 (m, 2H),

1.80−1.57 (m, 8H); UPLC−MS t_R,A= 0.20 min, [M + H]⁺= 169.2.

To a stirred solution of 1,9-diazaspiro[5.5]undecan-2-one triﬂuor-

oacetate (800 mg, 4.76 mmol) in 10 mL of DMF were added 2-

chloroquinoxaline (937 mg, 5.71 mmol) and K₂CO₃(3.3 g, 23.8

mmol), and the reaction mixture was stirred for 18 h at 80 °C under a

nitrogen atmosphere. The reaction mixture was quenched with ice-

cold water and extracted with ethyl acetate (2 × 50 mL), dried over

anhydrous Na₂SO₄, ﬁltered, and concentrated under reduced pressure.

The crude mixture was puriﬁed by ﬂash column chromatography (3%

methanol in chloroform) to yield the title compound 5 (1.0 g, 71%).

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.83 (s, 1H), 7.79 (d, J = 8.07

cold solution of tert-butyl 1-oxo-2,9-diazaspiro[5.5]undecane-9-carbox-

ylate (8) (480 mg, 1.79 mmol) in THF (3 mL). The resulting mixture

was stirred at 0 °C for 10 min, and TBAI (66.1 mg, 0.18 mmol) was

added. Then benzyl bromide (0.32 mL, 2.68 mmol) was added at 0

°C, and the reaction mixture was allowed to warm to room

temperature and heated at 45 °C for 20 h. The mixture was poured

into water, and the solution was extracted twice with ethyl acetate. The

combined organic layers were washed with water and brine, ﬁltered,

and dried over anhydrous sodium sulfate. The organic layer was

concentrated under reduced pressure. The crude product was puriﬁed

by ﬂash column chromatography (hexane/ethyl acetate 2:1) to yield

the title compound 9 (440 mg, 67%). ¹H NMR (400 MHz, DMSO-d₆)

δ ppm 7.28−7.35 (m, 2H), 7.19−7.26 (m, 1H), 7.15 (d, J = 7.04 Hz,

2H), 4.46 (s, 2H), 3.58−3.78 (m, 2H), 3.16 (t, J = 5.87 Hz, 2H), 3.02

(br s, 2H), 1.82−1.94 (m, 2H), 1.65−1.81 (m, 4H), 1.31−1.46 (m,

11H).

Hz, 1H), 7.50−7.64 (m, 3H), 7.30−7.42 (m, 1H), 4.12 (td, J = 4.86,

13.75 Hz, 2H), 3.46−3.63 (m, 2H), 2.12 (t, J = 6.11 Hz, 2H), 1.54−

1.82 (m, 8H). UPLC−MS t_R,A= 0.79 min, [M + H]⁺= 297.3.

1-(2,5-Dimethylbenzyl)-9-(quinoxalin-2-yl)-1,9-diazaspiro-

[5.5]undecan-2-one (6). To a solution of 9-(quinoxalin-2-yl)-1,9-

diazaspiro[5.5]undecan-2-one (5) (80 mg, 0.27 mmol) in THF (5

mL) was added NaH 95% (32 mg, 0.81 mmol), and the mixture was

stirred for 10 min at room temperature. Then 2,5-dimethylbenzyl

chloride (70 mg, 0.35 mmol) was added, and the reaction mixture was

heated at 60 °C for 18 h. Saturated aqueous NH₄Cl solution (40 mL)

was added, and the reaction mixture was extracted with ethyl acetate

(100 mL). The organic layer was dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure. The resulting crude product was

puriﬁed by reversed phase preparative HPLC (column Zorbax

eclipseXDB C18, ﬂow 20 mL/min, mobile phase 0.1% TFA in water

(A), acetonitrile (B) gradient) to yield the title compound 6 (49 mg,

22%). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.78 (s, 1H), 7.78 (d, J

= 7.82 Hz, 1H), 7.51−7.59 (m, 2H), 7.36 (ddd, J = 2.57, 5.81, 8.25 Hz,

1H), 6.90−6.95 (m, 1H), 6.82−6.88 (m, 1H), 6.69 (s, 1H), 4.48 (d, J

= 12.96 Hz, 2H), 4.34 (s, 2H), 3.11 (t, J = 12.10 Hz, 2H), 2.42 (t, J =

6.60 Hz, 2H), 2.21 (s, 3H), 2.09−2.19 (m, 2H), 2.04 (s, 3H), 1.77−

1.97 (m, 4H), 1.66 (d, J = 13.20 Hz, 2H). HRMS (ESI⁺) calcd for

tert-Butyl 4-Allyl-4-((1-tosyl-1H-indol-4-yl)methylamino)-

piperidine-1-carboxylate (10a). To a stirred mixture of 1-boc-

piperidin-4-one [79099-07-3] (0.25 g, 1.256 mmol), 4 Å molecular

sieves (0.25 g), and allyl boronic acid pinacol ester (0.255 g, 1.507

mmol) in toluene (10 mL) was added 1-tosyl-1H-indol-4-yl)-

methanamine (0.45 g, 1.507 mmol), and the reaction mixture was

heated at reﬂux for 16 h. The mixture was ﬁltered through a pad of

Celite. The ﬁltrate was concentrated under reduced pressure and the

residue was puriﬁed by column chromatography (10% ethyl acetate in

hexane) to yield the title compound 10a as a white solid (0.2 g, 70%).

LC−MS t_R,C= 0.341 min, [M + H]⁺= 524.0.

tert-Butyl 4-Allyl-4-((1-tosyl-1H-indol-3-yl)methylamino)-

piperidine-1-carboxylate (10b). To a solution of 1-tosyl-1H-

indole-3-carbaldehyde [50562-79-3] (1.90 g, 6.35 mmol) in 1,2-

dichloroethane (30 mL) were added tert-butyl 4-allyl-4-amino-

piperidine-1-carboxylate⁵⁸(1.52 g, 6.35 mmol) and acetic acid (381

mg, 6.35 mmol). The resulting solution was heated at 60 °C for 3 h.

Then NaBH(OAc)₃was added, and the reaction mixture was heated at

60 °C for 38 h. The mixture was cooled to room temperature.

Saturated NaHCO₃solution (30 mL) was added and extracted with

ethyl acetate (3 × 50 mL). The combined organic layers were washed

with water and brine, then dried over anhydrous Na₂SO₄, ﬁltered, and

concentrated under reduced pressure. The crude mixture was puriﬁed

by ﬂash column chromatography (20% ethyl acetate/hexane) to yield

the title compound 10b (2.0 g, 60%). LC−MS t_R,C= 0.34 min, [M +

H]⁺= 524.0.

tert-Butyl 4-Allyl-4-(N-((1-tosyl-1H-indol-4-yl)methyl)-

acrylamido)piperidine-1-carboxylate (11a). Acryloyl chloride

(0.360 g, 0.401 mmol) was added at 0 °C to a stirred solution of

tert-butyl 4-allyl-4-((1-tosyl-1H-indol-4-yl)methylamino)piperidine-1-

carboxylate (10a) (0.2 g, 0.382 mmol) and diisopropylethylamine

(0.32 mL, 1.91 mmol) in dichloromethane (5.0 mL). The reaction

mixture was stirred at 0 °C for 30 min and was then allowed to warm

to room temperature and stirred for 4 h. The reaction mixture was

concentrated under reduced pressure and the crude product was

puriﬁed by column chromatography (5% ethyl acetate in hexane) to

yield the title compound 11a as a white solid (0.16 g, 72%). LC−MS

t_R,C= 0.774 min, [M + H − boc]⁺= 477.9.

tert-Butyl 2-Oxo-1-((1-tosyl-1H-indol-4-yl)methyl)-1,9-

diazaspiro[5.5]undec-3-ene-9-carboxylate (12a). To a solution

of tert-butyl 4-allyl-4-(N-((1-tosyl-1H-indol-4-yl)methyl)acrylamido)-

piperidine-1-carboxylate (11a) (75 mg, 0.13 mmol) in dichloro-

methane (5.0 mL) under argon was added Grubbs second generation

catalyst (0.006 g, 0.006 mmol), and the reaction mixture was stirred at

room temperature overnight. The dark brown solution was

concentrated under reduced pressure and the crude product was

puriﬁed by column chromatography (25% ethyl acetate in hexane) to

C₂₆H₃₁N₄O (MH⁺) 415.249 53, found 415.249 24. UPLC−MS t_R,A

=

1.18 min, [M + H]⁺= 415.3.

1-tert-Butyl 4-Ethyl 4-(2-cyanoethyl)piperidine-1,4-dicar-

boxylate (7). To a solution of ethyl N-boc-piperidine-4-carboxylate

[124443-68-1] (10.0 g, 38.86 mmol) in THF (200 mL) was added

LDA (2 M solution in hexane, 38.86 mL, 77.72 mmol) at −78 °C, and

the mixture was stirred for 30 min. Then 3-bromopropionitrile (6.25 g,

46.63 mmol) was added at −78 °C. The resulting reaction mixture was

stirred at −60 °C for 4 h and quenched with saturated NH₄Cl

solution. Ethyl acetate was added, and the organic layer was washed

with water and brine. The organic layer was dried over Na₂SO₄,

ﬁltered, and concentrated. The crude product was puriﬁed by column

chromatography using 10% ethyl acetate in hexane to yield the title

compound 7 as a pale yellow liquid (5.0 g, 40%). ¹H NMR (400 MHz,

DMSO-d₆) δ ppm 4.08−4.16 (m, 2H), 3.60−3.77 (m, 2H), 2.82 (br s,

2H), 2.41 (t, J = 7.62 Hz, 2H), 1.87−1.96 (m, 2H), 1.82 (t, J = 7.82

Hz, 2H), 1.26−1.40 (m, 11H), 1.19 (t, J = 7.62 Hz, 3H). UPLC−MS

t_R,A= 1.07 min, [M + H]⁺= 311.3.

tert-Butyl 1-Oxo-2,9-diazaspiro[5.5]undecane-9-carboxylate

(8). A suspension of Raney Ni (5.0 g) in ethanolic ammonia (∼20% v/

v) and 1-tert-butyl 4-ethyl 4-(2-cyanoethyl)piperidine-1,4-dicarbox-

ylate (7) (8.5 g, 27.42 mmol) was hydrogenated at 100 psi for 48 h at

room temperature in an autoclave. After completion of the reaction,

the catalyst was ﬁltered oﬀ and washed with ethanol. The combined

ﬁltrate was concentrated under reduced pressure and triturated with n-

pentane to yield a solid which was puriﬁed by column chromatography

(6% methanol in chloroform) to furnish the title compound 8 as a

white solid (3.0 g, 41%). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.31

(br s, 1H), 3.65 (td, J = 4.25, 13.39 Hz, 2H), 2.92−3.11 (m, 4H),

1.72−1.84 (m, 2H), 1.58−1.72 (m, 4H), 1.37 (s, 9H), 1.26−1.35 (m,

2H). LC−MS t_R,D= 2.889 min, [M + H]⁺= 269.1.

yield the title compound 12a as a solid (0.060 g, 84%). LC−MS t_R,C

=

0.523 min, [M + H]⁺= 549.8

tert-Butyl 2-Oxo-1-((1-tosyl-1H-indol-4-yl)methyl)-1,9-

diazaspiro[5.5]undecane-9-carboxylate (13a). To a solution of

tert-butyl 2-oxo-1-((1-tosyl-1H-indol-4-yl)methyl)-1,9-diazaspiro[5.5]-

undec-3-ene-9-carboxylate (12a) (0.12 g, 0.218 mmol) in methanol

(6.0 mL) was added 10% Pd/C, and the reaction mixture was stirred

tert-Butyl 2-Benzyl-1-oxo-2,9-diazaspiro[5.5]undecane-9-

carboxylate (9). NaH (64.4 mg, 2.68 mmol) was added to an ice-

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for 6 h under hydrogen (1 atm pressure) at room temperature. The

reaction mixture was ﬁltered through a pad of Celite and washed with

methanol. The ﬁltrate was concentrated, and the product 13a was

isolated as a white solid (0.120 g, 99%). LC−MS t_R,C= 0.511 min, [M

+ H]⁺= 551.9.

12.42 Hz, 2H), 2.43 (t, J = 6.53 Hz, 2H), 2.14 (s, 6H), 2.09 (br s, 2H),

1.83−1.75 (m, 4H), 1.55 (d, J = 12.80 Hz, 2H). HRMS (ESI⁺) calcd

for C₂₄H₃₀N₅O (MH⁺) 404.244 52, found 404.244 49. UPLC−MS t_R,A

= 1.01 min, [M + H]⁺= 404.4.

1-((1H-Indol-4-yl)methyl)-9-(benzo[d]oxazol-2-yl)-1,9-

diazaspiro[5.5]undecan-2-one (17). Following the procedures

described for compound 16, the title compound 17 was synthesized

1-((1H-Indol-3-yl)methyl)-9-(quinoxalin-2-yl)-1,9-diazaspiro-

[5.5]undecan-2-one (14). The title compound 14 was synthesized

from tert-butyl 4-allyl-4-((1-tosyl-1H-indol-3-yl)methylamino)-

piperidine-1-carboxylate (10b) according to the procedures described

1

from 13a and 2-chlorobenzoxazole (52% over three steps). H NMR

(400 MHz, DMSO-d₆) δ ppm 11.06 (br s, 1H), 7.32 (d, J = 7.83 Hz,

1H), 7.16−7.25 (m, 3H), 7.10 (dt, J = 0.98, 7.70 Hz, 1H), 6.91−7.00

(m, 2H), 6.63 (d, J = 7.09 Hz, 1H), 6.39−6.45 (m, 1H), 4.78 (br s,

2H), 3.97 (dd, J = 3.42, 12.96 Hz, 2H), 3.18−3.30 (m, 2H), 2.43 (t, J

= 6.60 Hz, 2H), 2.06−2.16 (m, 2H), 2.00 (dt, J = 4.52, 13.02 Hz, 2H),

1.81 (td, J = 6.24, 11.98 Hz, 2H), 1.63 (d, J = 13.20 Hz, 2H). HRMS

(ESI⁺) calcd for C₂₅H₂₇N₄O₂(MH⁺) 415.212 56, found 415.212 85.

UPLC−MS t_R,A= 0.97 min, [M + H]⁺= 415.2.

9-(Quinoxalin-2-yl)-2,9-diazaspiro[5.5]undecan-1-one (18).

To a solution of tert-butyl 1-oxo-2,9-diazaspiro[5.5]undecane-9-

carboxylate (8) (2.5 g, 9.32 mmol) in dichloromethane (20 mL)

was added 4 M HCl in dioxane (5 mL). The solution was stirred for 18

h at room temperature. The mixture was evaporated to dryness. The

residue was crystallized from THF/heptane 3:1 to yield 2,9-

diazaspiro[5.5]undecan-1-one hydrochloride as a solid (1.6 g, 82%).

To a solution of 2,9-diazaspiro[5.5]undecan-1-one hydrochloride

(1.0 g, 4.89 mmol) in DMF (20 mL) were added potassium carbonate

(2.03 g, 14.66 mmol) and 2-bromoquinoxaline (1.12 g, 5.37 mmol).

The suspension was heated at 60 °C for 18 h. The reaction mixture

was quenched with water and extracted with dichloromethane (2 ×

100 mL). The combined organic layers were dried over anhydrous

Na₂SO₄, ﬁltered, and concentrated under reduced pressure. The crude

product was puriﬁed by ﬂash column chromatography (dichloro-

methane/MeOH 9:1) to yield the title compound 18 (1.13 g, 76%).

¹H NMR (360 MHz, DMSO-d₆) δ ppm 8.84 (s, 1H), 7.83 (d, J = 8.08

1

for compound 16. H NMR (400 MHz, DMSO-d₆) δ ppm 10.79 (s,

1H), 8.78 (s, 1H), 7.80 (d, J = 7.83 Hz, 1H), 7.50−7.62 (m, 2H),

7.31−7.42 (m, 2H), 7.28 (d, J = 8.07 Hz, 1H), 7.05 (d, J = 2.20 Hz,

1H), 6.99 (t, J = 8.07 Hz, 1H), 6.79 (t, J = 7.95 Hz, 1H), 4.59 (s, 2H),

4.46 (d, J = 12.47 Hz, 2H), 3.11 (t, J = 12.23 Hz, 2H), 2.39 (t, J = 6.72

Hz, 2H), 1.97−2.20 (m, 4H), 1.69−1.85 (m, 2H), 1.61 (d, J = 13.20

Hz, 2H). HRMS (ESI⁺) calcd for C₂₆H₂₈N₅O (MH⁺) 426.228 73,

found 426.228 84. UPLC−MS t_R,A= 1.03 min, [M + H]⁺= 426.3.

1-((1H-Indol-4-yl)methyl)-9-(quinoxalin-2-yl)-1,9-diazaspiro-

[5.5]undecan-2-one (15). According the procedures described for

compound 16, the title compound 15 was synthesized from 13a and 2-

chloroquinoxaline (45% over three steps). ¹H NMR (400 MHz,

DMSO-d₆) δ ppm 11.05 (br s, 1H), 8.74 (s, 1H), 7.77 (d, J = 7.83 Hz,

1H), 7.47−7.59 (m, 2H), 7.35 (ddd, J = 2.32, 6.05, 8.25 Hz, 1H),

7.12−7.25 (m, 2H), 6.95 (t, J = 7.70 Hz, 1H), 6.64 (d, J = 7.34 Hz,

1H), 6.37 (br s, 1H), 4.75 (br s, 2H), 4.44 (d, J = 12.47 Hz, 2H), 3.10

(t, J = 12.23 Hz, 2H), 2.44 (t, J = 6.72 Hz, 2H), 2.08−2.21 (m, 2H),

1.99 (dt, J = 4.40, 12.96 Hz, 2H), 1.83 (td, J = 6.24, 11.98 Hz, 2H),

1.66 (d, J = 12.96 Hz, 2H). HRMS (ESI⁺) calcd for C₂₆H₂₈N₅O

(MH⁺) 426.228 84, found 426.228 96. UPLC−MS t_R,A= 1.00 min, [M

+ H]⁺= 426.3.

1-((1H-Indol-4-yl)methyl)-9-(4,6-dimethylpyrimidin-2-yl)-

1,9-diazaspiro[5.5]undecan-2-one (16). (a) To a stirred solution

of tert-butyl 2-oxo-1-((1-tosyl-1H-indol-4-yl)methyl)-1,9-diazaspiro-

[5.5]undecane-9-carboxylate (13a) (0.12 g, 0.21 mmol) in dichloro-

methane (5.0 mL) was added TFA (0.5 mL) at 0 °C, and the reaction

mixture was stirred for 16 h at room temperature under a nitrogen

atmosphere. The reaction mixture was concentrated to yield 1-((1-

tosyl-1H-indol-4-yl)methyl)-1,9-diazaspiro[5.5]undecan-2-one triﬂuor-

oacetate as a olorless oil (0.11 g, 95%) which was used for the next

step without further puriﬁcation.

(b) To a stirred solution of 1-((1-tosyl-1H-indol-4-yl)methyl)-1,9-

diazaspiro[5.5]undecan-2-one triﬂuoroacetate (125 mg, 0.210 mmol)

in 1 mL of NMP were added 2-chloro-4,6-dimethylpyrimidine (37 mg,

0.252 mmol) and DBU (0.112 mL, 0.735 mmol), and the reaction

mixture was stirred for 25 min at 110 °C in the microwave. The

reaction mixture was quenched with saturated NaHCO₃solution and

extracted with TBME (2 × 25 mL). The organic layer was washed

with saturated NH₄Cl solution and brine and then dried over

anhydrous Na₂SO₄, ﬁltered, and concentrated under reduced pressure.

The crude mixture was puriﬁed by ﬂash chromatography (3% MeOH

in chloroform) to yield 9-(4,6-dimethylpyrimidin-2-yl)-1-((1-tosyl-1H-

indol-4-yl)methyl)-1,9-diazaspiro[5.5]undecan-2-one as a white foam

(67 mg, 57%). ¹H NMR (400 MHz, chloroform-d) δ ppm 7.82 (d, J =

8.28 Hz, 1 H), 7.74 (d, J = 8.53 Hz, 2 H), 7.54 (d, J = 3.76 Hz, 1 H),

7.22 (d, J = 7.78 Hz, 3 H), 6.89 (d, J = 7.53 Hz, 1 H), 6.62 (d, J = 4.27

Hz, 1 H), 6.26 (s, 1 H), 4.80 (br s, 2 H), 4.71 (d, J = 18.07 Hz, 2 H),

2.93−2.85 (m, 2 H), 2.60 (t, J = 6.78 Hz, 2 H), 2.35 (s, 3 H), 2.24 (s, 6

H), 2.15−2.12 (m, 2 H), 1.96−1.89 (m, 4 H), 1.63 (br s, 1 H), 1.60

(br s, 1 H), 1.53 (s, 1 H), 1.28−1.25 (m, 1 H). UPLC−MS t_R,A= 1.32

min, [M + H]⁺= 558.4.

Hz, 1H), 7.60 (d, J = 3.54 Hz, 2H), 7.32−7.45 (m, 2H), 4.27 (td, J =

4.36, 13.52 Hz, 2H), 3.39−3.53 (m, 2H), 3.08−3.21 (m, 2H), 1.95−

2.10 (m, 2H), 1.79−1.89 (m, 2H), 1.67−1.79 (m, 2H), 1.43−1.60 (m,

2H).

2-((1H-Indol-4-yl)methyl)-9-(quinoxalin-2-yl)-2,9-diazaspiro-

[5.5]undecan-1-one (19). To the suspension of 9-(quinoxalin-2-yl)-

2,9-diazaspiro[5.5]undecan-1-one (18) (100 mg, 0.34 mmol) and

NaH (17.8 mg, 0.74 mmol) in THF (5 mL) at 0 °C was added 4-

(bromomethyl)-1-tosyl-1H-indole (147 mg, 0.41 mmol). The mixture

was stirred at 50 °C for 18 h. Saturated ammonium chloride solution

was added, and the mixture was extracted with dichloromethane (2 ×

50 mL). The organic layer was washed with water and brine. The

combined organic layers were dried over anhydrous sodium sulfate,

ﬁltered, and evaporated to give a pale yellow oil. The crude product

was puriﬁed by ﬂash column chromatography (hexane/ethyl acetate

1

3:2) to yield the title compound 19 (80 mg, 55%). H NMR (600

MHz, DMSO-d₆) δ ppm 11.14 (br s, 1H), 8.84 (s, 1H), 7.81 (d, J =

8.28 Hz, 1H), 7.54−7.62 (m, 2H), 7.37 (ddd, J = 2.22, 5.95, 8.17 Hz,

1H), 7.28−7.34 (m, 2H), 7.03 (t, J = 7.57 Hz, 1H), 6.82 (d, J = 7.06

Hz, 1H), 6.43 (br s, 1H), 4.76 (s, 2H), 4.26−4.36 (m, 2H), 3.38−3.47

(m, 2H), 3.13 (t, J = 5.95 Hz, 2H), 2.07−2.17 (m, 2H), 1.80−1.88 (m,

2H), 1.67−1.76 (m, 2H), 1.53−1.63 (m, 2H). HRMS (ESI⁺) calcd for

C₂₆H₂₈N₅O (MH⁺) 426.228 75, found 426.228 84. LC−MS t_R,B= 2.70

min, [M + H]⁺= 426.4.

tert-Butyl 1-Oxo-2-((1-tosyl-1H-indol-3-yl)methyl)-2,9-

diazaspiro[5.5]undecane-9-carboxylate (20). To a solution of

diisopropylamine (1.238 mL, 8.60 mmol) in THF (40 mL) was added

n-butyllithium (6.01 mL, 9.61 mmol) at 0 °C, and the mixture was

stirred for 30 min at 0 °C. Then a solution of tert-butyl 1-oxo-2,9-

diazaspiro[5.5]undecane-9-carboxylate (8) (2.57 g, 9.28 mmol) in

THF (10 mL) was added within 3 min, and the mixture was stirred for

30 min at 0 °C. 3-(Bromomethyl)-1-tosyl-1H-indole [58550-81-5]

(3.2 g, 8.43 mmol) in THF (10 mL) was added dropwise to the

reaction mixture within 15 min. The mixture was stirred at 0 °C for 1 h

and allowed to warm to room temperature overnight. The reaction

mixture was quenched with ice-cold water and extracted with TBME

(2 × 150 mL). The combined organic layers were washed with 5%

(c) Cs₂CO₃(137 mg, 0.415 mmol) was added to a stirred solution

of 9-(4,6-dimethylpyrimidin-2-yl)-1-((1-tosyl-1H-indol-4-yl)methyl)-

1,9-diazaspiro[5.5]undecan-2-one (55 mg, 0.098 mmol) in methanol

(2 mL), and stirring was continued for 18 h at 78 °C. The solvent ws

removed under reduced pressure and the crude product was puriﬁed

by ﬂash column chromatography (ethyl acetate) to yield the title

1

compound 16 as a white foam (37 mg, 94%). H NMR (400 MHz,

DMSO-d₆) δ ppm 7.26 (t, J = 2.76 Hz, 1H), 7.20 (d, J = 8.28 Hz, 1H),

6.96 (t, J = 7.65 Hz, 1H), 6.62 (d, J = 7.03 Hz, 1H), 6.43 (br s, 1 H),

6.32 (s, 1H), 4.72 (br s, 2H), 4.54 (d, J = 13.80 Hz, 2H), 2.89 (t, J =

L

dx.doi.org/10.1021/jm4007627 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

aqueous citric acid and brine, dried over anhydrous Na₂SO₄, ﬁltered,

and concentrated to yield 20 (5.3 g, 100%). H NMR (400 MHz,

1H), 4.61 (s, 2H), 4.34 (td, J = 4.16, 13.45 Hz, 2H), 3.19−3.29 (m,

2H), 3.15 (t, J = 5.99 Hz, 2H), 1.91−2.04 (m, 2H), 1.74−1.83 (m,

2H), 1.60−1.71 (m, 2H), 1.35−1.48 (m, 2H). HRMS (ESI⁺) calcd for

C₂₂H₂₆N₅O (MH⁺) 376.213 17, found 376.213 19.

1

DMSO-d₆) δ ppm 7.88 (d, J = 8.28 Hz, 1H), 7.81 (d, J = 8.28 Hz, 2H),

7.74 (s, 1 H), 7.55 (d, J = 7.78 Hz, 1H), 7.36 (d, J = 8.03 Hz, 2H),

7.32 (t, J = 7.91 Hz, 1H), 7.25−7.21 (m, 1H), 4.58 (s, 2H), 3.75−3.63

(m, 2H), 3.09 (t, J = 5.77 Hz, 2H), 2.99 (br s, 2H), 2.30 (s, 3H),

1.90−1.80 (m, 2H), 1.72−1.57 (m, 4H), 1.39 (s, 9H), 1.36−1.28 (m,

2H). UPLC−MS t_R,A= 1.37 min, [M + H]⁺= 552.3.

2-((1H-Indol-3-yl)methyl)-2,9-diazaspiro[5.5]undecan-1-one

(21). (a) To a solution of tert-butyl 1-oxo-2-((1-tosyl-1H-indol-3-

yl)methyl)-2,9-diazaspiro[5.5]undecane-9-carboxylate (20) (5.3 g,

8.45 mmol) in dichloromethane (30 mL) was added TFA (4.93 mL,

63.4 mmol). The solution was stirred for 70 min at room temperature.

The mixture was evaporated to dryness. The residue was crystallized

from THF/heptane 3:1 to yield 2-((1-tosyl-1H-indol-3-yl)methyl)-2,9-

diazaspiro[5.5]undecan-1-one triﬂuoroacetate as white crystals (5.5 g,

quantitative). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.43 (br s, 2H),

7.89 (d, J = 8.28 Hz, 1H), 7.82 (d, J = 8.28 Hz, 2H), 7.78 (s, 1H), 7.56

(d, J = 7.78 Hz, 1H), 7.38−7.31 (m, 3H), 7.24−7.20 (m, 1H), 4.59 (s,

2H), 3.29−3.19 (m, 2H), 3.12 (t, J = 5.77 Hz, 2H), 3.07−2.95 (m,

2H), 2.30 (s, 3H), 2.10 (ddd, J = 14.24, 10.35, 4.02 Hz, 2H), 1.75−

1.59 (m, 4H), 1.58−1.48 (m, 2H). UPLC−MS t_R,A= 0.88 min, [M +

H]⁺= 452.3.

2-((1H-Indol-3-yl)methyl)-9-(4-methylpyrimidin-2-yl)-2,9-

diazaspiro[5.5]undecan-1-one (25). The title compound was

synthesized according to the procedure described for compound 26

from 2-((1H-indol-3-yl)methyl)-2,9-diazaspiro[5.5]undecan-1-one

1

(21) and 2-chloro-4-methylpyrimidine (74%). H NMR (400 MHz,

DMSO-d₆) δ ppm 8.19 d, J = 4.77 Hz, 1H), 7.52 (d, J = 7.78 Hz, 1H),

7.33 (d, J = 8.03 Hz, 1H), 7.27 (d, J = 2.26 Hz, 1H), 7.05 (t, J = 7.53

Hz, 1H), 6.96−6.92 (m, 1H), 6.47 (d, J = 4.77 Hz, 1H), 4.61 (s, 2H),

4.40−4.31 (m, 2H), 3.26−3.11 (m, 4H), 2.26 (s, 3H), 2.02−1.91 (m,

2H), 1.82−1.74 (m, 2H), 1.70−1.61 (m, 2H), 1.45−1.37 (m, 2H).

HRMS (ESI⁺) calcd for C₂₃H₂₈N₅O (MH⁺) 390.228 85, found

390.228 84. UPLC−MS t_R,A= 1.04 min, [M + H]⁺= 390.2.

2-((1H-Indol-3-yl)methyl)-9-(4-methoxypyrimidin-2-yl)-2,9-

diazaspiro[5.5]undecan-1-one (26). To the solution of 2-((1H-

indol-3-yl)methyl)-2,9-diazaspiro[5.5]undecan-1-one (21) (3.4 g,

11.32 mmol) in acetonitrile (10 mL) in a microwave tube were

added 2-chloro-4-methoxypyrimidine (2.17 g, 14.71 mmol), DIPEA

(6.99 mL, 39.6 mmol), and DBU (0.052 mL, 0.34 mmol). The tube

was sealed, and the reaction mixture was heated at 120 °C for 2 h

under microwave conditions. The solvent was removed under reduced

pressure. The resulting crude product was puriﬁed by ﬂash

chromatography (heptane/EtOAc 55/45 to heptane/EtOAc 13/87).

The product was crystallized from TBME to give the title compound

26 as white crystals (3.33 g, 72%). ¹H NMR (400 MHz, DMSO-d₆) δ

ppm 8.07 (d, J = 5.52 Hz, 1H), 7.52 (d, J = 7.78 Hz, 1H), 7.33 (d, J =

8.28 Hz, 1H), 7.27 (d, J = 2.26 Hz, 1H), 7.05 (t, J = 7.65 Hz, 1H),

6.96−6.92 (m, 1H), 6.03 (d, J = 5.52 Hz, 1H), 4.62 (s, 2H), 4.34 (ddd,

J = 13.43, 4.14, 4.02 Hz, 2H), 3.82 (s, 3H), 3.28−3.19 (m, 2H), 3.15

(t, J = 6.02 Hz, 2H), 2.04−1.93 (m, 2H), 1.81−1.74 (m, 2H), 1.70−

1.61 (m, 2H), 1.47−1.38 (m, 2H). HRMS (ESI⁺) calcd for

C₂₃H₂₈N₅O₂(MH⁺) 406.223 75, found 406.223 43. UPLC−MS t_R,A

= 0.96 min, [M + H]⁺= 406.4.

(b) The suspension of 2-((1-tosyl-1H-indol-3-yl)methyl)-2,9-

diazaspiro[5.5]undecan-1-one triﬂuoroacetate (15 g, 23.2 mmol) and

Cs₂CO₃(45.4 g, 139 mmol) in methanol (170 mL) was heated at

reﬂux for 2.5 h. The solution was diluted with water and extracted with

dichloromethane. The aqueous phase was adjusted to pH 11 by the

addition of saturated aqueous K₂CO₃solution, and extraction with

dichloromethane was repeated. The combined organic layers were

dried over sodium sulfate, ﬁltered, and evaporated to give 6.96g (99%)

1

of 21 as a beige foam. H NMR (400 MHz, DMSO-d₆) δ ppm 10.91

(br s, 1H), 7.42−7.61 (m, 1H), 7.32 (d, J = 8.2 Hz, 1H), 7.24 (d, J =

2.0 Hz, 1H), 7.04 (t, J = 7.6 Hz, 1H), 6.88−6.99 (m, 1H), 4.60 (s,

2H), 3.10 (t, J = 6.1 Hz, 2H), 2.51−2.88 (m, 4H), 1.88−2.10 (m, 3H),

1.60 (d, J = 5.5 Hz, 4H), 1.25−1.40 (m, 2H). LC−MS t_R,D= 2.44 min,

[M + H]⁺= 298.2.

2-((1H-Indol-3-yl)methyl)-9-(4,6-dimethylpyrimidin-2-yl)-

2,9-diazaspiro[5.5]undecan-1-one (27). The title compound was

synthesized according to the procedure described for compound 26

from 2-((1H-indol-3-yl)methyl)-2,9-diazaspiro[5.5]undecan-1-one

(21) and 2-chloro-4,6-dimethylpyrimidine (56%). ¹H NMR (400

MHz, DMSO-d₆) δ ppm 7.52 (d, J = 8.03 Hz, 1H), 7.33 (d, J = 8.03

Hz, 1H), 7.27 (d, J = 2.01 Hz, 1H), 7.05 (t, J = 7.53 Hz, 1H), 6.96−

6.92 (m, 1H), 6.36 (s, 1H), 4.61 (s, 2H), 4.42−4.33 (m, 2H), 3.23−

3.09 (m, 4H), 2.21 (s, 6H), 2.01−1.90 (m, 2H), 1.81−1.73 (m, 2H),

1.70−1.60 (m, 2H), 1.45−1.33 (m, 2H). HRMS (ESI⁺) calcd for

2-((1H-Indol-3-yl)methyl)-9-(quinoxalin-2-yl)-2,9-diazaspiro-

[5.5]undecan-1-one (22). The title compound was synthesized

according to the procedure described for compound 26 from 2-((1H-

indol-3-yl)methyl)-2,9-diazaspiro[5.5]undecan-1-one (21) and 2-

1

chloroquinoxaline (60%). H NMR (400 MHz, DMSO-d₆) δ ppm

10.92 (br s, 1H), 8.81 (s, 1H), 7.79 (d, J = 7.82 Hz, 1H), 7.55−7.61

(m, 2H), 7.52 (d, J = 7.82 Hz, 1H), 7.30−7.39 (m, 2H), 7.27 (d, J =

1.95 Hz, 1H), 7.05 (t, J = 7.62 Hz, 1H), 6.90−6.97 (m, 1H), 4.61 (s,

2H), 4.29 (td, J = 4.40, 13.49 Hz, 2H), 3.34−3.47 (m, 2H), 3.16 (t, J =

5.87 Hz, 2H), 2.03−2.16 (m, 2H), 1.74−1.84 (m, 2H), 1.60−1.72 (m,

2H), 1.45−1.57 (m, 2H). HRMS (ESI⁺) calcd for C₂₆H₂₈N₅O (MH⁺)

426.228 88, found 426.228 84. LC−MS t_R,B= 2.404 min, [M + H]⁺=

426.2.

2-((1H-Indol-3-yl)methyl)-9-(pyrazin-2-yl)-2,9-diazaspiro-

[5.5]undecan-1-one (23). The title compound was synthesized

according to the procedure described for compound 26 from 2-((1H-

indol-3-yl)methyl)-2,9-diazaspiro[5.5]undecan-1-one (21) and 2-

chloropyrazine (41%). ¹H NMR (400 MHz, DMSO-d₆) δ ppm

10.94 (br s, 1H), 8.30 (d, J = 1.47 Hz, 1H), 8.06 (dd, J = 1.59, 2.57 Hz,

1H), 7.78 (d, J = 2.45 Hz, 1H), 7.52 (d, J = 7.82 Hz, 1H), 7.33 (d, J =

8.07 Hz, 1H), 7.27 (d, J = 2.45 Hz, 1H), 7.05 (t, J = 7.58 Hz, 1H),

6.90−6.97 (m, 1H), 4.61 (s, 2H), 4.07 (td, J = 4.22, 13.33 Hz, 2H),

3.10−3.26 (m, 4H), 2.04 (ddd, J = 4.40, 11.25, 13.45 Hz, 2H), 1.72−

1.81 (m, 2H), 1.60−1.71 (m, 2H), 1.39−1.51 (m, 2H). HRMS (ESI⁺)

calcd for C₂₂H₂₆N₅O (MH⁺) 376.213 39, found 376.213 19.

C₂₄H₃₀N₅O (MH⁺) 404.244 49, found 404.244 76. UPLC−MS t_R,A

=

1.11 min, [M + H]⁺= 404.4].

9-(4,6-Dimethylpyrimidin-2-yl)-2-((1-methyl-1H-indol-3-yl)-

methyl)-2,9-diazaspiro[5.5]undecan-1-one (28). NaH (10 mg,

0.26 mmol, 60% in mineral oil) was added to an ice-cold solution of 2-

((1H-indol-3-yl)methyl)-9-(4,6-dimethylpyrimidin-2-yl)-2,9-

diazaspiro[5.5]undecan-1-one (27) (69 mg, 0.17 mmol) in THF (3

mL). The resulting mixture was stirred at 0 °C for 10 min. Then

methyl iodide (0.02 mL, 0.26 mmol) was added at 0 °C and the

reaction mixture was allowed to warm to room temperature over a

period of 18 h. The mixture was poured into water and the solution

was extracted twice with ethyl acetate. The combined organic layers

were washed with water and brine, ﬁltered, and dried over anhydrous

sodium sulfate. The organic layer was concentrated under reduced

pressure. The product was puriﬁed by preparative HPLC (column

AG/PP-C18-15/021, ﬂow 20 mL/min, mobile phase 0.1% TFA in

water (A), acetonitrile (B) gradient) to yield the title compound 28

2-((1H-Indol-3-yl)methyl)-9-(pyrimidin-2-yl)-2,9-diazaspiro-

[5.5]undecan-1-one (24). The title compound was synthesized

according to the procedure described for compound 26 from 2-((1H-

indol-3-yl)methyl)-2,9-diazaspiro[5.5]undecan-1-one (21) and 2-

chloropyrimidine (44%). ¹H NMR (400 MHz, DMSO-d₆) δ ppm

10.93 (br s, 1H), 8.34 (d, J = 4.65 Hz, 2H), 7.51 (d, J = 7.82 Hz, 1H),

7.33 (d, J = 8.07 Hz, 1H), 7.27 (d, J = 2.44 Hz, 1H), 7.05 (dt, J = 1.10,

7.52 Hz, 1H), 6.94 (dt, J = 0.98, 7.46 Hz, 1H), 6.58 (t, J = 4.77 Hz,

1

(18 mg, 25%). H NMR (400 MHz, DMSO-d₆) δ ppm 7.53 (d, J =

8.07 Hz, 1H), 7.37 (d, J = 8.31 Hz, 1H), 7.25 (s, 1H), 7.12 (t, J = 8.19

Hz, 1H), 6.93−7.03 (m, 1H), 6.36 (s, 1H), 4.59 (s, 2H), 4.37 (td, J =

3.97, 13.33 Hz, 2H), 3.73 (s, 3H), 3.09−3.24 (m, 4H), 2.21 (s, 6H),

1.89−2.01 (m, 2H), 1.72−1.82 (m, 2H), 1.59−1.71 (m, 2H), 1.33−

1.45 (m, 2H). HRMS (ESI⁺) calcd for C₂₅H₃₂N₅O (MH⁺) 418.260 36,

found 418.260 14. UPLC−MS t_R,A= 1.27 min, [M + H]⁺= 418.3.

M

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9-(4,6-Dimethylpyrimidin-2-yl)-2,9-diazaspiro[5.5]undecan-

1-one (29). (a) To a solution of tert-butyl 1-oxo-2,9-diazaspiro[5.5]-

undecane-9-carboxylate (8) (20.0 g, 72.3 mmol) in dichloromethane

(200 mL) was added TFA (56.3 mL, 723 mmol) at 0 °C. The solution

was allowed to warm to room temperature and stirred for 1 h. The

mixture was evaporated to dryness. The residue was dried under high

vacuum to yield 2,9-diazaspiro[5.5]undecan-1-one triﬂuoroacetate as a

The white solid (188 mg) was then dissolved in MeOH (3 mL),

and Cs₂CO₃(439 mg, 1.35 mmol) was added. The mixture was stirred

at 70 °C for 90 min and then cooled to room temperature. Brine was

added and the aqueous phase extracted with EtOAc. Then the

combined organic phases were washed with brine, dried over Na₂SO₄,

ﬁltered and the solvent was removed under reduced pressure to give a

pale yellow residue. Crystallization from a minimum of hot Et₂O/

1

solid (41.9 g, quantitative). H NMR (400 MHz, DMSO-d₆) δ ppm

TBME yielded 32 as a pale yellow solid (100 mg, 73%). H NMR

8.41 (br s, 2H), 7.46 (br s, 1H), 3.18−3.32 (m, 2H), 2.95−3.14 (m,

4H), 1.98−2.10 (m, 2H), 1.61−1.75 (m, 4H), 1.46−1.58 (m, 2H).

UPLC−MS t_R,A= 0.21 min, [M + H]⁺= 169.2

(400 MHz, DMSO-d₆) δ ppm 11.47 (br s, 1 H), 8.17 (d, J = 4.30 Hz, 1

H), 7.90 (d, J = 7.43 Hz, 1 H), 7.40 (d, J = 2.35 Hz, 1 H), 7.01 (dd, J =

8.02, 4.50 Hz, 1 H), 6.35 (s, 1 H), 4.58 (s, 2 H), 4.31−4.42 (m, 2 H),

3.12−3.22 (m, 4 H), 2.21 (s, 6 H), 1.88−2.00 (m, 2 H), 1.73−1.81

(m, 2 H), 1.66 (d, J = 5.87 Hz, 2 H), 1.39 (d, J = 13.69 Hz, 2 H).

HRMS (ESI⁺) calcd for C₂₃H₂₉N₆O (MH⁺) 405.239 74, found

405.239 61. LC−MS t_R,D= 2.34 min, [M + H]⁺= 405.2.

(b) To a solution of 2,9-diazaspiro[5.5]undecan-1-one triﬂuor-

oacetate (1.4 g, 4.96 mmol) in ethanol (11 mL) in a microwave tube

were added 2-chloro-4,6-dimethylpyrimidine (0.875 g, 6.0 mmol),

DIPEA (4.3 mL, 25 mmol), and DMAP (30 mg, 0.25 mmol). The

tube was sealed, and the suspension was heated at 160 °C for 2 h

under microwave conditions. The solvent was removed under reduced

pressure. The resulting crude product was taken up in ethanol and

ﬁltered and the solid washed with ethanol. The ﬁltrate was

concentrated. The precipitate from the ﬁltrate was ﬁltered and washed

with ethanol, and the combined solids were taken up in ethyl acetate.

The ethyl acetate layer was washed with water and brine and dried

over anhydrous sodium sulfate. The organic layer was ﬁltered and

concentrated under reduced pressure to give 1.02 g (75%) of 29 as a

9-(4,6-Dimethylpyrimidin-2-yl)-2-((4-phenyl-1H-pyrazol-3-

yl)methyl)-2,9-diazaspiro[5.5]undecan-1-one (34). The title

compound was synthesized according to the procedure described for

compound 35 starting from 9-(4,6-dimethylpyrimidin-2-yl)-2,9-

diazaspiro[5.5]undecan-1-one (29) and 3-(bromomethyl)-4-phenyl-

1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole (33a)⁵⁹(91% over

1

two steps). H NMR (600 MHz, DMSO-d₆) δ ppm 12.82 (br s, 1H),

7.64−7.99 (m, 1H), 7.26−7.39 (m, 4H), 7.16−7.24 (m, 1H), 6.34 (s,

1H), 4.67 (br s, 2H), 4.16−4.30 (m, 2H), 3.14−3.26 (m, 2H), 3.00−

3.13 (m, 2H), 2.19 (s, 6H), 1.75−1.87 (m, 2H), 1.54−1.75 (m, 4H),

1.24−1.33 (m, 2H). HRMS (ESI⁺) calcd for C₂₅H₃₁N₆O (MH⁺)

431.255 39, found 431.255 31. LC−MS t_R,D= 2.804 min, [M + H]⁺=

431.2.

1

white solid. H NMR (600 MHz, DMSO-d₆) δ ppm 7.34 (br s, 1H),

6.36 (s, 1H), 4.19−4.38 (m, 2H), 3.23 (t, J = 11.10 Hz, 2H), 3.03−

3.15 (m, 2H), 2.21 (s, 6H), 1.81−1.92 (m, 2H), 1.75−1.81 (m, 2H),

1.63−1.73 (m, 2H), 1.40 (d, J = 13.32 Hz, 2H). UPLC−MS t_R,A= 0.69

min, [M + H]⁺= 275.3.

9-(4,6-Dimethylpyrimidin-2-yl)-2-((5-phenyl-2H-1,2,3-tria-

zol-4-yl)methyl)-2,9-diazaspiro[5.5]undecan-1-one (35). To a

suspension of 9-(4,6-dimethylpyrimidin-2-yl)-2,9-diazaspiro[5.5]-

undecan-1-one (29) (150 mg, 0.541 mmol) and TBAI (20.2 mg,

0.054 mmol) in dry THF (5 mL) at 0 °C under argon was added NaH

(60%, 44 mg, 1.08 mmol). After the mixture was stirred for 20 min, a

solution of 4-(bromomethyl)-5-phenyl-2-((2-(trimethylsilyl)ethoxy)-

methyl)-2H-1,2,3-triazole (33b)⁵⁹(222 mg, 0.595 mmol) in dry THF

(0.5 mL) was added. The mixture was allowed to cool to room

temperature and stirred for 4 h. Water was added, and the reaction

mixture was extracted with dichloromethane. The organic layer was

washed with water and brine and dried over anhydrous sodium sulfate,

ﬁltered, and concentrated under reduced pressure. The resulting crude

product was used without further puriﬁcation in the next step (400

mg, 99%). UPLC−MS t_R,A= 1.56 min, [M + H]⁺= 562.5].

9-(4,6-Dimethylpyrimidin-2-yl)-2-((5-methoxy-1H-indol-3-

yl)methyl)-2,9-diazaspiro[5.5]undecan-1-one (30). The title

compound was synthesized according to the procedure described for

compound 32 starting from 9-(4,6-dimethylpyrimidin-2-yl)-2,9-

diazaspiro[5.5]undecan-1-one (29) and 3-(bromomethyl)-5-me-

thoxy-1-tosyl-1H-indole⁵⁹(88% over two steps). ¹H NMR (400

MHz, DMSO-d₆) δ ppm 10.80 (s, 1H), 7.20−7.28 (m, 2H), 7.06 (d, J

= 2.38 Hz, 1H), 6.71 (dd, J = 2.45, 8.72 Hz, 1H), 6.38 (s, 1H), 4.60 (s,

2H), 4.39 (td, J = 3.89, 13.30 Hz, 2H), 3.69 (s, 3H), 3.08−3.25 (m,

4H), 2.23 (s, 6H), 1.93−2.05 (m, 2H), 1.74−1.83 (m, 2H), 1.61−1.72

(m, 2H), 1.37−1.48 (m, 2H). HRMS (ESI⁺) calcd for C₂₅H₃₂N₅O₂

(MH⁺) 434.255 05, found 434.255 35. UPLC−MS t_R,A= 1.06 min, [M

+ H]⁺= 434.3.

2-((1H-Indazol-3-yl)methyl)-9-(4,6-dimethylpyrimidin-2-yl)-

2,9-diazaspiro[5.5]undecan-1-one (31). The title compound was

synthesized according to the procedure described for compound 32

starting from 9-(4,6-dimethylpyrimidin-2-yl)-2,9-diazaspiro[5.5]-

undecan-1-one (29) and 3-(bromomethyl)-1-tosyl-1H-indazole⁵⁹

(75% over two steps). ¹H NMR (400 MHz, DMSO-d₆) δ ppm

12.86 (s, 1H), 7.67 (d, J = 8.03 Hz, 1H), 7.47 (d, J = 8.53 Hz, 1H),

7.32 (t, J = 7.53 Hz, 1H), 7.06 (t, J = 7.40 Hz, 1H), 6.36 (s, 1H), 4.81

(s, 2H), 4.37 (td, J = 3.98, 13.36 Hz, 2H), 3.11−3.25 (m, 4H), 2.21 (s,

6H), 1.88−2.02 (m, 2H), 1.74−1.85 (m, 2H), 1.61−1.73 (m, 2H),

1.35−1.48 (m, 2H). HRMS (ESI⁺) calcd for C₂₃H₂₉N₆O (MH⁺)

405.239 74, found 405.239 98. UPLC−MS t_R,A= 0.99 min, [M + H]⁺

= 405.3.

The residue (395 mg, 0.527 mmol) was dissolved in EtOH (3 mL),

and aqueous 6 M HCl (3.0 mL, 18.0 mmol) was added. The reaction

mixture was stirred at 90 °C for 1 h. The reaction mixture was allowed

to warm to room temperature. Saturated aqueous NaHCO₃solution

was added and the mixture was extracted with TBME (2×). The

combined organic phases were washed with water and brine, dried

over anhydrous sodium sulfate, ﬁltered, and evaporated to give a pale

yellow oil. The crude product was puriﬁed by ﬂash chromatography

(10% EtOAc in heptane to 100% EtOAc). The product was

crystallized from diisopropyl ether to give the title compound 35 as

1

white crystals (76 mg, 32% over two steps). H NMR (400 MHz,

DMSO-d₆) δ ppm 7.64 (d, J = 7.28 Hz, 2H), 7.31−7.50 (m, 3H), 6.35

(s, 1H), 4.77 (s, 2H), 4.15−4.37 (m, 2H), 3.11−3.26 (m, 4H), 2.20 (s,

6H), 1.79−1.92 (m, 2H), 1.61−1.78 (m, 4H), 1.26−1.42 (m, 2H).

HRMS (ESI⁺) calcd for C₂₄H₃₀N₇O (MH⁺) 432.250 63, found

432.250 84. UPLC−MS t_R,A= 0.98 min, [M + H]⁺= 432.4.

9-(4,6-Dimethylpyrimidin-2-yl)-2-((1-methyl-4-phenyl-1H-

pyrazol-3-yl)methyl)-2,9-diazaspiro[5.5]undecan-1-one (36).

The title compound was synthesized according to the procedure

described for compound 37 starting from 9-(4,6-dimethylpyrimidin-2-

yl)-2-((4-phenyl-1H-pyrazol-3-yl)methyl)-2,9-diazaspiro[5.5]undecan-

1-one (34) (74% yield). ¹H NMR (600 MHz, DMSO-d₆) δ ppm 7.92

(s, 1H), 7.28−7.37 (m, 4H), 7.18−7.26 (m, 1H), 6.36 (s, 1H), 4.65 (s,

2H), 4.19−4.32 (m, 2H), 3.82 (s, 3H), 3.20 (t, J = 12.82 Hz, 2H), 3.09

(t, J = 5.65 Hz, 2H), 2.21 (s, 6H), 1.75−1.85 (m, 2H), 1.56−1.71 (m,

4H), 1.24−1.31 (m, 2H). HRMS (ESI⁺) calcd for C₂₆H₃₃N₆O (MH⁺)

2-((1H-Pyrrolo[2,3-b]pyridin-3-yl)methyl)-9-(4,6-dimethyl-

pyrimidin-2-yl)-2,9-diazaspiro[5.5]undecan-1-one (32). To the

suspension of 9-(4,6-dimethylpyrimidin-2-yl)-2,9-diazaspiro[5.5]-

undecan-1-one (29) (100 mg, 0.36 mmol) and TBAI (13 mg, 0.04

mmol) in anhydrous THF (4 mL) at 0 °C was added NaH (95%, 18

mg, 0.73 mmol). After the mixture was stirred for 20 min, 3-

(bromomethyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine⁵⁹(172 mg, 0.40

mmol) was added and stirring was continued at room temperature

for 2.5 h. Water was added, and the aqueous layer was extracted with

ethyl acetate. The organic layer was washed with water and brine. The

combined organic layers were dried over anhydrous sodium sulfate,

ﬁltered, and evaporated to give a pale yellow oil. The crude product

was puriﬁed by ﬂash column chromatography (hexane/ethyl acetate

3:2) to yield a white solid (188 mg, 92%). LC−MS t_R,D= 2.59 min, [M

+ H]⁺= 559.2.

N

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445.271 04, found 445.270 74. LC−MS t_R,D= 2.903 min, [M + H]⁺=

445.2.

presence of up to eight increasing concentrations of test compounds.

All concentration−response curves were run in triplicate.

Mouse PK Experiments. Pharmacokinetic Study. A cassette

dosing approach (up to ﬁve compounds and one reference from the

same chemical series) was chosen in order to obtain pharmacokinetic

parameter and information about the brain penetration properties of a

series of chemically similar compounds in a timely fashion with a

higher throughput. A reference compound with known PK properties

was always included to validate the cassette dosing. For iv injection, 1

mg/kg of each test compound was individually solubilized in N-

methylpyrrolidone, then mixed together and ﬁnally diluted in blank

plasma. The ﬁnal formulation mixture (NMP/blank plasma 10/90, v/

v) was administered at 5 mL/kg body weight into the saphenous vein

of male C57BL/6 mice, weighing between 20 and 30 g (from Iﬀa-

Credo, France). Blood was collected by sublingual bleeding (∼70 μL/

mouse) or at sacriﬁce (∼300 μL/mouse) at 5 and 30 min and 1, 2, 4,

6, 8, and 24 h pd. The brain was collected after decapitation at 5 min

and at 1, 4, and 24 h pd. For oral dosing, 3 mg/kg of each test

compound was individually suspended in carboxymethylcellulose 0.5%

w/v in water/Tween 80 (99.5%/0.5%, v/v). Suspensions were mixed

together and applied by oral gavage to mice to deliver a ﬁnal dose

volume of 10 mL/kg body weight. The blood and brain samples were

collected at the same time points as after intravenous administration

with the exception that the ﬁrst blood and brain samples were

collected after 15 min pd.

9-(4,6-Dimethylpyrimidin-2-yl)-2-((2-methyl-5-phenyl-2H-

1,2,3-triazol-4-yl)methyl)-2,9-diazaspiro[5.5]undecan-1-one

(37). To a solution of 9-(4,6-dimethylpyrimidin-2-yl)-2-((5-phenyl-

2H-1,2,3-triazol-4-yl)methyl)-2,9-diazaspiro[5.5]undecan-1-one (35,

250 mg, 0.556 mmol) and methyl iodide (0.078 mL, 0.834 mmol)

in dry THF (4 mL) at 0 °C under argon was added NaH (60%, 28 mg,

0.695 mmol). After 90 min at 0 °C the reaction mixture was allowed to

warm to room temperature and stirred for 4 h. Saturated aqueous

NaHCO₃solution was added, and the mixture was extracted with

TBME (2×). The combined organic layers were washed with water

and brine, dried over anhydrous sodium sulfate, ﬁltered, and

evaporated to give a pale yellow residue. The crude product was

puriﬁed by ﬂash chromatography (10% EtOAc in heptane to 100%

EtOAc) to yield the title compound and a regioisomer. The product

was crystallized from diisopropyl ether to give the title compound 37

1

as white crystals (74 mg, 30%). H NMR (400 MHz, DMSO-d₆) δ

ppm 7.61 (dd, J = 1.25, 8.03 Hz, 2H), 7.33−7.46 (m, 3H), 6.34 (s,

1H), 4.74 (s, 2H), 4.27 (td, J = 4.14, 13.30 Hz, 2H), 4.14 (s, 3H),

3.12−3.26 (m, 4H), 2.20 (s, 6H), 1.77−1.89 (m, 2H), 1.61−1.76 (m,

4H), 1.27−1.39 (m, 2H). HRMS (ESI⁺) calcd for C₂₅H₃₂N₇O (MH⁺)

446.266 46, found 446.266 28. UPLC−MS t_R,A= 1.19 min, [M + H]⁺

= 446.3.

B. Biology. All animals were maintained under standard housing

conditions with access to standard pelleted food and water ad libitum,

including throughout experiments. The animal experiments were

conducted in accordance with Swiss national animal welfare

regulations, under the ethically approved animal experimentation

licenses authorized by the Cantonal Veterinary Authority of Basel City

and the Federal Veterinary Oﬃce of Switzerland.

Calcium Accumulation in Cells (FLIPR). Chinese hamster ovary

cells and human embryo kidney cells expressing human and mouse

OX₁R or OX₂R receptors respectively, grown in Dulbecco’s minimum

essential medium (DMEM)/F12/10% fetal calf serum (FCS) were

seeded at 8000 cells/well in 384 well black-walled clear bottom, poly-

D-lysine coated plates. After 24 h, the medium was removed and cells

were washed once with phosphate buﬀered saline and serum-deprived

overnight in assay buﬀer (130 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂,

0.8 mM MgSO₄, 0.9 mM NaH₂PO₄, 25 mM glucose, 20 mM HEPES,

pH 7.4) containing bovine serum albumin (1% w/v).

On the day of the experiment, the cells seeded in black plates were

treated with assay buﬀer containing the Ca²⁺sensitive ﬂuorescent dye

Fluo4-AM (2 μM) and probenecid (0.1 mM). After 1 h, plates were

washed twice with, and resuspended in, assay buﬀer containing

probenecid (0.1 mM) using a multiplate washer. The plates were

placed into a FLIPR II (ﬂuorometric imaging plate reader, FLIPR-384,

Molecular Devices, Sunnyvale, CA, USA), and baseline ﬂuorescence

(ﬂuorescence light units, FLU) was measured (ﬁve measurements, 2 s

each, laser excitation 488 nm at 0.6−1 W, CCD camera exposure 0.4

s) before addition of buﬀer alone (basal) or containing test

compounds (either test compound alone, agonist alone, or agonist

in the presence of various concentrations of a test compound).

Fluorescence measurements were then continued every 1 s for 120 s

followed by every 4 s for 240 s.

Similarly, mice in the satellite PK group for the in vivo eﬃcacy

studies received 50 mg/kg test compound suspended in methyl-

cellulose 0.5% w/v in water by oral gavage at a dose volume of 10 mL/

kg body weight. Blood and brain samples were collected after

decapitation at 15 and 60 min after dosing. All PK experiments were

run with n = 3 mice per sampling time point. The blood and brain

samples were stored at −20 °C until analysis by LC/MS/MS.

Bioanalytical Method. The brain samples were homogenized with

2 mM KH₂PO₄buﬀer (approximately 1/2, w/v). A structurally similar

internal standard was added to the blood or brain homogenate

samples, and the test compounds were extracted by quenching with a

4-fold volume of acetonitrile. After centrifugation an aliquot of the

supernatant was directly injected into the LC/MS/MS system (CTC

PAL autosampler, Rheos Allegro LC pump, TSQ Quantum MS, all

from Thermo Scientiﬁc, Waltham, MA, U.S.). The speciﬁc analytical

conditions, i.e., type of the LC column, mobile phase, ionization mode,

ion source parameter, and MRM transitions, were adapted to the

respective cassette dosing experiment to achieve optimal chromato-

graphic separation and sensitive detection of the test compounds.

Quantitative drug concentrations were obtained based on a six-level

calibration curve, using quadratic ﬁtting of a weighed plot of the log−

log transformed peak area ratios vs the blood concentrations. Usually,

the LLOQ ranged between 0.4 and 2 ng/mL in blood and between 1.2

and 6 ng/g in brain.

PK Data Analysis. The absorption and disposition parameters were

estimated by a noncompartmental analysis of the mean blood

concentration (n=3) versus time proﬁle after oral and intravenous

administration. The apparent terminal phase rate constant was

determined by choosing the last three data points from the log−

linear regression of the blood concentrations versus time curve and

was used for the extrapolation of the AUC to inﬁnity. The absolute

oral bioavailability was calculated by dividing the dose-normalized

AUC after oral administration by the respective AUC after intravenous

administration assuming linear pharmacokinetics between the doses of

3 and 1 mg/kg, respectively.

The measurements were typically made in two sequences: In the

ﬁrst round, compounds were tested alone to conﬁrm that they do not

display any signiﬁcant agonist activity. Compounds were tested usually

in a concentration range from 10⁻⁹to 10⁻⁵M. In the second round,

performed 1 h later (to allow for equilibration), orexin A was tested

either in the absence (calibration curves, orexin A agonist controls) or

in the presence of test compounds to determine antagonism.

Inhibition data is expressed as pK_I[−log M], converted by the

Cheng and Prusoﬀ correction (K_I= IC₅₀/[1 + (L/EC₅₀)]), where IC₅₀

is the 50% inhibition value determined in concentration−response

inhibition curves, EC₅₀is the half maximal activation concentration

determined for orexin A in concentration−response curves, and L is

the concentration of orexin A used in inhibition experiments

performed with a submaximal concentration of orexin A in the

Locomotor Paradigm in Mouse. For measuring the eﬀects of

compounds on locomotor activity, a computerized motility measure-

ment system was used (Moti 4.25, TSE Systems, Bad Homburg,

Germany). This system automatically measures locomotor activity in

transparent boxes (20 cm × 32 cm × 17 cm) by counting the

interruptions of horizontal infrared beams spaced 5.7−8.4 cm apart in

a frame set at the ﬂoor level of the cage.

C57Bl/6 mice (Janvier, Le Genest Saint Isle, France) adapted to a

12h/12h light/dark cycle with lights oﬀ at 15:00 were individually

placed into these cages at about 9:00 and were allowed to habituate for

O

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Journal of Medicinal Chemistry

Article

1.5 days. Immediately before lights-oﬀ (15:00) on the following day,

animals were treated with either vehicle control (0.5% methylcellulose

in water, w/v), 26 (in MEPC5 40%), or suvorexant (0.5%

methylcellulose in water, w/v). Fifteen hours later, the animals were

brought back to their home cages.

there was a statistically signiﬁcant eﬀect of treatment or a signiﬁcant

interaction between treatment and hour. Post hoc Fisher’s least

signiﬁcant diﬀerence tests were applied to determine hour by hour

where there were signiﬁcant diﬀerences between the vehicle and

compound days.

One minute of inactivity was deﬁned as a minute without any

interruptions of the infrared beams. Previous studies demonstrated

that this measure is highly correlated with sleep.⁵³To analyze

compounds eﬀects, the diﬀerence in inactivity in the 4 h after

compound administration (15:00 to 19:00) and the inactivity during

the same 4 h at 24 h earlier (during the habituation phase) were

calculated for each animal. Treatment group mean values were then

compared using an analysis of variance (one-way ANOVA) with post

hoc Dunnett’s tests (comparisons with vehicle).

AUTHOR INFORMATION

Corresponding Authors

*C.B.: phone, +41 61 6967739; e-mail, claudia.betschart@

novartis.com. For

*S.H.: phone, +41 61 6968456; e-mail, samuel.hintermann@

novartis.com.

■

Present Addresses

Mouse Sleep Assay. Implantation of Electrocorticogram/Electro-

encephalogram (EEG) and Electromyogram (EMG) Electrodes. Male

C57Bl/6 mice were administered temgesic (0.05 mg/kg sc) 1 h before

surgery. Mice were anesthetized with ketamine/xylazine (110 mg/kg,

10:1, ip) and placed in a stereotaxic frame. The skull was exposed, and

four miniature stainless steel screws (SS-5/TA Science Products

GmbH, Hofheim Germany) attached to 36-gauge Teﬂon coated solid

silver wires were placed in contact with the frontal and parietal cortex

(3 mm posterior to bregma, 2 mm from the sagittal suture) through

bore holes. The frontal electrodes served as reference. The wires were

crimped to a small six-channel connector (CRISTEK Micro Strip

Connector) that was aﬃxed to the skull with dental acrylic. EMG

signals were acquired by a pair of multistranded stainless steel wires

(7SS-1T, Science Products GmbH, Hofheim, Germany) inserted into

the neck muscles and also crimped to the headmount. After surgery,

mice were singly housed and allowed to recover in their cage placed on

a heating pad. Temgesic, 0.05 mg/kg, sc, was given 8 and 16 h after

surgery to prevent pain. After 24 h, the mice were housed with their

former cage mates and allowed to recover for 2 weeks. Mice were

continuously kept on a 12 h/12 h light/dark cycle with lights on at

03:00.

^⊥D.H.: Department of Pharmacology & Therapeutics, Faculty

of Medicine, Dentistry and Health Sciences, School of

Medicine, The University of Melbourne, Parkville, Victoria

3010, Australia. E-mail: d.hoyer@unimelb.edu.au

^#C.E.G.: Institute for Synaptic Physiology, Center for

Molecular Neurobiology Hamburg, Falkenried 94, 20251

Hamburg, Germany. E-mail: christine.gee@zmnh.uni-

hamburg.de.

^∞M.F.: Institute for Pharmacology and Toxicology, Otto-von-

Guericke University Magdeburg, Leipziger Strasse 44, D-39120

Magdeburg, Germany. E-mail: markus.fendt@med.ovgu.de.

^×V.C.: Department of Medicinal Chemistry, Lupin Pharma-

ceuticals, Inc., Lupin Research Park, Genesis Square, Phase-II,

Hinjewadi, Pune-411057, India. E-mail: vinodchaudhari@

lupinpharma.com.

^●L.H.J.: The Florey Institute of Neuroscience and Mental

Health, The University of Melbourne, Kenneth Myer Building,

Genetics Lane on Royal Parade, Victoria 3010, Australia. E-

mail: ljacobson@unimelb.edu.au.

Sleep Studies. Mice were habituated to individual cages in the

sound-attenuated recording chamber for 6−10 days (lights on 03:00,

lights oﬀ 15:00, max 80 lx) and a constant temperature of about 23 °C.

Mice had access to food and water ad libitum and access to one

nesting paper and a piece of wood. Mice were weighed and attached to

recording cables that connected their head mounts to a commutator

(G-4-E, Gaueschi) allowing free movement in the experiment boxes

on the morning of the ﬁrst day of the experiment. All further

manipulations and oral applications were performed in a time window

of 5−15 min before lights oﬀ and start of the recordings. On day 1, the

mice were manipulated and habituated to the oral application syringe

just before lights oﬀ. On day 2, they received vehicle (methylcellulose

0.5%, 10 mL/kg, per os). On day 3, 26 or suvorexant was administered

per os. Recordings began with lights oﬀ at 15:00 (hour 0) and

continued for 23 h. The experimental chamber was secured about 5

min prior to lights oﬀ, and the mice were undisturbed during the

recordings. One hour before lights oﬀ, recordings were stopped and

the chamber was opened to care for the mice and perform any

manipulations necessary. On day 4, the mice were replaced in groups

in their normal housing cages. EEG/EMG signals were ampliﬁed using

a Grass model 78D ampliﬁer (Grass Instrument CO., Quincy, MA,

USA), analogue ﬁltered (EEG, 0.3−30 Hz; EMG, 5−30 Hz) and

acquired using Harmonie V5.2 (acquisition frequency of 200 Hz with

calibration the ﬁrst day, record duration of 23 h). Animals were

videorecorded during data collection, using an infrared video camera,

and locomotor activity was detected using infrared sensors (InfraMot

infrared activity sensor 30-2015 SENS, TSE Systems) placed in the

roof of the boxes. Activity signals were acquired at 10 s intervals by the

software Labmaster V2.4.4. EEG/EMG and activity recordings were

imported into and scored in 10 s epochs using the rodent scoring

module of Somnologica into wake, NREM sleep, and REM sleep.

Epochs during which there were state transitions were scored as the

state present for at least 50% of the epoch. The time in each state per

Author Contributions

^○C.B. and S.H. contributed equally.

The manuscript was written through contributions of all

authors. All authors have given approval to the ﬁnal version of

the manuscript.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank Richard Felber, Sandrine Liverneaux, Andrea Hasler,

Alfred Tanner, Thomas Gut, Marc Weibel, Fatma Limam,

Marjorie Bourrel, and Michael Wright for synthetic support,

Christian Guenat for the determination of high resolution mass,

Thomas Durst, Stefan Imobersteg, Hugo Burki, Edi Schuep-

̈

bach, Daniel Langenegger, and Dominique Monna for in vitro

and in vivo studies, Agnese Vit and Karen Beltz for compound

formulation support, and Eric Legangneux for helpful

discussions.

ABBREVIATIONS USED

■

AUC_bl, area under the blood concentration time curve; [bl],

blood concentration; [br] brain concentration; Cl_bl, total blood

clearance; C_max, observed maximum concentration in blood;

DORA, dual orexin receptor antagonist; EMG, electromyo-

gram; f_u, free fraction; FLIPR, ﬂuorometric imaging plate

reader; LC/MS/MS, liquid chromatography coupled to tandem

mass spectrometry; LipE, lipophilicity eﬃciency; LLOQ, lower

limit of quantiﬁcation; NREM, nonrapid eye movement sleep;

OX₁R, orexin (hypocretin) receptor type 1; OX₂R, orexin

(hypocretin) receptor type 2; OXR, orexin receptor; pd,

hour was calculated, and the mean

SEM is shown. Restricted

maximum likelihood analysis (REML) was applied to determine if

P

dx.doi.org/10.1021/jm4007627 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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1998, 18, 9996−19915.

postdosing; REM, rapid eye movement sleep; V_ss, apparent

volume of distribution at steady state

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dx.doi.org/10.1021/jm4007627 | J. Med. Chem. XXXX, XXX, XXX−XXX

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Identification of a novel series of orexin receptor antagonists with a distinct effect on sleep architecture for the treatment of insomnia

DOI: 10.1021/jm4007627

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Article abstract of DOI:10.1021/jm4007627

Full text of DOI:10.1021/jm4007627

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