Discovery of 5″-chloro-n-[(5,6-dimethoxypyridin-2- yl)methyl]-2,2':5',3″-Terpyridine-3'-carboxamide (mk-1064): A selective orexin 2 receptor antagonist (2-sora) for the treatment of insomnia

Article abstract of DOI:10.1002/cmdc.201300447

The field of small-molecule orexin antagonist research has evolved rapidly in the last 15 years from the discovery of the orexin peptides to clinical proof-of-concept for the treatment of insomnia. Clinical programs have focused on the development of antagonists that reversibly block the action of endogenous peptides at both the orexin 1 and orexin 2 receptors (OX1R and OX2R), termed dual orexin receptor antagonists (DORAs), affording late-stage development candidates including Merck's suvorexant (new drug application filed 2012). Full characterization of the pharmacology associated with antagonism of either OX1R or OX2R alone has been hampered by the dearth of suitable subtype-selective, orally bioavailable ligands. Herein, we report the development of a selective orexin 2 antagonist (2-SORA) series to afford a potent, orally bioavailable 2-SORA ligand. Several challenging medicinal chemistry issues were identified and overcome during the development of these 2,5-disubstituted nicotinamides, including reversible CYP inhibition, physiochemical properties, P-glycoprotein efflux and bioactivation. This article highlights structural modifications the team utilized to drive compound design, as well as in vivo characterization of our 2-SORA clinical candidate, 5''-chloro-N- [(5,6-dimethoxypyridin-2-yl)methyl] -2,2':5',3''-terpyridine-3'-carboxamide (MK-1064), in mouse, rat, dog, and rhesus sleep models.

Full text of DOI:10.1002/cmdc.201300447

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DOI: 10.1002/cmdc.201300447
Discovery of 5’’-Chloro-N-[(5,6-dimethoxypyridin-2-
yl)methyl]-2,2’:5’,3’’-terpyridine-3’-carboxamide (MK-1064):
A Selective Orexin 2 Receptor Antagonist (2-SORA) for the
Treatment of Insomnia
Anthony J. Roecker,*[a] Swati P. Mercer,[a] John D. Schreier,[a] Christopher D. Cox,[a]
Mark E. Fraley,[a] Justin T. Steen,[a] Wei Lemaire,[b] Joseph G. Bruno,[b] C. Meacham Harrell,[c]
Susan L. Garson,[c] Anthony L. Gotter,[c] Steven V. Fox,[d] Joanne Stevens,[d]
Pamela L. Tannenbaum,[d] Thomayant Prueksaritanont,[e] Tamara D. Cabalu,[e] Donghui Cui,[e]
Joyce Stellabott,[f] George D. Hartman,[a] Steven D. Young,[a] Christopher J. Winrow,[c]
John J. Renger,[c] and Paul J. Coleman[a]
The field of small-molecule orexin antagonist research has
evolved rapidly in the last 15 years from the discovery of the
orexin peptides to clinical proof-of-concept for the treatment
of insomnia. Clinical programs have focused on the develop-
ment of antagonists that reversibly block the action of endog-
enous peptides at both the orexin 1 and orexin 2 receptors
(OX1R and OX2R), termed dual orexin receptor antagonists
(DORAs), affording late-stage development candidates includ-
ing Merck’s suvorexant (new drug application filed 2012). Full
characterization of the pharmacology associated with antago-
nism of either OX1R or OX2R alone has been hampered by the
dearth of suitable subtype-selective, orally bioavailable ligands.
Herein, we report the development of a selective orexin 2 an-
tagonist (2-SORA) series to afford a potent, orally bioavailable
2-SORA ligand. Several challenging medicinal chemistry issues
were identified and overcome during the development of
these 2,5-disubstituted nicotinamides, including reversible CYP
inhibition, physiochemical properties, P-glycoprotein efflux and
bioactivation. This article highlights structural modifications
the team utilized to drive compound design, as well as in vivo
characterization of our 2-SORA clinical candidate, 5’’-chloro-N-
[(5,6-dimethoxypyridin-2-yl)methyl]-2,2’:5’,3’’-terpyridine-3’-car-
boxamide (MK-1064), in mouse, rat, dog, and rhesus sleep
models.
Introduction
Primary insomnia is defined as difficulty initiating/maintaining
sleep or finding sleep non-restorative in the absence of psy-
chological or physical ailments, and it affects approximately
50% of the general population at some point in their lives.[1]
The current standard of care for insomnia is treatment with
non-benzodiazepine GABA modulators such as zolpidem.
While this method of treatment is effective for many insomnia
patients, significant side effects have been noted including
next day residual somnolence, rebound insomnia, amnesia, ef-
fects on motor coordination, and parasomnias.[1,2] These side
effects have resulted in the issuance of revised labeling and, in
some cases, black box warnings.[3] Recently, the US Food and
Drug Administration (FDA) also recommended that the dose of
zolpidem for women should be decreased by half due to the
risk of next-day somnolence.[4] These concerns have encour-
aged further efforts to discover and develop treatments for in-
somnia with differentiated mechanisms of action from current
therapies.
[a] Dr. A. J. Roecker, S. P. Mercer, J. D. Schreier, Dr. C. D. Cox, Dr. M. E. Fraley,
J. T. Steen, Dr. G. D. Hartman, Dr. S. D. Young, Dr. P. J. Coleman
Department of Medicinal Chemistry
Merck Research Laboratories
P.O. Box 4, Sumneytown Pike, West Point, PA 19486 (USA)
E-mail: anthony_roecker@merck.com
[b] W. Lemaire, J. G. Bruno
Department of In Vitro Pharmacology (Merck, West Point)
The orexin peptides, orexin A and orexin B, were originally
discovered in 1998 by two independent research groups.[5]
These peptides were then associated with activity on two
orphan G protein-coupled receptors, orexin 1 (OX1R) and
orexin 2 (OX2R). Initially thought to be involved in feeding be-
haviors, the peptides were named after the Greek word
“orexis” meaning appetite. Since that time, the effects on ap-
petite have been called into question, but a strong link has
been established between the orexin system and effects on
[c] C. M. Harrell, S. L. Garson, Dr. A. L. Gotter, Dr. C. J. Winrow, Dr. J. J. Renger
Department of Neuroscience (Merck, West Point)
[d] S. V. Fox, J. Stevens, Dr. P. L. Tannenbaum
Department of In Vivo Pharmacology (Merck, West Point)
[e] Dr. T. Prueksaritanont, Dr. T. D. Cabalu, Dr. D. Cui
Department of Drug Metabolism (Merck, West Point)
[f] J. Stellabott
Department of Basic Pharmaceutical Sciences (Merck, West Point)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300447.
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the sleep/wake cycle from studies in orexin knockout mice,[6]
narcoleptic canines with OX2R receptor mutations,[7] and narco-
leptic humans owning low cerebrospinal fluid (CSF) levels of
orexin peptides and a loss of hypothalamic orexin neurons.[8]
These studies and the pharmacology associated with orexin re-
ceptor antagonists have been the topic of several recent re-
views.[9] Additionally, intracerebroventricular injection of orexin
peptides has been shown to stimulate wakefulness and de-
creases slow wave sleep in rats.[10] These findings strongly sug-
gest that the orexin peptides are wake-promoting agents, and,
by extension, antagonism of these peptides might offer an al-
ternative treatment for insomnia. This strategy is therefore dif-
ferentiated from that of GABA modulators, which initiate sleep
through potentiation of the effects of a ubiquitous inhibitory
neurotransmitter. Recently, we have invested further efforts in
preclinical differentiation demonstrating that orexin antago-
nists have improved therapeutic margins versus cognitive end-
points in rats and rhesus monkeys compared with GABA mod-
ulators.[11] This differentiated mechanism of action has pro-
duced high levels of interest across the scientific and medical
communities.
have a reportedly mild phenotype with no changes in sleep-
stage time and minor sleep fragmentation, suggesting a rela-
tively minor role for OX1R in sleep regulation.[17] OX1R and
OX2R have partially overlapping (lateral hypothalamus, locus
coeruleus, dorsal raphe, peduncular pontine nucleus) and dis-
tinct (tuberomamillary nucleus [TMN] for OX2R; prefrontal
cortex for OX1R) localization throughout the brain. Selective ex-
pression of OX2R in histaminergic neurons of the TMN supports
a primary role of this receptor in driving arousal through stim-
ulation of histamine release.[9a] In fact, DORA-induced effects
on sleep time are nearly eliminated in mice lacking OX2R, indi-
cating that selective OX2R blockade might be sufficient for
sleep-inducing effects of orexin receptor antagonism.[18]
The implications of additional OX1R antagonism provided by
DORAs relative to selective orexin 2 antagonists (2-SORAs) are
currently unclear, and previous tool compounds used to delin-
eate these issues have off-target activities[9b] and hydrolytic sta-
bility issues potentially contributing to mixed results.[19] In one
study, co-administration of the 1-SORA ligand, SB-408124, with
the 2-SORA ligand, JNJ-10397049, measurably diminished the
sleep-promoting effect of the 2-SORA ligand in rats suggesting
improved sleep efficacy for 2-SORA versus DORA com-
pounds.[20] A different group found improved efficacy for the
DORA, almorexant, relative to the highly selective 2-SORA,
EMPA, at similar OX2R occupancies.[21] Clearly, a need exists for
improved tools to study the underlying pharmacology for re-
versible and selective blockade of OX1R and OX2R. Herein, we
describe our efforts toward the discovery of a series of
improved 2-SORA ligands culminating in the discovery of clini-
cal candidate, 5’’-chloro-N-[(5,6-dimethoxypyridin-2-yl)methyl]-
2,2’:5’,3’’-terpyridine-3’-carboxamide (MK-1064, 3, Figure 1).[22]
Several companies have disclosed medicinal chemistry pro-
grams targeting the discovery and development of dual orexin
receptor antagonists (DORAs).[12] Medicinal chemistry efforts
from our laboratories describing the discovery of two DORAs,
MK-4305 (suvorexant, 1)[13] and MK-6096 (2),[14] have been pub-
lished (Figure 1). Suvorexant has achieved clinical proof-of-con-
cept for the treatment of primary insomnia, and a new drug
application (NDA) for suvorexant was filed with the FDA in
2012.[15] The clinical success of antagonists of both OX1R and
OX2R has clearly energized the field of orexin research.[16] How-
ever, questions still exist surrounding subtype-selective antago-
nism of the individual receptors.
Results and Discussion
Genetic and pharmacological evidence indicates a primary
role of OX2R in orexin-mediated arousal. Targeted mutation of
OX2R in mice and genetic loss of the receptor in canines is as-
sociated with a narcoleptic phenotype including hypersom-
nia.[6b,7] In mice, this phenotype is nearly identical, but not
quite as pervasive as that displayed by knockouts lacking the
gene encoding the orexin ligand,[6b] while OX1R knockouts
A recent publication from our group described the discovery
of 4, a novel 2,5-disubstituted nicotinamide 2-SORA ligand
identified by hit-to-lead efforts following high-throughput
screening of the Merck sample collection (Figure 2).[23] Com-
pound 4 demonstrates low-picomolar affinity for the human
OX2R as measured by a radioligand-displacement binding
assay (expressed as Ki values) and high selectivity over OX1R
(>1000-fold). Compound 4 has excellent OX2R potency and
moderate OX1R selectivity in a fluorometric imaging plate
Figure 1. Merck DORAs, MK-4305 and MK-6096, and 2-SORA MK-1064.
Figure 2. Previously reported 2,5-disubstituted nicotinamide 2-SORA (4).
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reader (FLIPR) assay that provides a functional readout of
orexin receptor antagonism in CHO cells engineered to overex-
press the human receptors (expressed as IC50 values).[13a] This
novel 2-SORA ligand demonstrated sleep efficacy similar to
DORA molecules (decreased active wake, increased delta and
REM sleep) when dosed orally to rats during their active phase.
Although 2-SORA 4 demonstrated sleep efficacy in a rat
electroencephalographic (EEG) model through oral dosing, sev-
eral key issues precluded advanced characterization. First, the
level of functional selectivity for OX2R might not be high
enough to be confident that at plasma concentrations for
sleep efficacy compound 4 would not inhibit OX1R to some
extent. Therefore, one focus of the optimization effort was to
maximize the selectivity over OX1R within this series. We tar-
geted 100-fold selectivity over OX1R in the FLIPR assay as an
initial goal. Second, compound 4 is a potent reversible inhibi-
tor of CYP3A4 (IC50 =300 nm).[24] Third, although highly perme-
able (Papp=32ꢁ10ꢀ6 cmsꢀ1), compound 4 is a P-glycoprotein
(Pgp) substrate with an efflux ratio of 6.4 (human monolayer
preparations; substrate ratio ꢁ3).[25] This might limit central
nervous system (CNS) penetration, thus requiring significantly
higher doses and plasma concentrations for sleep efficacy. Fi-
nally, compound 4 has bioactivation risk derived from the di-
methoxyphenyl motif.[26] This theoretical liability was confirmed
through the identification of high levels of glutathione adducts
in human and rat liver microsomal incubations (see below).
The aromatic rings of the nicotinamide scaffold are labeled A
through D to facilitate the discussion of the profile optimiza-
tion effort targeting the issues outlined above.
modified slightly from previous efforts to place the diversity in-
corporating step at the end of the route (Scheme 1). Commer-
cially available methyl-2-chloro-5-iodonicotinate (5) was trans-
formed into 6 via selective Suzuki coupling, hydrolysis, and
amide formation in acceptable yield. This common intermedi-
ate was utilized to produce final compounds through addition-
al Suzuki couplings (7, 8, and 10), Stille couplings (9), or nucle-
ophilic substitution reactions (11). The 3,5-dimethylphenyl A-
ring in compound 7 was used for SAR purposes targeting
lower molecular weight and decreased Pgp susceptibility
(hPgp ratio: 2; Papp: 31ꢁ10ꢀ6 cmsꢀ1) compared with the 3,5-
dichloro A-ring in compound 4. As shown in Table 1, the posi-
tion of the nitrogen of the C-ring has very little impact on
binding and functional potency on OX2R with compounds 7, 8,
and 9. Selectivity over OX1R was also in a similar range; howev-
er, the reversible inhibition of CYP3A4 was vastly different for
these three compounds. The 3- and 4-pyridyl motifs were
potent reversible inhibitors of CYP3A4, while the 2-pyridyl sub-
stituent significantly decreased this liability (IC50 =38 mm). Inter-
estingly, replacing the pyridyl substituents with phenyl (10) or
morpholine (11) motifs also decreased the reversible CYP3A4
potency, although the former possessed less functional selec-
tivity than the lead compound 4, and the latter demonstrated
weaker binding and functional potency. Hence, the 2-pyridyl
motif was selected for additional SAR studies.
While the A- and D-rings were explored previously, it
became apparent with leading compounds such as 9 that ele-
vated logD (3.2) and high human plasma protein binding
(PPB=99.5%) were nonoptimal. A target for human PPB was
not specified in this effort, however, measurable free fraction
(>1%) is generally thought to be a desirable profile element
in CNS programs.[27] Notably, compound 9 maintained the de-
sirable Pgp profile of earlier lead compound 7 (Table 2). This
Our previous disclosure around the nicotinamide series of 2-
SORA compounds focused on the structure–activity relation-
ships (SARs) of the A- and D-rings of the series leaving the
C-ring intact. In order to explore this SAR, the synthesis was
Scheme 1. Synthesis of C-ring analogues. Reagents and conditions: a) 3,5-dimethylphenylboronic acid, PdCl2(dppf), Cs2CO3, DMF/water (19:1), 258C, 18 h
(83%); b) KOSiMe3, THF, 258C, 24 h (100%); c) EDC, HOAt, Et3N, 3,4-dimethoxybenzylamine, DMF, 458C, 3 h (62%); d) 2-(tri-n-butylstannyl)pyridine, Pd(PPh3)4,
CsF, CuI, DMF, 1608C, 20 min, mW (67%); e) morpholine, DMF, 1408C, 15 min, mW (58%); f) PdCl2(dppf), Cs2CO3, DMF/water, 3-(4,4,5,5-tetramethyl-1,3,2-dioxa-
borolan-2-yl)pyridine, 1608C, 20 min, mW (62% for 7), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (20% for 8), phenylboronic acid (22% for 10);
Abbreviations: dppf = 1,1’-(diphenylphosphino)ferrocene; EDC = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; HOAt = 1-hydroxy-7-azabenzotriazole.
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OX1R, adequate PPB, and de-
creased Pgp susceptibility. Com-
pound 15 demonstrated the
need for an additional substitu-
ent on the pyridine A-ring due
to significant decrease in OX2R
potency. Despite significant im-
Table 1. Structure–activity relationships and reversible CYP3A4 inhibition data for C-ring analogues.
provements
in
reversible
Compd
OX2R[a]
IC50 [nm]
OX1R[a]
IC50 [nm]
SI[b]
(Ki/IC50)
CYP3A4[c]
IC50 [mm]
Ki [nm]
Ki [nm]
CYP3A4 inhibition, selectivity
over OX1R, and physical proper-
ties, bioactivation concerns still
remained for compounds such
as 14. These challenges were ad-
dressed in the D-ring efforts de-
scribed below.
(7)
0.7
17
17
8
462
705
748
660/41
349/44
467/23
1100/18
412/61
0.1
1
(8)
1.5
0.3
0.2
8.3
523
140
(9)
182
38
Indeed, incubation of com-
pound 14 in human liver micro-
somes in the presence of gluta-
thione (GSH) resulted in the
trapping of significant levels of
adducts consistent with experi-
ments on compound 4. Similar
bioactivation issues were en-
countered during the DORA pro-
gram,[13a] hence, an in vitro glu-
tathione trapping assay was
(10)
(11)
14
56
220
245
13
3467
3400
>50
[a] Ki values were determined using a radioligand-displacement binding assay and IC50 values were determined
using a fluorometric imaging plate reader (FLIPR) assay; each value is the mean of at least two or more inde-
pendent determinations; for assay details, see Ref. [13a]; [b] Selectivity index (SI) for OX1R over OX2R as calcu-
lated from the Ki and IC50 values; [c] Reversible CYP3A4 inhibition; see the Supporting Information for assay
details.
parameter was closely monitored as greater polarity was intro- available in house and utilized to select compounds that de-
duced into analogues. Efforts were then focused on further ex- creased this metabolic liability. Precedent suggested that oxi-
ploration of A-ring SAR as shown in Table 2 with the aim of im- dation of the dimethoxyphenyl moiety could unveil quinone
proving physical properties. De-
letion of a methyl group in com-
Table 2. Structure–activity relationships, and P-glycoprotein (Pgp), LogD, and protein binding data for A-ring
analogues.
pound 12 diminished functional
potency on OX2R by twofold,
while increasing selectivity over
OX1R. This modification lowered
logD by 0.4 units and decreased
PPB. In an attempt to further im-
prove physical properties, pyridyl
analogues such as 13, 14, and
15 were synthesized. Compound
13 maintained the intrinsic and
functional potency of compound
12 and similar levels of selectivi-
ty over OX1R while decreased
logD by 1.3 units and human
PPB to 62%. Unfortunately, the
incorporation of a basic pyridine
in compound 13 resulted in in-
creased Pgp susceptibility. In
order to modulate Pgp activity,
the methyl group in compound
13 was replaced with a chlorine
atom to decrease the basicity of
the A-ring pyridine motif. Grati-
fyingly, compound 14 demon-
strated acceptable on target po-
tency, improved selectivity over
Compd
OX2R[a]
OX1R[a]
SI[b]
hPgp[c]
Papp[c]
LogD hPPB[d]
Ki [nm] IC50 [nm] Ki [nm] IC50 [nm] (Ki/IC50) (BA/AB) [10ꢀ6 cmsꢀ1
]
(HPLC)
[%]
(9)
0.3
8
140
182
467/23
1.1
26
3.2
99.5
(12)
(13)
1.4
0.6
19
28
2550
763
985 1821/52
1092 1272/39
0.7
4.4
29
28
2.8
1.5
98.5
62
(14)
(15)
0.7
5.7
20
68
2113
1308 3019/65
1.7
3.4
29
30
1.9
1.3
94
39
13500 >10000 2328/147
[a] Ki values were determined using a radioligand-displacement binding assay and IC50 values were determined
using a fluorometric imaging plate reader (FLIPR) assay; each value is the mean of at least two or more inde-
pendent determinations; for assay details, see Ref. [13a]; [b] Selectivity index (SI) for OX1R over OX2R as calcu-
lated from the Ki and IC50 values; [c] Human P-glycoprotein (hPgp) susceptibility in human preparations and
passive permeability (Papp); for assay details, see Ref. [25]; [d] Human plasma protein binding (hPPB); data is
the % bound, and values were determined by standard methods; see the Supporting Information for details.
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intermediates that can be readily conjugated by organic nucleo-
philes.[28] Initially, analogues were synthesized that either re-
moved or replaced the methoxy groups in compound 14,
however, these generally increased logD and decreased either
potency for OX2R or selectivity over OX1R (data not shown).
Armed with the knowledge that the 3,4-dimethoxy motif was
important to the overall profile, several nitrogen-containing
heterocycles such as compounds 16, 17, and 3 were designed
and synthesized. The synthetic strategy to afford these ana-
logues was similar to Scheme 1, and the slight modifications
are shown in Schemes 2 and 3. Incorporation of a nitrogen
into the D-ring in compound 16 had the desired effect in de-
creasing GSH adducts in vitro (85% reduction), although intrin-
sic and functional potency on OX2R decreased significantly,
while Pgp susceptibility increased (Table 3). Dimethoxypyrazine
analogue 17 demonstrated similar potency to 14 with im-
proved selectivity[29] and limited impact on Pgp efflux; howev-
er, in vitro bioactivation was not decreased as much as in com-
pound 16. Finally, the pyridine isomer found in compound 3
provided the most balanced overall profile possessing excel-
lent potency on OX2R, high levels of selectivity over OX1R, ac-
ceptable Pgp susceptibility in human preparations, high levels
of passive permeability, and low bioactivation potential in
vitro. On the strength of these data, compound 3 was selected
for additional profiling.
Scheme 3. Synthesis of heterocyclic benzylamines. Reagents and conditions:
a) CuCN, DMF, 150–1858C, 15–40 min, mW (79% for 21; 58% for 23; 66% for
25); b) Pd(OH)2, H2, HCl (concd), MeOH, 258C, 1.5–2 h (94% for 21; 92% for
23; 49% for 25); c) bromine, saturated aq NaHCO3/DCM, 08C, 2 h (49%).
rat and dog. Bile duct-cannulated rat studies were also per-
formed utilizing radiolabeled compound 3, and these experi-
ments suggested excellent oral absorption with nearly 100%
of the dose being recovered in urine (~20%) and bile (~79%)
after 48 h. Qualitatively, the metabolites observed in rat in vitro
(hepatocytes) and in vivo studies were very similar.
As described previously, the drug development team utilized
an ex vivo humanized transgenic rat occupancy assay to assist
in the determination of target engagement.[13a] Due to the low
levels of endogenous orexin expressed centrally, it was neces-
sary to produce a colony of rats that overexpressed human
OX2R to reliably measure receptor occupancy. Compound 3
demonstrated 90% occupancy of OX2R at 695 nm, 203 nm, and
52 nm in the plasma, brain, and CSF, respectively, utilizing
a rising intravenous (IV) dosing paradigm (Figure 3).[30] The
CSF-to-plasma ratio (0.07) for this experiment was somewhat
lower than the free fraction in rat (0.19) consistent with low to
moderate Pgp efflux activity. Rat, monkey, and mouse Pgp ex-
periments showed higher ratios than in human with BA/AB
ratios of 6.1, 5.5, and 8.4 compared with 2.4 in Table 3. From
these data, the impact of Pgp efflux on central penetration in
human subjects was predicted to have less of an impact on ef-
ficacy than in preclinical species.
The pharmacokinetic parameters of compound 3 are shown
in Table 4. This compound has moderate clearance in all three
species with half-life values in an appropriate range for once-
nightly dosing for the treatment of insomnia. Gratifyingly, com-
pound 3 possessed moderate oral bioavailability (%F) similar
to its predecessor compound 4 demonstrating 54%, 48%, and
16% bioavailability in rat, dog, and rhesus monkey, respective-
ly. Additional in vitro metabolic studies performed in rat, dog,
and human liver microsomes (HLM) demonstrated that the
major route of metabolism was O-dealkylation of the 5-me-
thoxy group to afford the phenolic product, which was further
converted to its sulfate and glucuronide in hepatocytes. CYP3A
was determined to be the primary enzyme responsible for this
transformation through immuno-inhibition studies in HLM. All
metabolites found in human in vitro studies were also seen in
Additional drug metabolism studies revealed that
compound 3 is not a potent reversible inhibitor of
CYP3A4 (IC50 =28 mm), CYP2C9 (IC50 >100 mm), or
CYP2D6 (IC50 >100 mm). Compound 3 does not acti-
vate human PXR (EC50 >30 mm; 7% activation com-
pared with rifampicin control). Compound 3 does
have moderate time-dependent inhibition (TDI) of
CYP3A4 with a Ki value of 9.5 mm and a Kinact value of
0.11 minꢀ1 [31]
however, if target human exposures are
;
low, the potential risk for human drug–drug interac-
tions from this mechanism could be minimal. In
order to assess this possibility, compound 3 was ad-
vanced into in vivo testing in preclinical models of
sleep efficacy.
Scheme 2. Representative synthesis of A- and D-ring analogues. Reagents and conditions:
a) 5-(chloropyridin-3-yl)boronic acid, PdCl2(dppf), Cs2CO3, DMF/water (22:1), 258C, 18 h
(99%); b) 2-(tri-n-butylstannyl)pyridine, Pd(PPh3)4, CsF, CuI, DMF, 808C, 2 h (52%);
c) NaOH (10m), THF/MeOH (5:1), 1358C, 15 min, mW; d) 21, EDC, HOAt, iPr2NEt, DMF,
608C, 2 h (79% over 2 steps).
The preclinical sleep facility at Merck has a range
of species for the assessment of in vivo efficacy of
novel compounds measuring electrocorticogram
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their wake period. Sleep promo-
tion was observed in all species
with similar changes in vigilance
state following treatment; at-
tenuated active wake is accom-
panied by significant increases in
both slow-wave sleep (SWS) and
REM sleep following treatment.
Generally, these sleep-promoting
responses to compound 3 are
similar to that observed for
DORAs suvorexant and MK-6096
in that decreases in active wake
are accompanied by significant
increases in slow wave and REM
sleep.[13b,14b]
Table 3. Structure–activity relationships, and P-glycoprotein (Pgp), glutathione (GSH) trapping/bioactivation
data for D-ring analogues.
Compd
OX2R[a]
OX1R[a]
SI[b]
(Ki/IC50)
hPgp[c]
Papp[c]
GSH/IS
ratio[d]
Ki [nm] IC50 [nm] Ki [nm] IC50 [nm]
(BA/AB) [10ꢀ6 cmsꢀ1
]
(14)
(16)
(17)
(3)
0.7
6.8
2.1
0.5
20
70
31
18
2113
13620
7640
1308
5200
4100
1789
3019/65
2003/74
3638/132
3168/99
1.7
3.1
1.8
2.4
29
30
34
34
0.93
0.14
0.27
0.14
Table 5 displays the exposure
data from the sleep studies de-
scribed above as well as binding
potencies from each of the spe-
cies tested. At 100 mgkgꢀ1, MK-
1064 showed robust efficacy in
mouse models with an AUC
value of 33.5 mm·h, a Cmax value
of 10.2 mm, and a value Cmax,u of
2.0 mm with a short Tmax value.
1584
[a] Ki values were determined using a radioligand-displacement binding assay and IC50 values were determined
using a fluorometric imaging plate reader (FLIPR) assay; each value is the mean of at least two or more inde-
pendent determinations; for assay details, see Ref. [13a]; [b] Selectivity index (SI) for OX1R over OX2R as calcu-
lated from the Ki and IC50 values; [c] Human P-glycoprotein (hPgp) susceptibility in human preparations and
passive permeability (Papp); for assay details, see Ref. [25]; [d] GSH/internal standard (IS) ratio (human); for
assay details, see Ref. [13a].
High levels of efficacy in rat (20 mgkgꢀ1, p.o.) were
achieved at similar total and unbound exposures. In
order to assure that there were no potency devia-
tions within species, membranes were prepared ex-
pressing the species-specific receptors.[32] Notably,
the OX2R potency and selectivity over OX1R remained
consistent for mouse and human, as well as the
other species tested. From this consistent level of po-
tency across species and the rat occupancy data
(Occ90 plasma=695 nm; CSF=52 nm; see above), it
was concluded that high levels (>90%) of OX2R oc-
cupancy were achieved in the mouse and rat sleep
experiments. It should also be noted that these
Table 4. Pharmacokinetics of compound 3 in rat, dog, and rhesus monkey after intra-
venous and oral administration.
Intravenous (IV) administration[a]
Oral (PO) administration[b]
Species
Dose
[mgkgꢀ1
Cl
Vdss
[Lkgꢀ1
t1/2
[h]
Dose
[mgkgꢀ1
AUC Cmax
] [mm·h] [mm] [%]
F
]
[mLminꢀ1 kgꢀ1
]
]
Rat
Dog
Rhesus
2
1
2
39
16
12
0.8
1.4
0.8
0.3
1.0
0.8
5
3
5
2.5
3.4
2.2
1.5 54
1.0 48
0.9 16
[a] Vehicle=1:1:1 N,N-dimethylacetamide (DMA)/PEG 200/H2O for dog (n=3) and
rhesus (n=3); DMSO for rat (n=2); [b] Dosed as the free base in 20% d-a-tocopherol
polyethylene glycol 1000 succinate (vitamin E–TPGS); n=3 for all species.
(ECoG) and electromyogram (EMG) activities for polysomno-
graphic analysis. Figure 4 illustrates the time course of respon-
ses to compound 3 in radiotelemetry-implanted mice, rats,
dogs, and rhesus monkeys following oral administration during
doses in rodent were not the minimally efficacious doses
tested, but representative of the efficacy observed.[33]
In dog, efficacy was achieved at a decresed AUC (0.5 mm·h)
and Cmax (0.3 mm) compared with rodent, and the Cmax,u value
Figure 3. Plots of occupancy versus plasma, brain, and cerebrospinal fluid (CSF) in transgenic rats for compound 3.
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based on functional FLIPR po-
tencies and Cmax,u values within
the experiments. From our previ-
ous DORA efforts, suvorexant
demonstrated sleep efficacy in
dog at a 3 mgkgꢀ1 oral dose
with an AUC value of 4.0 mm·h,
a Cmax value of 0.82 mm, and
a Cmax,u value of approximately
8 nm.[13b] Rhesus monkey efficacy
was achieved with suvorexant at
a 10 mgkgꢀ1 oral dose with an
AUC value of 8.8 mm·h, a Cmax
value of 1.44 mm, and a Cmax,u
value of approximately 14 nm.
These data suggest that in
higher order species (dog and
rhesus), sleep efficacy similar to
DORAs can be achieved from se-
lective antagonism of OX2R.
Figure 4. Effects of compound 3 on sleep parameters across mouse, rat, canine, and non-human primate.[a] Sleep-
promoting responses to compound 3 in mice (n=14), rats (n=5), dogs (n=6), and rhesus monkeys (n=6) follow-
ing active-phase treatment with the indicated dosages. Mean time spent in each sleep stage during 30 min inter-
vals is plotted for both vehicle (*) and compound 3 (*) dosed orally in either 20% vitamin E–TPGS (mouse, rat,
rhesus monkeys) or 10 mm sodium citrate (canine) relative to Zeitgeber time (ZT), where ZT 0:00 refers to lights
on for all species. Dose times are indicated by a bold arrow. Treatments were administered in a crossover design
such that each subject received drug and vehicle (5 days consecutive treatment for mouse and canine; 7 days for
rat; 1 day for monkeys). Mean time in vigilance state in 30 min analysis intervals during vehicle and compound
treated conditions were averaged over all days of treatment are compared using a linear mixed effects model for
repeated measures t test, where significant responses are indicated by grey lines where tick marks indicate signifi-
cance level (short, medium, long, p <0.05, 0.01, 0.001).
Based on the in vitro and in
vivo efficacy data described
above and the assumption that
oxidative metabolism is the
dominant excretion mode, com-
pound 3 was predicted to be
a low-clearance compound (1–
4 mLminꢀ1 kgꢀ1) in humans with
an appropriate half-life (2–16 h)
to support insomnia treatment.
Compound 3 was highly selec-
was also decreased at 0.09 mm. Sleep effects in rhesus monkey
were observed at similar total concentrations compared with
dog and the Cmax,u value of 0.13 mm. These results could be
due to diminished Pgp susceptibility compared with rodent
(see above), or a delayed Tmax (in rhesus) giving rise to more
coverage over the course of the experiment. It should be
noted that hPPB (97.4%), binding potency (Ki =0.5 nm), and
Pgp susceptibility (human BA/AB=2.4; rhesus BA/AB=5.5 at
0.5 mm) most closely align with the rhesus data. Therefore,
human efficacy might most closely align with the rhesus data.
In all of these experiments, high levels of OX2R occupancy are
expected with very low-to-moderate antagonism of OX1R
tive over a range of targets in a Panlabs screen (165 assays)
with only two activities of note: guinea pig adenosine receptor
activity (Ki =2.5 mm) and human histamine 1 activity (Ki =
2.7 mm). These data represent a greater than 5000-fold margin
over the OX2R human binding activity for compound 3. Com-
pound
3 is a non-hygroscopic, modestly water soluble
(0.015 mgmLꢀ1 at neutral pH), moderate molecular weight
(462 gmolꢀ1), crystalline material that is well tolerated in pre-
clinical safety studies in a five-day rat toxicology study, achiev-
ing an AUC value of 973 mm·h and a Cmax value of 71 mm
(750 mgkgꢀ1 dose). No treatment-related changes in organ
weight, serum biochemistry or histomorphology were noted in
this study. Cardiovascular studies in conscious dogs
(170 mgkgꢀ1, p.o.) revealed no conduction changes
(PR, QRS, QT/QTc) and no effects on heart rate and
Table 5. Exposure data and binding potencies from sleep studies across species.
blood pressure. Compound 3 was also not genotoxic
Species
Dose[a]
AUC
[mm·h]
Tmax
[h]
Cmax
[mm]
PPB[b]
[%]
Cmax,u
[mm]
Ki [nm][c]
in preclinical studies. Based on the data presented
above, compound 3 was approved for further devel-
opment and designated MK-1064. Additional preclini-
cal and clinical studies concerning this compound
will be reported in due course.
[mgkgꢀ1
]
OX2R
OX1R
Mouse
Rat
Dog
100
20
1
33.5
38
0.5
8.6
2.0
4.7
0.7
3.6
10.1
7.2
0.3
80.3
81.1
71.4
93.6
2.0
1.3
0.09
0.13
1.0
0.8
0.8
0.6
1559
1069
1300
815
Rhesus
10
2.0
[a] For vehicle details, see the footnotes of Table 4; [b] Species-specific plasma protein
binding (PPB); data is the % bound, and values were determined by standard meth-
ods; see the Supporting Information for details; [c] Ki values were determined using
a radioligand-displacement binding assay; each value is the mean of at least two or
more independent determinations; for assay details, see Ref. [13a].
Conclusions
We have described the development of 5’’-chloro-N-
[(5,6-dimethoxypyridin-2-yl)methyl]-2,2’:5’,3’’-terpyri-
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in 20% vitamin E–TPGS and dosed at 20 mgkgꢀ1 in a dose volume
of 1.67 mLkgꢀ1 (p.o.). Rats were treated for 7 days of consecutive
vehicle and compound treatments during the active (dark) phase
7 h prior to lights off (ZT 17:00) in a balanced crossover design
(2 days of baseline (all); 2 days vehicle run in (all); 7 days vehicle or
compound treatment arm; 7 days compound or vehicle treatment
arm).
dine-3’-carboxamide (MK-1064), one of the most potent and
selective 2-SORA compounds reported to date. This effort re-
quired the resolution of numerous issues, including reversible
CYP3A4 inhibition, challenging physiochemical properties, cen-
tral nervous system (CNS) penetration, and bioactivation po-
tential relative to the initial lead. MK-1064 demonstrated effica-
cy in preclinical sleep models across species and was well tol-
erated in preclinical safety studies. MK-1064 represents a novel
2-SORA ligand with a desirable profile for the treatment of clin-
ical sleep disorders.
In adult male beagles (~12 kg; Marshall BioResources, North Rose,
NY, USA), compound 3 was combined with excipient, hydroxyprop-
yl methylcellulose acetate succinate (HPMCAS-HG) and spray-dried
before resuspension in 10 mm sodium citrate (pH 5.5) vehicle and
dosed (p.o.) at 1 mgkgꢀ1 (dose volume: 5 mLkgꢀ1). Sleep respons-
es to compound 3 relative to vehicle were averaged from five con-
secutive days of treatment during the active phase 9 h prior to
lights off (ZT 03:00) in a block-repeated measures design: all sub-
jects received vehicle for 5 days followed by a 2 day vehicle treat-
ment washout followed by five consecutive days of compound
treatment.
Experimental Section
Biology
Ethical statement: All animal studies were performed according to
the US National Research Council’s Guide for the Care and Use of
Laboratory Animals (http://www.nap.edu/catalog.php?record_id=
12910), and experimental protocols were reviewed by the Merck
Animal Care and Use Committee. All efforts were made to mini-
mize animal use and suffering.
In adult male rhesus monkeys (~11 kg; Macaca mulatta), responses
to 10 mgkgꢀ1 doses of compound 3 were measured following
treatment with either vehicle (20% vitamin E TPGS) or compound
in a dose volume of 10 mLkgꢀ1 (p.o.). Monkeys were dosed during
their active (light) phase 6.5 h prior to lights off (ZT 05:30) in
a one-day block crossover design: one day vehicle (all); one day
compound (all). Compound and control conditions were separately
averaged, and the mean difference in sleep stages relative to vehi-
cle was determined for the 2 h following treatment.
Radioligand binding assay: This assay was performed in an identical
manner as described in Ref. [13a]. For non-human species assays,
membranes that express the mouse, rat, dog, or rhesus monkey
orexin 2 receptor (OX2R) and the Ile408-Val variant of the orexin 1
receptor (hOX1R) were prepared in CHO cells according to the
method described by Kunapuli et al.[34]
FLIPR, bioactivation, and rat occupancy assays: These assays were
performed in an identical manner as described in Ref. [13a].
Chemistry
General comments: All solvents used were commercially available
anhydrous grade, and reagents were utilized without purification
unless otherwise noted. Nonaqueous reactions were carried out in
oven- or heat-gun-dried glassware under a nitrogen atmosphere.
Magnetic stirring was used to agitate the reactions, which were
monitored for completion by either thin-layer chromatography
(TLC) using silica gel 60 plates (Merck KGaA) or LC/MS. A Smith-
Creator microwave from Personal Chemistry was used for micro-
wave heating, and a CombiFlash system utilizing RediSep cartridg-
es by Teledyne Isco was utilized for silica gel column chromatogra-
phy with fraction collection at 254 nm. Reverse-phase HPLC purifi-
cation was carried out on a Waters HPLC (XBridge Prep C18 5 mm
19ꢁ150 mm column) utilizing a gradient of 0.1% trifluoroacetic
acid (TFA) in water/CH3CN with sample collection triggered by pho-
todiode array detection. The reported yields are for isolated com-
pounds of ꢁ95% purity, unless otherwise noted; reactions were
not extensively optimized. Purity analysis was carried out by HPLC
with a Waters 2690 separations module equipped with a YMC Pro
50ꢁ3 mm I.D. C-18 column interfaced with a Waters Micromass
ZMD spectrometer utilizing a gradient of 0.05% TFA in water/
Sleep assays in mice, rats, canines and nonhuman primates: Sleep
was quantitated by polysomnography in mice and rats subcutane-
ously implanted with radio-telemetric physiologic monitors (Data
Sciences International, Arden Hills, MN, USA) to simultaneously
record continuous electrocorticogram (ECoG) and electromyogram
(EMG) activities as previously described (Ref. [13b] and Ref. [14b]).
In telemetry-implanted dogs and rhesus monkeys, polysomnogra-
phy was performed via ECoG, EMG and electrooculoram (EOG) as
described in Ref. [13b] and Ref. [11], respectively. Sleep scoring and
the difference relative to vehicle in time spent in active wake, REM
sleep, and slow wave sleep (SWS) (mice, rats) or SWS I and SWS II
(dogs, rhesus) during the 2 h following compound treatment has
been detailed elsewhere (Ref. [13b] and Ref. [35]). All species were
acclimated to 12:12 light/dark cycles prior to the initiation of stud-
ies.
In adult male C57/BL6 wild-type mice (~30 grams; Taconic Farms,
Germantown, NY, USA), the current studies evaluated compound 3
(free base form) at 100 mgkgꢀ1 in a dose volume of 6.67 mLkgꢀ1
(p.o.) formulated in 20% d-a-tocopherol polyethylene glycol 1000
succinate (vitamin E–TPGS). Compound and vehicle were adminis-
tered during the active (dark) phase 4 h prior to lights on (Zeitgeb-
er time [ZT] 20:00, where ZT 00:00 is lights on), for 5 days in a bal-
anced crossover design (5 days of vehicle or compound; 2 day
washout; 5 days of compound or vehicle, where groups were re-
versed relative to the first arm of the crossover). Compound and
vehicle conditions for each animal were combined and averaged
over a 24 h time scale before determination of compound-induced
effects relative to vehicle during the 2 h immediately following
treatment.
1
CH3CN with UV detection at 215 and/or 254 nm. H and 13C NMR
spectra were recorded on a Varian INOVA 400, 500 or 600 MHz
spectrometer, and all chemical shifts (d) are referenced to an inter-
nal standard of tetramethylsilane and the CDCl3 residual solvent
peak, respectively, unless otherwise noted. High-resolving power
accurate mass measurement electrospray (ES) and atmospheric
pressure chemical ionization (APCI) mass spectral data were ac-
quired by use of a Bruker Daltonics 7T Fourier transform ion cyclo-
tron resonance mass spectrometer (FT-ICR MS).
2-Chloro-N-(3,4-dimethoxybenzyl)-5-(3,5-dimethylphenyl)pyri-
dine-3-carboxamide (6): To a solution of methyl 2-chloro-5-iodo-
pyridine-3-carboxylate (5, 3.00 g, 10.1 mmol) in DMF (34 mL)/water
In adult male Sprague–Dawley rats (~600 g; Taconic Farms, Ger-
mantown, NY, USA), compound 3 (free base form) was formulated
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(1.8 mL) was added 3,5-dimethylphenylboronic acid (1.97 g,
13.1 mmol), PdCl2(dppf) (1.11 g, 1.51 mmol), and Cs2CO3 (8.21 g,
25.2 mmol), and the reaction was stirred at 258C for 18 h. The reac-
tion mixture was partitioned between EtOAc (150 mL) and water
(150 mL). The organic phase was washed with water (2ꢁ100 mL)
and brine (2ꢁ100 mL), dried over MgSO4, filtered, and concentrat-
ed in vacuo. The residue was purified by column chromatography
(0!25% EtOAc/hexane) to afford methyl 2-chloro-5-(3,5-dimethyl-
0.097 mmol) and morpholine (85 mg, 0.97 mmol) in DMF (490 mL)
was heated to 1408C in a microwave reactor for 15 min. The reac-
tion was partitioned between EtOAc (10 mL) and water (10 mL).
The organic phase was washed with water (3ꢁ10 mL), dried over
MgSO4, filtered and concentrated in vacuo. The reaction was puri-
fied by column chromatography (5!75% EtOAc/hexane) to afford
11 as a white solid (26 mg, 58%): 1H NMR (400 MHz, CDCl3): d=
9.06–8.98 (m, 1H), 8.58 (dd, J=9.6, 2.4 Hz, 2H), 7.22 (s, 2H), 7.03 (s,
1H), 6.98–6.92 (m, 2H), 6.86 (d, J=8.4 Hz, 1H), 4.60 (d, J=5.6 Hz,
2H), 3.89 (s, 3H), 3.88 (s, 3H), 3.62–3.54 (m, 4H), 3.20–3.12 (m, 4H),
2.38 ppm (s, 6H); 13C NMR (100 MHz, CDCl3): d=165.5, 159.1, 149.6,
149.0, 148.0, 138.9, 138.8, 136.8, 133.0, 130.9, 129.8, 124.9, 121.2,
120.8, 111.8, 111.5, 66.8, 56.2, 51.8, 43.9, 21.6 ppm; HRMS-ESI: m/z
[M+H]+ calcd for C27H31N3O4: 462.2387, found: 462.2374.
phenyl)pyridine-3-carboxylate as
a white solid (2.32 g, 83%):
1H NMR (500 MHz, CDCl3): d=8.69 (d, J=2.5 Hz, 1H), 8.32 (d, J=
2.5 Hz, 1H), 7.18 (s, 2H), 7.09 (s, 1H), 3.99 (s, 3H), 2.40 ppm (s, 6H);
13C NMR (100 MHz, CDCl3): d=165.3, 150.1, 148.6, 139.2, 138.7,
136.0, 135.5, 130.7, 126.6, 125.1, 53.1, 21.5 ppm; MS (ESI+): m/z=
276.0 [M+H]+.
To a solution of 2-chloro-5-(3,5-dimethylphenyl)pyridine-3-carboxyl-
ate (1.80 g, 6.53 mmol) in THF (33 mL) at 258C was added KOSiMe3
(1.26 g, 9.79 mmol). The reaction was stirred at RT until completion
was reached after 24 h and Et2O (25 mL) was added to fully precipi-
tate the product. The reaction was filtered to afford potassium 2-
chloro-5-(3,5-dimethylphenyl)pyridine-3-carboxylate as an off-white
solid (1.96 g, 100%), which was used in subsequent reactions with-
out further purification.
N-(3,4-Dimethoxybenzyl)-5-(3,5-dimethylphenyl)-2,3’-bipyridine-
3-carboxamide (7): A solution of 6 (50 mg, 0.12 mmol), 3-(4,4,5,5-
tetramethyl-1,3,2-dioxaboralan-2-yl)pyridine (37 mg, 0.18 mmol),
Cs2CO3 (99 mg, 0.30 mmol), and PdCl2(dppf) (13 mg, 0.018 mmol)
in DMF (600 mL)/water (20 mL) was heated to 1608C in a microwave
reactor for 20 min. The reaction was partitioned between EtOAc
(10 mL) and water (10 mL). The organic phase was washed with
water (3ꢁ10 mL), dried over MgSO4, filtered and concentrated in
vacuo. The reaction was purified by column chromatography (0!
20% MeOH/EtOAc) to afford 7 as a white solid (34 mg, 62%):
1H NMR (400 MHz, CDCl3): d=8.91 (d, J=2.4 Hz, 1H), 8.84 (d, J=
2.0 Hz, 1H), 8.53 (dd, J=5.2, 1.6 Hz, 1H), 8.14 (d, J=2.4 Hz, 1H),
7.94 (dt, J=8.0, 2.0 Hz, 1H), 7.26–7.20 (m, 3H), 7.09 (s, 1H), 6.75 (d,
J=8.0 Hz, 1H), 6.68–6.60 (m, 2H), 6.15 (t, J=5.2 Hz, 1H), 4.39 (d,
J=5.6 Hz, 2H), 3.85 (s, 3H), 3.82 (s, 3H), 2.41 ppm (s, 6H); 13C NMR
(100 MHz, CDCl3): d=168.1, 151.3, 149.9, 149.4, 148.9, 139.1, 136.4,
136.3, 136.0, 135.2, 134.6, 131.9, 130.6, 129.7, 125.1, 123.3, 120.7,
111.7, 111.4, 56.2, 56.1, 44.5, 21.6 ppm; HRMS-ESI: m/z [M+H]+
calcd for C28H27N3O3: 454.2125, found: 454.2138.
To a suspension of potassium 2-chloro-5-(3,5-dimethylphenyl)pyri-
dine-3-carboxylate (500 mg, 1.67 mmol) in DMF (8.3 mL) was
added 3,4-dimethoxybenzylamine (363 mg, 2.17 mmol), EDC
(480 mg, 2.50 mmol), HOAt (341 mg, 2.50 mmol), and Et3N
(506 mg, 5.0 mmol), and the reaction was heated to 458C for 3 h.
The reaction mixture was cooled and partitioned between EtOAc
(50 mL) and saturated aq NaHCO3 (50 mL). The organic phase was
washed with saturated aq NaHCO3 (2ꢁ50 mL), water (2ꢁ50 mL),
and brine (2ꢁ50 mL), dried over MgSO4, filtered and concentrated
in vacuo. The residue was purified by column chromatography
(0!80% EtOAc/hexane) to afford 6 as a white solid (422 mg,
1
62%): H NMR (500 MHz, CDCl3): d=8.64 (d, J=2.5 Hz, 1H), 8.31 (d,
N-(3,4-Dimethoxybenzyl)-5-(3,5-dimethylphenyl)-2,4’-bipyridine-
3-carboxamide (8): A solution of 6 (50 mg, 0.12 mmol), 4-(4,4,5,5-
tetramethyl-1,3,2-dioxaboralan-2-yl)pyridine (37 mg, 0.18 mmol),
Cs2CO3 (99 mg, 0.30 mmol), and PdCl2(dppf) (13 mg, 0.018 mmol)
in DMF (600 mL)/water (20 mL) was heated to 1608C in a microwave
reactor for 20 min. The reaction was partitioned between EtOAc
(10 mL) and water (10 mL). The organic phase was washed with
water (3ꢁ10 mL), dried over MgSO4, filtered and concentrated in
vacuo. The reaction was purified by column chromatography (0!
20% MeOH/EtOAc) to afford 8 as a white solid (11 mg, 20%):
1H NMR (500 MHz, CDCl3): d=8.96 (d, J=2.0 Hz, 1H), 8.64 (d, J=
6.0 Hz, 2H), 8.14 (d, J=2.5 Hz, 1H), 7.61 (d, J=4.5 Hz, 2H), 7.25 (s,
2H), 7.10 (s, 1H), 6.76 (d, J=8.5 Hz, 1H), 6.67 (d, J=2.0 Hz, 1H),
6.58 (dd, J=8.5, 2.5 Hz, 1H), 5.78 (m, 1H), 4.39 (d, J=5.5 Hz, 2H),
3.86 (s, 3H), 3.82 (s, 3H), 2.41 ppm (s, 6H); 13C NMR (100 MHz,
CDCl3): d=168.0, 151.6, 150.3, 149.5, 149.4, 149.0, 146.3, 139.2,
136.7, 136.2, 135.3, 132.0, 130.8, 129.4, 125.2, 123.5, 120.6, 111.6,
111.5, 56.2, 56.1, 44.7, 21.6 ppm; HRMS-ESI: m/z [M+H]+ calcd for
C28H27N3O3: 454.2125, found: 454.2120.
J=2.0 Hz, 1H), 7.19 (s, 2H), 7.08 (s, 1H), 6.96–6.92 (m, 2H), 6.86 (d,
J=9.0 Hz, 1H), 4.64 (d, J=5.5 Hz, 2H), 3.90 (s, 3H), 3.89 (s, 3H),
2.39 ppm (s, 6H); 13C NMR (100 MHz, CDCl3): d=164.7, 149.5, 149.2,
148.9, 145.5, 139.2, 138.3, 136.7, 135.5, 130.8, 130.7, 130.0, 125.1,
120.4, 111.5, 111.4, 56.2, 56.1, 44.5, 21.5 ppm; MS (ESI+): m/z=
411.0 [M+H]+.
N-(3,4-Dimethoxybenzyl)-5-(3,5-dimethylphenyl)-2,2’-bipyridine-
3-carboxamide (9): A solution of 6 (50 mg, 0.12 mmol), Pd(PPh3)4
(14 mg, 0.012 mmol), CsF (55 mg, 0.36 mmol), CuI (4.6 mg,
0.024 mmol),
and
2-(tri-n-butylstannyl)pyridine
(100 mg,
0.24 mmol) was made in DMF (600 mL), and the reaction was
heated to 1608C in a microwave reactor for 20 min. The reaction
was partitioned between EtOAc (10 mL) and water (10 mL). The or-
ganic phase was washed with water (3ꢁ10 mL), dried over MgSO4,
filtered and concentrated in vacuo. The reaction was purified by
column chromatography (65!100% EtOAc/hexane) to afford 9 as
1
a white solid (37 mg, 67%): H NMR (400 MHz, CDCl3): d=8.91 (d,
J=2.0 Hz, 1H), 8.36 (d, J=4.8 Hz, 1H), 8.25 (d, J=2.0 Hz, 1H), 7.98
(d, J=8.0 Hz, 1H), 7.80 (dt, J=7.6, 1.6 Hz, 1H), 7.63–7.58 (m, 1H),
7.28–7.23 (m, 3H), 7.08 (s, 1H), 6.86–6.78 (m, 3H), 4.51 (d, J=
5.6 Hz, 2H), 3.88 (s, 3H), 3.81 (s, 3H), 2.40 ppm (s, 6H); 13C NMR
(100 MHz, CDCl3): d=168.5, 157.1, 152.6, 149.3, 148.7, 148.5, 148.1,
139.1, 137.2, 136.6, 136.4, 136.3, 132.2, 130.6, 130.5, 125.2, 124.4,
123.7, 120.6, 111.7, 111.4, 56.2, 56.1, 44.5, 21.6 ppm; HRMS-ESI: m/z
[M+H]+ calcd for C28H27N3O3: 454.2125, found: 454.2133.
N-(3,4-Dimethoxybenzyl)-5-(3,5-dimethylphenyl)-2-phenylpyri-
dine-3-carboxamide (10): A solution of 6 (50 mg, 0.12 mmol), phe-
nylboronic acid (44 mg, 0.36 mmol), Cs2CO3 (119 mg, 0.37 mmol),
and PdCl2(dppf) (13 mg, 0.018 mmol) in DMF (600 mL)/water (20 mL)
was heated to 1608C in a microwave reactor for 20 min. The reac-
tion was partitioned between EtOAc (10 mL) and water (10 mL).
The organic phase was washed with water (3ꢁ10 mL), dried over
MgSO4, filtered and concentrated in vacuo. The reaction was puri-
fied by column chromatography (0!75% EtOAc/hexane) to afford
10 as a white solid (12 mg, 22%): 1H NMR (400 MHz, CDCl3): d=
N-(3,4-Dimethoxybenzyl)-5-(3,5-dimethylphenyl)-2-(morpholin-4-
yl)pyridine-3-carboxamide (11):
A
solution of
6
(40 mg,
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1
8.92 (d, J=2.0 Hz, 1H), 8.22 (d, J=2.0 Hz, 1H), 7.68–7.64 (m, 2H),
7.42–7.36 (m, 3H), 7.08 (s, 1H), 6.71 (d, J=8.0 Hz, 1H), 6.55 (d, J=
2.0 Hz, 1H), 6.53 (dd, J=8.0, 2.0 Hz, 1H), 5.63 (t, J=5.6 Hz, 1H),
4.32 (d, J=5.6 Hz, 2H), 3.85 (s, 3H), 3.79 (s, 3H), 2.41 ppm (s, 6H);
13C NMR (100 MHz, CDCl3): d=168.4, 154.5, 149.3, 149.2, 148.8,
139.1, 138.9, 136.7, 135.7, 135.5, 131.1, 130.4, 129.7, 129.2, 129.1,
128.9, 125.2, 120.6, 111.5, 111.3, 56.2, 56.1, 44.6, 21.6 ppm; HRMS-
ESI: m/z [M+H]+ calcd for C29H29N2O3: 452.2173, found: 453.2174.
MeOH/EtOAc) to afford the 3 as a white solid (3.4 g, 79%): H NMR
(500 MHz, CDCl3): d=8.95 (d, J=2.5 Hz, 1H), 8.80 (s, 1H), 8.66 (d,
J=2.0 Hz, 1H), 8.38 (d, J=5.0 Hz, 1H), 8.26 (d, J=2.5 Hz, 1H), 8.04
(d, J=8.0 Hz, 1H), 7.96 (bs, 1H), 7.78 (t, J=3.0 Hz, 1H), 7.47 (bs,
1H), 7.23 (dd, J=7.5, 5.5 Hz, 1H), 7.01 (d, J=7.5 Hz, 1H), 6.85 (d,
J=7.5 Hz, 1H), 4.55 (d, J=5.5 Hz, 2H), 3.88 (s, 3H), 3.83 ppm (s,
3H); 13C NMR (100 MHz, CDCl3): d=168.0, 155.9, 154.1, 153.8,
148.6, 148.4, 148.0, 145.8, 143.6, 143.2 136.9, 136.2, 134.1, 133.6,
132.7, 132.6, 131.5, 123.9, 123.8, 117.7, 114.7, 55.9, 53.5, 44.6 ppm;
HRMS-ESI: m/z [M+H] calcd for C24H20ClN5O3: 462.1327, found:
462.1331.
Methyl 5’,6-dichloro-3,3’-bipyridine-5-carboxylate (18): To a solu-
tion of 5 (30.0 g, 101 mmol) in DMF (403 mL)/water (18 mL) was
added (5-chloropyridin-3-yl)boronic acid (16.2 g, 103 mmol), PdCl2-
(dppf) (7.38 g, 10.1 mmol), and Cs2CO3 (99.0 g, 303 mmol), and the
reaction was stirred at 258C for 18 h. The reaction mixture was par-
titioned between EtOAc (1 L) and water (1 L). The organic phase
was washed with water (2ꢁ500 mL) and brine (2ꢁ500 mL), dried
over MgSO4, filtered, and concentrated in vacuo. The residue was
purified by trituration with MeOH (~200 mL) followed by EtOAc/
Et2O (1:1, 500 mL) to afford 18 as a beige solid (28.4 g, 99%):
1H NMR (500 MHz, CDCl3): d=8.78–8.70 (m, 2H), 8.67 (d, J=2.5 Hz,
1H), 8.35 (d, J=2.5 Hz, 1H), 7.89 (t, J=2.5 Hz, 1H), 4.01 ppm (s,
3H); 13C NMR (100 MHz, CDCl3): d=164.5, 150.4, 149.7, 149.0,
145.7, 138.7, 134.1, 132.7, 132.5, 131.1, 127.0, 53.2 ppm; MS (ESI+):
m/z=282.9 [M+H]+.
2,3-Dimethoxy-6-aminomethylpyridine (21): To a solution of com-
mercially available 6-iodo-2,3-dimethoxypyridine (20, 24.0 g,
91 mmol) in DMF (181 mL) was added CuCN (9.73 g, 109 mmol),
and the reaction was heated to 1508C for 20 min in a microwave
reactor. The mixture was partitioned between water (400 mL) and
EtOAc (400 mL). The organic phase was washed with brine (3ꢁ
200 mL), dried over Na2SO4, filtered and concentrated in vacuo.
The crude material was purified by column chromatography (0!
40% EtOAc/hexane) to yield the 5,6-dimethoxypyridine-2-carboni-
trile as an off-white crystalline powder (11.7 g, 79%): MS (ESI+):
m/z=165.0 [M+H]+.
To a solution of 2,3-dimethoxy-6-cyanopyridine (5.1 g, 31.1 mmol)
in MeOH (260 mL) was added Pearlman’s catalyst (2.18 g,
3.11 mmol, 20%/w) and 12m HCl (20.0 mL, 249 mmol). The system
was then stirred under an atmosphere of hydrogen for 1.5 h at RT.
The reaction mixture was filtered through a pad of Celite, and
MeOH was removed in vacuo. The crude mixture was basified
using saturated aq Na2CO3 (100 mL) and then extracted with
CHCl3/EtOH (4:1, 4ꢁ200 mL). The organic phase was washed with
brine (2ꢁ50 mL), dried over Na2SO4, filtered and concentrated in
vacuo to yield the 21 as a bone-colored semisolid (4.8 g, 94%):
1H NMR (500 MHz, CDCl3): d=7.00 (d, J=8.0 Hz, 1H), 6.76 (d, J=
8.0 Hz, 1H), 4.04 (s, 3H), 3.87 (s, 3H), 3.81 ppm (s, 2H); 13C NMR
(100 MHz, CDCl3): d=154.1, 14.8, 142.8, 117.8, 113.5, 56.0, 53.7,
47.0 ppm; MS (ESIꢀ): m/z=152.1 [MꢀNH2]ꢀ.
2,3-Dimethoxy-5-aminomethylpyrazine (23): To commercially
available 5-bromo-2,3-dimethoxypyrazine (22, 1.66 g, 12.1 mmol) in
DMF (25 mL) was added CuCN (1.20 g, 13.4 mmol), and the mixture
was heated to 1858C for 20 min in the microwave reactor. The mix-
ture was cooled, and partitioned between EtOAc (50 mL) and
water (50 mL). The organic phase was washed with brine (2ꢁ
50 mL), dried over Na2SO4, filtered and concentrated in vacuo. The
crude material was purified by column chromatography (0!65%
EtOAc/hexane) to afford 5,6-dimethoxypyrazine-2-carbonitrile
(1.17 g, 58%).
Methyl 5’’-chloro-2,2’:5’,3’’-terpyridine-3’-carboxylate (19): To
a solution of 18 (7.6 g, 26.8 mmol) in DMF (107 mL) was added 2-
tri-n-butylstannylpyridine (13.8 g, 37.6 mmol), CsF (12.2 g,
81.0 mmol), CuI (1.0 g, 5.4 mmol, 0.2 equiv), and Pd(PPh3)4 (3.1 g,
2.7 mmol), and the system was heated to 808C for 2 h. The reac-
tion mixture was cooled, diluted with EtOAc (300 mL), and filtered
through a pad of Celite. The reaction mixture was washed with
water (3ꢁ100 mL) and brine (1ꢁ100 mL), dried over MgSO4, fil-
tered and concentrated. The residue was purified by column chro-
matography (10!100% EtOAc/hexane), followed by normal phase
chiral chromatography (Chiracel OD, 10 cm column; hexane/iPrOH
[1:1], flow rate=150 mLminꢀ1) to afford 19 as a white solid (4.5 g,
1
52%): H NMR (400 MHz, CDCl3): d=8.95 (d, J=2.0 Hz, 1H), 8.80 (d,
J=2.0 Hz, 1H), 8.66 (d, J=3.0 Hz, 1H), 8.65–8.61 (m, 1H), 8.23 (d,
J=8.0 Hz, 1H), 8.13 (d, J=2.0 Hz, 1H), 7.95 (t, J=2.5 Hz, 1H), 7.87
(td, J=7.6, 1.6 Hz, 1H), 7.34 (ddd, J=7.6, 4.8, 1.2 Hz, 1H), 3.85 ppm
(s, 3H); 13C NMR (100 MHz, CDCl3): d=169.1, 155.3, 155.2, 148.7,
148.6, 148.3, 145.8, 136.9, 135.3, 134.1, 133.5, 132.7, 131.2, 128.9,
124.0, 122.8, 52.6 ppm; MS (ESI+): m/z=326.0 [M+H]+.
5’’-Chloro-N-[(5,6-dimethoxypyridin-2-yl)methyl]-2,2’:5’,3’’-terpyr-
idine-3’-carboxamide (3): To a solution of 19 (3.05 g, 9.36 mmol)
in THF/MeOH (22 mL/4.5 mL) was added 10n aq NaOH (1.12 mL,
11.2 mmol), and the system was heated to 1358C for 15 min in
a microwave reactor. The reaction mixture was cooled, concentrat-
ed, and azeotroped with EtOAc (2ꢁ100 mL) and toluene (3ꢁ
100 mL) to afford sodium 5’’-chloro-2,2’:5’,3’’-terpyridine-3’-carbox-
ylate as a white solid (3.12 g, quant.): MS (ESI+): m/z=311.9
[M+H]+.
To 5,6-dimethoxypyrazine-2-carbonitrile (1.17 g, 7.1 mmol) in MeOH
(59 mL) was added Pearlman’s catalyst (0.50 g, 0.71 mmol, 20%/w)
and 12m HCl (4.65 mL, 57 mmol). The system was then stirred
under an atmosphere of hydrogen for 1.5 h at RT. The reaction
contents were filtered through a pad of Celite, followed by remov-
al of MeOH in vacuo. The crude mixture was then dissolved in
CH2Cl2 (100 mL), basified using saturated aq Na2CO3 (50 mL), and
then extracted several times with CH2Cl2 (4ꢁ100 mL). The organic
phase was washed with brine (2ꢁ50 mL), dried over Na2SO4, fil-
tered and concentrated in vacuo to yield 23 as a bone-colored
To a suspension of sodium 5’’-chloro-2,2’:5’,3’’-terpyridine-3’-car-
boxylate (3.12 g, 9.35 mmol) in DMF (47 mL) was added 1-(5,6-di-
methoxy-pyridin-2-yl)methanamine (21, 1.65 g, 9.82 mmol), EDC
(3.58 g, 18.7 mmol), HOAt (2.55 g, 18.7 mmol), and DIPEA (4.83 g,
37.4 mmol), and the system was heated to 608C for 2 h. The reac-
tion mixture was cooled and diluted with EtOAc (300 mL). The re-
action mixture was washed with saturated aq NaHCO3 (2ꢁ100 mL),
water (2ꢁ100 mL) and brine (1ꢁ100 mL), dried over MgSO4, fil-
tered and concentrated in vacuo. The residue was purified by
column chromatography (10!100% EtOAc/hexane then 0!23%
1
solid (1.1 g, 92%): H NMR (500 MHz, CDCl3): d=7.56 (s, 1H), 4.05
(s, 3H), 4.02 (s, 3H), 3.81 ppm (s, 2H); 13C NMR (100 MHz, CDCl3):
d=150.2, 149.6, 144.9, 128.7, 54.1, 53.9, 44.6 ppm; MS (ESIꢀ):
m/z=152.8 [MꢀNH2]ꢀ.
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1-(5,6-Dimethoxypyridin-3-yl)methanamine (25): To a solution of
commercially available 2,3-dimthoxypyridine (24, 2.5 g, 18.0 mmol)
in CH2Cl2/saturated aq NaHCO3 (80 mL/40 mL) at 08C was added
Br2 (0.93 mL, 18.0 mmol), and the reaction mixture was stirred for
2 h at 258C. The reaction mixture was quenched with solid Na2SO3
(~10 g), and the aqueous phase was extracted with CH2Cl2 (3ꢁ
100 mL). The combined organic phase was dried over MgSO4, fil-
tered and concentrated in vacuo. The residue was purified by
column chromatography (0!20% EtOAc/hexane) to afford 5-
bromo-2,3-dimethoxypyridine as an oil after concentration (2.1 g,
49%): MS (ESI+): m/z=218.0 [M+H]+.
Keywords: antagonists · insomnia · medicinal chemistry ·
neurotransmitters · orexin receptors
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a solution of 5,6-dimethoxypyridine-3-carbonitrile (50 mg,
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Supporting Information
Experimental procedures for the synthesis of compounds 12–17,
as well as reproductions of the 1H and 13C NMR spectra for key
compounds can be found in the Supporting Information available
on the WWW under http://dx.doi.org/10.1002/cmdc.201300447.
Acknowledgements
The authors would like to thank Rodney A. Bednar, Duane R.
Reiss, Kathy L. Murphy, and Broc Flores for technical support
toward the publication of this manuscript, Jacquelyn Binns, Alan
Savitz, Spencer Tye, and Scott M. Doran for polysomnography
support, and Ashleigh Bone, Jim Destefano, Tami Montgomery,
and Dr. Colena Johnson for surgical support. The authors also
thank Dr. Chuck Ross and Ms. Joan Murphy for HRMS analysis,
and Thomas S. Reger for helpful discussions regarding this manu-
script.
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Received: November 4, 2013
Published online on December 27, 2013
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