Design and Synthesis of Non-Peptide, Selective Orexin Receptor 2 Agonists

Article abstract of DOI:10.1021/acs.jmedchem.5b00988

Orexins are a family of neuropeptides that regulate sleep/wakefulness, acting on two G-protein-coupled receptors, orexin receptors 1 (OX1R) and 2 (OX2R). Genetic and pharmacologic evidence suggests that orexin receptor agonists, especially OX2R agonist, will be useful for mechanistic therapy of the sleep disorder narcolepsy/cataplexy. We herein report the discovery of a potent (EC₅₀ on OX2R is 0.023 μM) and OX2R-selective (OX1R/OX2R EC₅₀ ratio is 70) agonist, 4′-methoxy-N,N-dimethyl-3′-[N-(3-{[2-(3-methylbenzamido)ethyl]amino}phenyl)sulfamoyl]-(1,1′-biphenyl)-3-carboxamide 26.

Full text of DOI:10.1021/acs.jmedchem.5b00988

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Brief Article
Design and Synthesis of Non-Peptide, Selective Orexin Receptor 2 Agonists
Takashi Nagahara, Tsuyoshi Saitoh, Noriki Kutsumura, Yoko Irukayama-Tomobe, Yasuhiro Ogawa, Daisuke
Kuroda, Hiroaki Gouda, Hidetoshi Kumagai, Hideaki Fujii, Masashi Yanagisawa, and Hiroshi Nagase
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00988 • Publication Date (Web): 12 Aug 2015
Downloaded from http://pubs.acs.org on August 14, 2015
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Journal of Medicinal Chemistry
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Design and Synthesis of Non-Peptide, Selective Orexin Receptor 2
Agonists
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‡
†
†
†
Takashi Nagahara, Tsuyoshi Saitoh, Noriki Kutsumura, Yoko IrukayamaꢀTomobe, Yasuhiro
ǁ
†
§
§
†,
‡
Ogawa, Daisuke Kuroda, Hiroaki Gouda, Hidetoshi Kumagai, Hideaki Fujii, Masashi Yanꢀ
agisawa, Hiroshi Nagase*
ǁ
†
,
,†,‡
†International Institute for Integrative Sleep Medicine (WPIꢀIIIS), University of Tsukuba, 1ꢀ1ꢀ1 Tennodai, Tsukuꢀ
ba, Ibaraki 305ꢀ8575, Japan
‡Department of Medicinal Chemistry, School of Pharmacy, Kitasato University, 5ꢀ9ꢀ1 Shirokane, Minatoꢀku, Toꢀ
kyo 108ꢀ8641, Japan
§
School of Pharmacy, Showa University, 1ꢀ5ꢀ8 Hatanodai, Shinagawaꢀku, Tokyo 142ꢀ8555, Japan
ǁ
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390ꢀ8584,
USA
ABSTRACT: Orexins are a family of neuropeptides that regulate sleep/wakefulness, acting on two G proteinꢀcoupled
receptors, orexin receptorsꢀ1 (OX1R) and ꢀ2 (OX2R). Genetic and pharmacologic evidence suggests that orexin receptor
agonists, especially OX2R agonist, will be useful for mechanistic therapy of the sleep disorder narcolepsy/cataplexy. We
herein report the discovery of a potent (EC50 on OX2R = 0.023 ꢀM) and OX2Rꢀselective (OX1R/OX2R EC50 ratio = 70)
agonist, 4’ꢀmethoxyꢀN,Nꢀdimethylꢀ3’ꢀ[Nꢀ(3ꢀ{[2ꢀ(3ꢀmethylbenzamido)ethyl]amino}phenyl)sulfamoyl]ꢀ(1,1’ꢀbiphenyl)ꢀ3ꢀ
carboxamide 26.
7
Introduction
lepsy phenotype. Moreover, OX1Rꢀnull mice exhibit no
appreciable sleep/wakefulnessꢀrelated phenotype,8 sugꢀ
Orexins (orexin A and B, also known as hypocretin 1 and
gesting that OX2R, rather than OX1R, plays a predomiꢀ
nant role in sleep/wake regulation, and an intact OX2Rꢀ
mediated signaling is sufficient to prevent the symptoms
of narcolepsy/cataplexy. The importance of OX2R sigꢀ
naling in sleep/wakefulness regulation has been also
demonstrated pharmacologically by the finding that
OX2Rꢀselective or OX1R/OX2R dual antagonists induce
sleep, whereas OX1Rꢀselective antagonists are largely
devoid of effects on sleep.9
2
) are a pair of lateral hypothalamic neuropeptides origꢀ
inally identified as the endogenous ligands for two (then
orphan) G proteinꢀcoupled receptors, orexin receptorsꢀ1
(OX1R) and ꢀ2 (OX2R). They are derived from the single
1
precursor peptide preproꢀorexin (also termed preproꢀ
hypocretin), of which the mRNA had also been identified
by a differential cloning approach as a transcript highly
enriched in the hypothalamus.2,3 Orexin A,
a Cꢀ
terminally amidated 33ꢀresidue peptide with two intraꢀ
molecular disulfide bridges and an Nꢀterminal pyrogluꢀ
tamate residue, shows similar high potency for both
OX1R and OX2R, whereas orexin B, a 28ꢀaminoꢀacid, Cꢀ
terminally amidated linear peptide, exhibits a ≈10ꢀfold
selectivity for OX2R over OX1R.
A vast majority (>90%) of human patients with narcoꢀ
lepsy/cataplexy has been shown to lack detectable levels
of orexin peptides in their cerebrospinal fluid.1 Orexin
deficiency in narcolepsy patients is due to a highly selecꢀ
0,11
tive loss of orexinꢀproducing neurons in the lateral hypoꢀ
12,13
thalamus,
which is likely caused by an autoimmune
An essential role of the orexin system in the regulation
of sleep and wakefulness was initially demonstrated by
the discoveries that OX2Rꢀdeficient dogs and preproꢀ
14
mechanism. These findings indicate that human narcoꢀ
lepsy/cataplexy is essentially an orexinꢀdeficiency synꢀ
drome, and imply that a replacement of orexin signaling
(especially OX2R signaling) can be a mechanistic theraꢀ
py for the disorder. Indeed, genetic or pharmacologic
replacement of orexin peptides effectively ameliorates
the narcolepsy/cataplexy phenotype of a mouse model in
which orexin neurons are genetically and postnatally
ablated, providing a proof of concept for orexin replaceꢀ
ment therapy.1 However, since orexin peptides do not
penetrate the bloodꢀbrain barrier efficiently,1 it would
be difficult to use the neuropeptides themselves as a
therapeutic drug.
4
5
orexin knockout mice both exhibit symptoms highly
similar to the sleep disorder narcolepsy/cataplexy. Narꢀ
colepsy/cataplexy is a chronic neurological disorder
characterized by nonꢀrapid eye movement (REM) sleepꢀ
related symptoms such as excessive daytime sleepiness
and “sleep attacks”, as well as REM sleepꢀrelated signs
including cataplexy (sudden bilateral loss of muscle tone
often caused by a strong emotion), hypnagogic hallucinaꢀ
5ꢀ17
8,19
6
tion and sleep paralysis. Whereas OX1R/OX2R double
null mice exhibit a severe narcoleptic phenotype indisꢀ
tinguishable from that seen in preproꢀorexin knockout
mice, OX2Rꢀnull mice show a somewhat milder narcoꢀ
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We expect that OX2R receptor agonists would be useꢀ pound 8 with the methoxy group was improved to 0.800
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ful not only for mechanistic drug therapy of narcolepꢀ
sy/cataplexy, but also for other pathologic conditions
with excessive sleepiness, since it is well known that
orexins exert potent wakeꢀinducing effects upon central
ꢀM, meanwhile the introductions of the ester group or
the fluorine led to decreased activity (the compounds 9
and 14). Interestingly, the introduction of the amide
groups remarkably increased both potency and efficacy,
and the 3ꢀpyridyl group also increased the activity (the
compounds 10–12 and 15).
15,20,21
administration.
Although the role of endogenous
2
2
orexin pathways is unclear in narcolepsy without cataꢀ
plexy (typeꢀ2 narcolepsy), OX2R agonists might be useꢀ
ful in this condition as well. In addition, OX2R agonists
may potentially be useful in the prevention and treatꢀ
ment of obesity and metabolic syndrome, since it has
recently been reported that genetic or pharmacologic
enhancement of OX2R signaling has a netꢀnegative efꢀ
fects on body weight homeostasis and prevents high fat
dietꢀinduced obesity in mice.23 Indeed, it has also been
reported that human patients with narcolepsy have highꢀ
er body mass index and are more prone to metabolic
Table 1. Structure activity relationships between
the substituent Ar on the A-ring and calcium flux
activity mediated by OX2R in vitro assay.
0
1
2
3
4
5
6
7
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9
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0
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0
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0
1
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7
8
9
0
Calcium influxa
Compounds
EC50 (ꢀM)
E
max (%)
48b
2
4,25
syndrome than ageꢀmatched control individuals.
7
8
> 10
Motivated by these observations, we have initiated an
effort to discover and optimize smallꢀmolecule, nonꢀ
peptide orexin receptor agonists. We have conducted a
highꢀthroughput screen of a library of ~250,000 drugꢀ
like compounds for OX2R agonist activity, and started a
systematic medicinal chemistry effort based on the strucꢀ
tural motifs of initial “hit” compounds from the screen.
Here we report design and synthesis of novel potent,
highly OX2Rꢀselective agonists through such efforts.
0.800
> 10
54c
b
9
49
98
74
c
10
11
0.182
0.050
c
1
2
0.235
85b
Results and Discussion
73b
47b
88b
We commenced with the screening of the universal
chemical library.26 We paid attention to the sulfonamide
group of the resulting hit compound 1, bearing two aroꢀ
matic rings (Aꢀring and Bꢀring) connected to each other
by a linker moiety at a certain length (Figure 1). We conꢀ
verted the compound 1 into the tertiary sulfonamide deꢀ
rivatives, termed type I compounds as shown in Figure 1.
However, none of the obtained derivatives showed any
activity, which led us to synthesize the secondary sulfonꢀ
amides 2–6, termed as type II compounds. As a result,
the compounds 2–4 showed weak OX2R agonistic activiꢀ
ty in NFATꢀluciferase assay but were not effective in the
calcium influx assay. These results suggested that the
proton on the secondary sulfonamide group might play a
role to afford agonistic activity. In addition, we found
that the conversion of the 1,2ꢀphenylenediamine moiety
in the compound 4 into 1,3ꢀ and 1,4ꢀphenylenediamine
moieties (the compounds 5 and 6) led to noticeable imꢀ
provement in activities. When we changed the isopropyl
group on the Aꢀring of more active derivative 5 into the
phenyl group and removed the methyl group, the obꢀ
tained compound 7 showed higher activity (NFATꢀ
Luciferase activity: 41% (Efficacy at 10 ꢀM), calcium inꢀ
13
14
15
> 1.0
> 10
0.268
a
The value is the average of n = 5. Emax expressed as a perꢀ
centage of OXA maximum. Efficacy at 10 ꢀM. Efficacy at 1
M.
b
c
ꢀ
Next, we focused on the compound 11 with the dimethyl
amide group, which showed the highest activity in Table
, and attempted to change the Bꢀring of 11. The synthetꢀ
1
ic method of those compounds 11 and 22–28 is shown
in Scheme 1. First, 3ꢀfluoronitrobenzene 16 was easily
transformed into the compound 17 through the three
steps (i. treatment with ethylenediamine; ii. protection of
the primary amine by a Boc group; iii. protection of the
secondary nitrogen by a benzyl group). After the iron
reduction of nitro group in 17, the obtained aniline deꢀ
rivative was treated with sulfonyl chloride 18 to afford
the sulfonamide 19. Then, the Suzuki–Miyaura coupling
of 19 with 3ꢀmethoxycarbonylphenyl boronic acid pinaꢀ
col ester and the following removal of the benzyl group
afforded the corresponding biaryl compound 20. Hyꢀ
drolysis of the methyl ester, followed by amidation with
dimethylamine hydrochloride provided the compound
flux: 48% (Efficacy at 10 ꢀM)) as shown in Table 1. Finalꢀ
ly, after examination of the position of the substituent R1
on the aryl group (Ar) in the compound 7, we found that
substituent at the 3ꢀposition favored OX2R agonistic
activity relative to the 2ꢀ or 4ꢀpositions. We then examꢀ
ined the introduction of other effective functional
2
1. Finally, the removal of Boc group, followed by amiꢀ
dation of 21 with a variety of benzoic acid derivatives
using BOP reagent afforded the corresponding comꢀ
pounds 11 and 22–28 (for experimental section, see
supporting information).
1
groups, shown as R , into the 3ꢀposition on the aryl
group (Table 1, for experimental section, see supporting
information). As a result, the efficacies for OX2R in both
NFATꢀLuciferase activity and calcium influx were further
increased in many cases. The EC50 value of the comꢀ
Scheme 1. Synthesis of the compounds 11 and 22–
2
8.
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OMe
2
2
H
> 10
N.D.b
74
N.T.c
5.227
9.248
N.T.c
N.C.d
105
Bn
N
18
1
2
3
4
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7
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9
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6
O2N
F
a, b, c
O2N
Br
2
SO Cl
NHBoc
11
2ꢀMeO 0.050
3ꢀMeO 0.033
4ꢀMeO 1.047
46
49
d, e
2
2
2
2
3
4
5
6
54
280
16
17
94
N.C.d
OMe
H
N
OMe
Bn
f, g
H
H
N.T.c
N.C.d
2ꢀMe
3ꢀMe
2ꢀCl
0.800
0.023
0.031
0.094
85
N
MeO2C
N
N
Br
S
NHBoc
S
NHBoc
O2
O2
98
1.616
100
50
70
198
60
19
20
j, k
27
90
78
6.134
5.720
OMe
h, i
H
H
28
3ꢀCl
73
Me2NOC
N
N
S
NHBoc
O2
aThe value is the average of n = 5. OXA EC50 (OX1R) = 1.5
nM. EC50 (OX2R) = 1.0 nM. Emax expressed as a percentage
0
1
2
3
4
5
6
7
8
9
0
1
2
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7
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9
0
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0
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0
1
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3
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7
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9
0
21
OMe
2: R2 = Hꢀ
11: R2 = 2ꢀMeOꢀ 26: R2 = 3ꢀMeꢀ
25: R2 = 2ꢀMeꢀ
2
of OXA maximum. N.D. = not detected. N.T. = not tested.
b
c
O
H
N
H
R2
Me2NOC
N
d
S
N
3: R2 = 3ꢀMeOꢀ 27: R2 = 2ꢀClꢀ
4: R2 = 4ꢀMeOꢀ 28: R2 = 3ꢀClꢀ
N.C. = not calculated.
2
2
O2
H
Next, we converted the sulfonamide group into the carꢀ
boxyamide group in 26 to afford the compound 29 to
examine the effect of the sulfonamide moiety to the
OX2R agonistic activity. Surprisingly, the carboxyamide
derivative 29 was inactive toward both OX1R and OX2R,
which supports our aforementioned postulation that the
sulfonamide moiety is one of the most important strucꢀ
tural determinants for the OX2R agonistic activity (Figꢀ
ure 2).
Reagents and conditions: a) ethylenediamine, 100 °C;
b) Boc O, Et N, CH Cl , rt, 79% in two steps; c) BnBr, TBAI,
0% aq. NaOH, CH Cl , rt, 78%; d) Fe powder, NH Cl,
EtOH, H O, reflux, 99%; e) 18, pyridine, CH Cl , rt, 92%; f)
ꢀmethoxycarbonylphenylboronic acid pinacol ester,
2
3
2
2
5
2
2
4
2
2
2
3
2 2 3 2
PdCl (dppf), Na CO , dioxane, H O, reflux, 87%; g) Pd/C,
H
2
, MeOH, rt, 99%; h) 1 M aq. NaOH, MeOH, 60 °C; i)
Me NH—HCl, Et N, BOP rgt., CH Cl , rt, 73% in two steps; j)
HClꢀMeOH, 50 °C; k) ArCO H, Et
2
3
2
2
2
3
N, BOP rgt., CH
2
Cl
2
, rt.
OMe
O
H
H
Me2NOC
N
N
Me
2
S
N
The introduction of the substituent R on the Bꢀring afꢀ
forded a substantial influence on both potency and effiꢀ
cacy as shown in Table 2. Removal of the methoxy group
on the Bꢀring in 11 led to a significant loss in both potenꢀ
cy and efficacy against OX2R (Compound 22: EC50 = >
10 ꢀM in OX2R). Changing the position of the methoxy
group from the 2ꢀ to the 3ꢀposition on the Bꢀring slightly
increased the potency and selectivity but not the efficacy
O
O
H
26
OX2R: EC50 = 0.023 ꢁM, Emax = 98%
OX1R: EC50 = 1.616 ꢁM, Emax = 100%
OMe
H
N
O
H
Me2NOC
N
Me
N
H
O
29
OX2R: not active
OX1R: not active
(
Compound 23: EC50 = 0.033 ꢀM, Emax = 54% in OX2R).
Figure 2. Structures of the sulfonylamide derivative 26
and the carbonylamide derivative 29.
On the other hand, substitution of the methoxy group at
the 4ꢀposition led to a greater than 20ꢀfold decrease in
potency (Compound 24: EC50 = 1.047 ꢀM, Emax = 94% in
OX2R). Introduction of a methyl group to the 2ꢀposition
provided a decrease in potency (Compound 25: EC50
.800 ꢀM, Emax = 85% in OX2R), while a methyl group at
the 3ꢀposition increased both potency and efficacy
(Compound 26: EC50 = 0.023 ꢀM, Emax = 98% in OX2R).
Introduction of a chlorine to the 2ꢀ or 3ꢀpositions proꢀ
vided almost the same results as that of the methoxy
group (Compounds 27 and 28). The compounds 11, 23,
6, 27, and 28 showed not only potent activity, but also
high selectivity for OX2R over OX1R (Table 2). In CHO
cells overexpressing hOX1R and HEKꢀ293 cells overexꢀ
Recently, the Xꢀray crystal structure of the human OX2R
bound to
vorexant
a
dual orexin receptor antagonist, suꢀ
was solved at 2.5 Å. Using this Xꢀray
2
7,28
29
=
0
structure, the binding mode of 26 with OX2R was examꢀ
ined by molecularꢀdocking calculations (see supporting
information). The resulting binding mode of 26 (Figure
3) suggested that the dimethyl carbamoyl group on 26
was located deep in the ligandꢀbinding site of OX2R, and
its carbonyl group formed a hydrogen bond with N324
(transmebrane hexix 5, TM5). The compound 26 also
used a nitrogen atom of the ethylenediamine moiety to
form an additional hydrogen bond with the mainꢀchain
carbonyl group of C210 (TM3). The biphenyl group of 26
was located in the hydrophobic pocket consisting of V138
(TM3), F227 (TM5), Y317 (TM6), I320 (TM6), and V353
(TM7) to make hydrophobic interactions. The sulfonylꢀ
amide group of 26 was in proximity to T111 (TM2), H350
(TM7), and Y354 (TM7) of OX2R (see supporting inforꢀ
mation’s Figure 2). Interestingly, 26 directed two oxꢀ
ygens of sulfonylamide group to hydroxyl groups of T111
(TM2) and Y354 (TM7) and imidazole ring of H350
(TM7), respectively. The sulfonylamide group of 26 may
form hydrogen bonds in the structural changes associatꢀ
ed with receptor activation. This may explain that the
carboxyamide derivative 29 was inactive, because the
direction of oxygen was different between carboxyamide
and sulfonylamide groups. T111 (TM2) of OX2R is muꢀ
tated to Ser in OX1R. This mutation might be related to
2
1
25
pressing hOX2R, compound 26 displaced [ I]orexinꢀA
in a concentration dependent manner: 26 bound to
hOX2R and hOX1R with K = 0.14 ꢁM and 0.77 ꢁM, reꢀ
i
spectively.
Table 2. Structure activity relationships between
the substituent R2 on the B-ring and calcium flux
activity mediated by OX2R in vitro assay.
Calcium influxa
Comꢀ
pounds
OX2R
OX1R
Selectivity
R2
EC50
ꢀM)
E
max
EC50
(ꢀM)
E
(%)
max
OX1R EC50
OX2R EC50
/
(
(%)
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high selectivity for OX2R of compounds in Table 2. The
Page 4 of 7
Figure 4. Superposition of suvorexant (green) and the
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
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3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
benzene groups in the middle and terminal portions of
26 also formed hydrophobic interactions with V114
(TM2), W120 (ECL1), and I130 (TM3) and with Y343
(TM7) and F346 (TM7). The neighborhood of the Bꢀring
of the compound 26 was given in supporting inforꢀ
mation’s Figure 3. This also may explain that compounds
compound 26 (purple) in the ligandꢀbinding site of OX2R.
In Table 2, the most potent compound was 3ꢀmethyl deꢀ
rivative 26. Meanwhile, the most selective compound for
OX2R was 3ꢀmethoxy derivative 23. However, the comꢀ
pounds 11 and 22–28 in Table 2 were barely soluble in
water, which led to difficulty in confirming in vivo activiꢀ
ty, although every compound showed satisfactory activiꢀ
ties and potencies in vitro. Therefore, we introduced the
23, 26, and 28 with a substituent group at the 3ꢀ
position possessed potent activity, because we could see
large space in the direction of 3ꢀposition to accommoꢀ
date the substituent group. Figure 4 compares the bindꢀ
ing modes of suvorexant and 26 with OX2R. As reported
2
ꢀdimethylamino group on the Bꢀring to give more polar
derivative 30 (Figure 5), and then converted 30 into
dihydrochloride salt 31, which could be dissolved in saꢀ
line at 1.3 M. To evaluate the pharmacological effect of
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29
in the literature, suvorexant adopted a πꢀstacked
horseshoeꢀlike conformation to direct its 7ꢀmembered
ring to TM5 and TM6 of OX2R. This binding mode was
believed to be responsible for the OX2R antagonist activꢀ
ity of suvorexant, as the 7ꢀmembered ring seems to inꢀ
hibit inward movements of TM5 and TM6 relative to the
rest of the TM bundle, which may be a general trigger for
GPCR activation. In contrast, 26 binds to OX2R with an
extended conformation, and we detected some spatial
allowance between 26 and TM5 and TM6 of OX2R.
Therefore, we could consider that the binding of 26 to
OX2R may induce the inward movements of TM5 and
TM6, which would allow 26 to have OX2R agonist activiꢀ
ty.
3
1 in vivo, we administered it intracerebroventricularly
in the light phase and observed the effect on sleep/wake
states by recording EEG/EMG in C57BL/6J mice. 31
promoted wakefulness in a doseꢀdependent manner
(
Figure 6). 260 nmol of 31 increased wake time to 53
min, to a similar degree reported with 3.0 nmol orexinꢀA
(58 min). In contrast, 31 did not increase wakefulness
in OX1R/OX2R double knockout (DKO) mice, demonꢀ
strating that the effect of 31 requires orexin receptors as
expected. The intraperitorial injection of 31 afforded the
similar effects. The details will be reported in separate
papers.
16
Figure 5. Structures of 30 and its waterꢀsoluble dihydroꢀ
chloride salt 31.
Figure 3. Binding mode of the compound 26 with the
OX2R determined by our docking procedure. Hydrogenꢀ
bonding interactions are indicated by red dashed lines.
Figure 6. Effect of intracerebroventricular injection of 31
on sleep/wake states in wildꢀtype and OX1R/OX2R DKO
mice. Cumulative time spent in wake within 2 h after saline
(
sal.) or 31 administration at 6 h into the light phase (ZT6).
*, P < 0.05; **, P <0.001 saline vs. 31 in wildꢀtype mice.
n.s.: not significant.
Conclusions
We paid attention to the sulfonamide group of hit comꢀ
pound 1 and synthesized more than 1000 compounds.
Those compounds were tested against a CHO cell line
expressing the human orexin receptor using the NFATꢀ
luciferase assay and the calcium influx assay. We found
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Journal of Medicinal Chemistry
General Synthetic Procedure for the Compounds
the first lead compound 5, then focused on the transforꢀ
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
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2
2
2
2
2
2
2
2
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3
3
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3
4
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4
4
5
5
5
5
5
5
5
5
5
5
6
mation of the Aꢀring and the Bꢀring in 5. As a result, the
first, potent and selective agonist 26 for OX2R was disꢀ
covered. The conversion from the sulfonamide group in
26 to the carbonylamide group and the evaluation of the
obtained 29 suggested that the sulfonamide moiety is
one of the most important structural features for the
OX2R agonistic activity. In addition, we also developed
the dimethylamino derivative 30 and its dihydrochloride
salt, which could be dissolved in saline at 1.3 M. The in
vivo assay of the dihydrochloride salt showed a definite
wakeꢀpromoting effect. These results would be helpful
for further design of OXR agonists.
in Table 2.
A mixture of 21 (2.55 mmol) in 10% hydrogen chloride
methanol solution (10 mL) was stirred at 50 °C for 2 h.
The reaction mixture was concentrated in vacuo, and
then the residue was purified by amineꢀsilica gel column
chromatography
(ammoniaꢀsaturated
chloroꢀ
form/methanol = 100/0 to 95/5) to afford the desired
primary amine. Then, a mixture of the primary amine
(1.0 equiv), the benzoic acid derivative (1.0 equiv), trimeꢀ
thylamine (3.3 equiv), and BOP reagent (1.1 equiv) in
dichloromethane (0.5 M) was stirred at room temperaꢀ
ture for 12 h under argon atmosphere. The mixture was
quenched with saturated aq. sodium hydrogen carꢀ
bonate, and extracted with chloroform. The extract was
0
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3
4
5
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7
8
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0
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2
3
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8
9
0
1
2
3
4
5
6
7
8
9
0
Experimental Section
Synthesis.
dried over Na
2
SO and concentrated in vacuo. The resiꢀ
4
Methyl
3’-
{N-[3-({2-[(tert-
due was purified by silica gel column chromatography
(ethyl acetate) to give the desired compound.
butoxycarbon-
yl)amino]ethyl}amino)phenyl]sulfamoyl}-4'-
methoxy-(1,1'-biphenyl)-3-carboxylate (20).
mixture of 19 (2.36 g, 4.00 mmol), 3ꢀ
4’-Metoxy-N,N-dimethyl-3’-[N-(3-{[2-(3-
methylbenzami-
do)ethyl]amino}phenyl)sulfamoyl]-(1,1’-
biphenyl)-3-carboxamide (26).
A
methoxycarbonylphenylboronic acid pinacol ester (1.26
g, 4.80 mmol), 2.0 M aq. sodium carbonate (4.0 mL,
Yield: 77% in two steps; colorless solid; MP 137–139 °C;
8.00 mol), and 1,1’ꢀbis(diphenylphosphino)ferroceneꢀ
ꢀ
1
1
IR (KBr) 3408, 2919, 1605, 1502, 1281, 1151 cm ; H
NMR (400 MHz, CDCl ) δ = 2.34 (3H, s), 3.02 (3H, brs),
.13 (3H, brs), 3.19–3.22 (2H, m), 3.58–3.63 (2H, m),
.04 (3H, s), 4.47 (1H, brs), 6.22 (1H, dd, J = 8.0, 2.0
palladium(II)dichloride dichloromethane complex (163
mg, 0.20 mmol) in dioxane (4.0 mL) was stirred under
argon atmosphere at 100 °C for 18 h. After being diluted
with ethyl acetate, the insoluble material was filtrated
through a pad of Celite. The filtrate was concentrated in
vacuo, and then the residue was purified by silica gel
column chromatography (hexane/ethyl acetate = 67/33
to 50/50) to give the desired coupling product (2.25 g,
3
3
4
Hz), 6.32 (1H, dd, J = 8.4, 2.0 Hz), 6.45 (1H, t, J = 2.0
Hz), 6.88 (1H, brs), 6.92 (1H, t, J = 8.0 Hz), 7.04 (1H, d,
J = 8.4 Hz), 7.13–7.18 (1H, m), 7.21–7.34 (3H, m), 7.42
(
1H, t, J = 8.0 Hz), 7.50–7.58 (4H, m), 7.68 (1H, dd, J =
8.4, 2.4 Hz), 8.12 (1H, d, J = 2.4 Hz); 13C NMR (100
MHz, CDCl ) δ = 21.3, 35.4, 39.3, 39.6, 44.9, 56.6, 103.7,
87%) as a yellow amorphous powder. A mixture of the
3
compound (2.25 g, 3.48 mmol) in methanol (20 mL) was
stirred in the presence of catalytic 10% Pd/C at room
temperature for 5 h under hydrogen pressure (balloon
pressure). After filtration through a pad of Celite, the
filtrate was concentrated in vacuo. The residure was puꢀ
rified by silica gel column chromatography (chloroꢀ
form/methanol = 90/10) to give 20 (1.94 g, 99%) as a
pale yellow amorphous powder.
109.2, 110.3, 112.7, 124.1, 125.6, 125.7, 126.5, 127.7, 127.8,
128.3, 128.8, 129.6, 129.8, 132.2, 132.7, 133.0, 134.1,
1
37.0, 137.7, 138.2, 139.4, 149.1, 155.9, 168.8, 171.3;
+
HRMSꢀESI: m/z [M + Na] calcd for C32
34
H N
4
O
5
SNa:
6
09.2148; found: 609.2134. Purity was > 99% as asꢀ
sessed by HPLC (254 nm) and NMR.
ASSOCIATED CONTENT
Supporting Information.
General information and detailed experimental procedures,
synthetic protocols, and chemical data. The Supporting Inꢀ
formation is available free of charge via the Internet at
http://pubs.acs.org.”
tert-Butyl
{2-[(3-{[3’-(dimthylcarbamoyl)-4-
methoxy-(1,1’-biphenyl)]-3-
sulfonamido}phenyl)amino]ethyl}carbamate
(21).
To a solution of 20 (1.94 g, 3.49 mmol) in methanol (30
mL) was added 1.0 M aq. sodium hydroxide (10.5 mL,
1
0.5 mmol) and the mixture was stirred at 60 °C for 3 h.
AUTHOR INFORMATION
Corresponding Author
After being neutralized by 1.0 M aq. hydrochloric acid,
the mixture was extracted with chloroform/methanol (=
1
0/1), washed with brine, dried over Na
2
SO
4
, and conꢀ
*Phone: +81ꢀ29ꢀ853ꢀ6437.
Eꢀmail: nagase.hiroshi.gt@u.tsukuba.ac.jp
centrated in vacuo. The residual colorless amorphous
powder was dissolved in dichloromethane (30 mL), and
then triethylamine (1.59 mL, 11.4 mmol), dimethylamine
hydrochloride (533 mg, 6.54 mmol), and BOP reagent
ACKNOWLEDGMENT
This work was supported by JSPS Funding Program for
WorldꢀLeading Innovative R&D on Science and Technology
(M.Y. and H.N.), JSPS KAKENHI (GrantꢀinꢀAid for Young
Scientists (B)) Grant Number 15K16557 (T.S.), and JSPS
KAKENHI (GrantꢀinꢀAid for Scientific Research (C)) Grant
Number 15K07900 (H.G.). IIIS is supported by the World
Premier International Research Center (WPI) initiative,
Japan.
(
1.59 g, 3.60 mmol) were added. The mixture was stirred
for 12 h at room temperature under argon atmosphere.
The reaction mixture was quenched with saturated aq.
sodium hydrogen carbonate, and extracted with chloroꢀ
form. The extract was dried over Na
2
SO and concentratꢀ
4
ed in vacuo. The residue was purified by silica gel colꢀ
umn chromatography (hexane/ethyl acetate = 5/95 to
0/100) to give the desired compound (1.45 g, 73% in two
ABBREVIATIONS
steps) as a colorless amorphous.
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Journal of Medicinal Chemistry
Page 6 of 7
OX1R, orexin receptorꢀ1; OX2R, orexin receptorꢀ2; REM,
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rapid eye movement; NFAT, nuclear factor of activated Tꢀ
cells; Boc, tertꢀbutoxycarbonyl; BOP, [(benzotriazolꢀ1ꢀ
yloxy)ꢀtris(dimethylamino)phosphonium hexafluorophosꢀ
phate; MP, melting point.
(
7
(
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a
Figure 1. Drug design approach from the hit compound 1 to the lead compound 5. NFATꢀLuciferase activity mediated by
b
2+
c
OX2R (Efficacy at 5 ꢀM). Ca influx mediated by OX2R (Efficacy at 5 ꢀM). NFATꢀLuciferase activity mediated by OX2R
d
2+
(Efficacy at 10 ꢀM). Ca influx mediated by OX2R (Efficacy at 10 ꢀM).
Insert Table of Contents artwork here
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