Diaryl urea analogues of SB-334867 as orexin-1 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2011.03.048

As a part of our program to develop OX1-CB1 bivalent ligands, we required a better understanding of the basic structure-activity relationships (SARs) of orexin antagonists. A series of SB-334867 analogues were synthesized and evaluated in calcium mobilization assays. SAR results suggest that the 2-methylbenzoxazole moiety may be replaced with a disubstituted 4-aminophenyl group without loss of activity and an electron-deficient system is generally preferred at the 1,5-naphthyridine moiety for OX1 antagonist activity. In particular, substitution of larger potential linkers such as n-hexyl provided compound 33 with equivalent activity at the OX1 receptor compared to the lead compound SB-334867. These compounds should be of value in the development of ligands targeting the orexin-1 receptor and its potential heterodimers.

Full text of DOI:10.1016/j.bmcl.2011.03.048

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2980–2985
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Diaryl urea analogues of SB-334867 as orexin-1 receptor antagonists
⇑
⇑
David A. Perrey, Brian P. Gilmour, Scott P. Runyon, Brian F. Thomas , Yanan Zhang
Research Triangle Institute, 3040 Cornwallis Road, Research Triangle Park, NC 27709, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
As a part of our program to develop OX1–CB1 bivalent ligands, we required a better understanding of the
basic structure–activity relationships (SARs) of orexin antagonists. A series of SB-334867 analogues were
synthesized and evaluated in calcium mobilization assays. SAR results suggest that the 2-methylbenzox-
azole moiety may be replaced with a disubstituted 4-aminophenyl group without loss of activity and an
electron-deficient system is generally preferred at the 1,5-naphthyridine moiety for OX1 antagonist
activity. In particular, substitution of larger potential linkers such as n-hexyl provided compound 33 with
equivalent activity at the OX1 receptor compared to the lead compound SB-334867. These compounds
should be of value in the development of ligands targeting the orexin-1 receptor and its potential
heterodimers.
Received 27 January 2011
Revised 11 March 2011
Accepted 14 March 2011
Available online 21 March 2011
Keywords:
Orexin
Antagonist
SB-334867
Ó 2011 Elsevier Ltd. All rights reserved.
Structure–activity relationships
Orexin-A and -B (also known as hypocretins 1 and 2) are two
hypothalamic neuropeptides that were independently discovered
by two groups in 1998.1,2 Orexin-A (33 amino acids) and orexin-B
(28 amino acids) are highly conserved across mammalian species
and are derived by proteolytic cleavage from a common 130-amino
acid precursor produced in the hypothalamus named prepro-orex-
in. Orexin-A and -B are the endogenous ligands for two G protein-
coupled receptors (GPCRs), orexin 1 (OX1R) and orexin 2 (OX2R).
Orexin-A is equipotent at both receptors, whereas orexin-B displays
moderate selectivity for the OX2R.2 Orexin-expressing neurons are
located predominantly in a small area in the hypothalamus and lo-
cus coeruleus.2–5 However, the nerve fibers of orexin neurons pro-
ject throughout the central nervous system (CNS), suggesting that
orexins have multiple CNS functions.3,6–8 In fact, the orexin system
has already been shown to modulate a variety of important biolog-
ical processes, including sleep/wake cycles,9,10 feeding,2 drug addic-
tion and reward,11–13 as well as energy homeostasis.2
A significant body of evidence indicates that many GPCRs can
form heterodimers or oligomers, and these heterodimers/oligo-
mers often display unique binding, distinct phenotypic trafficking,
and altered signaling properties than their individual mono-
mers.14–16 In particular, OX1R has an overlapping pattern of distri-
bution with the cannabinoid receptor 1 (CB1) receptor in a number
of brain regions including the lateral hippocampus.17 In addition,
the OX1R has been shown to form heterodimers in vitro with the
CB1 receptor, as demonstrated by co-expression, co-immunopre-
cipitation and resonance energy transfer studies.18 Biochemical,
pharmacological and functional evidence also suggests a crosstalk
between OX1R and CB1R in a Chinese Hamster Ovary (CHO) heter-
ologous expression system, where potency of direct activation of
OX1R to activate the mitogen-activated protein kinase pathway
was affected by the CB1 receptor.19 Evidence supporting the
in vivo relevance of OX1–CB1 dimerization includes the observa-
tion that in pre-fed rats, pretreatment with subeffective doses of
the CB1 antagonist SR141716 attenuates the orexigenic actions of
orexin A.20
Considering both the physiological functions of the orexin sys-
tem and the potential to modulate OX1–CB1 heterodimers with
small molecules we have pursued a program to develop bivalent li-
gands,21 which feature an OX1R antagonist and a CB1 antagonist
linked together by a spacer. Although several structural classes of
orexin antagonists have been developed,22–26 there is little struc-
ture–activity data available that can be used to develop bivalent li-
gands. Therefore, identifying a region of bulk tolerance without
altering OXR activity is critical. SB-334867 (1), developed by
GlaxoSmithKline (GSK), was the first selective OX1 antagonist de-
scribed (Fig 1).27 It has ꢀ50-fold higher affinity for OX1R than for
OX2R and was selective for OXR over more than 50 GPCRs and
ion channels.27 GSK later reported other OX1 selective antagonists,
including SB-408124 (2), SB-410220 (3) and SB-674042 (4)
(Fig. 1);28 however, SB-334867 remains the most widely studied
OX1 antagonist and represents a viable pharmacological tool for
evaluating the physiological role of the OX1R specific pathway in
vivo. As a part of our program to construct OX1–CB1 bivalent li-
gands to study GPCR heterodimerization we required a better
understanding of the basic structure–activity relationships (SARs)
of SB-334867. Here we describe the synthesis and biological eval-
uation of a series of diaryl urea analogues of SB-334867.
⇑
Corresponding authors. Tel.: +1 919 541 6552; fax: +1 919 541 6499 (B.F.T.);
tel.: +1 919 541 1235; fax: +1 919 541 6499 (Y.Z.).
2-Methylquinoline analogue (5) was synthesized following a
slightly modified literature procedure as shown in Scheme 1.29
E-mail addresses: bft@rti.org (B.F. Thomas), yzhang@rti.org (Y. Zhang).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.048

D. A. Perrey et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2980–2985
2981
NO2
N
N
O
O
O
NH2
HN
HN
a
b
F
F
HN
N
N
H
HN
N
N
H
X
R2
O
N
R1
X
N
R1
N
9-13
R3
N
1 (SB-334867)
2 (SB-408124)
NH2
R4
N
N
HN
HN
HN
N
c
O
R2
R2
O
HN
O
O
N
HN
N
N
H
X
X
N
S
F
O
R1
N
R1
N
14
15-17; 19-23; 33
F
F
H
N
R4
d
3 (SB-410220)
4 (SB-674042)
O
HN
HN
Figure 1. Structures of SB334867 and other OX1 selective antagonists.
R2
O
X
2-Methylbenzoxazole acid (8) was obtained in three steps from
commercially available methyl 3-hydroxy-4-nitrobenzoate. Reduc-
tion of the nitro group gave 6 followed by condensation with ethyl
acetimidate to form benzoxazole ring 7, and subsequent ester
hydrolysis provided 8. Curtius rearrangement of 8 using diphenyl-
phosphoryl azide formed an in situ isocyanate, which was treated
with 2-methyl-4-aminoquinoline to afford the desired urea 5.
Compounds 9–23 and 33 were synthesized by combining the req-
uisite 4-aminoquinoline derivative with the appropriate 4-nitro-
phenylisocyanate (9–13). The resulting 4-nitro compounds were
reduced using 10% palladium on carbon and subjected to reductive
alkylation (15–17, 19–23, 33) or BOP-mediated amidation (18)
(Scheme 2). Similarly, 24–28, 30–32 and 34–37 were prepared by
condensation of the appropriate 4-aminoquinoline with the arylis-
ocyanate. Formation of thiourea 26 required sodium hydride
activation of the aminoquinoline followed by condensation with
4-dimethylaminophenylisothiocyanate at ꢁ20 °C. Amide 29 was
prepared by BOP coupling of 8-fluoro-2-methyl-4-aminoquinoline
with 4-dimethylaminophenylacetic acid. Compounds 38 and 39
were obtained through formation of the phenyl carbamate of ami-
noquinoline (40) followed by displacement of the phenoxide with
the appropriate amine (Scheme 3). All target compounds were fully
characterized by NMR and mass spectroscopy,30 and then tested in
a calcium mobilization based functional assay.31
N
R1
18
Scheme 2. Reagents and conditions: (a) 4-NO2-Ph-NCO, toluene, 80 °C; (b) H2
(40 psi), Pd/C, EtOH; (c) aldehyde, Na(OAc)3BH, 1,2-DCE; (d) acid, BOP, Et3N, THF.
tively, with other aromatic systems (Table 1). The naphthyridine in
SB-334867 was first replaced with 2-methylquinoline (5), which
led to a modest reduction in activity (12-fold). Since 2-methyl-4-
aminoquinoline 5 had reasonable activity and was commercially
available, this group was employed as the core scaffold for evaluat-
ing substitutions on the 2-methylbenzoxazole moiety. The 4-nitro
derivatives (9–13) were inactive as antagonists at OX1R. Aniline
analogue 14 was also inactive under the test conditions in both
OX1 and OX2 assays. When 14 was dimethylated (15), activity
was restored albeit 7-fold weaker than SB-334867. Introduction
of an ortho methyl (16) or methoxy (17) group to 15 significantly
decreased potency. Monoalkylation, acylation or dialkylation with
long chain alkyl groups (18–22) resulted in markedly reduced
activity. Finally, replacing the 2-methylbenzoxazole ring in SB-
334867 with a 4-dimethylaminophenyl group (23) restored the
activity and selectivity for the OX1R, which is consistent with pre-
vious data for SB-408124 and SB-410220.28
The primary goal of this study was to develop SAR surrounding
the SB-334867 scaffold that could be used to identify suitable sites
to attach linkers for bivalent ligand development. Since little SAR is
known regarding this class of ligands at OX receptors we broad-
ened our scope to evaluate multiple positions and substitutions.
Initial attempts to develop SAR around SB-334867 included replac-
ing the naphthyridine and the 2-methylbenzoxazole rings, respec-
Since the 4-dimethylaminophenyl group retained comparable
activity as the 2-methylbenzoxazole (5 vs 15) this moiety was used
to further probe the SAR of the naphthyridine ring (Table 2). Con-
sidering electron rich 2-methylquinoline had reduced activity (5),
electron deficient aromatic rings were explored. Introduction of
an 8-fluoro group to quinoline 24 or 2-methylquinoline 25 pro-
vided analogues with excellent potency, as well as selectivity for
H2N
N
O2N
HO
b
a
O
CO2Me
HO
CO2Me
CO2Me
6
7
N
O
O
N
H
N
O
HN
N
c
d
CO2H
8
5
Scheme 1. Reagents and conditions: (a) H2, Pd/C, EtOH; (b) ethyl acetimidate hydrochloride, EtOH; (c) 2 N NaOH, MeOH; (d) 2-methyl-4-aminoquinoline, PO(OPh)2N3, Et3N,
toluene, DMF.

2982
D. A. Perrey et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2980–2985
R2
HN
NH2
N
HN
X
a
R1
R1
N
24-28; 30-32; 34-37
R2
O
Ph
O
HN
HN
NH2
N
c
b
HN
O
R1
R1
R1
N
N
40
38-39
Scheme 3. Reagents and conditions: (a) arylisocyanate, toluene, 80 °C or arylisothiocyanate, NaH, DMF, ꢁ20 °C; (b) PhOCOCl, Et3N, CH2Cl2; (c) R2-NH2, Et3N, THF.
Table 1
Activity of quinoline urea analogues against OX1R and OX2R
R3
N
NO2
N
O
R4
O
O
O
HN
N
N
H
HN
N
N
HN
N
N
H
R1
X
R1
X
R2
R2
Me
5
9-13
14-23
No
R1
R2
R3
R4
X
Ke (nM)
OX1a
OX2b
1
45 12
1194
5
9
682.6 231
>10,000
>10,000
>10,000
>10,000
>10,000
Nt
Nt
Nt
Nt
H
H
Cl
Me
OMe
H
H
NO2
NO2
NO2
NO2
NO2
H
Me
Me
Me
Hexanoyl
3-Ph-propyl
3-Ph-propyl
Hexyl
Hexyl
Me
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
>10,000
>10,000
Nt
H
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
H
Me
Me
Me
H
H
Me
H
356.7 89
2561 266
3945 2517
>10,000
3278 639
>10,000
Me
OMe
H
H
H
H
H
H
>10,000
398 16
Me
Me
46.3
8
Nt = not tested.
a
Values are the mean of at least three independent experiments in duplicate.
Values are the mean of at least two independent experiments in duplicate.
b
the OX1R. The corresponding thiourea 26 was significantly less ac-
tive suggesting a steric limitation exists at this region. In addition,
the position of the trifluoromethyl substituent was critical, as
shown by the loss of activity in 27 compared to 28. Finally, removal
of the urea as shown in the corresponding amide provided ana-
logue 29 with no antagonist activity, confirming the importance
of the urea group for optimal potency.
Having identified an 8-fluoroquinoline core with improved
activity, the effects of substitution on the 2-methylbenzoxazole
ring was re-examined (Table 3). While the 4-dimethylaminophenyl
derivative (24) showed the best activity, its conformationally con-
strained analogue indoline 32 had almost identical potency. The
piperidine derivative (30) was 100-fold less potent than 24 and
activity was completely abolished when N-methylpiperazine (31)
was substituted for the dimethylamino group, indicating that cyc-
lic hydrophobic structures at this position are not well tolerated.
When a longer n-hexyl chain was substituted for methyl on the
aniline nitrogen (33), a reduction in potency compared to 24 was
seen but it retained comparable activity with SB-334867, suggest-
ing flexibility in the hydrophobic chain on the aniline nitrogen is
critical for potent activity. This finding was significant since com-
pound 33 contained a potential site for linker attachment for biva-
lent ligands. Favorable antagonist activity was also observed for
oxygen-containing substituents. Although the dimethoxy analogue
(36) was an weak antagonist and the 3,4,5-trimethoxy derivative
(37) was inactive, constraining the two oxygens as in the five or
six-membered rings of 34 and 35 gave good activity. The simple
4-ethyl analogue (38) showed surprisingly good activity. Finally,
cyclohexyl derivative (39) was completely inactive.
All target compounds showed a strong selectivity for OX1R over
OX2R, with the majority showing no activity at OX2R and those
that did show activity on OX2 possessed at least a 30-fold selectiv-

D. A. Perrey et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2980–2985
2983
Table 2
Activity of 4-dimethylaminophenyl quinoline ureas against OX1R and OX2R
N
X
HN
Y
R1
N
R2
No
R1
R2
X
Y
Ke (nM)
OX1a
OX2b
24
25
26
27
28
29
8-F
8-F
8-F
6-CF3
8-CF3
8-F
H
Me
H
H
H
O
O
S
O
O
O
NH
NH
NH
NH
NH
2.4 1.7
65 33
1115.1
>10,000
131 33
>10,000
171 25
>10,000
>10,000
>10,000
>10,000
>10,000
H
CH2
a
Values are the mean of at least three independent experiments in duplicate.
Values are the mean of at least two independent experiments in duplicate.
b
Table 3
Activity of 8-fluoroquinoline urea analogues against OX1R and OX2R
O
R
HN
N
H
N
F
No
R
Ke (nM)
OX1a
OX2b
N
24
2.4 1.7
171 25
N
30
395.6 111
>10,000
N
N
31
32
>10,000
>10,000
N
4.9
2
491 90
(continued on next page)

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D. A. Perrey et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2980–2985
Table 3 (continued)
No
R
Ke (nM)
OX1a
OX2b
N
33
64.2 12
2410 250
O
34
35
13.4
10.3
9
5
1617 50
306 30
O
O
O
O
36
37
965 419
>10,000
>10,000
O
O
O
O
>10,000
38
39
50.5 18
3199 207
>10,000
4563 1797
a
Values are the mean of at least three independent experiments in duplicate.
Values are the mean of at least two independent experiments in duplicate.
b
ity for OX1R. All compounds were also evaluated for agonist activ-
ity and none of the analogues demonstrated any appreciable ago-
Acknowledgments
nist stimulation at 10 lM.
This work was supported by National Institute on Drug Abuse,
National Institute of Health, USA (Grant No. DA026582). We thank
Ms. Tiffany Langston, Mr. Keith Warner and Ms. Allyson Smith for
their valuable technical assistance. We also thank Dr. Hernan A.
Navarro and Dr. James B. Thomas for their help in the calcium
mobilization assay development.
In conclusion, we have synthesized a series of analogues of SB-
334867 in order to define critical receptor tolerances to substitu-
tion for potential use as bivalent ligands. We have characterized
our analogues using a calcium mobilization functional assay and
identified several important structural features. SAR results sug-
gest that the 2-methylbenzoxazole moiety may be replaced with
a disubstituted 4-aminophenyl group without loss of activity and
an electron-deficient system is generally preferred at the 1,5-naph-
thyridine moiety for OX1 antagonist activity. In particular, substi-
tution of a larger n-hexyl chain for methyl provided compound
33 with roughly equal activity at the OX1 receptor compared to
the lead compound SB-334867 suggesting a region of bulk toler-
ance that can be exploited at this position. Further modification
of this scaffold is underway to optimize OX potency and develop
bivalent ligands containing OX1 receptor ligands. Future com-
pounds based on these scaffolds should be of value in the develop-
ment of ligands targeting the orexin-1 receptor and its potential
heterodimers.
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