Disubstituted piperidines as potent orexin (hypocretin) receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2012.04.122

A series of orexin receptor antagonists was synthesized based on a substituted piperidine scaffold. Through traditional medicinal chemistry structure-activity relationships (SAR), installation of various groups at the 3-6-positions of the piperidine led to modest enhancement in receptor selectivity. Compounds were profiled in vivo for plasma and brain levels in order to identify candidates suitable for efficacy in a model of drug addiction.

Full text of DOI:10.1016/j.bmcl.2012.04.122

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3890–3894
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Disubstituted piperidines as potent orexin (hypocretin) receptor antagonists
Rong Jiang, Xinyi Song, Purva Bali, Anthony Smith, Claudia Ruiz Bayona, Li Lin, Michael D. Cameron,
⇑
Patricia H. McDonald, Paul J. Kenny, Theodore M. Kamenecka
Department of Molecular Therapeutics, and Translational Research Institute, The Scripps Research Institute, Scripps Florida, 130 Scripps Way #2A1, Jupiter, FL 33458, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of orexin receptor antagonists was synthesized based on a substituted piperidine scaffold.
Through traditional medicinal chemistry structure–activity relationships (SAR), installation of various
groups at the 3–6-positions of the piperidine led to modest enhancement in receptor selectivity. Com-
pounds were profiled in vivo for plasma and brain levels in order to identify candidates suitable for effi-
cacy in a model of drug addiction.
Received 28 March 2012
Revised 26 April 2012
Accepted 27 April 2012
Available online 4 May 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Orexin
GPCR
Addiction
Orexins/hypocretins (Orexin-A and B, or hypocretin-1 and 2),
simultaneously discovered by two groups in 1998, are a pair of
hypothalamic neuropeptides with substantial amino acid identi-
ties.1,2 Though produced by a small population of neurons in the
posterior and lateral hypothalamus (LH), orexins exert multiple
functions particularly in areas related to energy homeostasis, sleep,
arousal and brain reward when binding to their respective G-pro-
tein coupled receptors OX1 and OX2 (Hcrt1 and Hcrt2 receptors,
respectively). Due to the pharmacological potential from the mod-
ulation of these receptors, a significant effort has been poured into
this area of research mostly in the area of insomnia.3–9 The most
advanced candidate Almorexant (a dual OX1–OX2 antagonist) from
Actelion/GlaxoSmithkline(GSK) for the treatment of sleep disor-
ders was dropped in late stage clinical development for safety con-
reinstatement of extinguished nicotine, alcohol and cocaine seek-
ing, and attenuates stress-induced reinstatement of extinguished
cocaine and alcohol seeking.11–15 Injection of SB-334867 directly
into the ventral tegmental area (VTA), a key brain area in drug
addiction, attenuated the rewarding effects of morphine, as mea-
sured in a conditioned place preference (CPP) procedure and also
mediated cue-induced cocaine seeking behavior.16 These data sug-
gest that orexin receptors, particularly those in the VTA, regulate
the rewarding effects of drugs of abuse and support an important
role for orexin transmission in drug-seeking and drug-taking
behaviors. Thus, blockade of OX1 receptors with OX1 selective
antagonists may provide a new mechanism and a promising ther-
apeutic treatment for a variety of addiction related disorders.
The first OX1 selective antagonist reported in the literature was
cerns. Merck is also advancing
a
dual OX1–OX2 antagonist
SB-334867 (Fig. 1,1).17,18 It has a reported OX1 IC50 = 40 nM (Ca2+
)
(Suvorexant) for sleep and is currently in PhIII.10 Both of these drug
candidates are dual OX1–OX2 antagonists with roughly equal po-
tency on each receptor.
and is >100-fold selective for OX1 versus OX2. It was developed by
GSK by modification of lead compounds from high throughput
screening and is widely used in vitro and in vivo for OX1 target
A growing body of evidence indicates that OX1 receptors may
play an important role in the behavioral adaptations associated
with chronic drug exposure that may contribute to the develop-
ment of addiction. Recently, compelling evidence has shown that
activation of OX1 in the brain plays a critical role in reward-seek-
ing, drug relapse and addiction.11 Chemical activation of LH orexin
neurons reinstates extinguished morphine seeking behavior in
rats, an effect blocked by the selective OX1 receptor antagonist
SB-334867.11 Blockade of OX1 transmission also decreases nico-
tine, and alcohol self-administration and attenuates cue-induced
N
O
HN
R
HN
O
4
5
3
2
n
H
N
H
N
N
6
Ar2
Ar2
N
X
N
X
N
Ar1
O
Ar1
O
n=0,1
1
2
3
OX1 pKb 7.4
⇑
Corresponding author.
E-mail address: kameneck@scripps.edu (T.M. Kamenecka).
Figure 1. Orexin antagonist scaffolds.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.122

R. Jiang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3890–3894
3891
Table 1
R
R
R
f-h
k
2-Substitutedpiperidine (R@H) orexin receptor antagonists based on 2
NHXAr2
NHBoc
NHBoc
N
N
H
N
N
H
N
Ar2
Ar1
Ph
18
17
16
O
N
X
j
Ar1
O
R
R
R
R
#
Ar1
Ar2
X
OX1 IC50 (nM) OX2 IC50 (nM)
a
a
a or b
c,d
or
NHBoc
N
N
Y
N
CO2H
N
CN
4
CO
CO
CO
3
5
4
2
23
4
Ph
N
10
11
e
N
S
R
R
N
R
5
6
7
N
4-F-Ph
g
f
NH2
NHBoc
NHBoc
N
H
O
S
14
13
12
O
Ar1
O
Ar1
N
Ph
O
N
S
h or i
CO <1
<1
R = alkyl, haloalkyl, amino, hydroxy, alkoxy
Ar1 = Ar2 = aryl, heteroaryl
X = CO or bond
4-F-Ph
R
S
Y = halogen
NHXAr2
Ar1 15
8
9
CO
—
6
8
8
N
N
S
N
N
Ph
Ph
N
O
CF3
13
Scheme 1. Synthesis of 2,3- and 2,6-disubstitutedpiperidine orexin receptor
antagonists. Reagents: (a) ZnCN2, Pd(PPh3)4; (b) SOCl2, NH4OH, Tf2O, DCM; (c) H2,
Pd/C, HOAc; (d) (BOC)2O, Et3N, DCM; (e) H2, HOAc, Nishimura’s catalyst; (f)
Ar1CO2H, HATU, DIEA, DCM; (g) TFA, DCM; (h) Ar2CO2H, HATU, DIEA, DCM; (i)
Ar2Br, xantphos, Cs2CO3, DMAC; (j) (1) BnBr, CH3CN; (2) NaBH4, CH3OH; (k)
Pd(OH)2, H2, CH3OH.
a
Values are means of at least three experiments.
validation. However, the undesirable pharmacokinetic profile
(t1/2 = 0.4 h, 10% oral bioavailability) and potential for off-target
activity at 5HT2B and 5HT2C hampered its progress beyond discov-
ery phase.18 Recently, another group further optimized this scaf-
fold to dial out OX2 completely, though no data is given with
regards to off-target activity or pharmacokinetics.19 Evaluation of
both the primary and patent literature revealed that several orexin
receptor antagonists have been developed based on a pyrrolidine
or piperidine core with differentially substituted appendages at
the N-1 and C-2 positions (Fig. 1,2).6 When our research investiga-
tion began, there were scant reports of disubstituted piperidine
antagonists (3). It wasn’t clear if this was because ring substitution
wasn’t tolerated, or the chemistry simply hadn’t yet advanced to
this stage. We wondered if ring substitution could alter the chair
topography of the piperidine ring, and subsequently affect selectiv-
ity for OX1 versus OX2. Recently, a patent application from Rottap-
harm S.P.A. published validating just such a strategy.20 Herein we
report the results of our investigation into substituted piperidines
as orexin receptor antagonists.
To get a baseline and establish controls for comparison, we ini-
tially synthesized a variety of differentially substituted piperidines
wherein we modified the N-1 acyl group and the substitution at C-2.
These molecules have been reported mostly in the patent literature
and contain little in vitro functional data.21–24 Compounds were
synthesized as described in the applications and screened in a
functional cell-based assay using CHO cells stably expressing OX1
(or OX2 as a counterscreen) which is based on OXA-stimulated intra-
cellular calcium mobilization using a combination of calcium-sensi-
tive dyes and a fluorescent imaging plate reader (FLIPR) (Table 1).25
These 2-substituted piperidines bearing a variety of heterocy-
cles are potent dual OX1–OX2 receptor antagonists (Table 1). The
most popular amides from the patent literature were chosen for
N-1 substitution. The 2-biphenyl aryl amide and both phenyl-
substituted thiazole isomers provided potent compounds (4–9).
The 8-quinoline and 4-benzofuran amides, as well as the trifluo-
romethylpyridine anilines were chosen as C-2 substitutions. Com-
pound 7 is the racemic version of SB649868, GSK’s initial dual
OX1–OX2 antagonist that advanced as far as PhII clinical trials
before being pulled for preclinical toxicology findings.26
OH
O
a
b
O
NH
Ph
N
N
H
CO2Me
R
Ph
OH
R
N
c
d
e-h
H
N
Ar2
N
CO2Me
CO2Me
N
O
O
Ar1
Ar1
Ar1
O
O
R = OH or OMe
R = OH or OMe
i or j-k
X
Y
X
Y
e-h
H
N
Ar2
N
N
CO2Me
O
Ar1
O
Ar1
O
X = F, Y = H
X = Y = O
X = Y = F
X = F, Y = H
X = Y = F
Scheme 2. Synthesis of 2,4-disubstitutedpiperidine orexin receptor antagonists.
Reagents: (a) OHCCO2H, CH3CN, 4A sieves; (b) H2, Pd/C, MeOH; (c) Ar1CO2H, HATU,
DIEA, DCM; (d) For R = OMe, MeI, NaH, DMF; (e) LiBH4, THF; (f) Phthalimide, DIAD,
Ph3P, THF; (g) N2H4, MeOH; (h) Ar2CO2H, HATU, DIEA, DMAC; (i) DAST, DCM; (j)
Dess–Martin periodinane, DCM; (k) Deoxo–Fluor, DCM.
To probe the effects of additional substitution, we introduced
substituents at positions 3–6 of the piperidine ring using the chem-
istry highlighted in Schemes 1–3.
We commenced the first round of SAR by introduction of substi-
tution at the 3-position of the piperidine ring. This required access
to 3-substituted 2-cyanopyridines, which were fairly easy to se-
cure (10). Stepwise reduction and protection of the primary amine

3892
R. Jiang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3890–3894
OEt
O
EtO
EtO
a
b
HCl
CO2Et
HCl
N
H
CO2H
N
H
N
CO2Et
O
Ar1
33
34
35
F
F
F
F
F
F
c,d
h
e-g
H
N
35
Ar2
NH2
N
N
CO2Et
N
R
N
N
O
Ar1
O
Ar1
O
Ar1
O
36
37
38
EtO
O
H
N
H
N
Ar2
e-g
h or i; c
j,d or k
Ar2
NH2
35
N
X
X
Ar1 40
41
O
Ar1
O
Ar1
X = bond or CO
39
O
Scheme 3. Synthesis of 2,5-disubstitutedpiperidines. Reagents: (a) SOCl2, EtOH; (b) Ar1CO2H, HATU, DIEA, DMAC; (c) TFA, DCM; (d) Deoxo-Fluor, DCM; (e) LiBH4, THF; (f)
Phthalimide, DIAD, Ph3P, THF; (g) N2H4, CH3OH; (h) Ar2CO2H, HATU, DIEA, DMAC; (i) Ar2Br, Pd(OAc)2, xantphos, Cs2CO3; (j) NaBH4, CH3OH; (k) RNH2, NaBH(OAc)3, DCM.
Table 2
at C-2 followed by saturation of the pyridine ring using Nishim-
2,3-Disubstitutedpiperidine orexin receptor antagonists
ura’s catalyst afforded the 2,3-disubstituted piperidines (12). Com-
pounds were almost exclusively 2,3-cis-oriented.27–29 Standard
R
H
N
Ar2
manipulations afforded final products (Table 2). Synthesis of the
2,3-trans analogs was achieved from a common intermediate
through a step-wise reduction of the pyridine ring after activation,
followed by hydrogenation of the N-benzyl tetrahydropiperidine
(16) to the deprotected piperidine (17). We were pleased to find
that 3-substitution was indeed tolerated as most compounds
exhibited good in vitro potency (Table 2).
A pair of bis-amide analogs showed almost 100-fold selectivity
for OX1 versus OX2 (19 and 23) in the calcium flux assay, however,
this trend was difficult to track as many analogs showed a more
modest 10- to 20-fold selectivity. In the bis-amides, the bulkier
CF3 group provided no advantage over a standard methyl group
(25 vs 20), nor did it appear that cis or trans methyl substitution
made a difference (27 vs 26) in this bis-amide series. Interestingly,
a 3-methyl substituted mono amide with an aminobenzoxazole in
the side chain afforded a compound with an enhanced selectivity
for OX1 versus OX2 (28). More analogs of this type are currently
being synthesized to see if OX2 activity can be completely abol-
ished from this series.
2,4-Disubstituted piperidine analogs were synthesized follow-
ing standard literature chemistry as described in Scheme 2. Only
a few analogs were made, as initial results weren’t promising with
regards to selectivity (Table 3). It’s not clear how a simple fluorine
substitution for hydrogen (30 vs 4) could result in an almost 50-
fold drop in activity, but these results encouraged us to move on
and to look at the C-5 and C-6 positions. In fact, all 2,4-disubsti-
tuted derivatives synthesized were less potent than their corre-
sponding 2-substituted analogs.
2,5-Disubstituted compounds could be made following chemis-
try as outlined in Scheme 1, but we also developed an alternate
synthetic route, as we desired to expand the range of groups at this
position on the ring. To that end, we synthesized 5-ketopipecolic
acid (33) as a starting material as described in the literature.30
Chemistry as outlined in Scheme 3 led to 2,5-disubstituted analogs
(Table 4). Compounds resulting from reductive amination of the 5-
ketopiperidine (40) gave mostly trans-2,5-disubstituted analogs
resulting from axial delivery of hydride in the reduction step.31
Compounds in this series also tolerated substitution of the
piperidine ring, and exhibited good in vitro activity (Table 4). There
didn’t appear to be much benefit, however, with regards to selec-
tivity towards OX1 versus OX2. Some compounds showed a modest
N
X
Ar1
O
Ar1
Ar2
X
R
OX1 IC50
(nM)
OX2 IC50
(nM)
a
a
#
N
S
Me
19
20
21
22
23
24
25
CO
CO
CO
CO
CO
CO
CO
8
658
8.7
N
(cis)
4-F-Ph
O
N
Me
0.8
15
9
S
(cis)
Ph
Ph
O
N
S
Me
221
40
(cis)
N
S
CF3
(cis)
N
Ph
Ph
O
N
S
Me
145
7
>10,000
50
(cis)
S
N
N
S
Me
N
(cis)
Ph
Ph
O
CF3
(cis)
10
40
N
S
Me
26
27
28
CO
CO
—
2
2
6
58
N
N
(cis)
Ph
Ph
Ph
N
S
Me
82
(trans)
N
N
S
Me
447
O
(cis)
a
Values are means of at least three experiments.
5- to 10-fold selectivity for OX1 (43, 44, 46), whereas others
showed no selectivity at all.
Finally, the effect of installing a group at C-6 was examined
(Table 5). Compounds were again synthesized following the gen-
eral protocol as described in Scheme 1. Following saturation of

R. Jiang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3890–3894
3893
Table 3
Table 5
2,4-Disubstitutedpiperidine orexin receptor antagonists
2,6-Diubstitutedpiperidine orexin receptor antagonists
R
H
N
Ar2
H3C
Ar1
N
X
H
N
Ar2
N
O
O
Ar1
O
Ar1
Ar2
X
OX1 IC50 (nM) OX2 IC50 (nM)
a
a
#
a
a
#
Ar1
Ar2
R
OX1 IC50 (nM) OX2 IC50 (nM)
O
N
S
48
CO
0.8
8
4-F-Ph
4-F-Ph
29
4-OH (cis)
4-F (trans)
4,4-diF
40
62
Ph
N
N
S
49
50
51
CO 12
151
96
N
30
31
32
146
>2000
541
NTb
NT
Ph
N
N
N
N
N
S
N
S
CF3
—
—
14
6
S
N
S
N
4-F-Ph
4-F-Ph
N
Ph
Ph
125
O
4-OMe (cis) 390
a
Values are means of at least three experiments.
a
Values are means of at least three experiments.
NT = not tested.
b
Table 6
In vitro and in vivo parameters of selected orexin antagonists
Table 4
#
Microsome stabilitya
Mouse in vivob
2,5-Disubstitutedpiperidine orexin receptor antagonists
t1/2
Plasmac
(
lm)
Brain (
l
m)
bpd
R
H
1/NTe/NT
1/1/1
NT/2/3
1/1/1
1/3/3
NT/NT/NT
1/1/1
1/7/1
7/15/52
4/6/5
1/3/2
1/1/1
1.5
33.7
8.6
12.5
12.8
20.8
1.6
4.6
27.6
0.3
12.4
3
0.06
18.7
17.9
3
7.8
10.1
0.9
0.5
1.4
0.1
14.3
1
4
N
Ar2
5
7
9
N
X
55
208
24
61
49
56
11
5
Ar1
O
21
22
23
28
45
46
49
50
51
#
Ar1
Ar2
X
R
OX1 IC50
(nM)
OX2 IC50
(nM)
a
a
42
43
44
45
CO F,F
2
2
Ph
Ph
Ph
N
33
115
33
CO NH(CH2)2OH
CO OH
27
2
137
13
<1
N
a
b
c
In mouse/rat/human liver microsomes.
Dosed 50 mg/kg ip.
Drug levels at l h timepoint.
Brain penetration.
N
d
e
NT = not tested.
O
N
S
CO
F
1
Ph
N
S
The microsomal stability of most compounds examined in all
46
47
CO NH2
121
7
2000
7
three species (mouse, rat, human) was poor. Compounds were
metabolized quite quickly. Even the racemate of GSK’s PhII com-
pound (7) was rapidly metabolized in microsomes. When com-
pounds were dosed to mice (10 mg/kg, ip), there was little drug
in plasma at t = 1 h, and even less in the brain.33 Hence, the dose
was increased to 50 mg/kg in an effort to increase plasma and brain
levels that would support a compound’s use in vivo. Data from
these higher dosing experiments is shown in Table 6. For several
analogs, plasma and brain levels at t = 1 h were quite high. In these
cases, it is possible the metabolic mechanisms leading to low drug
levels are being overwhelmed. However, this still does not explain
the disconnect between microsomal stability and the fact that the
enantiomer of 7 advanced into man. Nonetheless, these plasma
and brain levels are likely sufficient to provide receptor coverage
within the parameters of the in vivo study. Given the enhanced
OX1 selectivity of 23 and 28, as well as their favorable drug plasma
and brain levels at t = 1 h, these compounds may be useful in vivo
candidates. Results of their in vivo efficacy in a model of drug
addiction will be reported elsewhere.
N
Ph
Ph
N
N
S
CF3
—
F
a
Values are means of at least three experiments.
the pyridine ring, a single diastereomer was produced as indicated
by 1H NMR analysis (also single peak by reverse-phase analytical
HPLC). It is presumed to be cis based on literature precedent.27,32
No effort was made to synthesize the 2,6-trans isomer. A 6-methyl
group was tolerated and imparted a modest 10-fold selectivity for
OX1 versus OX2 (48–51). Synthesis of bulkier 6-substituted analogs
is still on-going to see if more selectivity can be derived.
While chemical SAR has been on-going, in parallel, we have
been routinely profiling compounds through drug metabolism in
order to identify a compound suitable for in vivo use. We have
been profiling compounds in microsomal stability, as well as
looking for plasma and brain exposure in mice following ip dosing.
Data is compiled in Table 6.
In summary, we have described a series of orexin receptor
antagonists based on substituted piperidines. SAR focusing on the

3894
R. Jiang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3890–3894
16. Narita, M.; Nagumo, Y.; Hashimoto, S.; Khotib, J.; Miyatake, M.; Sakurai, T.;
Yanagisawa, M.; Nakamachi, T.; Shioda, S.; Suzuki, T. J. Neurosci. 2006, 26, 398.
17. Smart, D.; Sabido-David, C.; Brough, S. J.; Jewitt, F.; Johns, A.; Porter, R. A.;
Jerman, J. C. Br. J. Pharmacol. 2001, 132, 1179.
18. Porter, R. A.; Chan, W. N.; Coulton, S.; Johns, A.; Hadley, M. S.; Widdowson, K.;
Jerman, J. C.; Brough, S. J.; Coldwell, M.; Smart, D.; Jewitt, F.; Jeffrey, P.; Austin,
N. Bioorg. Med. Chem. Lett. 2001, 11, 1907.
19. Perrey, D. A.; Gilmour, B. P.; Runyon, S. P.; Thomas, B. F.; Zhang, Y. Bioorg. Med.
Chem. Lett. 2011, 21, 2980.
20. Stasi, P. L.; Rovati, L., WO2011006960, Rottapharm S.P.A.
21. Aissaoui, H.; Boss, C.; Koberstein, R.; Ferlen, T.; Trachsel, D., WO2009/104155,
Actelion Pharmaceuticals Ltd.
3-position revealed that it is possible to synthesize compounds
with enhanced selectivity for the OX1 receptor relative to OX2. Sub-
stitution at other piperidine positions afforded potent compounds,
with reduced or no selectivity for OX1 versus OX2. Further explora-
tion of substitutions at the piperidine 3-position are still on-going.
Evaluation of compounds such as 23 and 28 in an animal model of
drug addiction will be reported in due course.
Acknowledgment
22. Aissaoui, H.; Boss, C.; Gude, M.; Koberstein, R.; Sifferlen, T., WO2008/087611,
Actelion Pharmaceuticals Ltd.
Supported by the National Institute on Drug Abuse (DA023915
to P.J.K.).
23. Branch, C.; Nash, D. J.; Stemp, G., WO2003/051368, Smithkline Beechem PLC.
24. Branch, C.; Coulton, S.; Johns, A.; Nash, D. J.; Porter, R. A.; Stemp, G., WO2004/
026866, Glaxo Group Limited.
25. Measurement of [Ca2+]i using a FLIPR: CHO-OX1or CHO-OX2 cells will be seeded
into black-walled clear-base 384-well plates (Costar) at a density of 20,000
cells per well in F12-K medium supplemented with 10% FBS and selection
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and after 2 h blood and brain were collected. Plasma was generated and the
samples were frozen at À800 °C. The plasma and brain were mixed with
acetonitrile (1:5 v/v or 1:5 w/v, respectively). The brain sample was sonicated
with a probe tip sonicator to break up the tissue, and samples were analyzed
for drug levels by LC–MS/MS. Plasma drug levels were determined against
standards made in plasma and brain levels against standards made in blank
brain matrix. All procedures were approved by the Scripps Florida IACUC.