Synthesis of (3,4-dimethoxyphenoxy)alkylamino acetamides as orexin-2 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2008.09.038

The discovery and synthesis of a series of (dimethoxyphenoxy)alkylamino acetamides as orexin-2 receptor antagonists from a small-molecule combinatorial library using a high-throughput calcium mobilization functional assay (HEK293-human OX2-R cell line) is described. Active compounds show a good correlation between high-throughput single concentration screening data and measured IC₅₀s. Specific examples exhibit IC₅₀ values of ～20 nM using human orexin A as the peptide agonist for the orexin-2 receptor.

Full text of DOI:10.1016/j.bmcl.2008.09.038

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5420–5423
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of (3,4-dimethoxyphenoxy)alkylamino acetamides as orexin-2
receptor antagonists
a
a
a
a
Andrew G. Cole a, , Ilana L. Stroke , Lan-Ying Qin , Zahid Hussain , Srilatha Simhadri ,
Marc-Raleigh Brescia a, Frank S. Waksmunski a, Barbara Strohl a, John E. Tellew b, John P. Williams b,
John Saunders b, Kenneth C. Appell a, Ian Henderson a, Maria L. Webb a
*
a Pharmacopeia, Inc., PO Box 5350, Princeton, NJ 08543, USA
b Neurocrine Biosciences, Inc. 12790 El Camino Real, San Diego, CA 92130, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery and synthesis of a series of (dimethoxyphenoxy)alkylamino acetamides as orexin-2 recep-
tor antagonists from a small-molecule combinatorial library using a high-throughput calcium mobiliza-
tion functional assay (HEK293—human OX2-R cell line) is described. Active compounds show a good
correlation between high-throughput single concentration screening data and measured IC50s. Specific
examples exhibit IC50 values of ꢀ20 nM using human orexin A as the peptide agonist for the orexin-2
receptor.
Received 6 August 2008
Revised 5 September 2008
Accepted 9 September 2008
Available online 12 September 2008
Keywords:
Hypocretin
Orexin
Ó 2008 Elsevier Ltd. All rights reserved.
GPCR
Combinatorial chemistry
ECLiPSTM
The orexins (hypocretins), orexin A and orexin B, are neuropep-
tides produced in the hypothalamus.1,2 These peptides bind to and
activate the two G protein-coupled receptors (GPCR’s) orexin-1
(OX1-R/hypocretin R1) and orexin-2 (OX2-R/hypocretin R2). For
OX1-R, orexin A has a 10-fold greater affinity than orexin B,
whereas both peptides bind to OX2-R with similar affinity.1 While
the Orexin receptors share common ligands, they differ in several
respects. Recombinantly-expressed OX1-R signals via Gq, and
OX2-R via Gq or Gi/o, and OX1-R and OX2-R are also expressed
in different regions of the brain.3,4 The orexin receptor system
has been implicated in feeding behavior and metabolism. Exoge-
nous administration of orexin A increases arousal and food intake
in rats and the stimulation of food intake has been shown to be
blocked by a specific OX1-R antagonist.1,8–12 Recently, the orexin
receptor system has also been implicated in sleep and wakefulness.
Knockout of the orexins, or a double knockout of OX1-R and OX2-R,
results in sleep-onset REM periods, cataplexy, and short sleep la-
tency in mice; deficiency in OX1-R alone leads only to fragmented
sleep, whereas OX2-R deficiency leads to narcoleptic symptoms
that are similar to but less severe than those manifested by the
double-receptor knockout mice, with only mild cataplexy.5–7
Naturally-occurring mutations in OX2-R have been identified in
narcoleptic dogs, whereas narcolepsy in humans has been
associated primarily with decreased hypocretin levels in brain/
cerebrospinal fluid and with the loss of hypocretin neurons.13–18
Interestingly, low hypocretin levels in narcoleptic patients have
also been associated with decreased metabolic rate and obesity de-
spite the associated hypophagia. Experimental ablation of hypocre-
tin neurons in mice has a similar effect.19,20 The role of orexins
acting at OX2-R suggests that this receptor might be a suitable tar-
get for pharmacological intervention for the treatment of narco-
lepsy (agonists) and insomnia (antagonists). There has been
considerable interest in the discovery and development of hypo-
cretin receptor antagonists such as ACT-078573 (an OX1-R/OX2-
R antagonist)21 which is currently the subject of clinical trials for
the treatment of sleep disorders. We describe here the identifica-
tion of novel small-molecule antagonists of OX2-R.
O
O
N
O
N
H
CF3
* Corresponding author. Tel.: +1 609 452 3653; fax: +1 609 655 4187.
E-mail addresses: acole@pcop.com, acole4@verizon.net (A.G. Cole).
ACT-078573
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.038

A. G. Cole et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5420–5423
5421
R3
N
O
R3
N
O
n
n
O
O
O
O
R1
R1
X
Y
X
Y
R4
R2
R2
1
2
Figure 1. General structure of OX2-R active library (1) and subset of OX2-R antagonists (2).
TM
A high-throughput screen of 90 distinct ECLiPS (Encoded Combi-
natorial Libraries on Polymeric Support)22 libraries comprising >4
million compounds was conducted employing a 384-well FLIPR-
based calcium flux assay designed to identify small-molecule antag-
onists of the human OX2-R receptor. A library of ꢀ140,000 com-
pounds, synthesized through the incorporation of four points of
diversity (R1–R4) and based on a substituted phenoxyalkylaminoace-
tamide motif (1), resulted in the identification of a set of (3,4-dimeth-
oxyphenoxy) alkylaminoacetamides (2) as potent OX2-R antagonists
(Fig. 1).
O
4
NH2
NH2
a, b
c
NH2
N
H
3
4
Br
NO2
O
O
L
Br
NH
O
=
N
H
NO2
Br
5
4
The combinatorial library was synthesized utilizing amino-
N
H
methyl-terminated TentagelÒ (3) as the polymeric support
5
(Scheme 1). Initial derivatization of 3 with N-a-N-e-bis-Fmoc-ly-
Scheme 1. Reagents and conditions: (a) N-a-N-e-bis-Fmoc-Lys, HOBt monohy-
drate, DIC, CH2Cl2, DMF, 25 °C; (b) piperidine, DMF, 25 °C; (c) 4-(bromomethyl)-3-
nitrobenzoic acid, HOBt monohydrate, DIC, CH2Cl2, DMF, 25 °C.
sine followed by Fmoc deprotection to generate 4 increases the
loading capacity of the resin by doubling the number of amino
groups for further functionalization. This was followed by acyla-
O
O
n
H
a
c
b
N
OH
Br
L
NH
R1
L
N
R1
L
Br
L
N
R1
X
Y
6
5
7
8
R2
R3
O
R3
N
O
O
R3
N
n
n
n
N
O
f
O
e
OH
Ar4
d
L
N
R1
Ar4
HN
R1
L
N
R1
X
Y
X
Y
X
Y
10
1
R2
9
R2
R2
Scheme 2. Reagents and conditions: (a) R1-NH2, THF, 25 °C; (b) bromoacetic acid, DIC, DMF, 25 °C; (c) R2-NH2, DMSO, 25 °C; (d) R3’-CHO, NaCNBH3, 1% AcOH/MeOH, 25 °C; (e)
Ar4OH, TMAD, PBu3, THF/CH2Cl2, 25 °C; (f) h
t (365 nm), 2% v/v TFA/MeOH, 50 °C.
R15
R12
R21
50
40
30
R18
R22
R28
R2
10
frequency
R1
20
10
R31
R2
R310
R2
O
O
R34
X
Y
0
R1
1
2
N
O
3
4
5
6
n
7
8
R3
9
O
10
R3
11
12
13
14
2
15
16
17
18
19
Synthon Number
20
21
22
R314
23
Figure 2. Synthon frequency of active compounds (2) identified in OX2-R screening of the combinatorial library.

5422
A. G. Cole et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5420–5423
tion with 4-(bromomethyl)-3-nitrobenzoic as a linker to generate
5. This linker allows photo-mediated cleavage of the final
compounds from solid phase, and is compatible with the chemistry
required to conduct the library synthesis.
Synthesis of the library, using commercially available reagents,
was initiated by the generation of a resin-bound secondary amine
6 via alkylation of a series of primary amines (R1NH2) with 5
(Scheme 2). Subsequent bromoacetylation of 6 provided 7, which
was used to alkylate a series of aminoalcohols (R2-NH2) to yield
sured potencies are detailed in Table 1 (ethylene-based systems)
and Table 2 (propylene-based systems).
Analysis of the potencies for the synthesized compounds indi-
cated that the isobutyl carboxamide (11–14) is the least potent of
the three most frequently occurring R1 substituents. This observa-
tion is in good agreement with the screening activities observed in
library screening. Incorporation of the unsubstituted benzylic com-
ponent (R15, 15–18) at R1 appears to provide, on average, a fivefold
increase in potency in comparison to the 2-chloro-substituted ben-
zylic component(R18, 19–22), potentially indicatinga preference for
an electron-neutral aromatic system or an adverse steric or confor-
mational effect provided by the 2-chloro substitution. A moderate
preference for the ethylene-based amino ether (15–18) in compari-
8. Incorporation of the third point of diversity was achieved via
0
reductive alkylation of 8 with a series of aromatic aldehydes (R3 -
CHO) to give 9.
Library synthesis was completed through Mitsunobu coupling
of the resin-bound alcohol 9 with a series of substituted phenols
(Ar4-OH) utilizing N,N,N’,N’-tetramethylazodicarboxamide (TMAD)
and tri-n-butyl phosphine (PBu3) to generate 10. The use of TMAD/
PBu3 in the Mitsunobu coupling allowed efficient conversion of
both primary and secondary alcohols to the corresponding aryl
ethers. Photo-mediated cleavage of 10 to release 1 from the solid
support was achieved by irradiating a suspension of 10 in 2% v/v
trifluoroacetic acid/methanol at 365 nm.
High-throughput screening was conducted by monitoring hu-
man orexin A-stimulated calcium release in a HEK293/OX2-R cell
line following stimulation with 20 nM (EC80) of human orexin-A.
No agonist activity was observed for the compounds described.
As a specificity control, compounds were also tested in the pres-
Table 1
Ethylene-based OX2-R antagonists
O
O
R3
N
R1
O
O
Compound
R1
R3
OX2-R pIC50 SEMa
6.52 0.04
11 (R12, R31)
N
H
12 (R12, R34)
13 (R12, R310)
14 (R12, R314)
15 (R15, R31)
16 (R15, R34)
17 (R15, R310)
18 (R15, R314)
19 (R18, R31)
20 (R18, R34)
21 (R18, R310)
6.25 0.06
6.64 0.10
6.49 0.01
7.60 0.06
7.71 0.01
7.73 0.08
6.96 0.14
6.82 0.02
6.86 0.01
7.09 0.08
6.71 0.06
ence of 8 lM ATP in place of orexin-A for effect on activation of
N
H
endogenous purinergic receptors in the same HEK293/OX2-R cell
line. Compounds derived from the portion of the combinatorial li-
brary in which Ar4 was present as the 3,4-dimethoxyphenyl substi-
tuent were shown to exhibit good OX2-R antagonist activity and
showed no effect on challenge with ATP. Structural determination
of the active compounds (>60% reduction in calcium release) pres-
ent within this portion of the library was conducted (defining the
R1–R3 substituents) via analysis of the encoding molecules incor-
porated during ECLiPSTM library synthesis.
Analysis of the frequency of the individual combinatorial syn-
thons that were present in the active compounds is shown graphi-
cally in Figure 2. Preference for specific synthons was observed at
each of the combinatorial variants. Synthon R1 corresponds to incor-
poration of a secondary amino substituent at the glycinyl carboxy
group. A strong preference is observed for synthons R12, R15 and
R18, which correspond to isobutyl, benzyl and 2-chlorobenzyl sub-
stitutions, respectively. Analysis of the activities in the screen for
the active compounds reveals that fragment R12 (isobutyl) is only
present in active structures that demonstrated inhibition of calcium
release by 60–70% in the screen, whereas R15 and R18 are identified
in compounds with superior activity. Synthon R2 defines the ami-
noether core of the library. Within this combinatorial element, a
strong preference is observed for synthons R21, R22, R28 and R210,
F
F
N
H
F
S
S
S
N
H
N
H
N
H
F
F
N
H
F
N
H
corresponding to ethylene,
a-methyl ethylene, b-methyl ethylene
N
and propylene-based amino ether cores. Screening activity data sug-
gested comparable potencies for the ethylene-based systems and
slightly weaker potency for the propylene-based aminoether sys-
tems. Synthon R3 corresponds to the reductive amination performed
to generate the tertiary amino center of the aminoether and a selec-
tion of substituentsare present within the data set (Fig. 2), indicating
that a variety of substitutions will be toleratedwithin this position of
the molecule. The most frequently occurring synthons at the R3 po-
sition are R31 (benzyl), R310 (3,4-difluorobenzyl), R314(2-thiophene
methyl) and R317 (3-thiophene methyl).
H
Cl
N
H
F
F
Cl
Cl
Cl
N
H
F
To correlate percent activity derived from library screening
with accurate IC50 values,23a selection of compounds were synthe-
sized in multi-milligram quantities by parallel synthesis following
the solid-phase synthetic route outlined in scheme 2. The mea-
N
H
22 (R18, R314)
a
pIC50 standard error mean upon challenge with 20 nM Ox-A, based on P3
independent determinations.

A. G. Cole et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5420–5423
5423
Table 2
2. de Lecea, L.; Kilduff, T. S.; Peyron, C.; Gao, X.; Foye, P. E.; Danielson, P. E.;
Fukuhara, C.; Battenberg, E. L.; Gautvik, V. T.; Bartlett, F. S. 2nd.; Frankel, W. N.;
van den Pol, A. N.; Bloom, F. E.; Gautvik, K. M.; Sutcliffe, J. G. Proc. Natl. Acad. Sci.
U.S.A. 1998, 95, 322.
3. Zhu, Y.; Miwa, Y.; Yamanaka, A.; Yada, T.; Shibahara, M.; Abe, Y.; Sakurai, T.;
Goto, K. J. Pharmacol. Sci. 2003, 92, 259.
Propylene-based OX2-R antagonists
O
R3
N
O
O
O
R1
4. Marcus, J. N.; Aschkenasi, C. J.; Lee, C. E.; Chemelli, R. M.; Saper, C. B.;
Yanagisawa, M.; Elmquist, J. K. J. Comp. Neurol. 2001, 435, 6.
5. Chemelli, R. M.; Willie, J. T.; Sinton, C. M.; Elmquist, J. K.; Scammell, T.; Lee, C.;
Richardson, J. A.; Williams, S. C.; Xiong, Y.; Kisanuki, Y.; Fitch, T. E.; Nakazato,
M.; Hammer, R. E.; Saper, C. B.; Yanagisawa, M. Cell 1999, 98, 437.
6. Willie, J. T.; Chemelli, R. M.; Sinton, C. M.; Yanagisawa, M. Annu. Rev. Neurosci.
2001, 24, 429.
7. Willie, J. T.; Chemelli, R. M.; Sinton, C. M.; Tokita, S.; Williams, S. C.; Kisanuki, Y.
Y.; Marcus, J. N.; Lee, C.; Elmquist, J. K.; Kohlmeier, K. A.; Leonard, C. S.;
Richardson, J. A.; Hammer, R. E.; Yanagisawa, M. Neuron 2003, 38, 715.
8. Hagan, J. J.; Leslie, R. A.; Patel, S.; Evans, M. L.; Wattam, T. A.; Holmes, S.;
Benham, C. D.; Taylor, S. G.; Routledge, C.; Hemmati, P.; Munton, R. P.;
Ashmeade, T. E.; Shah, A. S.; Hatcher, J. P.; Hatcher, P. D.; Jones, D. N.; Smith, M.
I.; Piper, D. C.; Hunter, A. J.; Porter, R. A.; Upton, N. Proc. Natl. Acad. Sci. U.S.A
1999, 96, 10911.
Compound
R1
R3
OX2-R pIC50 SEMa
7.29 0.03
23 (R12, R31)
N
H
F
F
24 (R12, R310)
6.97 0.06
6.88 0.07
N
H
S
9. Methippara, M. M.; Alam, M. N.; Szymusiak, R.; McGinty, D. Neuroreport 2000,
11, 3423.
25 (R12, R314)
N
H
10. Piper, D. C.; Upton, N.; Smith, M. I.; Hunter, A. J. Eur. J. Neurosci. 2000, 12, 726.
11. Huang, Z. L.; Qu, W. M.; Li, W. D.; Mochizuki, T.; Eguchi, N.; Watanabe, T.;
Urade, Y.; Hayaishi, O. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 9965.
12. Rodgers, R. J.; Halford, J. C.; Nunes de Souza, R. L.; Canto de Souza, A. L.; Piper, D.
C.; Arch, J. R.; Upton, N.; Porter, R. A.; Johns, A.; Blundell, J. E. Eur. J. Neurosci.
2001, 13, 1444.
a
pIC50 standard error mean upon challenge with 20 nM Ox-A, based on P3
independent determinations.
13. Lin, L.; Faraco, J.; Li, R.; Kadotani, H.; Rogers, W.; Lin, X.; Qiu, X.; de Jong, P. J.;
Nishino, S.; Mignot, E. Cell 1999, 98, 365.
14. Reilly, C. E. J. Neurol. 1999, 246, 985.
15. Hungs, M.; Fan, J.; Lin, L.; Lin, X.; Maki, R. A.; Mignot, E. Genome Res. 2001, 11,
531.
16. Peyron, C.; Faraco, J.; Rogers, W.; Ripley, B.; Overeem, S.; Charnay, Y.;
Nevsimalova, S.; Aldrich, M.; Reynolds, D.; Albin, R.; Li, R.; Hungs, M.;
Pedrazzoli, M.; Padigaru, M.; Kucherlapati, M.; Fan, J.; Maki, R.; Lammers, G.
J.; Bouras, C.; Kucherlapati, R.; Nishino, S.; Mignot, E. Nat. Med. 2000, 6, 991.
17. Nishino, S.; Ripley, B.; Overeem, S.; Lammers, G. J.; Mignot, E. Lancet 2000, 355,
39.
18. Thannickal, T. C.; Moore, R. Y.; Nienhuis, R.; Ramanathan, L.; Gulyani, S.;
Aldrich, M.; Cornford, M.; Siegel, J. M. Neuron 2000, 27, 469.
19. Schuld, A.; Hebebrand, J.; Geller, F.; Pollmacher, T. Lancet 2000, 355, 1274.
20. Hara, J.; Beuckmann, C. T.; Nambu, T.; Willie, J. T.; Chemelli, R. M.; Sinton, C. M.;
Sugiyama, F.; Yagami, K.; Goto, K.; Yanagisawa, M.; Sakurai, T. Neuron 2001, 30,
345.
son to the propylene-based amino ether (23–25) is also evident from
the synthesized compounds. Alkylation to provide the tertiary
amino system as determined by R3 gives compounds of excellent po-
tency (IC50 < 30 nM) for R31 (benzyl, 15), R34 (4-fluorobenzyl, 16)
and R310 (3,4-difluorobenzyl, 17). However, based on the screening
data, a number of substitutions appear to be tolerated at the R3 posi-
tion, providing the potential for further optimization and the inves-
tigation of components that may provide increased antagonist
activity. It is noteworthy that the 3,4-dimethoxyphenyl substituent
(Ar4), was present in all active compounds. Minor deviation from the
3,4-dimethoxy phenyl system to provide the 3-methoxy, 4-methoxy
and 3,4-methylenedioxy analogs resulted in compounds exhibiting
21. Brisbare-Roch, C.; Dingemanse, J.; Koberstein, R.; Hoever, P.; Aissaoui, H.;
Flores, S.; Mueller, C.; Nayler, O.; van Gerven, J.; de Haas, S. L.; Hess, P.; Qiu, C.;
Buchmann, S.; Scherz, M.; Weller, T.; Fischli, W.; Clozel, M.; Jenck, F. Nat. Med.
2007, 13, 150.
22. (a) Ohlmeyer, M. H. J.; Swanson, R. N.; Dillard, L.; Reader, J. C.; Asouline, G.;
Kobayishi, R.; Wigler, M.; Still, W. C. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 10922;
(b) Nestler, H. P.; Bartlett, P. A.; Still, W. C. J. Org. Chem. 1994, 59, 4723.
IC50 values of > 2 lM.
In conclusion, potent antagonists of OX2-R were identified in a
ꢀ140,000 member combinatorial library using a 384-well high-
throughput functional calcium-mobilization screen. Correlation
between library screening data (% inhibition) and measured IC50
values was excellent. Analysis of the specific substituents present
in the active compounds identifies areas of the molecules where
key functionality is required. The toleration of multiple tertiary
amine substituents in conjunction with varying chain lengths
and substitutions of the aminoalcohol-based spacer provides the
opportunity for a number of cyclic constraints to be applied to
the molecules. The optimization of this series of compounds as
OX2-R antagonists and the scope of constraint applications will
be reported elsewhere.
23. HEK293/OX2-R cells were seeded at 20,000 cells (50
-lysine)-treated 384-well plates (Costar, black clear-bottom cell culture-
lL) per well in poly-
(D
treated) in culture medium (Dulbecco’s modified Eagle’s medium containing
10% heat-inactivated fetal bovine serum, 1% Gluta-Max (Invitrogen), 1%
nonessential amino acids, 1% 1 U/mL Penicillin/1
sodium pyruvate, 10 mM HEPES, and 200 g/mL G418). After 24 h at 37 °C, 5%
CO2, culture medium was removed and replaced with 25 L per well of dye/
lg/mL Streptomycin, 1 mM
l
l
buffer mix (equal parts of 2ꢁ concentrated Molecular Devices Calcium 3 no-
wash dye and buffer consisting of Hanks’ Balanced Salt Solution, 20 mM HEPES,
pH 7.5, 0.2% BSA), with probenecid to a final concentration of 2.5 mM. Plates
were incubated at 37 °C, 5% CO2 for 30 min, and then equilibrated to room
temperature for 30–90 min. Test compounds were diluted in Hanks’ Balanced
Salt Solution (HBSS), 20 mM HEPES, pH 7.5, 0.1% BSA, 2.5 mM probenecid
(assay buffer), with 2% DMSO. Prior to each assay run, FLIPR 384 tips
(Molecular Devices) were presoaked in 1% BSA/HBSS/20 mM HEPES, pH 7.5.
References and notes
For the antagonist assay, 12.5
l
L test compound was added to dye-loaded cells
1. Sakurai, T.; Amemiya, A.; Ishii, M.; Matsuzaki, I.; Chemelli, R. M.; Tanaka, H.;
Williams, S. C.; Richardson, J. A.; Kozlowski, G. P.; Wilson, S.; Arch, J. R.;
Buckingham, R. E.; Haynes, A. C.; Carr, S. A.; Annan, R. S.; McNulty, D. E.; Liu, W.
S.; Terrett, J. A.; Elshourbagy, N. A.; Bergsma, D. J.; Yanagisawa, M. Cell 1998, 92,
573.
in the FLIPR384, followed within 30 min by addition of 12.5
lL of human orexin
A (Bachem) in assay buffer to a final concentration of 20 nM. Fluorescence was
monitored for 125 s after additions.