Discovery and Characterization of ACT-335827, an Orally Available, Brain Penetrant Orexin Receptor Type1 Selective Antagonist

Article abstract of DOI:10.1002/cmdc.201300003

Stress relief: Orexin neuropeptides regulate arousal and stress processing through orexin receptor type1 (OXR-1) and 2 (OXR-2) signaling. A selective OXR-1 antagonist, represented by a phenylglycine-amide substituted tetrahydropapaverine derivative (ACT-335827), is described that is orally available, penetrates the brain, and decreases fear, compulsive behaviors and autonomic stress reactions in rats. Copyright

Full text of DOI:10.1002/cmdc.201300003

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DOI: 10.1002/cmdc.201300003
Discovery and Characterization of ACT-335827, an Orally
Available, Brain Penetrant Orexin Receptor Type 1
Selective Antagonist
Michel A. Steiner,* John Gatfield, Catherine Brisbare-Roch, Hendrik Dietrich,
Alexander Treiber, Francois Jenck, and Christoph Boss*[a]
Orexins are wake-promoting neuropeptide transmitters that
bind to two distinct and differentially expressed excitatory
G protein-coupled receptors, orexin receptor type 1 (OXR-1)
and 2 (OXR-2).[1] The simultaneous blockade of both receptors
decreases wakefulness in rats, dogs and human,[2] and has led
to the development of dual orexin receptor antagonists as
novel insomnia treatments.[2–4] Effects of selective blockade of
either OXR-1 or OXR-2 alone have remained ambiguous due to
the lack of selective small-molecule antagonists suitable as
pharmacological tools for in vivo interventions. Most investiga-
tions on the role of OXR-1 for physiological and behavioral
function have relied on 1-(2-methylbenzoxazol- 6-yl)- 3-
[1,5]naphthyridin- 4-yl urea (SB-334867),[5,6] an OXR-1 antagonist
with moderate selectivity across neuronal targets.[5,7] Here, we
report the synthesis of a novel, potent, and brain-penetrating
OXR-1 antagonist that is orally available and, at pharmacologi-
cally active doses, highly selective for OXR-1 across a wide
panel of common neuronal targets. The compound elicits anx-
iolytic-like effects without promoting sleep.
Scheme 1. Compound library synthesis via a multicomponent Ugi-type reac-
tion and lead compound 5 identified from the initial library screening.
The initial OXR antagonist screening library was prepared by
a multicomponent Ugi-type reaction, starting from commer-
cially available tetrahydropapaverin 1, an aldehyde 2, and an
isocyanate 3, to yield the screening compounds 4 as mixtures
of 4 diastereoisomers (Scheme 1). This library was screened for
orexin receptor antagonistic activity in a high-throughput fluo-
rescence imaging plate reader (FLIPR) intracellular calcium re-
lease assay using recombinant CHO cells expressing OXR-1 and
OXR-2 and applying dilution series of antagonists 10 min prior
to orexin-A (OxA) stimulation. With this assay, we identified
compound 5 as a promising starting point for further medici-
nal chemistry investigations.[8,9]
A first optimization cycle identified compound 6 (Scheme 2),
an aminoindane derivative, which exhibited significantly im-
proved activity on the OXR-1 while retaining a similar selectivi-
ty profile as initial hit compound 5. However, compound 6 did
not show significant brain penetration in rats. Further optimi-
zation efforts by substituting the a-amino acid carbon atom,
as indicated in the initial preparation scheme, for a simple
[a] Dr. M. A. Steiner, Dr. J. Gatfield, Dr. C. Brisbare-Roch, Dr. H. Dietrich,
Dr. A. Treiber, Dr. F. Jenck, Dr. C. Boss
Actelion Pharmaceuticals Ltd.
Gewerbestrasse 16, 4123 Allschwil (Switzerland)
E-mail: michel.steiner@actelion.com
christoph.boss@actelion.com
Scheme 2. Second generation (not brain penetrant) lead compound 6, and
third generation (brain penetrant) lead compounds 7 and 8 identified
through optimization cycles.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300003.
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Table 1. Benzyl replacements for the piperonyl moiety (R1).
Ox1
Ox2 654
9
5
261
5
207
43
5280
5
272
13
2542
77
8675
9
147
7
679
16
231
IC50[a] [nm]
[a] IC50 value determined using a fluorescence imaging plate reader (FLIPR) intracellular calcium release assay with a 10 min pre-incubation period with the
antagonists. Data represent geomeans of one up to 57 measurements.
phenyl ring (i.e., replacing the “glycine core” by a “phenyl-gly-
cine core”) resulted in compound 7, which showed a similarly
good OXR-1 selectivity (Scheme 2). Following the separation of
diastereomers of compound 7 by chromatography using chiral
stationary phases, (S/R)-diastereomer 8 was identified as being
the most potent stereoisomer (chirality was assessed based on
comparative data generated with structurally related com-
pounds such as almorexant[10,11] in our parallel efforts to identi-
fy dual OXR antagonists). An in
termined by calcium release assays with either 10 or 120 min
antagonist pre-incubation time. A butyl chain or an isopropyl
group in the R1 position was the most promising substituent.
All derivatives were additionally characterized in an in vivo rat
BBB penetration experiment, indicating that the derivative
with an isopropyl substituent in position R1 showed the most
promising properties.
vivo blood–brain barrier (BBB)
penetration experiment in rat re-
Table 2. Alkyl and cycloalkyl replacements for the piperonyl moiety of both the methoxy (A) and methyl (B)
derivatives.
vealed excellent brain penetra-
tion for 8, despite an unfavora-
ble in vitro safety profile.
Based on the properties of
compound 8, structure–activity
relationship (SAR) investigations
focused on replacing the piper-
onyl-moiety with differently sub-
stituted benzyl groups (R1;
Table 1) in an attempt to elimi-
nate unwanted drug–drug inter-
actions and metabolic instability.
All synthesized compounds were
preferential/selective OXR-1 an-
tagonists, however ortho substi-
tution at the benzyl unit usually
resulted in the least active com-
pounds as compared with meta-
and para-analogues, which ex-
hibited similar activities. Combin-
ing the meta and para substitu-
A) Methoxy derivatives
Ox1 (10)
Ox2 (10)
Ox1 (120)
9
26
58
3
244
16
7
365
4
6
417
9
4
191
29
3
92
21
28
2510
39
11
281
71
IC50[a] [nm]
Ox2 (120) 561
987
440
271
4180
1970
3140
3890
[Plasma][b] [ngmLÀ1
[Brain][b] [nggÀ1
]
223
69
563
163
850
530
1457
880
741
208
690
247
983
362
1193
800
]
B) Methyl derivatives
Ox1 (10)
34
13
17
12
10
62
8
14
493
4
33
124
n.d.
n.d.
58
107
n.d.
n.d.
41
47
18
41
34
38
22
333
60
n.d.
n.d.
Ox2 (10)
Ox1 (120)
Ox2 (120)
IC50[a] [nm]
162
391
336
ent into
a
3,4-disubstituted
[Plasma][b] [ngmLÀ1
[Brain][b] [nggÀ1
]
64
34
96
46
129
44
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
92
60
n.d.
n.d.
system did not improve potency.
As a next step, we replaced
the benzyl units by smaller alkyl
moieties (R1; Table 2A). All deriv-
atives remained preferential/se-
lective OXR-1 antagonists as de-
]
[a] IC50 value determined using a fluorescence imaging plate reader (FLIPR) intracellular calcium release assay
with a 10 min or 120 min pre-incubation period with the antagonists. Data represent geomeans of one up to
57 measurements. [b] An in vivo blood–brain barrier (BBB) experiment was performed in rat at a dose of
100 mgkgÀ1 and a time period of 3 h. Not determined (n.d.). Data given are for the S/R diastereoisomers.
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In a third step, we replaced two aromatic methoxy substitu-
ents by two methyl groups in combination with the set of
alkyl- and cycloalkyl substituents used for the preparation of
the compounds summarized in Table 2A. The methyl-substitut-
ed set of derivatives differentially evolved towards OXR antag-
onistic activity (Table 2B). First, the activity was generally de-
creased. Second, the selectivity towards OXR-1 was lower, lost,
or even reversed (see for example the cyclobutyl derivative).
Third, brain penetration was diminished. With these results
and the limited chemical methodologies available for the con-
struction of tetrahydroisoquinoline systems, such as Pictet–
Spengler- or Bischler–Napieralski reactions, which work best
under conditions of strongly electron-donating methoxy sub-
stituents on the phenethylamine precursors, we did not at-
tempt a replacement of all four aromatic methoxy groups.
The most promising compound (9), designated as ACT-
335827, was selected for further in vitro and in vivo characteri-
zation (Scheme 3). To determine the specific mode of antago-
Scheme 3. (R)-2-((S)-1-(3,4-Dimethoxybenzyl)-6,7-dimethoxy-3,4-dihydroiso-
quinolin-2(1H)-yl)-N-isopropyl-2-phenylacetamide (ACT-335827; 9). For a de-
tailed description of the synthesis, see the Supporting Information. IC50
values and brain penetration data were determined as described in Table 2.
Figure 1. CHO cells expressing a) OXR-1 and b) OXR-2 were pre-incubated
with dilution series of ACT-335827 for 120 min followed by the addition of
a dilution series of orexin-A (OxA). After 60 min of stimulation, cells were
lysed and the inositol-1-phosphate (IP1) content was determined. Data
shown are the mean ÆSEM of n=4 experiments.
nism of ACT-335827 at OXR-1 and OXR-2, concentration–re-
sponse curves (CRCs) to OxA were generated in the presence
of increasing concentrations of ACT-333827 using inositol-1-
phosphate accumulation assays in CHO cells expressing either
OXR-1 or OXR-2 (Figure 1). ACT-335827 caused equidistant
rightward shifts in the CRCs of OxA in both cell lines, dem-
onstrating a competitive mode of antagonism by ACT-
335827 at both OXR-1 and OXR-2. Schild Kb values were
41 nm at OXR-1 and 560 nm at OXR-2, confirming the se-
with brain/plasma ratios of 0.29 to 0.93 (Table 3), indicating
sufficient exposure for pharmacological testing. A broad mo-
lecular target screen, performed by MDS Pharma Services
Table 3. Plasma and brain concentrations of ACT-335827 after oral dosing in
Wistar rats.[a]
Dose:
100 mgkgÀ1
[B]
300 mgkgÀ1
[B]
[nggÀ1
]
lectivity of the compound as determined by calcium re-
lease measurements. ACT-335827 was also characterized
using radioligand displacement assays[12] and displayed
IC50 values of 120 nm (OXR-1) and 2300 nm (OXR-2).
[c]
[c]
Time[b]
[h]
[P]
[ngmLÀ1
B/P Bfree
ratio [nm]
[P]
[ngmLÀ1
B/P Bfree
ratio [nm]
]
[nggÀ1
]
]
1
3
6
1874Æ327 783Æ211 0.42 30
2326Æ167 862Æ140 0.37 33
1712Æ206 489Æ45 0.29 19
3380Æ375 2526Æ486 0.75 97
4167Æ261 3820Æ725 0.92 147
4635Æ276 4318Æ707 0.93 166
The free fraction of ACT-335827 was 3–4% in rat and
~1% in human plasma, the free fraction in a rat brain ho-
mogenate binding assay was ~2%. Rat brain concentra-
tions after oral dosing at 100 mgkgÀ1 were above
489 nggÀ1 (calculated free concentration:19 nm), and at
300 mgkgÀ1 rat brain concentrations were above
2526 nggÀ1 (97 nm free), for a duration of more than 6 h
24
n.d.
n.d.
n.d.
–
38Æ9
12Æ4
0.32
1
[a] Data represent the mean ÆSEM of n=4–6 animals per dose and time point.
Vehicle = 0.5% methyl cellulose. [b] Time after oral gavage. [c] Brain free fraction
(Bfree) concentrations are calculated based on 2% free in a brain homogenate
binding assay. Abbreviations: plasma (P), brain (B), brain free (Bfree), not deter-
mined (n.d.).
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(Taipei, Taiwan), revealed ACT-335827 exhibited no considera-
ble activities at 10 mm against a panel of more than 110 differ-
ent enzymes, receptors and ion channels—only binding to the
melatonin MT1 receptor revealed >50% inhibition (58%).
Almorexant, a dual OXR antagonist, decreases conditioned
fear and autonomous nervous system reactions to stress.[13,14]
The possibility that these effects partly result from OXR-1 inhibi-
tion was tested in rats orally gavaged with 30, 100 or
300 mgkgÀ1 of ACT-335827. In the fear-potentiated startle
paradigm, where rats react with an enhanced startle reflex
when exposed to noise stimuli during a light cue (conditioned
stimulus), which they have been trained to associate with an
unpleasant electric foot shock, ACT-335827 at 300 mgkgÀ1 se-
lectively decreased fear-induced startle reactions (Figure 2)
with a similar magnitude to almorexant.[14]
stress-induced hyperthermia at 300 mgkgÀ1 and tachycardia at
100 and 300 mgkgÀ1, without affecting locomotion and blood
pressure (Figure 3). A dual OXR antagonist recently identified
by Actelion was tested in the same paradigm and similarly de-
creased social stress-induced hyperthermia and tachycardia.[15]
Almorexant also decreases cardiovascular responses to psycho-
logical stressors such as novelty or contextual fear.[13]
ACT-335827 did not affect social interaction under nonstress
conditions, indicating intact sensorimotor function (see Sup-
porting Information). In the elevated plus maze assay, an anxi-
ety paradigm based on approach–avoidance, where both the
OXR-1 antagonist SB-334867[16] as well as almorexant were
found to be inactive,[14] ACT-335827 had also no effect (see
Supporting Information). In the rat schedule-induced polydip-
sia model of obsessive compulsive disorder, ACT-335827 at
300 mgkgÀ1 decreased excessive adjunctive, non-goal-directed,
compulsive drinking behavior, without influencing goal-direct-
ed behavior (head entries), similar to the selective serotonin-re-
uptake inhibitor fluoxetine (Figure 4a–d). ACT-335827 did not
affect water intake in the home cage (Figure 4e).
Sleep promotion is believed to result from either OXR-2
blockade or the simultaneous blockade of both OXRs.[1,2] Ac-
cordingly, blocking OXR-1 with 300 mgkgÀ1 of ACT-335827 in
telemetrized rats, allowing electro-encephalographic/myo-
graphic recordings, revealed no influence on the duration and
proportion of sleep and wake stages (Figure 5). As expected,
this is in contrast to the dual OXR antagonist almorexant,
which increases both non-REM and REM sleep.[2] However, simi-
lar to almorexant,[17,18] ACT-335827 did not impair forepaw grip
strength, spontaneous and forced locomotion, or cognitive
function (see Supporting Information).
Figure 2. Rats were treated (po), with vehicle (0 mgkgÀ1) or increasing doses
of ACT-335827, 1 h before testing in the fear-potentiated startle paradigm.
Mean startle amplitudes were assessed upon presentation of the fear-elicit-
ing conditioned light stimulus (CS) or under dark baseline control conditions
(no CS). Data shown are the mean ÆSEM of n=16 animals in a within-sub-
jects cross-over design. # <0.05 vs. 0 mgkgÀ1 (CS).
In summary, our structure–activity characterization of
a novel series of selective OXR-1 antagonists, represented by
(phenyl)glycine-amide substituted tetrahydropapaverine deriv-
atives, has led to the identification of ACT-335827 (9) as
a potent, selective and brain-penetrating agent. The com-
pound was highly selective for OXR-1 across common neuronal
targets, showed a pharmacokinetic profile suitable for oral in
vivo application, and exerted certain anxiolytic-like effects in
the rat without affecting motor or cognitive function, similar to
Control startle reactions to noise in the dark (without condi-
tioned light cue) remained unaffected by ACT-335827. In a resi-
dent–intruder paradigm involving social interaction stress be-
tween two male rats, ACT-335827 significantly decreased
Figure 3. Telemetrized rats were treated (po), with vehicle (0 mgkgÀ1) or increasing doses of ACT-335827, 1 h before testing in the social stress-induced hyper-
thermia paradigm. a) Average locomotion, b) body temperature, c) heart rate, and d) blood pressure were assessed during the stress procedure lasting 1 h.
Dotted lines represent the respective baseline values before treatment and stress induction. Data shown are the mean ÆSEM of n=11 animals in a within-
subjects cross-over design; * <0.001 vs. vehicle control (0 mgkgÀ1).
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Figure 4. Rats were treated (po), with vehicle (0 mgkgÀ1) or ACT-335827 (300 mgkgÀ1) or fluoxetine (30 mgkgÀ1), 1 h before testing in the schedule-induced
polydipsia paradigm. The average fluid intake (panels a and b) and the number of head entries (panels c and d) into the food dispenser were monitored
during the 2 h test session. Control home cage water intake with ACT-335827 was assessed over the 12 h dark period (panel e). Data shown are the mean
ÆSEM of n=11–15 animals in a within-subjects cross-over design; * <0.05 vs. control (0 mgkgÀ1).
Figure 5. Telemetrized rats were treated (po) with vehicle or ACT-335827 (300 mgkgÀ1) at the end of the light phase, and the time spent in a) active wake,
b) quiet wake, c) non-REM sleep, and d) REM sleep was assessed during the following 12 h. Data shown are the mean ÆSEM of n=8 animals in a within-sub-
jects cross-over design. ACT-335827 had no significant effect on the duration of wake and sleep stages.
the dual OXR antagonist almorexant. However, in contrast to
Keywords: anxiety
· neurotransmitters · orexin receptor
almorexant, ACT-335827 did not decrease wakefulness. ACT-
335827 may serve as a novel pharmacological tool to further
explore the specific role of OXR-1 signaling in physiology and
behavior.
antagonists · peptides · sleep
[1] T. Sakurai, M. Mieda, Trends Pharmacol. Sci. 2011, 32, 451–462.
[2] C. Brisbare-Roch, J. Dingemanse, R. Koberstein, P. Hoever, H. Aissaoui, S.
Flores, C. Mueller, O. Nayler, J. van Gerven, S. L. de Haas, P. Hess, C. Qiu,
S. Buchmann, M. Scherz, T. Weller, W. Fischli, M. Clozel, F. Jenck, Nat.
Med. 2007, 13, 150–155.
Experimental Section
[3] P. Bettica, L. Squassante, S. Zamuner, G. Nucci, H. Danker-Hopfe, E. Ratti,
Sleep 2012, 35, 1097–1104.
[4] C. J. Winrow, A. L. Gotter, C. D. Cox, S. M. Doran, P. L. Tannenbaum, M. J.
Breslin, S. L. Garson, S. V. Fox, C. M. Harrell, J. Stevens, D. R. Reiss, D. Cui,
P. J. Coleman, J. J. Renger, J. Neurogenet. 2011, 25, 52–61.
See the Supporting Information for descriptions of the methods
and detailed results with additional figures concerning the chemi-
cal, biological, and pharmacological experiments. All experimental
procedures involving animals were approved by the Basel-Land-
schaft Veterinary Office and strictly adhered to Swiss federal regula-
tions on animal experimentation.
[5] A. L. Gotter, A. L. Webber, P. J. Coleman, J. J. Renger, C. J. Winrow, Phar-
macol. Rev. 2012, 64, 389–420.
[6] R. A. Porter, W. N. Chan, S. Coulton, A. Johns, M. S. Hadley, K. Widdows-
on, J. C. Jerman, S. J. Brough, M. Coldwell, D. Smart, F. Jewitt, P. Jeffrey,
N. Austin, Bioorg. Med. Chem. Lett. 2001, 11, 1907–1910.
[7] S. R. Morairty, F. G. Revel, P. Malherbe, J. L. Moreau, D. Valladao, J. G.
Wettstein, T. S. Kilduff, E. Borroni, PLoS One 2012, 7, e39131.
[8] R. Koberstein, H. Aissaoui, D. Bur, M. Clozel, W. Fischli, F. Jenck, C. Muel-
ler, O. Nayler, T. Sifferlen, A. Treiber, T. Weller, Chimia 2003, 57, 270–275.
[9] 1,2,3,4-Tetrahydroisoquinoline Derivatives, H. Aissaoui, M. Cappi, M.
Clozel, W. Fischli, R. Koberstein, (Actelion Pharmaceuticals Ltd, Allschwil,
Switzerland), PCT Int. Pat. Appl. WO/2001/068609, 2001.
[10] Substituted 1,2,3,4-Tetrahydroisoquinoline Derivatives, T. Weller, R. Kober-
stein, H. Aissaoui, M. Clozel, W. Fischli, (Actelion Pharmaceuticals Ltd,
Allschwil, Switzerland), PCT Int. Pat. Appl. WO/2005/118548, 2005.
[11] C. Boss, C. Brisbare-Roch, F. Jenck, H. Aissaoui, R. Koberstein, T. Sifferlen,
T. Weller, Chimia 2008, 62, 974–979.
Acknowledgements
For expert technical and conceptual support, the authors thank
Ursula Fusco-Hug, Pascal Rebmann, Stephane Delahaye, Stepha-
nie Hertzog, Julia Friedrich, Isabelle Weber, Cedric Fischer, Malvi-
na Louis, Helene Roellinger, Hugues Lecourt, Carla Sciarretta, Re-
bekka Pietrek, Monica Gaiffi, Thomas Sasse, Celia Mꢀller-Grand-
jean, Katalin Menyhart, Markus von Raumer, Bruno Capeleto, and
Roberto Bravo (all employees of Actelion Pharmaceuticals Ltd,
Allschwil, Switzerland).
[12] C. D. Cox, M. J. Breslin, D. B. Whitman, J. D. Schreier, G. B. McGaughey,
M. J. Bogusky, A. J. Roecker, S. P. Mercer, R. A. Bednar, W. Lemaire, J. G.
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2013, 8, 898 – 903 902

CHEMMEDCHEM
COMMUNICATIONS
www.chemmedchem.org
Bruno, D. R. Reiss, C. M. Harrell, K. L. Murphy, S. L. Garson, S. M. Doran, T.
Prueksaritanont, W. B. Anderson, C. Tang, S. Roller, T. D. Cabalu, D. Cui,
G. D. Hartman, S. D. Young, K. S. Koblan, C. J. Winrow, J. J. Renger, P. J.
Coleman, J. Med. Chem. 2010, 53, 5320–5332.
(Actelion Pharmaceuticals Ltd, Allschwil, Switzerland), PCT Int. Pat. Appl.
WO/2012/025877, 2012.
[16] R. J. Rodgers, F. L. Wright, N. F. Snow, L. J. Taylor, Behav. Brain Res. 2013,
243, 213–219.
[17] M. A. Steiner, H. Lecourt, D. S. Strasser, C. Brisbare-Roch, F. Jenck, Neuro-
psychopharmacology 2011, 36, 848–856.
[13] T. M. Furlong, D. M. Vianna, L. Liu, P. Carrive, Eur. J. Neurosci. 2009, 30,
1603–1614.
[18] H. Dietrich, F. Jenck, Psychopharmacology 2010, 212, 145–154.
[14] M. A. Steiner, H. Lecourt, F. Jenck, Psychopharmacology 2012, 223, 465–
75.
[15] Proline Sulfonamide Derivatives as Orexin Receptor Antagonists, C. Boss,
C. Brotschi, J. Gatfield, M. Gude, B. Heidmann, T. Sifferlen, J. T. Williams,
Received: January 2, 2013
Published online on April 15, 2013
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