Discovery of 1H-pyrazolo[3,4-b]pyridines as potent dual orexin receptor antagonists (DORAs)

Article abstract of DOI:10.1016/j.bmcl.2015.10.055

Compound rac-1 was identified by high throughput screening. Here we report SAR studies and MedChem optimization towards the highly potent dual orexin receptor antagonists (S)-2 and (S)-3. Furthermore, strategies to overcome the suboptimal physicochemical properties are highlighted and the pharmacokinetic profiles of representative compounds is presented.

Full text of DOI:10.1016/j.bmcl.2015.10.055

Bioorganic & Medicinal Chemistry Letters 25 (2015) 5555–5560
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 1H-pyrazolo[3,4-b]pyridines as potent dual orexin
receptor antagonists (DORAs)
a
a
b
b
Dirk Behnke a, , Simona Cotesta , Samuel Hintermann , Markus Fendt , Christine E. Gee ,
Laura H. Jacobson b,f, Grit Laue c, Arndt Meyer a, Trixie Wagner a, Sangamesh Badiger d, Vinod Chaudhari d,
Murali Chebrolu d, Chetan Pandit d, Daniel Hoyer b,e,f, Claudia Betschart a
⇑
a Global Discovery Chemistry, Novartis Institutes for BioMedical Research, WKL-136.3.26, CH-4002 Basel, Switzerland
b Neuroscience, Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland
c Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland
d Medicinal Chemistry, Aurigene Discovery Technologies Limited, 39-40, KIADB Industrial Area, Phase II, Electronic City, Hosur Road, Bangalore 560100, India
e School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Parkville, Victoria 3010, Australia
f The Florey Institute of Neuroscience and Mental Health, Parkville Campus, Kenneth Myer Building at Genetics Lane, on Royal Parade, University of Melbourne, Parkville,
3010, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Compound rac-1 was identified by high throughput screening. Here we report SAR studies and MedChem
optimization towards the highly potent dual orexin receptor antagonists (S)-2 and (S)-3. Furthermore,
strategies to overcome the suboptimal physicochemical properties are highlighted and the pharmacoki-
netic profiles of representative compounds is presented.
Received 13 August 2015
Revised 16 October 2015
Accepted 18 October 2015
Available online 19 October 2015
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
Orexin
GPCRs
Insomnia
Antagonists
The orexin receptor 1 (OX1R) and the orexin receptor 2 (OX2R)
are two G-protein coupled receptors widely expressed in the brain.
The two endogenous neuropeptide agonists, orexin-A and orexin-B
(also known as hypocretin 1 and hypocretin 2), are selectively
expressed in a small cluster of neurons in the lateral hypothala-
mus.1,2 Activation of the orexin system by these peptides promotes
wakefulness in animals and humans.3–5 Consequently, the discov-
ery of OX1R and OX2R antagonists has become a major focus of
research for the treatment of (primary) insomnia over the past
years. Positive clinical proof of concept (PoC) were achieved with
Merck’s dual OX1,2R antagonists (DORAs) suvorexant (MK-4305)
and filorexant (MK-6096), Actelion’s almorexant (ACT-078573)
and Glaxo SmithKline’s SB-649868 (Fig. 1).6–11 In 2014 suvorexant
(tradename BELSOMRAÒ) was approved in the U.S. and Japan as the
first orexin antagonist for the treatment of primary insomnia.
In 2007, a drug discovery project was initiated at Novartis with
the aim to identify dual orexin receptor antagonists. Here we
report the discovery, SAR and optimization of a series of novel
substituted 1H-pyrazolo[3,4-b]pyridines as potent DORAs.
rac-1 was identified as a promising hit in a high throughput
screen (HTS) of the Novartis compound collection. It showed dual
potency on both orexin receptors in the cell-based functional Cal-
cium accumulation assay (as measured by FLIPR, fluorometric
imaging plate reader).12 Receptor binding was used as secondary
screen to confirm that rac-1 binds with similar potency to both
orexin receptors (Fig. 2).13 The series was considered a good
starting point for further optimization.
The initial SAR studies focused mainly on the amide motif of the
molecule exploring the influence of modifications on potency and
selectivity for both the hOX1R and hOX2R. A diverse set of deriva-
tives with differently substituted aryl/heteroaryl substituents was
synthesized (Table 1). The most promising results were obtained
with chiral benzylic aryl/heteroaryl amine substituents. The para
methoxy derivative rac-2 and the pyrazole compound rac-3 were
found to be most potent on the hOX2R with rac-2 showing a slight
trend for hOX2R selectivity. Compound rac-2 containing a para-
methoxy substituent were more potent on both orexin receptors
than rac-4 with a methoxy substituent in ortho position. A compar-
ison of rac-2 with the meta methoxy substituted compound rac-5
showed equivalent affinity for the hOX1R and 10-fold improved
affinity for the hOX2R. Compounds with electron poor heteroaryls
⇑
Corresponding author. Tel.: +41 61 696 29 59; fax: +41 61 696 23 30.
E-mail address: dirk.behnke@novartis.com (D. Behnke).
http://dx.doi.org/10.1016/j.bmcl.2015.10.055
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

5556
D. Behnke et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5555–5560
benzoate 14 with acetonitrile using sodium hydride as base
yielded the -cyano ketone 15. Ring closure to the corresponding
a
Cl
N
O
N
N
N
amino pyrazole 16 was achieved with methyl hydrazine in metha-
nol in moderate yield. For derivatives with other substituents at
the 3-position of the pyrazolopyridine core, the sequence was
started with the corresponding aryl/heteroaryl ester analogs of
14. Microwave-assisted reaction of 16 with ethyl 4,4,4-trifluoro-
3-oxobutanoate in acetic acid gave compound 17. The alkylation
of 17 with ethyl 2-bromoacetate using sodium hydride in DMF
selectively gave the O-alkylation product 18. Compound 18 was
subsequently subjected to ester hydrolysis yielding compound
19, which was the precursor for the last amide coupling step. If
available, enantiomeric pure amines with known absolute configu-
ration were used for the amide formation, otherwise chiral separa-
tions were performed after the final step (e.g., for compound (S)-3).
In such cases the absolute configuration was assigned based on the
biological activity.
N
N
N
O
O
O
N
N
N
F
Filorexant
(MK-6096)
Suvorexant
(MK-4305)
CF3
H
N
O
N
O
O
N
NH
O
N
O
O
S
F
The overall in vitro profiles of compounds (S)-2 and (S)-3 are
illustrated in Table 2. Both compounds are potent dual orexin
receptor antagonists as measured in the functional Calcium as well
as radioligand binding assays. Although high potencies were
achieved, the physicochemical properties were less than optimal.
Both (S)-2 and (S)-3 have high lipophilicity (HT-log P of 4.60 and
4.20) resulting in low solubility measured in a high throughput for-
mat at pH 6.8. Permeation measured in a PAMPA assay was high,
indicting they are class II compounds based on the Biopharmaceu-
tics Classification System (BCS).16 The CYP inhibition profile of
compound (S)-3 was better than that of compound (S)-2, for which
SB-649868
Almorexant
(ACT-078573)
Figure 1. DORAs that have achieved positive clinical proof of concept.
such as the pyridine analogs rac-6 and rac-7 were less favored. The
highly potent hOX2R antagonists rac-2 and rac-3 were subjected to
chiral separation yielding compounds (S)-2/(R)-2 and (S)-3/(R)-3. In
parallel, compounds (S)-2 and (R)-2 were synthesized using com-
mercially available enantiomerically pure (S) and (R)-1-(4-methox-
yphenyl)ethanamine. Compound (S)-2 was found to be the active
enantiomer whereas compound (R)-2 lost activity on both orexin
receptors. Likely, the (R)-configuration of the methyl group forced
the compounds into a conformation that is unfavorable for binding
to the orexin receptors. Compound 8 was more than one order of
magnitude less potent than (S)-2 on both receptors, further sup-
porting the importance of the properly configured methyl group.
By analogy, the absolute configuration in compounds (S)-3 and
(R)-3 obtained by chiral separation of the enantiomers were
assigned based on their potency on hOX1R and hOX2R. Removal
of the core N–Me in (S)-9 gave compound (S)-10 which was
equipotent on both hOX1R and hOX2R suggesting that the N–H
cannot engage in a hydrogen bond interaction with the orexin
receptors. Interestingly, replacing the ether oxygen in the side
chain by NH was detrimental for potency on the hOX2R (10-fold
loss) whereas for hOX1R only a marginal shift was observed,
resulting in an hOX1R preferring antagonist (see compound (S)-
11 vs (S)-9). Methylation of the pyrazole of (S)-3 resulted in com-
pound (S)-12 where dual potency could be maintained in a desired
range and an active transport mechanism as the most likely reason
for unfavorable brain penetration of (S)-3 was significantly
suppressed (see also paragraph in vivo characterization and PK
properties). More drastic changes at the core to reduce lipophilicity
as exemplified by compound (S)-13 led to loss of potency on both
receptors compared to (S)-2 and (S)-3.
CYP3A4 and CYP2C9 were inhibited with IC50’s of 8 and 5 lM,
respectively. Both compounds had high in vitro clearances in rat,
human and mouse liver microsomes and high plasma protein
binding, leading to plasma free-fractions of less than 1% in mouse.
Compounds (S)-2 and (S)-3 had no hERG flag and an excellent
in vitro selectivity and off-target profile.
With highly potent compounds in hand, in vivo characterization
was initiated. The blood pharmacokinetic (PK) and brain penetra-
tion properties of compounds (S)-2 and (S)-3 were tested in the
mouse (Table 3) in a cassette dosing setup; i.e., five experimental
compounds and
a reference compound were administered
together intravenously at 1 mg/kg and per os at 3 mg/kg. Com-
pound (S)-2 exhibited a medium blood clearance, a moderate vol-
ume of distribution and a relatively short terminal half-life. The
absolute oral bioavailability was very low (F = 3%) but the brain/
blood concentration ratio (br/bl = 1.15) indicated favorable brain
penetration. For compound (S)-3 the blood clearance was low
and, in contrast to compound (S)-2, the oral bioavailability was
moderate (F = 21%), and as such the AUC of (S)-3 after oral dosing
was higher than that of compound (S)-2. The brain/blood concen-
tration ratio of 0.02 for compound (S)-3 indicated a very low brain
penetration (if at all). A possible reason might be P-glycoprotein
(P-gp) activity as measured in the MDCK assay. Compound (S)-3
showed a BA/AB efflux ratio of 23 in human MDCK cells stably
expressing MDR1 and is therefore
a human P-glycoprotein
The synthesis of compound (S)-3 as a representative example
substrate, which is likely to also be the case in mice. The efflux
of compound (S)-3 could be considerably reduced for compound
(S)-12 (Table 1) with an efflux ratio of 3.
for this class is shown in Scheme 1.14 Ester condensation of methyl
Compound (S)-3 showed a limited correlation between in vitro
and in vivo clearance (high in vitro, low in vivo). A potential
hypothesis for this disconnect is a restrictive binding to plasma
proteins (very high plasma protein binding of >99% in mice), which
might protect the compound from metabolism in vivo.
The next round of optimization aimed to improve the physico-
chemical properties, in particular reducing lipophilicity and
improving solubility, with the goal of generating compounds with
improved PK properties. In order to achieve this, we initially
focused on the optimization and profiling of compound (S)-9.
CF3
N
FLIPR: hOX1R Ki = 120 nM
hOX2R Ki = 78 nM
H
N
N
binding: hOX1R Ki = 88 nM
hOX2R Ki = 168 nM
N
O
O
1
rac-
Figure 2.

D. Behnke et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5555–5560
5557
Table 1
Amide moiety SAR
R3
N
R4
N
H
N
R1
X
N
R2
O
Compd
R1
R2
R3
R4
X
hOX1R Calcium Ki (nM)
hOX2R Calcium Ki (nM)
Ratio hOX1R/hOX2R
rac-1
Me
CF3
Ph
O
O
O
O
120
78
1.54
14.50
1.33
1.73
O
rac-2
rac-3
rac-4
Me
Me
Me
CF3
CF3
CF3
Ph
Ph
Ph
29
4
2
N
HN
3
O
274
158
O
rac-5
Me
CF3
Ph
O
20
26
0.77
rac-6
rac-7
Me
Me
CF3
CF3
Ph
Ph
O
O
2667
970
No inh.
582
—
N
N
1.67
O
(S)-2
(R)-2
Me
Me
CF3
CF3
Ph
Ph
O
O
14
2
7.00
2.41
O
2287
949
N
HN
(S)-3
(R)-3
Me
Me
CF3
CF3
Ph
Ph
O
O
5
2
2.50
—
N
HN
>4000
>2000
O
8
Me
Me
H
CF3
CF3
CF3
CF3
CF3
CH3
Ph
O
800
175
200
357
7
83
9.64
1.62
4.65
0.31
0.70
4.95
(S)-9
(S)-10
(S)-11
(S)-12
(S)-13
Ph
O
108
43
Ph
O
Me
Me
Me
Ph
NH
O
1135
10
N
N
Ph
cyclo-propyl
O
109
22
N

5558
D. Behnke et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5555–5560
H2N
O
OMe
N
N
O
CN
CF3
(i)
(ii)
(iii)
N
N
O
N
H
14
15
16
17
CF3
CF3
(iv)
(v)
N
N
HO
O
N
N
O
N
O
N
O
O
18
19
CF3
N
(vi), (vii)
N
H
N
N
HN
N
O
O
3
(S)-
Scheme 1. Reagents and conditions: (i) NaH, DMSO, MeCN, rt, 66%; (ii) MeNHNH2, MeOH, 110 °C, 57%; (iii) CF3COCH2COOEt, AcOH, 150 °C, microwave, 64%; (iv) BrCH2COOEt,
NaH, DMF, rt, 77%; (v) aq LiOH, EtOH/THF, rt, 92%; (vi) (rac)-1-(3,5-dimethyl-1H-pyrazol-4-yl)ethanamine, HOBt, DIC, DMF, 80 °C; (vii) chiral separation on HPLC, 23% (2
steps).
Table 2
In vitro profile of compounds (S)-2 and (S)-3
Property
(S)-2
(S)-3
hOX1R Calcium Ki (nM)
hOX2R Calcium Ki (nM)
hOX1R binding Ki (nM)
hOX2R binding Ki (nM)
HT-log P15
14
2
8
4
5
2
3
8
4.60
4.20
HT-eq. sol (pH 6.8) (mM)
Log PAMPA
<0.004
À4
<0.004
À4.1
MDCKa BA/AB efflux ratio
CYP inh. 3A4/2D6/2C9 IC50
1
23
(l
M)
8/>20/5
147/190/478
>99/>99
>30/n.d.
0/108b
>20/>20/>20
603/433/330
96/>99
>30/>30
0/112b
Clint
PPB (%) h/m
hERG IC50
Number off-targets with IC50 < 1
(lL/min * mg) RLM/HLM/MLM
(lM) binding/QPatch
lM
a
PgP-mediated transport of compounds was tested in a medium-throughput assay in 24-well plates using MDCK cells stably expressing MDR1 (Madin–Darby Canine
Kidney cells retrovirally transduced with the human multidrug resistance gene 1; P-glycoprotein PgP).
b
Compounds were tested against a number of safety relevant off-targets (108 for (S)-2) and 112 for (S)-3) including receptors, proteases, kinases etc.; no off-target was
affected by (S)-2 and (S)-3 at IC50’s below 1 lM.
Table 3
Mouse in vivo PK data of selected compoundsb
Compd
Intravenous dose 1 mg/kgc
Oral dose 3 mg/kgd
Clbl (mL minÀ1 kgÀ1
)
Vss (L/kg)
t1/2term (h)
Cmax,bl d.n. (nM)
AUCbl d.n. (nM * h)
Ff (%)
[br]/[bl] at 1 h p.d. SDg
e
(S)-2
(S)-3
(S)-13
65
20
66
2.0
0.5
0.4
1.1
0.5
0.3
4
300
115
17
371
106
3
21
16
1.15 0.24
0.02 0.01
0.13 0.02
b
The absorption and disposition parameters were estimated by a non-compartmental analysis of the mean blood concentration (n = 3) versus time profile after oral and
intravenous administration; dosing was in a cassette of 6 compounds.
c
Dosed as a solution in NMP/blank plasma (10:90, v/v).
Dosed as a suspension in carboxymethylcellulose 0.5% w/v in water/Tween 80 (99.5%/0.5%, v/v).
AUC was extrapolated to infinity.
d
e
f
Absolute oral bioavailability was calculated by dividing the dose-normalized AUC after oral and intravenous dosing assuming linear pharmacokinetics between these 2
doses.
g
Mean brain/blood concentration ratio from n = 3; d.n. = dose-normalized, i.e., calculated to a dose of 1 mg * kgÀ1
.

D. Behnke et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5555–5560
5559
Table 4
Compounds with improved physicochemical properties
Compd
Structure
hOX1R Calcium Ki (nM)
hOX2R Calcium Ki (nM)
HT-log P
HT-eq. sol (pH 6.8) (mM)
N
CF3
N
(S)-20
660
367
3.50
0.04
H
N
N
N
N
O
O
N
(S)-21
(S)-22
4
5
3.30
3.90
0.043
0.115
H
N
N
N
O
N
O
N
N
52
15
H
N
N
N
N
O
O
N
F
O
O
F
N
N
rac-23
(S)-24
778
783
527
93
2.80
3.50
0.657
0.025
H
N
N
N
N
H
N
N
N
O
N
O
F
F
N
N
rac-25
63
59
3.90
0.248
H
N
N
N
O
N
O
and the amide NH. In addition, strong intermolecular aromatic
interactions are present (indicated by the arrows, Fig. 3). The core
phenyl in 3-position is involved in ‘edge to face’ interactions. The
low solubility of compound (S)-9 is therefore the result of a combi-
nation of high crystal lattice energy (reflected by the melting point
of 162–164 °C) and high lipophilicity (as shown by the HT-log P of
4.7). Unfortunately, attempts to reduce crystal lattice energy
through interruption of the hydrogen bond by N-alkylation led to
a loss in potency. Consequently, another strategy aimed at reduc-
ing stacking by adding non-aromatic moieties in the 3-position
(see compounds rac-23 and (S)-24). Results are shown in Table 4.
The comparison of the pyridyl compound (S)-20 with (S)-9 indi-
cates a significantly reduced lipophilicity (HT-log P of 4.7 for (S)-9
vs 3.50 for (S)-20) also resulting in improved solubility of 0.04 mM
at pH 6.8. The potencies on both orexin receptors slightly
decreased but could be restored by introducing a methyl group
in the para position of the amide aryl. This substitution in combi-
nation with the pyrimidine substituent in 3-position resulted in
the highly potent dual orexin receptor antagonist (S)-21 with
improved HT-log P of 3.30 and a solubility of 0.043 mM. The pyra-
zine analog (S)-22 showed better solubility but was less potent on
hOX1R and hOX2R. Replacement of the aromatic 3-substituent by a
cyclopropyl group was partially successful at improving the solu-
bility as shown for rac-23 but this improvement often occurred
at the expense of reduced potencies on hOX1R and hOX2R. A suc-
cessful strategy to keep the potency in a desired range but reduce
the lipophilicity was the modification of the CF3 group by CH3 and
CHF2 replacements. The most pronounced effect for lowering the
Figure 3. X-ray crystal interaction of compound (S)-9.
The in vitro biotransformation of compound (S)-9 after incuba-
tion with mouse and human liver microsomes and the in vivo
biotransformation in mouse revealed two major metabolic path-
ways: oxidation at the benzylic position of the amide part and oxi-
dation at the phenyl ring in 3-position of the core (data of
biotransformation studies not shown). Attempts to address the
metabolic weak spot at the amide benzylic position by introducing
non-aromatic moieties and by altering the length of the amide side
chain were less successful. Compounds (S)-20, (S)-21 and (S)-22
(Table 4) were therefore designed to reduce the potential for oxida-
tion at the phenyl ring by introducing electron poor aryls. These
variations, in parallel, were beneficial for improving the log P. A
small molecule X-ray analysis of compound (S)-917 shows an inter-
molecular hydrogen bond interaction between the amide carbonyl

5560
D. Behnke et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5555–5560
log P was achieved with the CHF2 group and the lipophilicity usu-
ally decreased in the order CF3 > CH3 > CHF2. Although the solubil-
ity could be improved for some compounds (see e.g., rac-23 and
rac-25 in Table 4), this alteration did not translate in an improved
in vitro metabolism. In general, all modifications mentioned above
aimed at improving the physicochemical properties were not
References and notes
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. S.;
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.
2. De Lecea, L.; Kilduff, T. S.; Peyron, C.; Gao, X.-B.; Foye, P. E.; Danielson, P. E.;
Fukuhara, C.; Battenberg, E. L. F.; Gautvik, V. T.; Bartlett, F. S., II; 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.
accompanied by reduced metabolism in vitro (Clint
(lL/min * mg)
in RLM: 924 for (S)-20, 660 for rac-23, 924 for (S)-24).
In conclusion, we have identified a series of 1H-pyrazolo[3,4-b]
pyridines as potent orexin receptor antagonists by high throughput
screening. Fast exploration of the SAR led to the dual orexin recep-
tor antagonists (S)-2 and (S)-3 with high functional and binding
potencies at hOX1R and hOX2R. Compounds (S)-2 and (S)-3 have
shown a favorable off-target profile. PK studies revealed that the
compounds have low to moderate clearance. Bioavailability and
brain penetration were in a low to moderate range. The suboptimal
physicochemical properties could be improved and compounds
with improved solubility and lipophilicity features were identified.
Overall, however, combining favorable in vitro pharmacology and
in vivo PK properties remains challenging in this series. Compound
(S)-13 was found to be one of the best compromises between blood
exposure and sufficient brain penetration to achieve brain expo-
sure after oral dosing.
3. Sakurai, T.; Ohno, K. Front. Neuroendicrinol. 2008, 29, 70.
4. Roecker, A. J.; Coleman, P. J. Curr. Top. Med. Chem. 2008, 8, 977.
5. Coleman, P. J.; Renger, J. J. Expert Opin. Ther. Pat. 2010, 20, 307.
6. Winrow, C. J.; Gotter, A. L.; Cox, C. D.; Doran, S. M.; Tannenbaum, P. L.; Breslin,
M. J.; Garson, S. L.; Fox, S. V.; Harrell, C. M.; Stevens, J.; Reiss, D. R.; Cui, D.;
Coleman, P. J.; Renger, J. J. J. Neurogenet. 2011, 25, 52.
7. Herring, W. J.; Snyder, E.; Budd, K.; Hutzelmann, J.; Snavely, D.; Liu, K.; Lines, C.;
Roth, T.; Michelson, D. Neurology 2012, 79, 2265.
8. 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.
9. Hoyer, D.; Jacobson, L. H. Neuropeptides 2013, 47, 477.
10. Kuduk, S. D.; Winrow, C. J.; Coleman, P. J. Annu. Rep. Med. Chem. 2013, 48, 73.
11. Bettica, P.; Nucci, G.; Pyke, C.; Squassante, L.; Zamuner, S.; Ratti, E.; Gomeni, R.;
Alexander, R. J. Psychopharmacol. 2012, 26, 1058.
12. Ca2+ accumulation (as measured by FLIPR, fluorometric imaging plate reader)
was used as a functional readout of orexin receptor activity, for details see
WO2011076744.
13.
[
125I]-orexin A was used as radioligand in the binding assay, for details see:
Badiger, S.; Behnke, D.; Betschart, C.; Chaudhari, V.; Chebrolu, M.; Cotesta, S.;
Hintermann, S.; Meyer, A.; Pandit, C. Patent WO2011076744, 2011.
Acknowledgements
14. Experimental information: Detailed synthetic procedures are described. In
Badiger, S.; Behnke, D.; Betschart, C.; Chaudhari, V.; Chebrolu, M.; Cotesta, S.;
Hintermann, S.; Meyer, A.; Pandit, C. Patent WO2011076744, 2011.
15. Faller, B.; Grimm, H. P.; Loeuillet-Ritzler, F.; Arnold, S.; Briand, X. J. Med. Chem.
2005, 48, 2571.
16. Wu, C.-Y.; Benet, L. Z. Pharm. Res. 2005, 22, 11.
17. X-ray coordinates are deposited with the Cambridge Crystallographic Data
Centre: CCDC deposition number 1429189.
We thank J. Blanz, P. Ramstein, W. Gertsch and F. Blasco for the
biotransformation study of compound 13. Furthermore we thank
Richard Felber, Sandrine Liverneaux, Andrea Hasler, Alfred Tanner,
Thomas Gut, Marc Weibel, Fatma Limam, Marjorie Bourrel, Michael
Wright for synthetic support, Dominique Monna, Edi Schuepbach
and Daniel Langenegger for in vitro biological data, and Jürgen
Wagner for management support and helpful discussions.