Investigation of orexin-2 selective receptor antagonists: Structural modifications resulting in dual orexin receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2017.02.012

In an ongoing effort to explore the use of orexin receptor antagonists for the treatment of insomnia, dual orexin receptor antagonists (DORAs) were structurally modified, resulting in compounds selective for the OX₂R subtype and culminating in the discovery of 23, a highly potent, OX₂R-selective molecule that exhibited a promising in vivo profile. Further structural modification led to an unexpected restoration of OX₁R antagonism. Herein, these changes are discussed and a rationale for selectivity based on computational modeling is proposed.

Full text of DOI:10.1016/j.bmcl.2017.02.012

Accepted Manuscript
Investigation of orexin-2 selective receptor antagonists: structural modifications
resulting in dual orexin receptor antagonists
Jason W. Skudlarek, Christina N. DiMarco, Kerim Babaoglu, Anthony J.
Roecker, Joseph G. Bruno, Mark A. Pausch, Julie A. O'Brien, Tamara D. Cabalu,
Joanne Stevens, Joseph Brunner, Pamela L. Tannenbaum, W. Peter Wuelfing,
Susan L. Garson, Steven V. Fox, Alan T. Savitz, Charles M. Harrell, Anthony
L. Gotter, Christopher J. Winrow, John J. Renger, Scott D. Kuduk, Paul J.
Coleman
PII:
S0960-894X(17)30132-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.02.012
BMCL 24684
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 January 2017
3 February 2017
5 February 2017
Please cite this article as: Skudlarek, J.W., DiMarco, C.N., Babaoglu, K., Roecker, A.J., Bruno, J.G., Pausch, M.A.,
O'Brien, J.A., Cabalu, T.D., Stevens, J., Brunner, J., Tannenbaum, P.L., Peter Wuelfing, W., Garson, S.L., Fox,
S.V., Savitz, A.T., Harrell, C.M., Gotter, A.L., Winrow, C.J., Renger, J.J., Kuduk, S.D., Coleman, P.J., Investigation
of orexin-2 selective receptor antagonists: structural modifications resulting in dual orexin receptor antagonists,
Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.02.012
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Investigation of orexin-2 selective receptor antagonists:
structural modifications resulting in dual orexin receptor antagonists
Jason W. Skudlareka,*, Christina N. DiMarcoa, Kerim Babaoglub, Anthony J. Roeckera, Joseph G. Brunoc,
Mark A. Pauschc, Julie A. O’Brienc, Tamara D. Cabalud, Joanne Stevense, Joseph Brunnerf, Pamela L.
Tannenbaume, W. Peter Wuelfingg, Susan L. Garsonf, Steven V. Foxe, Alan T. Savitze, Charles M.
Harrellf, Anthony L. Gotterf, Christopher J. Winrowf, John J. Renger f, Scott D. Kuduk#, and Paul J.
Colemana
aDepartment of Medicinal Chemistry, MRL, Merck & Co., Inc., West Point, PA 19486, USA
bDepartment of Chemical Capabilities & Screening, MRL, Merck & Co., Inc., West Point, PA 19486, USA
cDepartment of In Vitro Pharmacology, MRL, Merck & Co., Inc., West Point, PA 19486, USA
dDepartment of Drug Metabolism, MRL, Merck & Co., Inc., West Point, PA 19486, USA
eDepartment of In Vivo Pharmacology, MRL, Merck & Co., Inc., West Point, PA 19486, USA
fDepartment of Neuroscience, MRL, Merck & Co., Inc., West Point, PA 19486, USA
gDiscovery Pharmaceutical Sciences, MRL, Merck & Co., Inc., West Point, PA 19486, USA
_______________
*
Corresponding author. Tel.: + 1-215-652-2748 ; fax: + 1-215-652-3971.
E-mail address: jason_skudlarek@merck.com
#Current affiliation Novira Therapeutics, Inc., a part of Janssen Pharmaceuticals, Doylestown, PA 18902, USA
Abstract
In an ongoing effort to explore the use of orexin receptor antagonists for the treatment of
insomnia, dual orexin receptor antagonists (DORAs) were structurally modified, resulting in compounds
selective for the OX2R subtype and culminating in the discovery of 23, a highly potent, OX2R-selective
molecule that exhibited a promising in vivo profile. Further structural modification led to an unexpected
restoration of OX1R antagonism. Herein, these changes are discussed and a rationale for selectivity based
on computational modeling is proposed.

Since 1999, the pharmaceutical industry has been targeting the design of orexin receptor
antagonists for the treatment of insomnia following the identification of orexin neuropeptides and
receptors. 1-2 Orexin secreting neurons in the lateral hypothalamus release excitatory neuropeptides (OX-
A and OX-B) that activate two G-protein-coupled receptors (GPCRs): orexin receptor 1 (OX1R) and
orexin receptor 2 (OX2R).3 Upon activation, these receptors promote wakefulness in a circadian manner,
with signals peaking during normal wake periods and falling dormant during sleep.4-5
Merck & Co., Inc., Kenilworth, NJ, USA received FDA and PMDA approval of suvorexant
(Belsomra®, Fig. 1) for the treatment of insomnia recently.6 In addition to suvorexant, several additional
DORAs and selective orexin receptor antagonists (SORAs) have also been disclosed by Merck,7-10 and
several excellent reviews of the orexin field have recently been published which describe additional
clinical compounds from SmithKline Beecham (now GlaxoSmithKline), Actelion, and Eisai.9,11-20
In a previously published article, we disclosed the identification of MK-8133 (1, Fig. 1), a potent
and selective orexin-2 receptor antagonist (OX2R SORA), for the treatment of insomnia.21 Therein, we
briefly described the general SAR trends which led us to a new series of potent OX2R SORAs, beginning
with the unexpected discovery that potent DORAs could be transformed into SORAs via specific
structural changes.22 In summation, it was found that piperidine ethers could be converted from DORAs
into OX2R SORAs (isoquinoline 2 and quinoline 3 respectively, Fig. 1) by moving the heteroatom from
the 2- to the 4-position. This observation inspired us to explore substituted pyridines as replacements (4
and 5, Fig. 1).

Figure 1.
Figure 1. Evolution of piperidine SORAs.
During our OX2R SORA optimization efforts, we discovered additional examples of structural
changes which restored OX1R antagonism. While this shifting between SORA and DORA profiles
should not be completely unexpected given the highly conserved ligand binding pockets of OX1R and
OX2R,23 at the time we were surprised by the subtlety of structural alterations required to effect such
variation. Herein, alongside a broader discussion of OX2R SORA SAR, we will focus on a subset of
these structures and attempt to rationalize the selectivity profiles of our ligands using computational
methods.
Compounds were evaluated for binding affinity (expressed as Ki values) for OX2R and OX1R via
an in vitro radioligand competition binding assay with membranes from CHO-K1 cells which overexpress
the human orexin receptors. Additionally, cell-based functional FLIPR (fluorometric imaging plate
reader) assays, in which Ca2+ flux was measured as a functional determinant of orexin receptor antagonist
activity, were also conducted for determination of receptor subtype selectivity (expressed as IC50
values).24 Historically, we have used the FLIPR selectivity as the more reliable arbiter for the
identification of selective compounds; the binding data is included herein because it is a more appropriate
comparator for structure-based hypotheses of selectivity.

Our earliest efforts at optimizing OX2R SORAs began with the identification of a 4-cyano-3-
methyl-2-pyridyl ether moiety bound to our 2-methyl-piperidine linker (a monocyclic replacement of the
isoquinoline ring of 2) and a 2-triazolobenzene amide functionality (6, Table 1). Whereas 2 presented a
DORA profile, 6 exhibited excellent selectivity for OX2R in both binding and FLIPR assays (285-fold
and 125-fold over OX1R, respectively), achieving a crucial benchmark for the program. Modification of
the amide substituent in 6 provided additional OX2R-selective antagonists shown in Table 1.
Table 1. Amide SAR.a
OX2R
FLIPR
(IC50,
nM)
OX1R
FLIPR
(IC50,
nM)
OX2R
BIND
(Ki, nM) (Ki, nM)
OX1R
BIND
BIND ratio
(OX1R/2R)
FLIPR ratio
(OX1R/2R)
Compound
R
6
0.4
114
285x
8
996
125x
3.0
1.7
1.3
3568
1887
1170
1189x
1110x
900x
21
32
13
5928
> 10,000
6123
282x
> 313x
471x
7
8
9
5.1
1662
326x
29
> 10,000
> 345x
10
8.9
2.0
5765
2517
648x
65
22
> 10,000
> 10,000
> 154x
> 455x
11
12
1259x

0.7
3.1
0.8
0.7
519
1155
757
741x
373x
946x
849x
9
21
16
6
1741
4956
1967
2102
193x
236x
123x
350x
13
14
15
16
594
3.4
1.6
790
572
232x
358x
29
3102
4588
107x
596x
17
18
7.7
2.6
1128
1611
434x
97x
26
69
4673
4446
180x
64x
19
20
16.6
0.7
0.6
10
12
14x
20x
38
38
71
74
2x
2x
21
22
a
Values represent the average of n ≥ 2 experiments.
Interestingly, the binding and FLIPR selectivities for 6 could be further maximized. For example,
incorporation of a 2-methoxypyridine amide (7) increased binding and FLIPR selectivity 4.2-fold and 2.3-
fold over 6, respectively. Replacement of the triazole with a cyclopropane in a similar analog (8) proved
even more selective in FLIPR (313-fold). Subsequent removal of the pyridine nitrogen (9) likewise
improved FLIPR selectivity to 471-fold while only slightly reducing (relative to 7 and 8) binding
selectivity to 900-fold. Similar selectivity was achieved by replacing the methoxy group with a bromide

(10) or by replacing the triazole with a pyridine (11). Finally, replacement of the triazole with a
thiomethyl (12) gave the most selective compound of all, with 1259-fold binding selectivity and >455-
fold FLIPR selectivity.
Simplification of the amide also provided potent OX2R selective molecules. A series of mono-
substituted benzamides exhibited excellent selectivity. Substitution with an ortho-cyclopropane (13),
trifluoromethyl (14), trifluoromethoxy (15), ethoxy (16), or pyrrolidine (17) functionality gave potent (6-
29 nM OX2R FLIPR IC50) and selective compounds. Incorporation of a 2-tetrazole in the same position
provided 18, which showed exceptional 596-fold selectivity over OX1R in the FLIPR assay. The
overwhelming trend for these and many other compounds in this series was towards >30-fold selectivity
over OX1R in FLIPR. However, most of these compounds exhibited ancillary liabilities such as PXR
inhibition (7, 9, and 16), time-dependent CYP inhibition (8, 12, and 17), or low solubility which
precluded further development.
An interesting SORA to DORA shift was found in the piperidine ether series with incorporation
of an acetamide functionality on two related pyridyl amides. While phenyl-substituted pyridyl amide 20
exhibits less potency and selectivity comparable to compounds 6-18 and especially pyridyl regioisomer
19 above, the addition of an acetamide in both 21 and 22 results in exquisite OX2R and OX1R potency in
both the binding and FLIPR assays, with only 2-fold FLIPR selectivity and 14- to 20-fold binding
selectivity. These exceptions were perplexing, but with the recent availability of the X-ray structures for
both orexin receptors23,25 we are able to model the key interactions that might be driving receptor
selectivities. When compounds 21 or 22, which vary only in the location of the pyridine nitrogen, are
docked26 into the crystal structures of OX1R and OX2R, the overall pose of the molecules is very similar
to the X-ray coordinates of suvorexant (Fig. 2A). The acetamide group fills the region adjacent to
S103/T111 (OX1R/OX2R), a key area of the pocket previously identified to be a hot spot for selectivity.23
The carbonyl of the acetamide is able to pick up a hydrogen bond with the OH of Ser 103 in OX1R (Fig.
2B). In OX2R this residue is a Thr 111 which, due to the presence of the methyl, has its OH too far away
to form a proper hydrogen bond (Fig. 2C). The presence of the acetamide is still favorable as the potency
shifts 10-fold for OX2R relative to OX1R. However, the combination of filling the selectivity pocket
along with the added H-bond in OX1R presumably causes the significantly greater shift in affinity for
OX1R.
Figure 2. A) Docking pose of 22 in cyan superimposed with the X-ray structure of suvorexant in OX1R
(PDB 4ZJ8) B) Docking pose for 22 in OX1R suggests a potential hydrogen bond between the acetamide
and S103. C) When 22 is docked in OX2R the distance from the acetamide to T111 is too far (3.8Å) for a
hydrogen bond.
As we learned more about the requirements for potency and selectivity, we sought to simplify 6
by removing the 3-methyl group from the pyridine, resulting in 23 (Fig. 3). This analog proved superior
to the methylated parent, with excellent selectivity (293-fold in FLIPR) and good PK properties.

Figure 3.
Figure 3. Comparison of 23 and MK-8133.
The in vivo potential for 23 to affect physiology and behavior was assessed in rodents implanted
with
radio-telemetry
recording
devices
to
determine
vigilance/sleep
state
via
electroencephalogram/electromyogram (EEG/EMG) polysomnography. In rats, oral administration of 23
at 0.5, 1, and 3 mg/kg resulted in dose-dependent attenuation of active wake relative to vehicle for up to 2
hours following treatment (Fig. 4a). Wake decreases were accompanied by increases in both rapid eye
movement (REM) and non-REM sleep. In wild type mice, compound 23 dosed at 30 mg/kg was also
effective in reducing wakefulness, most prominently during the 1.5 hours following administration (Fig.
4b). These changes were largely accompanied by increases in NREM sleep in mice, with only marginal
increases in REM sleep, a species-dependent difference that has also been observed for OX2R SORA
MK-1064.18 Importantly, these changes in active wake and NREM sleep were absent in mice lacking
OX2R, indicating that the effects of 23 are mediated selectively through this receptor.
Figure 4. Sleep promoting efficacy of 23 in rodents. The mean time spent in active wake, non-REM
sleep (NREM), and REM sleep during 30-min intervals is shown following oral administration of 23
(open circles) relative to vehicle (20% vitamin E TPGS, filled circles) (dose time, vertical grey bar). (a)

Rats were treated with 0.5 mg/kg (nꢀ=ꢀ16) , 1 mg/kg (nꢀ=ꢀ14) or 3 mg/kg (nꢀ=ꢀ12) doses of 23 during the
mid active phase (ZT18:00 [6 hrs after lights off]) for 3 consecutive days in a balanced cross-over design.
(b) Wildtype (n=6) and OX2R knockout mice (n=5) were treated with 30 mg/kg of 23 during the late
active phase (ZT18:00 [6 hrs after lights off]) for 5 consecutive days in a balanced cross-over design.
Data from each all days of treatment under each condition were averaged over a 24 hour period as
described previously.27 Time points at which significant differences exist between vehicle and 23
responses are indicated by grey vertical lines highlighted by tick marks (short, medium, long; pꢀ<ꢀ0.05,
0.01, 0.001, respectively; linear mixed-effects model for repeated measures).
Unfortunately, 23 also had low biorelevant solubility (0.0137 mg/mL simulated gastric fluid;
0.0135 mg/mL FASSIF) which would require non-conventional formulation in safety assessment
studies.28 When the 2-triazole was replaced with the 2-pyrimidine to give 1 (MK-8133) we observed
slight decreases in selectivity but improved pH 7 solubility (Fig. 3; biorelevant solubility was also
improved to 0.68 mg/mL simulated gastric fluid and 0.66 mg/mL FASSIF), a favorable attribute which
led to its clinical development, as shown above.21
An additional discovery made during this extended effort was a SORA to DORA shift effected by
replacement of the isonicotinonitrile functionality (23, Table 2) with a nicotinate methyl ester (26, Table
3). This single modification suprisingly resulted in a potent DORA with only 26-fold binding and 9-fold
FLIPR selectivity. Moreover, this trend of increased OX1R potency while maintaining OX2R potency
held for the corresponding amine- and sulfide-linked analogs (24 vs. 27 and 25 vs. 28, Tables 2 and 3).
We speculate that the ester functionality was able to gain a favorable interaction with OX1R specifically
in the ortho position, as the corresponding isonicotinate methyl ester (29, Table 3) maintained OX2R
selectivity, albeit to a lesser extent.
Table 2. Isonicotinonitrile SAR.a
OX2R
OX1R
BIND
OX2R
FLIPR
OX1R
FLIPR
FLIPR
ratio
Compound
BIND (Ki, BIND (Ki, ratio
X
nM)
1.6
0.9
1.7
nM)
829
287
324
(OX1R/2R) (IC50, nM) (IC50, nM) (OX1R/2R)
O
NH
S
518x
319x
191x
13
29
13
3830
1742
1411
295x
60x
109x
23
24
25
a
Values represent the average of n ≥ 2 experiments.

Table 3. Isonicotinate methyl ester SAR.a
OX2R
BIND BIND
(Ki,
nM)
2.5
0.4
0.8
OX1R
OX2R
FLIPR FLIPR
(IC50,
nM)
35
38
24
22
OX1R
BIND
ratio
(OX1R/2R)
FLIPR
ratio
(OX1R/2R)
X
R1
R2
Compound
(Ki,
nM)
66
12
6.3
311
(IC50,
nM)
309
69
48
1809
O
NH
S
CO2Me
CO2Me
CO2Me
H
H
H
H
26x
30x
8x
9x
2x
2x
82x
26
27
28
O
CO2Me 3.9
80x
29
a
Values represent the average of n ≥ 2 experiments.
SORA to DORA shifting was observed to have even greater molecular complexity as further
work was done to explore related molecules. In one such case, the corresponding 4-cyano-3-methyl ester
analog (30, Fig. 5) showed modest selectivity (112-fold in binding and 39-fold in FLIPR). We speculate
that, in the presence of both nitrile and ester functionalities, the methyl ester is still able to achieve
positive interactions with OX1R, but hindered with regards to positive OX2R interactions with the nitrile
or perhaps by steric requirements. However, the dimethylated lactone analog (31) maintained strong
SORA characteristics, with binding selectivity of 379-fold and FLIPR selectivity of 414-fold, indicating
that the size and shape of the 4-substituent are not as vital as previously hypothesized.
Figure 5.
Figure 5. Interchange of SORA and DORA profiles.

At this time, we are unable to explain the differences in selectivity for these closely-related
analogs using the previously described computational modeling. Every residue within close proximity to
the ester moiety is completely conserved between the two receptors. Given that there are no additional
favorable or unfavorable interactions, dynamics or solvation effects are likely to be involved in the
selectivity profile observed.
Ether-linked compounds were synthesized via the route shown in Scheme 1. Following
hydrogenation of 32 to give a 4:1 mixture of trans:cis piperidines (33) and protection with a
benzyloxycarbonyl group (34), SFC gave separation of the trans (2R, 5R) enantiomer (35a) and the cis
(2R, 5S) enantiomer (35b). A switch to a Boc protecting group provided 36, which was appended with
the appropriate pyridine, as exemplified with 2-fluoro-5-cyanopyridine (37). Removal of the Boc group
provided the requisite piperidine (38) for subsequent amidation reactions which were mediated by EDC or
T3P.29,30
Scheme 1. (a) H2, PtO2, MeOH, HCl, 55 ºC. (b) CbzCl, TEA, CH2Cl2. (c) Chiral SFC. (d) Boc2O, H2, Pd/C (10%), EtOAc. (e) NaH, DMSO, rt.
(f) HCl, dioxane, 0 ºC. (g) EDC, HOAT, Et3N, DMF or T3P, DIEA, DMF.
Amine-linked analogs were synthesized according to Scheme 2. Beginning with cis (2R, 5S)
enantiomer 35b, mesylation (39) and displacement with sodium azide (40), followed by reduction with
trimethylphosphine, provided amine 41. Protection with a Boc group and removal of the Cbz gave 42,
which was coupled with the appropriate carboxylic acid to give 43. Removal of the Boc group and
substitution reactions with the requisite fluoro-pyridines provided the targeted analogs.31
Scheme 2. (a) MsCl, DMAP, DIEA, CH2Cl2. (b) NaN3, DMF. (c) Me3P, THF. (d) Boc2O, DMAP, CH2Cl2. (e) H2, Pd/C (10%), 1:1
EtOAc:MeOH. (f) 2-(2H-1,2,3-triazol-2-yl)benzoic acid, EDC, HOAT, Et3N, DMF. (g) HCl, EtOAc. (h) Cs2CO3, DMSO.

Sulfur-linked analogs were made in like-fashion by SN2 displacement of cis mesylates (e.g. 39)
with the appropriate 2-thiopyridines.32
Summary
Structural modification of known DORAs gave potent and highly selective OX2R SORAs. This
effort led to the discovery of 23, which exhibited a promising in vivo EEG profile. Low biorelevant
solubility prevented further development of this compound and additional work focused on analogs with
improved physicochemical properties.
During this effort, several analogs exhibited unexpected SORA to DORA shifting. As illustrated
with the addition of the acetamide group in compounds 21 and 22, the compounds are clearly able to pick
up an additional interaction with OX1R, resulting in a shift from a 2-SORA to a DORA. However, the
exquisite selectivity gained by the addition of the nitrile group exemplified in compound 23 and the lack
of selectivity with an ester in 26 cannot be readily explained with static structures alone. Under the
assumption that the molecules adopt the same binding pose, there are no obvious differences between the
bindings of these molecules in the two different receptors. Therefore, either an alternative binding pose
exists which the docking was unable to find, or more likely there is a large dynamic and/or solvent effect
that is causing the large shifts in observed selectivity. Further exploration of this system with additional
crystal structures and molecular dynamics may be required to fully elucidate these mechanisms and will
be the focus of future work.
Acknowledgments
The authors wish to thank the West Point NMR and Mass Spectrometry teams for their assistance
in characterization of compounds in this manuscript. We also thank Mr. Jeff Fritzen and Dr. Carmela
Molinaro for preparation of key intermediates.
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