Discovery of dual orexin receptor antagonists with rat sleep efficacy enabled by expansion of the acetonitrile-assisted/diphosgene-mediated 2,4-dichloropyrimidine synthesis

Article abstract of DOI:10.1016/j.bmcl.2014.03.052

Recent clinical studies have demonstrated that dual orexin receptor antagonists (OX₁R and OX₂R antagonists or DORAs) represent a novel treatment option for insomnia patients. Previously we have disclosed several compounds in the diazepane amide DORA series with excellent potency and both preclinical and clinical sleep efficacy. Additional SAR studies in this series were enabled by the expansion of the acetonitrile-assisted, diphosgene-mediated 2,4-dichloropyrimidine synthesis to novel substrates providing an array of Western heterocycles. These heterocycles were utilized to synthesize analogs in short order with high levels of potency on orexin 1 and orexin 2 receptors as well as in vivo sleep efficacy in the rat.

Full text of DOI:10.1016/j.bmcl.2014.03.052

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2079–2085
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of dual orexin receptor antagonists with rat sleep efficacy
enabled by expansion of the acetonitrile-assisted/diphosgene-
mediated 2,4-dichloropyrimidine synthesis
a
b
b
b
Anthony J. Roecker a, , Swati P. Mercer , C. Meacham Harrell , Susan L. Garson , Steven V. Fox ,
Anthony L. Gotter b, Thomayant Prueksaritanont c, Tamara D. Cabalu c, Donghui Cui c, Wei Lemaire d,
Christopher J. Winrow b, John J. Renger b, Paul J. Coleman a
⇑
a Department of Medicinal Chemistry, Merck Research Laboratories, PO Box 4, Sumneytown Pike, West Point, PA 19486, United States
b Department of Neuroscience, Merck Research Laboratories, PO Box 4, Sumneytown Pike, West Point, PA 19486, United States
c Department of Drug Metabolism, Merck Research Laboratories, PO Box 4, Sumneytown Pike, West Point, PA 19486, United States
d Department of In Vitro Pharmacology, Merck Research Laboratories, PO Box 4, Sumneytown Pike, West Point, PA 19486, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Recent clinical studies have demonstrated that dual orexin receptor antagonists (OX1R and OX2R antag-
onists or DORAs) represent a novel treatment option for insomnia patients. Previously we have disclosed
several compounds in the diazepane amide DORA series with excellent potency and both preclinical and
clinical sleep efficacy. Additional SAR studies in this series were enabled by the expansion of the aceto-
nitrile-assisted, diphosgene-mediated 2,4-dichloropyrimidine synthesis to novel substrates providing an
array of Western heterocycles. These heterocycles were utilized to synthesize analogs in short order with
high levels of potency on orexin 1 and orexin 2 receptors as well as in vivo sleep efficacy in the rat.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 12 February 2014
Revised 14 March 2014
Accepted 17 March 2014
Available online 26 March 2014
Keywords:
Orexin
Antagonist
Sleep
Bioactivation
Pyrimidine
The discovery of novel orexin receptor antagonists has evolved
rapidly in the past decade from the generation of novel peptide
analogs1 to small molecule clinical candidates2 to the NDA filing
of suvorexant (1, Fig. 1) in 2012 for the treatment of patients suf-
fering from insomnia.3 Suvorexant (1) and DORA 2 antagonize the
action of wake-promoting orexin peptides (orexin A and B) at orex-
in 1 (OX1R) and orexin 2 (OX2R) receptors, hence, they are catego-
rized as dual orexin receptor antagonists (DORAs). Several reviews
have been published detailing the pharmacology and therapeutic
utility associated with orexin antagonism and are beyond the
scope of this manuscript.4 Herein we report the expansion of the
acetonitrile-assisted, diphosgene-mediated 2,4-dichloropyrimi-
dine synthesis as it applies to the discovery of orexin antagonists.
As shown in Figure 1, Suvorexant and DORA 2 are both potent
antagonists of OX1R and OX2R with nanomolar to subnanomolar
binding potency and high levels of functional antagonism as deter-
mined in the FLIPR (fluorometric imaging plate reader) assay.5
DORA 2 possessed similar in vivo efficacy in rat sleep studies
Cl
N
N
O
O
N
N
N
N
N
N
N
N
F
N
N
O
N
Me
Me
Me
(2)
(1, Suvorexant)
OX1R Ki (IC50) = 0.6 nM (50 nM)
OX2R Ki (IC50) = 0.4 nM (56 nM)
OX1R Ki (IC50) = 1.8 nM (27 nM)
OX2R Ki (IC50) = 0.2 nM (27 nM)
Figure 1. Potent, optimized diazepane-containing dual orexin receptor antagonists
(DORAs) suvorexant (1) and 2 that have demonstrated excellent sleep efficacy.
compared to suvorexant, however, bioactivation concerns were
confirmed through glutathione (GSH) trapping studies in micro-
somal incubations.2c Through additional diazepane core and
heterocycle modifications and GSH trapping experiments, the 6-
fluoroquinazoline moiety was determined to be the substructure
responsible for the observed bioactivation. These findings encour-
aged us to examine the SAR of potential 6-fluoroquinazoline
replacements. In order to effectively examine this new SAR, we
sought an efficient synthesis of appropriately functionalized
bicyclic pyrimidines.
⇑
Corresponding author. Tel.: +1 215 652 8257; fax: +1 215 652 3971.
E-mail address: anthony_roecker@merck.com (A.J. Roecker).
http://dx.doi.org/10.1016/j.bmcl.2014.03.052
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

2080
A. J. Roecker et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2079–2085
N
Cl-
N
H+
HN
O
+
CN
NH2
N
diphosgene
N
C
Me
Me
acetonitrile
N
N
N
N
C
N
O
N
N
100°C, 14 hours
HCl
Cl
Cl
+H2N
O
N
N
Me
Cl
N
N
N
N
N
N
Cl-
51% yield
Scheme 1. Mechanism proposed by Chi et al. for their acetonitrile-assisted fused 2,4-dichloropyrimidine synthesis.
Chi and co-workers published a synthesis of 2,4-dichloroquino-
zole substrates 3 and 7 provided fused dichloropyrimidines 4 and 8
in slightly reduced yields (39% and 24%, respectively) compared to
the pyrazole substrate exemplified in Scheme 1. Substituted thio-
phene scaffolds were also competent substrates under the reaction
conditions with precursors 11 and 15 affording products 12 and 16
in 52% and 65% yields, respectively. Substrate 18, 2-aminothioph-
ene-3-carbonitrile, provided the desired product with less overall
yield (19%). Finally, substituted furans 21 and 25 performed well
giving rise to products 22 and 26 in acceptable to good yields.
Standard nucleophilic substitution reactions (SNAr) of 2,
4-dichloropyrimidines using secondary amines such as our diaze-
pane substrates provide 4-substituted products with high regiose-
lectivity.8 In order to afford the 2-substituted analogs similar to
DORA 2, bicyclic 2,4-dichloropyrimidine substrates were subjected
to a variant of known conditions for selective dechlorination of the
4-position. Treatment with excess elemental zinc and ammonium
hydroxide in refluxing ethanol afforded a mixture of desired
dechlorinated-, aminated-, and ethoxy substituted products.9 In
lines and 2,4-dichloroquinazolines in a single step from 2-ethyny-
lanilines or anthranilonitriles, respectively, using diphosgene in
acetonitrile.6 A single example of a five-membered ring substrate,
5-amino-1-benzyl-1H-pyrazole-4-carbonitrile, demonstrated that
this methodology would also provide fused pyrimidine heterocy-
cles that could serve as interesting quinazoline replacements for
the diazepane series shown in Figure 1. The mechanism proposed
by Chi and co-workers for this transformation is depicted in
Scheme 1, and it involves isocyanate formation, ring closure as-
sisted by acetonitrile, and two subsequent chloride ion attacks to
afford the final products in modest yield. Our group sought to fur-
ther evaluate the scope of this reaction and to test its application
toward the synthesis of DORAs with diminished bioactivation po-
tential compared to DORA 2.7
A series of commercially available five-membered heterocyclic
2-aminocarbonitriles were subjected to the conditions described
in Scheme 1 and the results are shown in Table 1. Truncated pyra-
Table 1
Acetonitrile-assisted synthesis of 2,4-dichloropyrimidines and subsequent amination/dechlorination
diphosgene
zinc, NH4OH
(1.5 equiv.)
EtOH, reflux
or
acetonitrile
(~ 1M)
Cl
H2N
X
H
CN
NH2
Z
Z
Z
Z
N
N
N
+
Cl
Cl
Cl
Y
NH4OH, THF
sealed tube
100°C
15 hours
Y
Y
Y
X
N
N
N
X
X
25°C
Substrate
Product
Yield (%)
39
Amine substitution (yield)a
Dechlorination (yield)a
CN
Cl
H2N
N
N
Cl
N
N
N
N
NH2
CN
Cl
Cl
N
N
N
N
N
N
N
Me
Me
N
(6; 25%)
Me
Me
(3)
(4)
(5)
Cl
H2N
Me
Me
N
Me
Me
N
N
Cl
Cl
N
N
N
N
Me
(7)
NH2
Cl
Cl
Cl
Cl
N
24
52
N
N
N
N
Me
N
N
Me
Me
(10; 31%)
(8)
(9; 51%)
H2N
Me
CN
NH2
Cl
Me
Me
N
Me
N
N
N
S
S
N
N
S
S
(14; 43%)
(11)
(12)
(13; 7%/57%b)

A. J. Roecker et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2079–2085
2081
Table 1 (continued)
Substrate
Product
Yield (%)
65
Amine substitution (yield)a
Dechlorination (yield)a
CN
Cl
H2N
N
N
NH2
Cl
Cl
Me
S
NS
N
N
Me
Me
S
S
(15)
(16)
(17; 57%b)
CN
Cl
H2N
N
N
NH2
S
(18)
Cl
Cl
19
38
75
NS
N
N
S
S
(19)
(20; 18%b)
H2N
CN
NH2
Cl
Me
Me
N
Me
Me
N
N
Cl
N
Cl
Cl
O
O
N
N
O
O
(23; 23%)
H2N
(24)
Me
(21)
(22)
CN
NH2
Cl
Me
N
Me
Me
N
N
Cl
N
Me
Cl
Cl
Me
O
O
N
N
Me
Me
(27; 3%)
O
O
(28; 6%)
(25)
(26)
Reagents and conditions: (a) zinc (8 equiv), ammonium hydroxide (5 equiv), ethanol (0.15 M), reflux, 30 min; (b) ammonium hydroxide (5 equiv), THF (0.2 M), 25 °C; NS = not
synthesized.
Table 2
Potent diazepane-containing DORAsa,b
N
O
N
N
Het
N
N
Me
X
a
a
Heterocycle (compound #)
X
OX2R Kia (nM)
OX1R Kia (nM)
OX2R IC50 (nM)
OX1R IC50 (nM)
N
N
N
N
Me
2
49
92
70
85
Me
(29)
Me
N
N
Me
H
0.8
4.5
3.7
92
119
N
N
N
N
Me
(30)
Me
N
N
N
200
280
N
Me
(31)
Me
Me
Me
Me
Me
0.2
0.5
0.3
1.8
0.6
0.5
0.2
5
45
34
42
35
O
(32)
Me
N
Me
Me
67
O
N
(33)
N
37
S
N
(34)
NH2
N
Me
195
160
O
N
(35)
(continued on next page)

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A. J. Roecker et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2079–2085
Table 2 (continued)
a
a
Heterocycle (compound #)
X
OX2R Kia (nM)
OX1R Kia (nM)
OX2R IC50 (nM)
OX1R IC50 (nM)
NH2
N
Me
H
1.8
25
195
26
15
47
31
53
28
210
34
35
35
34
43
29
O
N
(36)
NH2
N
Me
H
0.5
0.9
0.4
0.7
0.3
0.3
0.7
9.2
0.4
1.5
0.3
1.2
S
N
(37)
NH2
N
S
N
(38)
NH2
N
Me
Me
H
S
N
(39)
NH2
N
Me
Me
S
N
(40)
NH2
N
Me
H
S
N
(41)
NH2
N
Me
S
N
(42)
a
See Ref. 2c for the details of these assays. All compounds listed were assayed as single enantiomers.
All data reported as an average value from n P2; for n >2, standard deviation is <40% in all cases.
b
order to provide additional SAR, the aminated products (Table 1,
row 4), targeting improved physiochemical properties, and dechlo-
rinated products (Table 1, row 5) were isolated and the yields are
shown in Table 1. It was also found that the aminated products
could be synthesized more efficiently using ammonium hydroxide
in tetrahydrofuran at ambient temperature (products 13, 17, and
20).10 Both diazepane cores for DORAs 1 and 2 and the heterocycles
above were used to synthesize the analogs displayed in Table 2.2c
These analogs were synthesized under standard SNAr conditions
of heterocycle (1.2 equiv), diazepane core (1.0 equiv), Hunig’s base
or triethylamine (2.5 equiv), in dimethylformamide (ꢀ0.3 M) at
100–180 °C in a microwave reactor for 10–40 min.
Analysis of the SAR for the new compounds revealed that pyraz-
olopyrimidine 29 showed diminished binding and functional
potency on both OX1R and OX2R when compared with lead DORA
2 as shown in Table 2. Substitution of the pyrazole motif with a
methyl group (30) improved OX1R binding potency, however, func-
tional potency on both receptors decreased slightly. Removal of the
methyl group on the benzamide generally decreases potency on
both receptors for compounds in this series,11 and this was ob-
served for des-methyl analog 31. Furopyrimidines 32 and 33 dem-
onstrated subnanomolar binding potency for both orexin receptors
and functional potency similar to DORA 1. Given the improved
functional potency for mono-methyl furopyrimidine 32, thienopyr-
imidine 34 was synthesized. This analog possessed exquisite bind-
ing potency on both OX2R (0.3 nM) and OX1R (0.2 nM) and
functional potency similar to DORA 2. Unfortunately, compound
34 suffered from high plasma protein binding (PPB: 99.4% human;
99.3% rat) and high log D (3.8; HPLC method12) which precluded
additional pharmacokinetic or in vivo studies.
H2N
N
O
Me
N
N
N
N
N
N
S
Me
DORA 42
Plasma Protein Binding
Human: 98.5%
Rat: 98.9%
Luckily, 4-amino substituted heterocycles were available from
the synthetic studies in Table 1 allowing the production of analogs
that might provide improved physiochemical properties (Table 2).
Amino-substituted furopyrimidines 35 and 36 displayed reduced
binding and functional potency when compared to des-amino ana-
log 32. Surprisingly, amino-substituted thienopyrimidines 37 to 42
all demonstrated low to subnanomolar binding affinity and excel-
lent functional potency for OX2R and OX1R. While analogs 37 and
Dog: 97.8%
CNS Properties
Pgp ratio (BA/AB): 1.2(h); 1.8(r)
Papp: 31 cm * 10-6/s
HPLC log D: 2.0
PSA: 96 Å2
Figure 2. Additional properties of DORA 42.

A. J. Roecker et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2079–2085
2083
Table 3
Pharmacokinetics of DORA 42 in rat and dog
IVa
Dose (mg/kg)
POb
Tmax (h)
Species
Dose (mg/kg)
Cl (mL/min/kg)
Vdss (L/kg)
T1/2 (h)
AUC (lM h)
Cmax
(
lM)
F (%)
Rat
2
0.5
NA
22
13
NA
0.3
0.7
NA
0.2
0.8
NA
10
3
25
2.8
3.1
6.1
0.3
0.6
0.5
3.8
1.2
2.5
17
34
15
Dog
Ratc
a
b
c
Vehicle = 100% DMSO in rat and dog (n = 2).
Dosed as the hydrochloride salt in 100% PEG200 (n = 2).
Dosed as hydrochloride salt in 20% VitE TPGS in water; satellite rats for sleep study pharmacokinetics (n = 3).
H2N
H2N
S
N
N
O
O
Me
N
N
N
Me
N
(a)
N
N
+
N
N
HN
N
Cl
N
N
S
Me
Me
13
43
42
Scheme 2. Gram scale synthesis of compound 42. Reagents and reaction conditions: (a) triethylamine, DMF, 185 °C, 40 min, microwave, 39% (7.5 mmol scale; 1.3 grams
produced). See Ref. 2c for the synthesis of diazepane core 43.
38 possessed excellent potency profiles, the unsubstituted thio-
phene motif is a known substrate for bioactivation.13 Precedent
suggested that substitution on the thiophene motif can reduce this
liability.14 Since DORA 42 demonstrated the best balance of bind-
ing potency, functional potency, and theoretical bioactivation po-
tential of the final four analogs (39–42), this compound was
selected for additional profiling (Fig. 2).
The 4-amino substituent present in DORA 42 improved free
fraction to measurable levels across species and decreased logD
by 1.8 units compared to compound 34 (Table 2). In addition, the
compound was highly permeable and not a substrate for rat or hu-
man P-glycoprotein efflux. Compound 42 possessed polar surface
area in an acceptable range for brain penetrant small molecules.15
Pharmacokinetic properties were evaluated for compound 42
(dosed as a hydrochloride salt) and these data are displayed in
Table 3. In the rat and the dog, this compound had moderate clear-
ance, a short half-life, and bioavailability of 17% and 34%, respec-
tively. Since this compound was suitable for oral administration,
it was scaled up for additional in vivo characterization in rat sleep
studies. The gram scale synthesis is shown in Scheme 2.
an internal standard (Fig. 4). Although precedent suggested that
substituted thiophene analogs could reduce bioactivation potential
relative to their unsubstituted counterparts,14 the direct compari-
son between phenyl and thiophene isosteres is less well studied.
The bioactivation potential of DORA 42 precluded further develop-
ment of thiophene-containing DORAs.
Expansion of an acetonitrile-assisted, diphosgene-mediated
synthesis of bicyclic 2,4-dichloropyrimidines enabled the expedi-
tious production of analogs of DORA 2. These efforts afforded
DORAs with excellent binding and functional potency on OX1R
and OX2R as well as improved physiochemical properties. DORA
42 was identified as an advanced compound with superb potency
and rat sleep efficacy comparable to DORA 2. Unfortunately, the
bioactivation potential of DORA 42 precluded further development
Figure 3 displays the results of sleep studies from active phase
radiotelemetric recordings in the rat over the two hour period di-
rectly following compound administration using methods previ-
ously described.16 Upon dosing the hydrochloride salt of
compound 42 in rats at 25 mg/kg (p.o. in 20% Vitamin E TPGS),
immediate and significant reduction in active wake and concomi-
tant increases in slow wave sleep (SWS) and REM sleep were ob-
served over the two hour period immediately following
treatment. The sleep efficacy of compound 42 in the rat was
accompanied by a Cmax of 2.5
lM, Tmax of 30 min, and an AUC of
6.1 M h (Table 3). The Cmax data for compound 42 coincided ni-
l
cely with results from ex vivo occupancy studies in transgenic rats
which overexpress human OX2R demonstrating 88% receptor occu-
Figure 3. Sleep architecture effects of DORA 42 and DORA 2 in rat sleep dosed
during their active phase. (a) Mean change in active wake, light sleep, SWS and REM
sleep time relative to vehicle (20% Vitamin E TPGS) in rats monitored for two hours
following p.o. administration of compound 2 (30 mg/kg) and compound 42 (25 mg/
kg). Treatment occurred at Zeitgeber Time 16:00 (4 h after lights off) in a balanced
cross-over design such that each subject received drug and vehicle (3 or 7 days of
consecutive treatment, compounds 2 and 42, respectively). Values represent the
mean of within-subject differences between vehicle and compound treatment
( SEM). Mean times for the compound condition in experiments evaluating
compound 2 and compound 42 experiments, respectively (min): AW: 58, 53; LS:
14, 12; SWS: 41, 46; REM: 7, 9. Data were analyzed using within-subject ANOVA to
determine main effects and one sample t-test to compare to vehicle (N = 14, 8 for
studies evaluating compounds 2 and 42, respectively; ⁄p <0.05, ⁄⁄p <0.01, ⁄⁄⁄p
<0.001).
pancy at a plasma concentration of 2.0 l
M.17 These findings follow
the same occupancy/efficacy relationship as compounds 1 and 2. In
fact, Compound 42 induced a sleep architecture profile nearly
identical to that observed for compound
2
administered at
30 mg/kg (Cmax: 2.0 lM; Tmax: 1.3 h; AUC: 4.5 lM h), as previously
described for this structure as well as compound 1.2c
Finally, DORA 42 and DORA 2 were directly compared for bioac-
tivation potential via our GSH trapping assay in rat and human
microsomal incubations.18 DORA 42 and DORA 2 demonstrated
similar levels of bioactivation in rat and human experiments as
determined by AUC measurements of all GSH adducts relative to

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A. J. Roecker et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2079–2085
Figure 4. GSH trapping studies in rat and human liver microsomes. Ratios are AUC measurements normalized to the AUC of an internal standard (IS = labetalol). AUC = area
under curve; RLM = rat liver microsomes; HLM = human liver microsomes.
Cox, C. D.; Breslin, M. J.; Whitman, D. B.; Bogusky, M. J.; McGaughey, G. B.;
Bednar, R. A.; Lemaire, W.; Doran, S. M.; Fox, S. V.; Garson, S. L.; Gotter, A. L.;
Harrell, C. M.; Reiss, D. R.; Cabalu, T. D.; Cui, D.; Prueksaritanont, T.; Stevens, J.;
Tannenbaum, P. L.; Ball, R. G.; Stellabott, J.; Young, S. D.; Hartman, G. D.;
Winrow, C. J.; Renger, J. J. ChemMedChem 2012, 7, 415; (e) Roecker, A. J.; Mercer,
S. P.; Schreier, J. D.; Cox, C. D.; Fraley, M. E.; Steen, J. T.; Lemaire, W.; Bruno, J. G.;
Harrell, C. M.; Garson, S. L.; Gotter, A. L.; Fox, S. V.; Stevens, J.; Tannenbaum, P.
L.; Prueksaritanont, T.; Cabalu, T. D.; Cui, D.; Stellabott, J.; Hartman, G. D.;
Young, S. D.; Winrow, C. J.; Renger, J. J.; Coleman, P. J. ChemMedChem 2014, 9,
312.
of thiophene-containing DORAs. Studies focused on additional
strategies to reduce bioactivation of diazepane amide DORAs will
be published in due course.
Acknowledgments
We thank Dr. Charles W. Ross III and Joan S. Murphy for analyt-
ical studies in support of this manuscript. We would like to thank
Michael J. Bogusky for NMR support. We would like to thank
Joseph G. Bruno and Rodney A. Bednar for assay support.
3. (a) 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. Neurogenetics 2011, 25, 52; (b) Herring, W. J.;
Snyder, E.; Budd, K.; Hutzelmann, J.; Snavely, D.; Liu, K.; Lines, C.; Roth, T.;
Michelson, D. Neurology 2012, 79, 2265.
References and notes
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5. Please see Ref. 2c for a complete description of the binding and FLIPR assays.
6. Lee, J. H.; Lee, B. S.; Shin, H.; Nam, D. H.; Chi, D. Y. Synlett 2006, 65.
7. Since the completion of the work detailed in this manuscript, two additional
references have appeared detailing similar transformations for some thiophene
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For additional assay details, please see Ref. 2c.
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microsomal preparations.
12. See the supporting information in Ref. 2e for the HPLC logD assay protocol.