Identification of highly selective and potent orexin receptor 1 antagonists derived from a dual orexin receptor 1/2 antagonist based on the structural framework of pyrazoylethylbenzamide

Article abstract of DOI:10.1016/j.bmc.2017.07.051

The design, synthesis, and structure activity relationships of the novel class of pyrazolylethylbenzamide orexin receptor 1-selective antagonists are described. Further derivatization of the prototype dual orexin receptor 1/2 antagonist lead (1) by instal

Full text of DOI:10.1016/j.bmc.2017.07.051

Accepted Manuscript
Identification of highly selective and potent orexin receptor 1 antagonists de-
rived from a dual orexin receptor 1/2 antagonist based on the structural frame-
work of pyrazoylethylbenzamide
Aya Futamura, Dai Nozawa, Yuko Araki, Yunoshin Tamura, Seiken Tokura,
Hiroshi Kawamoto, Yuichi Tokumaru, Sora Kakihara, Takeshi Aoki, Norikazu
Ohtake
PII:
S0968-0896(17)31177-X
http://dx.doi.org/10.1016/j.bmc.2017.07.051
BMC 13888
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 June 2017
15 July 2017
18 July 2017
Please cite this article as: Futamura, A., Nozawa, D., Araki, Y., Tamura, Y., Tokura, S., Kawamoto, H., Tokumaru,
Y., Kakihara, S., Aoki, T., Ohtake, N., Identification of highly selective and potent orexin receptor 1 antagonists
derived from a dual orexin receptor 1/2 antagonist based on the structural framework of pyrazoylethylbenzamide,
Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.07.051
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Identification of highly selective and potent orexin receptor 1 antagonists derived from a dual orexin
receptor 1/2 antagonist based on the structural framework of pyrazoylethylbenzamide
Aya Futamuraa,*, Dai Nozawaa, Yuko Arakia, Yunoshin Tamuraa, Seiken Tokuraa, Hiroshi Kawamotoa, Yuichi
Tokumarub, Sora Kakiharab, Takeshi Aokib, and Norikazu Ohtakea
aChemistry Laboratories, , Taisho Pharmaceutical Co., Ltd. 1-403 Yoshino-cho, Kita-ku, Saitama 331-9530, Japan
bPharmacology Laboratories, Taisho Pharmaceutical Co., Ltd. 1-403 Yoshino-cho, Kita-ku, Saitama 331-9530,
Japan
*Corresponding author: Aya Futamura
Phone: +81-48-669-3029
Fax: +81-48-652-7254
E-mail: a-futamura@so.taisho.co.jp
1

Abstract
The design, synthesis, and structure activity relationships of the novel class of pyrazolylethylbenzamide orexin
receptor 1-selective antagonists are described. Further derivatization of the prototype dual orexin receptor 1/2
antagonist lead (1) by installing a (S)-methyl group into the ethyl linker moiety between the pyrazole ring and
benzamide resulted in an increase of the antagonist potency against orexin receptor 1/2 receptors. Optimization of
the benzamide and pyrazole parts of compounds
2
and 9b led to the identification of
N-ethyl-5-fluoro-N-{(2S)-1-[5-(4-fluorophenyl)-2H-tetrazol-2-yl]propan-2-yl}-2-(pyrimidin-2-yl)benzamide (24),
which exhibited excellent antagonistic activity against orexin receptor 1 with an IC50 of 2.01 nM and a 265-fold
selectivity for orexin receptor 1 over orexin receptor 2.
Keywords:
Selective orexin receptor antagonist (SORA), Dual orexin receptor antagonist (DORA), Orexin 1
2

Abbreviations
OX1R, orexin 1 receptor; OX2R, orexin 2 receptor; DORA, dual orexin receptor antagonist; SORA, selective orexin
receptor antagonist; SI, selectivity index; BBB, blood-brain barrier; CNS, central nervous system; SAR,
structure-activity relationship; Boc, tert-butoxycarbonyl; DIPEA, N,N-diisopropylethylamine; HATU,
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo-[4,5-b]pyridinium 3-oxide hexafluorophosphate; T3P®,
propylphosphonic acid anhydride; Ms, methanesulfonyl; RHS, right hand side; CHO, Chinese hamster ovary.
1. Introduction
Orexins, consisting of orexin-A and -B (also known as hypocretin-1 and -2), are hypothalamic neuropeptides that
have been reported to originate from the common precursor protein prepro-orexin peptide1,2. They constitute the
endogenous ligands for two subtypes of orexin receptors, orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R),
which are seven-transmembrane G-protein-coupled receptors. OX1R and OX2R are expressed differentially,
although widely, in various regions of the brain, suggesting that each receptor subtype might have distinct
physiological functions. The orexin system has been shown to play roles in a variety of important neurobiological
processes, including the control of reward, sleep-wake regulation, feeding, and energy homeostasis. 3
Research on the orexin system has mainly been directed toward the development of new hypnotic medications.
Both dual orexin receptor antagonists (DORAs) and selective orexin 2 receptor antagonists (2-SORAs) have been
developed for the treatment of sleep disorders. Proof-of-concept for treating human insomnia has been achieved
with several DORAs, including almorexant4, SB-6498685 and suvorexant6, and suvorexant was the first compound
to be successfully launched in the market in both the United States and Japan in 2014 (Figure 1). Phase I trials have
been completed for MK-10647 and JNJ-428479228, both 2-SORAs, and MK-1064 has been shown to induce
3

dose-dependent increases in the sleep efficacy and total sleep time, including rapid eye movement (REM) and
non-rapid eye movement (NREM) sleep in healthy volunteers.9 Although a number of DORAs and 2-SORAs have
been developed, few selective OX1R antagonist (1-SORA) tool compounds have been reported.10,11 The first
selective OX1R antagonist to be reported was SB-334867, which is a diarylurea derivative.12 Most in vivo and in
vitro research conducted to investigate OX1R has utilized this compound. However, SB-334867 has been
demonstrated to also show affinity for the adenosine 2A (Ki = 0.67 μM) and 5-HT2c (Ki = 1.2 μM) receptors.13
SB-334867, which has moderate OX1R selectivity over OX2R, with a selectivity index (SI = IC50 in OX2R/IC50 in
OX1R) of 50, allowed the initial elucidation of the functions of OX1R.14 Subsequently, Actelion reported
ACT-335827 as a structurally distinct selective OX1R antagonist.15 ACT-335827 (SI: 70) was reported as an orally
available and BBB-penetrant 1-SORA. The OX1R has been reported to play a much less significant role in the
sleep-wake cycle than OX2R based on studies carried out on orexin receptor knockout mice.16,17 Consistent with
previous reports from studies conducted using selective OX1R antagonists, ACT-335827 did not promote sleep in
telemeterized rats.15
Figure 1. Representative DORAs, 2-SORAs, and 1-SORAs
As the biology related to the orexin receptors emerges, selective compounds from multiple chemotypes will help
4

to further understand the role and biology of each receptor in the onset of CNS disorders. For the treatment of
psychiatric disorders involving the OX1R, one might see benefit in compounds which are selective for OX1R over
OX2R, so that sedation is minimized.15
To move research on the pharmacology of orexin receptors forward and to clarify the functions of OX1R, we
explored highly potent and selective 1-SORAs, starting from DORA with a pyrazoylethylbenzamide structure18,
which was previously discovered as a new series of DORAs. Herein, we report our findings of research on the
SARs of OX1R and OX2R based on a structural framework of pyrazolylethylbenzamide and the identification of
new class of potent SORAs with high selectivity for OX1R over OX2R.
2. Chemistry
The pyrazole derivatives 2, 3, 4b, 5b, 7a, 8a, 7b–9b, 10, and 11 were synthesized as shown in Scheme 1.
Compounds 2, 7a, 8a, and 7b–9b were prepared from the commercially available N-Boc-L-alaninol 28, which was
converted into the corresponding mesylate and successively alkylated with 5-fluoro-2-(1H-pyrazol-3-yl)pyridine
(29a) or 5-fluoro-2-(1H-pyrazol-4-yl)pyridine (29b) to yield amine 30a or 30b, respectively. The resulting 30a and
30b were acidified to remove the Boc groups and then amidated with 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic
acid (31) in the presence of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid
hexafluorophosphate (HATU) to yield 7a and 7b, which were reacted with the corresponding alkyl iodides to obtain
2, 8a, 8b and 9b. Compound 5b was prepared by the same procedure as compound 8b, except that tert-butyl
N-(2-bromoethyl)carbamate (32) was used as the starting material. For the syntheses of compounds 3, 10, and 11,
compound 29a was alkylated with mesylates which were converted from commercially available chiral alcohols
34–36 in the same manner as that to obtain 30a, to obtain amines 37–39, which were then ethylated and deprotected
of the Boc group under acidic conditions to yield secondary amines 40–42. The obtained compounds 40–42 were
subsequently amidated with benzoic acid 31 to yield 3, 10, and 11.
5

Synthesis of , ,
,
,
–
,
–
,
,
Scheme 1.
2 3 4b 5b 7a 8a 7b 9b 10 11
Reagents and conditions: (a) MsCl, Et N, THF, 0 °C; (b) 5-fluoro-2-(1H-pyrazol-3-yl)pyridine (
) or
29a
3
5-fluoro-2-(1H-pyrazol-4-yl)pyridine (
), Cs CO , DMF, 80 °C; (c) 4M HCl-EtOAc, EtOAc, rt; (d) HATU,
29b
2
3
DIPEA, DMF, 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid ( ); (e) NaH, DMF, then alkyliodide, 0–60 °C.
31
Synthesis of
– , –
9c 9g 12 27
Scheme 2.
Reagents and conditions: (a) MsCl, Et3N, CHCl3, 0 °C; (b) NaN3, DMF, 80 °C; (c) 2-ethynyl-5-fluoropyridine, CuI,
6

L-ascorbic acid, 4-methylpiperidine, DMF, 80 °C; (d) NaH, DMF, then ethyliodide, 0–60 °C; (e) 4M HCl-EtOAc,
EtOAc, rt; (f) HATU, DIPEA, DMF, ; (g) NaCN, DMF, 80 °C; (h) (1) NH OH aq., EtOH, 80 °C, (2)
31
5-fluoropyridine-2-carboxylic acid or 4-fluorobenzoic acid, 1,1'-carbonyldiimidazole, DMF, 40–80 °C; (i) HATU,
DIPEA, DMF, ; (j) 5-fluoro-2-(1H-1,2,3,4-tetrazol-5-yl)pyridine ( ) or
or T3P®, DIPEA, CHCl ,
2
–
–
44 46
47 49
29e
3
5-(4-fluorophenyl)-1H-tetrazole, Cs2CO3, DMF, 80 °C.
The synthetic routes for triazole (9c), oxadiazole (9d, 9f, 12–19) and tetrazole derivatives (9e, 9g, 20–27) are
depicted in Scheme 2. Heterocyclic intermediates 30c–g were synthesized from alcohol 28. For the synthesis of
1,2,3-triazole intermediate 30c (for 9c), alcohol 28 was mesylated and subsequently reacted with sodium azide
(NaN3) to yield an azide intermediate. 1,3-Dipolar cycloaddition of the azide intermediate to
2-ethynyl-5-fluoropyridine yielded 30c. To synthesize 30d and 30f (for 9d, 9f, 12–19), alcohol 28 was converted
into its cyano derivative by mesylation and subsequent SN2 reaction with sodium cyanide (NaCN). The resulting
cyano derivative was then treated with hydroxylamine to obtain the amidoxime derivative. After O-acylation of the
amidoxime with the corresponding acids by using 1,1'-carbonyldiimidazole, cyclization was achieved by heating to
obtain the 1,2,4-oxadiazole intermediates 30d and 30f. Similarly, tetrazole intermediates 30e and 30g (for 9e, 9g,
20–27) were prepared with 5-fluoro-2-(1H-1,2,3,4-tetrazol-5-yl)pyridine (29e) or 5-(4-fluorophenyl)-1H-tetrazole
in the same manner as that to obtain 30a in Scheme 1. The resulting compounds 30c–g were subjected to
N-ethylation followed by deprotection of the Boc group to obtain 43c–g. Compounds 43c–g were amidated with the
corresponding benzoic acids using HATU or 1-propanephosphonic acid cyclic anhydride (T3P®) as a coupling
agent to obtain 9c–9g and 12–27.
3. Results and discussion
Among compounds of the pyrazolylethylbenzamide class reported previously, compound 1 was one of the
representative DORAs, which exhibited comparable inhibitory activity against both OX1R and OX2R, with IC50
values of 3.22 and 2.81 nM, respectively. This compound was generated by the ligand-based drug design approach
carrying the characteristic U-shaped conformation19, with which potent OXRs ligands, such as suvorexant, are
known to bind to the OXRs.18 Therefore, we first investigated the effects of restriction of the U-shaped
7

conformation on the inhibitory potency and selectivity. As shown in Figure 2, conformational analysis of the
derivatives which were introduced as substituents at the ethyl linker between the pyrazole ring and benzamide
suggested that the distribution of the pyridine moiety of 1 spread widely, while that of the methyl substituted
compounds 2 and 3 was limited; especially that of (S)-enantiomer 2 was localized to the bioactive U-shaped
conformation (Figure 2).20 Based on the above findings, we synthesized a number of compounds bearing an ethyl
linker and S)-alkyl-substituted ethyl linkers (Table 1) and compared their inhibitory activities on OX1R and OX2R.
The inhibitory activities (IC50) on OX1R and OX2R were assessed in functional assays by measuring the
intracellular Ca2+ mobilization in CHO cells overexpressing the human receptors.
Figure 2. Conformational analysis of the derivatives
Red and green meshes show the distribution of the pyridine moiety under the alignment of the 5-tolyl triazole
substructures. The conformations of the compounds were calculated using the replica-exchange molecular
dynamics (REMD) simulation.20
All of the six (S)-methyl substituted derivatives (7a, 7b, 8a, 8b, 2, 9b) exhibited more potent inhibitory activities
as compared to the corresponding unsubstituted derivatives (4a, 4b, 5a, 5b, 1, 6b) on both OX1R and OX2R.
Comparison of the OX1R inhibitory potency of (S)-methyl substituted compound 2 with that of (R)-methyl
substituted derivative 3 suggested that placement of the (S)-methyl group into the ethyl linker played an important
role in enhancing the inhibitory potency against both OX1R and OX2R, as expected. In addition, these (S)-methyl
8

substituted derivatives showed a trend towards higher OX1R selectivity (SI) as compared to the corresponding
unsubstituted derivatives. The (S)-ethyl-substituted derivative 10 and the (S)-isopropyl-substituted 11 showed
higher OX1R selectivity than the (S)-methyl-substituted 8a, while the OX1R inhibitory potencies of 10 and 11 were
weaker than that of 8a. Among the derivatives bearing a 3-(5-fluoropyridin-2-yl)pyrazole on the right hand side
(RHS), compound 2 with an ethyl group at the amide nitrogen exerted the most potent activity on OX1R (IC50: 1.62
nM), and compound 9b was the most potent derivative (IC50: 0.767 nM) among the compounds of the
4-(5-fluoropyridin-2-yl)pyrazole class (Table 1). Interestingly, compound 2 did not show OX1R selectivity (SI: 1.0),
while compound 9b showed slight selectivity (SI: 7.7).
SAR of linkage moiety
Table 1.
Compd
R1
R2
RHS
IC50 (nM)a
SI
Compd
R1
R2
RHS
IC50 (nM)a
SI
OX1R OX2R
OX1R OX2R
4a
4b
5a
5b
1
H
H
H
H
H
H
H
H
a
b
a
b
a
b
119
908
198
1460
11.9
51.7
2.81
14.2
1.7
1.6
7a
7b
8a
8b
2
H
H
(S)-Me
(S)-Me
(S)-Me
(S)-Me
(S)-Me
(S)-Me
(R)-Me
(S)-Et
a
b
a
b
a
b
a
a
a
10.1
245
58.4
840
5.8
3.4
3.0
4.8
1.0
7.7
2.9
6.6
16
Me
Me
Et
5.54
56.1
3.22
9.06
2.1
Me
Me
Et
1.90
4.39
1.62
0.767
86.6
4.39
61.5
5.68
21.2
1.61
5.87
250
0.92
0.87
1.6
6b
Et
9b
3
Et
Et
10
11
Me
Me
29.0
952
(S)-iPr
aThe experiments were performed two to three times, and each test was performed in duplicate or triplicate.
The different selectivity (SI) of compounds and
suggested that the heteroaromatic motif on the RHS may
9b
2
have an influence on conferring selectivity for OX1R over OX2R. Therefore, we further synthesized five-membered
9

heteroaromatic derivatives and evaluated the inhibitory potencies on OX1R and OX2R, to examine the selectivity
for OX R (Table 2). Indeed, the oxadiazole derivative and tetrazole derivative showed better selectivity for
9d
9e
1
OX R over OX R (SI:
= 9.0, = 16), without impairing the OX R antagonistic activity (IC :
9e
= 1.26 nM,
9d 9e
9d
1
2
1
50
= 1.10 nM). These results encouraged us to further modify compounds
and , in which two substituents on the
9e
9d
benzamide moiety (R3) were replaced and/or the nitrogen on the 4-fluoropyridyl group on the RHS was substituted
with a carbon atom to improve the selectivity and maintain the antagonist potency for OX1R.
SAR of RHS
Table 2.
ClogPb
Compd
RHS
IC50 (nM)a
OX2R
SI
OX1R
1.62
2
1.61
5.87
22.8
11.3
17.9
1.0
7.7
12
4.2
4.0
3.6
2.8
3.4
9b
9c
9d
9e
0.767
1.93
1.26
1.10
9.0
16
b
aThe experiments were performed two to four times, and each test was performed in duplicate or triplicate. The
ClogP values were calculated using software from Daylight Chemical Information Systems, Inc.
10

Table 3 shows the inhibitory potencies for OX1R and OX2R and the OX1R selectivity (SI) of the oxadiazole
derivatives ( ,
–
). These derivatives, except for , exhibited single digit nano-molar potency for OX R. With
14
9f 12 19
1
respect to the OX R selectivity, it is worthy of note that the three derivatives
,
and
showed greater than
19
14 16
1
50-fold selectivity for OX R over OX R. As for the tetrazole derivatives (
,
–
) (Table 4), two derivatives (
9g 21 27
24
1
2
and ) exhibited single digit nano-molar potency for OX R and greater than 200-fold selectivity for OX R over
27
1
1
OX2R. Interestingly, 5-fluorobenzene and 2-methylpyridine as the R3 moiety emerged as the common structures for
enhancing the OX1R selectivity, through modifications of
and . We speculated on why these structures
9e
9d
improved the selectivity for OX1R over OX2R. According to recent publications on the crystal structures of human
OX1R21 and OX2R22 bound to suvorexant, OX1R and OX2R are highly similar, but there are two divergent residues
between the subtypes at the orthosteric pocket (OX1R to OX2R: Ser103 to Thr111 and Ala127 to Thr135).
11

SAR of acyl moiety (RHS = 4-fluoropyridyl oxadiazole ring )
Table 3.
d
ClogPb
2.8
Compd
9d
R3
X
N
IC50 (nM)a
SI
9.0
13
OX1R
OX2R
1.26
3.24
1.07
1.40
14.7
1.26
11.3
12
N
43.2
2.73
8.85
887
2.0
9f
CH
CH
N
2.6
6.3
60
3.9
13
3.1
14
2.5
15
CH
23.6
19
3.6
12

16
17
18
19
CH
CH
CH
CH
1.58
1.25
1.82
3.58
80.1
11.6
40.5
254
51
9.3
22
71
2.8
3.4
3.0
2.0
aThe experiments were performed two to three times, and each test was performed in duplicate or triplicate. bThe ClogP values were calculated using
software from Daylight Chemical Information Systems, Inc.
SAR of acyl moiety (RHS = 4-fluoropyridyl tetrazole ring )
Table 4.
e
ClogPb
3.4
Compd
9e
R3
X
N
IC50 (nM)a
SI
16
12
3.1
20
50
45
OX1R
OX2R
1.10
10.6
0.820
1.81
9.45
3.06
17.9
20
N
129
2.57
36.0
471
139
2.6
9g
CH
CH
N
4.6
21
3.8
22
3.1
23
CH
4.3
13

24
25
26
27
CH
CH
CH
CH
2.01
2.45
1.84
6.15
532
61.0
119
265
25
3.5
4.1
3.7
2.7
65
1310
213
aThe experiments were performed two to three times, and each test was performed in duplicate or triplicate. bThe ClogP values were calculated using
software from Daylight Chemical Information Systems, Inc.
To clarify the reason for the increased OX1R selectivity conferred by replacement of the 5-tolyl substituent with
5-fluorobenzene, we calculated the binding modes of
and
with OX1R and OX2R by molecular docking
23
9g
simulation using the crystal structures.23 The binding modes of the compounds to OX1R and OX2R suggest that the
5-tolyl moiety in
and 5-fluorobenzene moiety in
were directed toward the Ser103 in OX R or toward Thr111
23
1
9g
in OX2R, which represent one of the two divergent positions between the subtypes (Figure 3). Since the binding
modes of
and
to OX2R are very similar, it might be considered that the antagonistic activity of
on OX R was attenuated by electrostatic factors, resulting in improved selectivity for OX R.
9g
23
23
fluorine-substituted
2
1
More precisely, it appears that the electronegative fluorine atom on the benzamide moiety of
leads to repulsion
23
in the location of the relatively negative electrostatic potential surface generated by Thr111 in OX2R, resulting in
improved selectivity for OX1R. On the other hand, we remain unable to explain the reason for the increased OX1R
selectivity of compounds such as compound obtained by the replacement of 5-tolyl with 6-methyl-2-pyridine.
27
14

Figure 3. The predicted binding modes of compounds 9g and 23 with OX1R and OX2R.
The surface areas of OX1R and OX2R of positive, neutral and negative electrostatic potential energy are shown in
blue, white and red. Compounds, 9g (gray carbon) and 23 (yellow carbon), are shown as balls and sticks. All
residues within 5 angstroms from the compounds are shown as lines (OX1R: orange carbon, OX2R: blue carbon).
a,b) Interactions between 9g and OX1R or OX2R. The distance between the carbon of methyl group in 9g (Me9g)
and the oxygen of hydroxyl group of S103 (OS103) in OX1R was 3.56 angstroms. These between Me9g and the
oxygen of hydroxyl group of T111 (OT111) or the carbon of methyl group of T111 (MeT111) in OX2R were 3.62 or
3.73 angstroms, respectively.
c,d) Interactions between 23 and OX1R or OX2R. The distance between fluorine in 23 (F23) and OS103 was 3.87
angstroms. These between F23 and OT111 or MeT111 were 3.66 or 3.83 angstroms, respectively.
4. Conclusion
In conclusion, we investigated the SARs in detail from the structural framework of pyrazolylethylbenzamide ,
1
which was previously revealed as a DORA, and identified the potent and highly selective OX R antagonist
with
24
1
15

an IC50 of 2.01 nM and 265-fold selectivity. The results of investigation of the SARs suggested that incorporation
of a (S)-methyl group into the ethyl linker enhanced the inhibitory potencies on OX1R and OX2R with OX1R
preference, and the acyl moiety was relatively susceptible to OX1R selectivity. Especially, replacement of the
5-tolyl moiety with 5-fluorobenzene or 2-methylpyridine enhanced the selectivity, and this feature might be
associated with electrostatic factors at the divergent positions between the OX1R and OX2R subtypes by molecular
docking simulations based on the X-ray structures of the orexin receptors in the case of replacement with
5-fluorobenzene.
The present SAR findings could provide helpful information to transform potent DORAs into 1-SORAs, which
could serve as probes in research on the pharmacological functions of OX1R. Existence of a link between stress
and anxiety related behaviors24,25 and activation of the OX1R is highly likely, and future clinical studies will
hopefully provide final confirmation about the efficacy, without inducing somnolence15, with appropriate 1-SORA
compounds such as compound
.
24
5. Experimental section
5.1. Biology
5.1.1. Antagonistic activity on human OX1 and OX2 receptors
The antagonistic activities of the test compounds were determined as described previously18. Chinese hamster
ovary cells stably expressing recombinant human OX1R or human OX2R were seeded into a 96-well black/clear
bottom plate at 2.4 × 104 cells/well one day before the experiment. The cells were incubated in loading buffer
[Hank’s balanced salt solution (HBSS) (pH 7.4) containing 20 mM HEPES, 0.1% BSA, 0.2 mg/ml amaranth, 2.5
mM probenecid, 0.02% pluronic F-127 and 0.15 μM of Fluo-4AM (Invitrogen)] at 37 °C for 1 h. Then, after
removal of the loading buffer, the cells were incubated with assay buffer [HBSS (pH 7.4) containing 20 mM
HEPES, 0.1% BSA, 0.2 mg/ml amaranth, 2.5 mM probenecid], with or without the test compounds at various
concentrations, for 30 min at room temperature. The changes in the intracellular Ca2+ concentrations were
determined by monitoring the changes in fluorescence using a Functional Drug Screening System (Hamamatsu
Photonics, Shizuoka, Japan) after the application of [Ala6, 12]orexin-A (Peptide Institute, Osaka, Japan) (final
16

concentration of 0.5 nM for human OX1R or 1 nM for human OX2R).
The concentration-response curves were fitted using nonlinear regression analyses; 50% inhibitory concentration
(IC50) values were calculated using the GraphPad Prism software (version 5.04; GraphPad Software Inc., San Diego,
CA). The experiments were performed two to four times, and each test was performed in duplicate or triplicate.
5.2. Chemistry
5.2.1. General methods
All solvents and reagents were obtained from commercial suppliers and used without further purification, unless
otherwise noted. The preparative HPLC purification conditions were as follows: Gilson preparative HPLC system;
column waters ODS sunfire, 50 mm × 30 mm; eluent A, water + 0.1% CF3CO2H; eluent B, acetonitrile + 0.1%
1
CF3CO2H; 10% B up to 95% B in 12 min; Flow rate 40 mL/min. H and 13C NMR spectra were recorded on a
JOEL 600 MHz, 500 MHz, and BRUKER 400 MHz NMR spectrometers, and all chemical shifts are reported in
parts per million (ppm) relative to tetramethylsilane (TMS,  0.00 ppm), used as the internal standard.
High-resolution mass spectral data were acquired by a Shimadzu LCMS-IT-TOF equipped with an ESI/APCI dual
ion source. Mass spectra (MS) were recorded on a Shimadzu LCMS-IT-TOF or Agilent LC-MS equipped with an
ESI/APCI dual ion source. The LC-MS was performed under the following conditions: Agilent 1290 infinity and
Agilent 6150; column Waters Acquity CSH C18, 1.7 μm, 2.1 mm × 50 mm; eluent A, water + 0.1% formic acid;
eluent B, acetonitrile + 0.1% formic acid; 20–99% B in 1.2 min, 99% B in 0.2 min; flow rate 0.8 ml/min; UV
detection (λ = 254 nm).
The N-alkylated benzamide orexin receptor antagonists described in this paper exist in multiple conformations as
a result of hindered rotations that were slow on the NMR timescale. The 1H and 13C NMR spectra of compounds 2,
3, 5b, 8a, 8b, 9b–9g, and 10–27 consisted of broad and complicated multiplets, analysis in detail of the coupling
constants of which was difficult. Thus, the NMR resonances of these compounds are not listed in numerical format.
1
Instead, we have included pictures of the H and 13C NMR spectra at 25 °C of these compounds in the
Supplementary data. 1, 4a, 5a, 6b, 29a, and 29b were prepared according to procedures reported in the literature.21
17

5.2.2. tert-Butyl {(2S)-1-[3-(5-fluoropyridin-2-yl)-1H-pyrazol-1-yl]propan-2-yl}carbamate (30a)
To a solution of 28 (10.0 g, 57.1 mmol) in THF (114 mL) was added Et3N (11.9 mL, 85.6 mmol) and
methanesulfonyl chloride (4.70 mL, 59.9 mmol) at 0 °C. The mixture was stirred at 0 °C for 1 h and then filtered
through
a
pad of KC flock®. The filtrate was concentrated under reduced pressure to obtain
(2S)-2-[(tert-butoxycarbonyl)amino]propyl methanesulfonate as a colorless oil. To a solution of the
methanesulfonate in DMF (100 mL) was added 5-fluoro-2-(1H-pyrazol-3-yl)pyridine 29a (8.40 g, 51.4 mmol) and
Cs2CO3 (37.2 g, 0.110 mol) at room temperature. After the mixture was stirred at 80 °C for 3 h, water was added,
and the reaction mixture was extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4,
filtered, and concentrated under reduced pressure. The resulting solid was washed with EtOAc to obtain the title
compound 30a as a colorless solid (0.50 g, 3% yield over 2 steps). 1H NMR (400 MHz, CDCl3) δ 8.46 (d, J = 2.81
Hz, 1H), 7.95 (dd, J = 4.46, 8.74 Hz, 1H), 7.39–7.47 (m, 2H), 6.84 (d, J = 2.20 Hz, 1H), 4.86 (br s, 1H), 4.18–4.33
(m, 2H), 4.01–4.14 (m, 1H), 1.42 (s, 9H), 1.16 (d, J = 6.72 Hz, 3H); MS (ESI/APCI dual): m/z 321 [M+H]+.
5.2.3. tert-Butyl {(2S)-1-[4-(5-fluoropyridin-2-yl)-1H-pyrazol-1-yl]propan-2-yl}carbamate (30b)
The title compound was prepared from 5-fluoro-2-(1H-pyrazol-4-yl)pyridine 29b according to the procedure as
described for compound 30a (44% yield in 2 steps, colorless solid). 1H NMR (400 MHz, CDCl3) δ 8.41 (d, J = 2.69
Hz, 1H), 7.93 (s, 1H), 7.89 (s, 1H), 7.34–7.48 (m, 2H), 4.96 (br s, 1H), 4.14–4.35 (m, 2H), 3.98–4.13 (m, 1H), 1.43
(s, 9H), 1.14 (d, J = 6.72 Hz, 3H); MS (ESI/APCI dual): m/z 321 [M+H]+.
5.2.4.
N-{(2S)-1-[3-(5-Fluoropyridin-2-yl)-1H-pyrazol-1-yl]propan-2-yl}-5-methyl-2-(2H-1,2,3-triazol-2-yl)benzami
de (7a)
To a solution of 30a (0.50 g, 1.6 mmol) in EtOAc (4.0 mL) was added hydrogen chloride in EtOAc (4 mol/L, 3.9
mL, 0.016 mol) at room temperature, and the resulting mixture was stirred for 15 h. The organic solvent was
removed under reduced pressure to obtain the primary amine as a colorless amorphous.
A mixture of the primary amine, 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid 31 (0.33 g, 1.6 mmol),
18

N,N-diisopropylethylamine
(DIPEA,
2.5
mL,
14.7
mmol),
and
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 0.91 g,
2.4 mmol) in DMF (3.2 mL) was stirred at room temperature for 17 h. The reaction was quenched by the addition
of water, and the mixture was extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4,
filtered, and concentrated under reduced pressure. The resulting residue was purified by preparative HPLC to obtain
the title compound 7a as a colorless solid (0.18 g, 28% over 2 steps). HRMS (ESI/APCI dual) for C21H21FN7O
1
[M+H]+, calcd: 406.1786, found: 406.1785; LC-MS t = 0.78 min, [M+H]+ = 406; H NMR (500 MHz, CDCl3) δ
8.45 (d, J = 2.68 Hz, 1H), 7.87 (dd, J = 4.59, 8.79 Hz, 1H), 7.74 (s, 2H), 7.64 (d, J = 8.03 Hz, 1H), 7.50 (d, J = 2.29
Hz, 1H), 7.37–7.45 (m, 2H), 7.32–7.36 (m, 1H), 6.83 (d, J = 2.68 Hz, 1H), 6.44 (br d, J = 7.64 Hz, 1H), 4.45–4.54
(m, 1H), 4.30 (dd, J = 5.35, 13.38 Hz, 1H), 4.21–4.27 (m, 1H), 2.37 (s, 3H), 1.12 (d, J = 6.88 Hz, 3H); 13C NMR
(125 MHz, CDCl3) δ 167.0, 159.8, 157.8, 151.3, 148.5, 148.5, 139.0, 137.4, 137.2, 135.5, 134.8, 132.2, 131.3,
131.0, 129.5, 124.3, 123.5, 123.3, 121.0, 121.0, 104.3, 55.8, 46.2, 20.9, 17.4.
5.2.5.
N-{(2S)-1-[4-(5-Fluoropyridin-2-yl)-1H-pyrazol-1-yl]propan-2-yl}-5-methyl-2-(2H-1,2,3-triazol-2-yl)benzami
de (7b)
The title compound was prepared from 30b according to the procedure as described for compound 7a (60% yield
in 2 steps, colorless solid). HRMS (ESI/APCI dual) for C21H21FN7O [M+H]+, calcd: 406.1786, found: 406.1767;
LC-MS t = 0.74 min, [M+H]+ = 406; 1H NMR (500 MHz, CDCl3) δ 8.40 (d, J = 3.06 Hz, 1H), 7.96 (s, 1H), 7.89 (s,
1H), 7.76 (s, 2H), 7.65 (d, J = 8.03 Hz, 1H), 7.32–7.45 (m, 4H), 6.44 (br d, J = 7.64 Hz, 1H), 4.43–4.52 (m, 1H),
4.31 (dd, J = 5.00, 13.40 Hz, 1H), 4.25 (dd, J = 5.00, 13.80 Hz, 1H), 2.41 (s, 3H), 1.12 (d, J = 6.88 Hz, 3H); 13C
NMR (125 MHz, CDCl3) δ 167.1, 158.9, 156.9, 148.2, 148.1, 139.0, 137.7, 137.5, 135.6, 134.8, 131.3, 130.9, 129.5,
129.1, 124.2, 123.6, 123.4, 122.7, 120.2, 120.2, 55.8, 46.2, 21.0, 17.3.
5.2.6.
N-{(2S)-1-[3-(5-Fluoropyridin-2-yl)-1H-pyrazol-1-yl]propan-2-yl}-N,5-dimethyl-2-(2H-1,2,3-triazol-2-yl)benz
19

amide (8a)
NaH (55% in mineral oil, 3.9 mg, 0.089 mmol) was added to a solution of 7a (0.030 g, 0.074 mmol) in DMF (0.74
mL), and the mixture was stirred at 80 °C for 1 h. After cooling in an ice bath, MeI (5.1 μL, 0.081 mmol) was
added dropwise, and the resulting mixture was stirred at room temperature for 1 h. The reaction was quenched by
the addition of water, and the mixture was extracted with EtOAc. The organic layer was washed with brine, dried
over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was purified by column
chromatography (20–100% EtOAc in hexanes) to obtain the title compound 8a as a colorless amorphous (0.017 g,
55%). HRMS (ESI/APCI dual) for C22H23FN7O [M+H]+, calcd: 420.1943, found: 420.1941; LC-MS t = 0.80–0.89
min, [M+H]+ = 420. Please see the Supplementary data for pictures of 500 MHz 1H and 125 MHz 13C NMR spectra
in CDCl3 at 25 °C.
5.2.7. Preparation of Compounds 8b, 2, and 9b
These compounds were synthesized from their corresponding precursors according to the procedure as described
for compound 8a.
5.2.7.1.
N-{(2S)-1-[4-(5-Fluoropyridin-2-yl)-1H-pyrazol-1-yl]propan-2-yl}-N,5-dimethyl-2-(2H-1,2,3-triazol-2-yl)benz
amide (8b)
The title compound was prepared from 7b (55% yield, colorless amorphous). HRMS (ESI/APCI dual) for
C22H23FN7O [M+H]+, calcd: 420.1943, found: 420.1941; LC-MS t = 0.75–0.83 min, [M+H]+ = 420. Please see the
Supplementary data for pictures of 500 MHz 1H and 125 MHz 13C NMR spectra in CDCl3 at 25 °C.
5.2.7.2.
N-Ethyl-N-{(2S)-1-[3-(5-fluoropyridin-2-yl)-1H-pyrazol-1-yl]propan-2-yl}-5-methyl-2-(2H-1,2,3-triazol-2-yl)
benzamide (2)
The title compound was prepared from 7a (73% yield, colorless amorphous). HRMS (ESI/APCI dual) for
20

C23H25FN7O [M+H]+, calcd: 434.2099, found: 434.2099; LC-MS t = 0.91 min, [M+H]+ = 434. Please see the
Supplementary data for pictures of 500 MHz 1H and 125 MHz 13C NMR spectra in CDCl3 at 25 °C.
5.2.7.3.
N-Ethyl-N-{(2S)-1-[4-(5-fluoropyridin-2-yl)-1H-pyrazol-1-yl]propan-2-yl}-5-methyl-2-(2H-1,2,3-triazol-2-yl)
benzamide (9b)
The title compound was prepared from 7b (88% yield, colorless solid). HRMS (ESI/APCI dual) for C23H25FN7O
[M+H]+, calcd: 434.2099, found: 434.2096; LC-MS t = 0.90–0.95 min, [M+H]+ = 434. Please see the
Supplementary data for pictures of 500 MHz 1H and 125 MHz 13C NMR spectra in CDCl3 at 25 °C.
5.2.8. tert-Butyl {2-[4-(5-fluoropyridin-2-yl)-1H-pyrazol-1-yl]ethyl}carbamate (33)
Compound 32 (0.50 g, 2.2 mmol) and Cs2CO3 (1.5 g, 4.5 mmol) were added to a solution of 29b (0.36 g, 2.2
mmol) in DMF (2 mL) at room temperature, and the resulting mixture was stirred at 80 °C for 3 h. After cooling to
room temperature, water was added, and the mixture was extracted with EtOAc. The organic layer was washed
with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was purified
by column chromatography (20–80% EtOAc in hexanes) to obtain the title compound 33 as a colorless solid (0.47
1
g, 69%). H NMR (400 MHz, CDCl3) δ 8.41 (d, J = 2.69 Hz, 1H), 7.94 (s, 1H), 7.88 (s, 1H), 7.34–7.47 (m, 2H),
4.94 (br s, 1H), 4.27 (t, J = 5.40 Hz, 2H), 3.57–3.68 (m, 2H), 1.43 (s, 9H); MS (ESI/APCI dual): m/z 307 [M+H]+.
5.2.9.
(4b)
N-{2-[4-(5-Fluoropyridin-2-yl)-1H-pyrazol-1-yl]ethyl}-5-methyl-2-(2H-1,2,3-triazol-2-yl)benzamide
The title compound was prepared from 33 according to the procedure as described for compound 7a (42% yield in
2 steps, colorless solid). HRMS (ESI/APCI dual) for C20H19FN7O [M+H]+, calcd: 392.1630, found: 392.1619;
LC-MS t = 0.69 min, [M+H]+ = 392; 1H NMR (500 MHz, CDCl3) δ 8.39 (d, J = 2.68 Hz, 1H), 7.95 (s, 1H), 7.89 (s,
1H), 7.75 (s, 2H), 7.63 (d, J = 8.03 Hz, 1H), 7.35–7.43 (m, 3H), 7.31–7.34 (m, 1H), 6.45 (br t, J = 5.35 Hz, 1H),
4.33 (t, J = 5.35 Hz, 2H), 3.83 (dd, J = 6.12, 11.47 Hz, 1H), 2.38 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 168.0,
21

158.9, 156.9, 148.1, 148.1, 138.9, 137.9, 137.7, 137.5, 135.6, 134.8, 131.4, 130.5, 129.5, 128.8, 124.2, 123.6, 123.5,
122.6, 120.2, 120.2, 51.0, 40.1, 20.9.
5.2.10.
N-{2-[4-(5-Fluoropyridin-2-yl)-1H-pyrazol-1-yl]ethyl}-N,5-dimethyl-2-(2H-1,2,3-triazol-2-yl)benzamide (5b)
The title compound was prepared from 4b according to the procedure as described for compound 8a (44% yield,
colorless solid). HRMS (ESI/APCI dual) for C21H21FN7O [M+H]+, calcd: 406.1786, found: 406.1784; LC-MS t =
1
0.80 min, [M+H]+ = 406. Please see the Supplementary data for pictures of 500 MHz H and 125 MHz 13C NMR
spectra in CDCl3 at 25 °C.
5.2.11. Preparation of Compounds 37, 38, and 39
These compounds were from their corresponding precursors according to the procedure as described for
compound 30a.
5.2.11.1. tert-Butyl {(2R)-1-[3-(5-fluoropyridin-2-yl)-1H-pyrazol-1-yl]propan-2-yl}carbamate (37)
1
The title compound was prepared from 34 (20% yield in 2steps, colorless solid). H NMR (600 MHz, CDCl3) δ
8.47 (d, J = 2.89 Hz, 1H), 7.95 (dd, J = 4.54, 8.67 Hz, 1H), 7.41–7.46 (m, 2H), 6.84 (d, J = 2.06 Hz, 1H), 4.87 (br s,
1H), 4.19–4.32 (m, 2H), 4.02–4.10 (m, 1H), 1.42 (s, 9H), 1.16 (d, J = 7.02 Hz, 3H).
5.2.11.2. tert-Butyl {(2S)-1-[3-(5-fluoropyridin-2-yl)-1H-pyrazol-1-yl]butan-2-yl}carbamate (38)
1
The title compound was prepared from 35 (33% yield in 2 steps, colorless solid). H NMR (400 MHz, CDCl3) δ
8.46 (d, J = 2.69 Hz, 1H), 7.95 (dd, J = 4.52, 8.80 Hz, 1H), 7.39–7.47 (m, 2H), 6.83 (d, J = 2.32 Hz, 1H), 4.84 (br d,
J = 5.75 Hz, 1H), 4.22–4.33 (m, 2H), 3.77–3.92 (m, 1H), 1.62–1.71 (m, 1H), 1.47–1.58 (m, 1H), 1.42 (s, 9H), 0.97
(t, J = 7.40 Hz, 3H); MS (ESI/APCI dual): m/z 335 [M+H]+.
5.2.11.3. {(2S)-1-[3-(5-Fluoropyridin-2-yl)-1H-pyrazol-1-yl]-3-methylbutan-2-yl}carbamate (39)
22

1
The title compound was prepared from 36 (22% yield in 2 steps, colorless solid). H NMR (400 MHz, CDCl3) δ
8.46 (d, J = 2.81 Hz, 1H), 7.94 (dd, J = 4.46, 8.74 Hz, 1H), 7.34–7.51 (m, 2H), 6.82 (d, J = 2.20 Hz, 1H), 4.82 (br d,
J = 9.54 Hz, 1H), 4.29 (d, J = 5.40 Hz, 2H), 3.69–3.93 (m, 1H), 1.65–1.87 (m, 1H), 1.38 (s, 9H), 1.02 (d, J = 6.72
Hz, 3H), 0.95 (d, J = 6.85 Hz, 3H); MS (ESI/APCI dual): m/z 349 [M+H]+.
5.2.12. (2R)-N-Ethyl-1-[3-(5-fluoropyridin-2-yl)-1H-pyrazol-1-yl]propan-2-amine hydrochloride (40)
NaH (60% in mineral oil, 0.056 g, 1.4 mmol) was added to a solution of 37 (0.30 g, 0.94 mmol) in DMF (5.0 mL),
and the mixture was stirred at room temperature for 30 min. EtI (90 μL, 1.1 mmol) was added dropwise to the
reaction mixture under an ice bath and the mixture was stirred at room temperature for 1 day. The reaction was
quenched by the addition of water, and the mixture was extracted with EtOAc. The organic layer was washed with
brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The resulting residue was purified by
column
chromatography
(20–100%
EtOAc
in
hexanes)
to
obtain
tert-butyl
{(2R)-1-[3-(5-fluoropyridin-2-yl)-1H-pyrazol-1-yl]propan-2-yl}carbamate as a colorless oil (0.26 g, 80%).
To the solution of tert-butyl {(2R)-1-[3-(5-fluoropyridin-2-yl)-1H-pyrazol-1-yl]propan-2-yl}carbamate (0.26 g,
0.75 mmol) in MeOH (5.0 mL) was added hydrogen chloride in EtOAc (4 mol/L, 5.0 mL, 20 mmol) at room
temperature, and the resulting mixture was stirred for 18 h. The precipitate was then collected by filtration to obtain
1
the title compound 40 as a colorless solid (0.17 g, 71% yield). H NMR (400 MHz, DMSO-d6) δ 9.15 (br s, 2H),
8.59 (d, J = 2.93 Hz, 1H), 8.01 (dd, J = 4.58, 8.86 Hz, 1H), 7.96 (d, J = 2.32 Hz, 1H), 7.81 (dt, J = 2.93, 8.80 Hz,
1H), 6.85 (d, J = 2.20 Hz, 1H), 4.60 (dd, J = 5.01, 14.18 Hz, 1H), 4.44 (dd, J = 6.60, 14.18 Hz, 1H), 3.63–3.78 (m,
1H), 2.89–3.12 (m, 2H), 1.25 (t, J = 7.15 Hz, 3H), 1.19 (d, J = 6.60 Hz, 3H); MS (ESI/APCI dual): m/z 249
[M+H]+.
5.2.13. Preparation of Compounds 41 and 42
These compounds were prepared from their corresponding precursors according to the procedure as described for
compound 40.
23

5.2.13.1. (2S)-1-[3-(5-Fluoropyridin-2-yl)-1H-pyrazol-1-yl]-N-methylbutan-2-amine hydrochloride (41)
The title compound was prepared from 38 (28% yield in 2 steps, colorless oil). 1H NMR (400 MHz, DMSO-d6) δ
9.04 (br s, 2H), 8.58 (d, J = 2.93 Hz, 1H), 8.06 (dd, J = 4.46, 8.86 Hz, 1H), 7.98 (d, J = 2.32 Hz, 1H), 7.80 (dt, J =
2.93, 8.74 Hz, 1H), 6.84 (d, J = 2.32 Hz, 1H), 4.47–4.64 (m, 2H), 3.41–3.64 (m, J = 5.10, 8.00 Hz, 1H), 2.60 (t, J =
5.26 Hz, 3H), 1.61–1.74 (m, 1H), 1.49 (quind, J = 7.45, 14.69 Hz, 1H), 0.97 (t, J = 7.52 Hz, 3H); MS (ESI/APCI
dual): m/z 249 [M+H]+.
5.2.13.2. (2S)-1-[3-(5-Fluoropyridin-2-yl)-1H-pyrazol-1-yl]-N,3-dimethylbutan-2-amine hydrochloride (42)
The title compound was prepared from 39 (26% yield in 2 steps, colorless oil). 1H NMR (400 MHz, DMSO-d6) δ
9.41 (br s, 1H), 9.10 (br s, 1H), 8.59 (d, J = 2.20 Hz, 1H), 7.98–8.13 (m, 2H), 7.73–7.92 (m, 1H), 6.80–6.93 (m,
1H), 4.45–4.56 (m, 2H), 3.45–3.61 (m, 1H), 2.45 (br t, J = 5.01 Hz, 3H), 1.99–2.23 (m, 1H), 1.00 (dd, J = 5.20,
6.91 Hz, 6H); MS (ESI/APCI dual): m/z 263 [M+H]+.
5.2.14. Preparation of Compounds 3, 10, and 11
These compounds were prepared from their corresponding precursors according to the procedure as described for
compound 7a.
5.2.14.1.
N-Ethyl-N-{(2R)-1-[3-(5-fluoropyridin-2-yl)-1H-pyrazol-1-yl]propan-2-yl}-5-methyl-2-(2H-1,2,3-triazol-2-yl)
benzamide (3)
The title compound was prepared from 40 and 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid (40% yield,
colorless amorphous). HRMS (ESI/APCI dual) for C23H25FN7O [M+H]+, calcd: 434.2099, found: 434.2100;
LC-MS t = 0.92 min, [M+H]+ = 434. Please see the Supplementary data for pictures of 500 MHz 1H and 125 MHz
13C NMR spectra in CDCl3 at 25 °C.
5.2.14.2.
24

N-{(2S)-1-[3-(5-Fluoropyridin-2-yl)-1H-pyrazol-1-yl]butan-2-yl}-N,5-dimethyl-2-(2H-1,2,3-triazol-2-yl)benza
mide (10)
The title compound was prepared from 41 and 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid (60% yield,
colorless amorphous). HRMS (ESI/APCI dual) for C23H25FN7O [M+H]+, calcd: 434.2099, found: 434.2096;
LC-MS t = 0.87–0.95 min, [M+H]+ = 434. Please see the Supplementary data for pictures of 500 MHz 1H and 125
MHz 13C NMR spectra in CDCl3 at 25 °C.
5.2.14.3.
N-{(2S)-1-[3-(5-Fluoropyridin-2-yl)-1H-pyrazol-1-yl]-3-methylbutan-2-yl}-N,5-dimethyl-2-(2H-1,2,3-triazol-2
-yl)benzamide (11)
The title compound was prepared from 42 and 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid (76% yield,
colorless amorphous). HRMS (ESI/APCI dual) for C24H27FN7O [M+H]+, calcd: 448.2256, found: 448.2265;
LC-MS t = 1.00 min, [M+H]+ = 448. Please see the Supplementary data for pictures of 500 MHz 1H and 125 MHz
13C NMR spectra in CDCl3 at 25 °C.
5.2.15. tert-Butyl {(2S)-1-[4-(5-fluoropyridin-2-yl)-1H-1,2,3-triazol-1-yl]propan-2-yl}carbamate (30c)
To a solution of 28 (19.9 g, 0.110 mol) in CHCl3 (100 mL) were added Et3N (31.4 mL, 0.230 mol) and
methanesulfonyl chloride (13.2 mL, 0.170 mol), and the mixture was stirred at room temperature for 1 h. The
reaction was quenched by the addition of saturated NaHCO3 aqueous solution, and the mixture was extracted with
CHCl3. The organic layer was concentrated to obtain (2S)-2-[(tert-butoxycarbonyl)amino]propyl methanesulfonate
as a colorless oil.
To a solution of the methanesulfonate in DMF (100 mL) was added NaN3 (8.10 g, 0.120 mol), and the resulting
mixture was stirred at 80 °C for 3 h. The reaction was quenched by the addition of water, and the mixture was
extracted with EtOAc. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure.
The resulting residue was purified by column chromatography (20–100% EtOAc in hexanes) to obtain tert-butyl
N-[(2S)-1-azidopropan-2-yl]carbamate as a colorless oil (18.8 g, 83% yield).
25

tert-Butyl N-[(2S)-1-azidopropan-2-yl]carbamate (0.30 g, 2.5 mmol) and 2-ethynyl-5-fluoropyridine (0.50 g, 2.5
mmol) were suspended in a mixture of 4-methylpiperidine (2.0 mL) and DMF (8.0 mL). CuI (0.024 g, 0.12 mmol)
and L-ascorbic acid (0.087 g, 0.50 mmol) were added to the mixture, and the mixture was stirred at 80 °C for 2 h.
After cooling to room temperature, water was added, and the mixture was extracted with EtOAc. The organic layer
was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue
was purified by column chromatography (20–100% EtOAc in hexanes) to obtain the title compound 30c as a
1
colorless solid (0.58 g, 73% yield). H NMR (600 MHz, CDCl3) δ 8.44 (d, J = 2.89 Hz, 1H), 8.18 (dd, J = 4.33,
8.46 Hz, 1H), 8.10 (s, 1H), 7.50 (dt, J = 2.89, 8.46 Hz, 1H), 4.67 (br s, 1H), 4.42–4.61 (m, 2H), 4.05–4.19 (m, 1H),
1.42 (s, 9H), 1.18 (d, J = 6.61 Hz, 3H); MS (ESI/APCI dual): m/z 322 [M+H]+.
5.2.16. tert-Butyl {(2S)-1-[5-(4-fluorophenyl)-1,2,4-oxadiazol-3-yl]propan-2-yl}carbamate (30f)
To a solution of 28 (5.0 g, 29 mmol) in THF (50 mL) were added Et3N (7.0 mL, 50 mmol) and methanesulfonyl
chloride (2.1 mL, 27 mmol), in an ice bath. After this mixture was stirred at 0 °C for 15 min, the mixture was
filtered through a pad of KC flock®. The filtrate was concentrated under reduced pressure to obtain
(2S)-2-[(tert-butoxycarbonyl)amino]propyl methanesulfonate as a colorless oil.
The mixture of sodium cyanide (1.6 g, 32 mmol) in DMF (29 mL) was stirred at 35 °C for 30 min. To the mixture
was added tetrabuthylammonium bromide (6.9 mL, 25 mmol). After this mixture was stirred for 2 h, a solution of
the methanesulfonate in DMF (2.5 mL) was added to the mixture. The resulting mixture was stirred at 50 °C for 5 h.
The reaction was quenched by the addition of saturated NaHCO3 aqueous solution, and the mixture was extracted
with EtOAc. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced
pressure to obtain tert-butyl N-[(1S)-2-cyano-1-methyl-ethyl]carbamate (3.2 g, 60% yield over 2 steps) as a
colorless solid.
To a mixture of the above carbamate (1.5 g, 8.1 mmol) in EtOH (8.0 mL) was added hydroxylamine (50% in
water, 0.96 mL, 16 mmol) at room temperature, and the mixture was stirred at 90 °C for 5 h. After cooling to room
temperature, the mixture was concentrated under reduced pressure, water was added, and the mixture was extracted
with EtOAc. The organic layer was washed with brine and concentrated under reduced pressure to obtain the crude
26

oil, which was used without further purification. To a mixture of the crude product and 4-fluorobenzoic acid (1.3 g,
9.0 mmol) in DMF (3.0 mL) was added 1,1'-carbonyldiimidazole (1.6 g, 9.8 mmol), and the resulting mixture was
stirred at 90 °C for 13 h. After adding a saturated aqueous solution of NaHCO3 to the mixture, the resulting
precipitate was filtered to obtain the title compound 30f as a colorless solid (1.4 g, 55% yield in 2 steps).
1H NMR (400 MHz, CDCl3) δ 8.09–8.18 (m, 2H), 7.17–7.25 (m, 2H), 4.89 (br s, 1H), 4.12–4.28 (m, 1H),
2.93–3.01 (m, 2H), 1.43 (s, 9H), 1.23 (d, J = 6.72 Hz, 3H); MS (ESI/APCI dual): m/z 322 [M+H]+.
5.2.17. tert-Butyl {(2S)-1-[5-(5-fluoropyridin-2-yl)-1,2,4-oxadiazol-3-yl]propan-2-yl}carbamate (30d)
The title compound was prepared from 5-fluoro-2-pyridinecarboxylic acid according to the procedure as described
for compound 30f (42% yield in 4 steps, colorless amorphous). 1H NMR (600 MHz, CDCl3) δ 8.67 (d, J = 2.89 Hz,
1H), 8.26 (dd, J = 4.13, 8.26 Hz, 1H), 7.61 (dt, J = 2.89, 8.26 Hz, 1H), 4.82 (br s, 1H), 4.15–4.29 (m, 1H),
2.88–3.13 (m, 2H), 1.42 (s, 9H), 1.25 (d, J = 6.61 Hz, 3H); MS (ESI/APCI dual): m/z 345 [M+Na]+.
5.2.18. 5-Fluoro-2-(1H-1,2,3,4-tetrazol-5-yl)pyridine (29e)
To a solution of 2-cyano-5-fluoropyridine (2.0 g, 0.016 mol) in water (33 mL) were added NaN3 (1.2 g, 0.019
mol) and ZnBr2 (3.7 g, 0.016 mol), and the resulting mixture was stirred at 110 °C for 3 h. After cooling to room
temperature, hydrochloric acid (1.2 mol/L, 66 mL, 0.079 mol) and EtOAc (70 mL) were added, and the mixture
was stirred for 1 h. The mixture was extracted with EtOAc, and the organic layer was dried over Na2SO4, filtered,
and concentrated under reduced pressure to obtain the title compound 29e as a colorless solid (1.5 g, 56% yield). 1H
NMR (400 MHz, CD3OD) δ 8.67 (d, J = 2.93 Hz, 1H), 8.31 (dd, J = 4.22, 8.62 Hz, 1H), 7.85 (dt, J = 2.87, 8.53 Hz,
1H); MS (ESI/APCI dual): m/z 166 [M+H]+.
5.2.19. Preparation of Compounds 30e and 30g
These compounds were prepared from the corresponding precursors according to the procedure as described for
compound 30a.
27

5.2.19.1. tert-Butyl {(2S)-1-[5-(5-fluoropyridin-2-yl)-2H-tetrazol-2-yl]propan-2-yl}carbamate (30e)
The title compound was prepared from 29e (20% yield in 2 steps, colorless solid). 1H NMR (400 MHz, CDCl3) δ
8.64 (d, J = 2.69 Hz, 1H), 8.27 (dd, J = 4.40, 8.68 Hz, 1H), 7.58 (dt, J = 2.87, 8.28 Hz, 1H), 4.64–4.88 (m, 3H),
4.22–4.40 (m, 1H), 1.40 (s, 9H), 1.23 (d, J = 6.85 Hz, 3H); MS (ESI/APCI dual): m/z 323 [M+H]+.
5.2.19.2. tert-Butyl {(2S)-1-[5-(4-fluorophenyl)-2H-tetrazol-2-yl]propan-2-yl}carbamate (30g)
The title compound was prepared from 5-(4-fluorophenyl)-1H-tetrazole (38% yield in 2 steps, colorless solid). 1H
NMR (400 MHz, CDCl3) δ 8.10–8.19 (m, 2H), 7.13–7.23 (m, 2H), 4.62–4.85 (m, 3H), 4.20–4.38 (m, 1H), 1.41 (s,
9H), 1.20 (d, J = 6.85 Hz, 3H); MS (ESI/APCI dual): m/z 322 [M+H]+.
5.2.20. (2S)-N-Ethyl-1-[4-(5-fluoropyridin-2-yl)-1H-1,2,3-triazol-1-yl]propan-2-amine (43c)
To a solution of 30c (0.27 g, 0.85 mmol) in DMF (10 mL) was added NaH (60% in mineral oil, 0.047 g, 1.2
mmol), and the resulting mixture was stirred at room temperature for 30 min. EtI (0.081 mL, 1.0 mmol) was added
dropwise to the reaction solution and the mixture was stirred at room temperature for 2 h. The reaction was
quenched by the addition of water, and the mixture was extracted with EtOAc. The organic layer was washed with
brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was purified by
column chromatography (20–100% EtOAc in hexanes) to obtain a crude oil (0.31 g, quant).
To the solution of the crude product (0.31 g, 0.88 mmol) in MeOH (5.0 mL) was added hydrogen chloride in
dioxane (4 mol/L, 2.0 mL, 8.0 mmol) at room temperature, and the resulting mixture was stirred for 15 h. The
reaction mixture was neutralized with a saturated aqueous solution of NaHCO3, and the mixture was extracted with
CHCl3. The organic layer was washed with water, dried over Na2SO4, filtered, and concentrated under reduced
1
pressure to obtain the title compound 43c as colorless oil (0.25 g, quant.). H NMR (600 MHz, CDCl3) δ 8.44 (d, J
= 2.89 Hz, 1H), 8.20 (dd, J = 4.54, 8.67 Hz, 1H), 8.16 (s, 1H), 7.50 (dt, J = 2.89, 8.46 Hz, 1H), 4.37 (dd, J = 1.86,
5.57 Hz, 2H), 3.18–3.26 (m, 1H), 2.70–2.77 (m, 1H), 2.66 (qd, J = 7.16, 11.15 Hz, 1H), 1.11 (d, J = 6.61 Hz, 3H),
1.09 (t, J = 8.67 Hz, 3H).
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5.2.21. Preparation of Compounds 43d, 43e, 43f, and 43g
These compounds were prepared from the corresponding precursors according to the procedure as described for
compound 43c.
5.2.21.1. (2S)-N-Ethyl-1-[5-(5-fluoropyridin-2-yl)-1,2,4-oxadiazol-3-yl]propan-2-amine (43d)
The title compound was prepared from 30d (13% yield in 2 steps, colorless solid). 1H NMR (600 MHz, CDCl3) δ
8.68 (d, J = 2.89 Hz, 1H), 8.25 (dd, J = 4.54, 8.67 Hz, 1H), 7.59–7.64 (m, 1H), 3.24–3.31 (m, 1H), 3.01 (dd, J =
5.99, 14.66 Hz, 1H), 2.87 (dd, J = 6.61, 14.45 Hz, 1H), 2.69–2.78 (m, 2H), 1.18 (d, J = 5.78 Hz, 3H), 1.13 (t, J =
7.22 Hz, 3H); MS (ESI/APCI dual): m/z 251 [M+H]+.
5.2.21.2. (2S)-N-Ethyl-1-[5-(5-fluoropyridin-2-yl)-2H-tetrazol-2-yl]propan-2-amine (43e)
1
The title compound was prepared from 30e (65% yield in 2 steps, colorless oil). H NMR (400 MHz, CDCl3) δ
8.64 (d, J = 2.81 Hz, 1H), 8.29 (dd, J = 4.40, 8.68 Hz, 1H), 7.55–7.62 (m, 1H), 4.71 (dd, J = 6.60, 13.57 Hz, 1H),
4.63 (dd, J = 6.11, 14.31 Hz, 1H), 3.37–3.47 (m, 1H), 2.59–2.79 (m, 2H), 1.15 (d, J = 6.48 Hz, 3H), 1.09 (t, J =
7.09 Hz, 3H); MS (ESI/APCI dual): m/z 251 [M+H]+.
5.2.21.3. (2S)-N-Ethyl-1-[5-(4-fluorophenyl)-1,2,4-oxadiazol-3-yl]propan-2-amine hydrochloride (43f)
The title compound was prepared from 30f (40% yield in 2 steps, colorless solid). 1H NMR (400 MHz, CDCl3) δ
9.88 (br s, 1H), 8.07–8.21 (m, 2H), 7.14–7.25 (m, 2H), 3.72–3.89 (m, 1H), 3.60 (dd, J = 4.16, 15.04 Hz, 1H), 3.32
(dd, J = 9.41, 15.04 Hz, 1H), 3.10–3.27 (m, 2H), 1.52–1.62 (m, 6H); MS (ESI/APCI dual): m/z 250 [M+H]+.
5.2.21.4. (2S)-N-Ethyl-1-[5-(4-fluorophenyl)-2H-tetrazol-2-yl]propan-2-amine hydrochloride (43g)
The title compound was prepared from 30g (91% yield in 2 steps, colorless solid). 1H NMR (400 MHz, CDCl3) δ
10.10 (br s, 1H), 8.10–8.16 (m, 2H), 7.13–7.23 (m, 2H), 5.38 (dd, J = 4.71, 14.00 Hz, 1H), 5.11 (dd, J = 8.01, 14.00
Hz, 1H), 3.90–4.04 (m, 1H), 3.21–3.32 (m, 1H), 3.09–3.20 (m, 1H), 1.56 (t, J = 7.21 Hz, 3H), 1.51 (d, J = 6.60 Hz,
3H); MS (ESI/APCI dual): m/z 250 [M+H]+.
29